Beta-amyloid precursor protein cleavage enzyme 1 (BACE1) and beta-amyloid precursor protein cleavage enzyme 2 (BACE2), members of aspartyl protease family, are close homologues and have high similarity in their protein crystal structures. However, their enzymatic properties differ leading to disparate clinical consequences. In order to identify the residues that are responsible for such differences, we used evolutionary trace (ET) method to compare the amino acid conservation patterns of BACE1 and BACE2 in several mammalian species. We found that, in BACE1 and BACE2 structures, most of the ligand binding sites are conserved which indicate their enzymatic property of aspartyl protease family members. The other conserved residues are more or less randomly localized in other parts of the structures. Four group-specific residues were identified at the ligand binding site of BACE1 and BACE2. We postulated that these residues would be essential for selectivity of BACE1 and BACE2 biological functions and could be sites of interest for the design of selective inhibitors targeting either BACE1 or BACE2. Members of aspartyl protease family are known to be associated with some pathological states such as breast cancer and pruritic inflammatory skin disease . Furtherβ) peptide by BACE1 leading to the preliminary constituent of amyloid plaques in the brains of individuals with the disease ..9].Superimposition of the crystal structures of BACE1 (1FKN) and BACE2 (2EWY) gave a root-mean-square deviation (RMSD) of 1.46 over 373 C-alpha atoms indicating that the structures of BACE1 and BACE2 are very similar to each other . AlthougWe defined the ligand binding site as all amino acid residues within 5 Å from the ligand. Twenty-eight of amino acid residues in BACE1 and 24 of amino acid residues in BACE2 were identified as ligand binding site. The ligand binding site residues of BACE1 and BACE2 together with their evolutionary trace status are summarized in We mapped the ligand binding sites and their ET status onto the chain A of crystal structures of BACE1 (1FKN) and BACE2 (2EWY) . Two outMembers of aspartyl protease family are known to have a loop structure that covers the active site of enzyme upon binding with the ligand. This structure is known as the flap region, and it is flexible and can adopt different structural conformations upon binding with the substrate. It is assumed that this structure will shield the active site from the solvent . The flaThe last group-specific residue at the ligand binding site is Asn233 of BACE1 substituted with Leu246 of BACE2. The observation indicated that, in BACE2, the binding site at this region is more hydrophobic than the one in BACE1. This difference can be exploited for the design of a selective drug targeting either BACE1 or BACE2.β). As a result, the design of selective BACE1 or BACE2 inhibitors is rather challenging. Analyzing the sequences of a group of selected BACE1 and BACE2 proteins and mapping the details on known 3D structure reveal the differences between these two groups that would be beneficial for the selective drug design.Designing a drug that could selectively inhibit BACE1 could avoid the unwanted side-effects of also inhibiting other aspartic protease family members including BACE2. BACE1 and BACE2 are close homologs and they are competing against each other for the same substrate (AET analysis on amino acid sequences and protein structures of BACE1 and BACE2 from several mammalian species enabled us to identify the distinctive features of BACE1 and BACE2 amino acid sequences. Mapping the ET analysis onto a known 3D structure of BACE1 and BACE2 revealed that their active sites are well conserved. Four group-specific residues were identified in the ligand binding sites of BACE1 and BACE2. The residues are Pro70, Ile110, Ile126, and Asn233 of BACE1 substituting Lys86, Leu126, Leu142, and Leu246 of BACE2, respectively. These group-specific residues would be the reason for cleavage site selectivity in BACE1 and BACE2 biological function and would be the potential residues for the design of selective and specific inhibitors targeting either BACE1 or BACE2.


The aim of our breast milk study was to investigate the transfer of the most potent peanut allergen Ara h 2 into human breast milk in a German cohort. Therefore, the time courses of appearance after ingestion, the potential risk of sensitization to peanuts via breast milk in Germany and, if possible, the way Ara h 2 will be processed ® and an ELISA against digestion resistant Ara h 2 (DRP-Ara h 2)). Natural Ara h 2 was digested by Enzynorm f® and Kreon® to mimic the effect of the combined gastric and duodenal digestion in vivo and, subsequently, analysed by N-terminal sequencing and MALDI TOF MS.Of 32 lacating, non-peanut allergic women, breast milk samples were collected at different time points after ingestion of 100 g dry roasted peanuts . Breast milk samples were analysed for peanut protein in SDS-PAGE, Western blot and ELISA  at different concentrations and time points of appearance. To assess the way Ara h 2 is processed in vivo, natural Ara h 2 was digested into several digestion resistant immunoreactive peptides <15 kDa after treatment with Enzynorm f® and Kreon®, and a 12 kDa fragment was identified by N-terminal sequencing and mass spectrometry corresponding to the middle part of Ara h 2.Ara h 2 was undetectable using Western blot. Performing the Neogen Veratox ELISAAfter maternal ingestion Ara h 2 is secreted into breast milk in our German cohort in 25 % of the volunteers, individually either rapidly  or delayed (after 8h or 12h) and in different concentrations. To study Ara h 2 or Ara h 2 peptides that survive digestion and pass into human breast milk antibodies against the 12 kDa fragment are now raised for enrichment strategies to characterize these sensitizing or tolerogenic peanut structures in our breast milk samples.


M1L mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron–sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own.Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron–sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1  •The mitochondrial glutathione redox state, but not the cytoplasmic glutathione redox state, is crucial for oxidant tolerance.•The mitochondrial thioredoxin acts as a back-up antioxidant system for the glutathione system.•Mitochondrial glutathione and thioredoxin play an overlapping role in redox regulation which is important during ageing and oxidative stress conditions. It is an essential metabolite in eukaryotes, and for example, mice deficient in glutathione biosynthesis die rapidly Saccharomyces cerevisiae as a model organism. Initial studies confirmed that GSH is an essential metabolite during normal non-stress conditions GSH1, encoding the first step in GSH biosynthesis, are inviable, but viability can be maintained by the addition of exogenous GSH. Importantly, gsh1 mutants undergo a limited number of cell divisions in the absence of GSH during which they utilize their pre-accumulated stores of GSH and hence the consequences of GSH depletion can be readily followed gsh1 mutant cannot be restored by growth under anaerobic conditions GSH1 also accumulate mitochondrial iron, which is a common phenotype in mutants that are defective in the maturation of cytosolic iron–sulphur (Fe–S) proteins Much progress has been made towards understanding the eukaryotic requirement for GSH using the yeast gsh1 mutants depleted of GSH are sensitive to oxidants The essential requirement for GSH, together with the pleiotropic phenotypes which are observed in mutants depleted of GSH, particularly defects in iron metabolism, has meant that it has been difficult to define the role of GSH as an antioxidant. Whilst M1L mutant to examine the requirement for mitochondrial GSH. We show that it is the mitochondrial form of Glr1, rather than the cytosolic form, which is required for oxidative stress tolerance. Our data indicate that the GSSG:2GSH redox couple plays an essential function as a mitochondrial antioxidant, with the mitochondrial thioredoxin system providing only a minor back-up role. Our findings indicate that in contrast to its apparent minor role in cytoplasmic thiol-redox control, glutathione plays an essential role in mitochondrial thiol-redox regulation.Glutathione reductase (Glr1) is an NADPH-dependent oxidoreductase which converts oxidized GSSG to reduced GSH using reducing power generated by the pentose phosphate pathway. Yeast Glr1 is not essential for normal aerobic growth, but is required for viability during exposure to oxidative stress conditions 22.1S. cerevisiae strains used in this study were isogenic derivatives of W303 (MATa ura3-52 leu2-3 leu2-112 trp1-1 ade2-1 his3-11 can1-100). Strains deleted for the mitochondrial thioredoxin (trx3::kanMX4), cytoplasmic thioredoxins (trx1::TRP1 trx2::URA3), glutathione reductase (glr1::TRP1) and GSH1 (gsh1::LEU2) have been described previously M1L) was made in GLR1 with a C-terminal Flag tag contained on plasmid pRS413 or pRS416. The FET3-lacZ reporter fusion has been described previously [25].The 2.2600=1.0, 0.1, 0.01 and 0.001) onto agar plates containing various concentrations of oxidants. Chronological life span experiments were performed in liquid SCD media supplemented with a five-fold excess of uracil, leucine, tryptophan, adenine and histidine to avoid any possible artefacts arising from the auxotrophic deficiencies of the strains.Strains were grown in rich YEPD medium  or minimal SCD medium . For growth on non-fermentable carbon sources, SGE and YEPGE contained 3% (v/v) glycerol and 1% (v/v) ethanol. Media were solidified by the addition of 2% (w/v) agar. Stress sensitivity was determined by growing cells to stationary phase and spotting diluted cultures  LSRFortessa™ cell analyser and data were analysed using BD FACSDiva 8.0.1 software. The degree of probe oxidation (OxD) was calculated as described previously [28].The redox state of Trx3 was measured by covalent modification with the thiol-reactive probe 4-acetamido-4′maleimidyldystilbene-2,2′-disulphonic acid  as described previously 2.4Arthrobacter luteus) with gentle agitation at room temperature for 30–60 min. Spheroplasting efficiency was determined at regular intervals by measuring the OD600 of small aliquots of cells in 1 ml of 0.6% (g/v) SDS. Cell homogenization was achieved using a tissue grinder . Protein concentrations were measured using a NanoDrop ND-8000 spectrophotometer. Cell fractionation was verified by Western blot analysis using mitochondrial  and cytosolic (αPgk1) specific antibodies. Protein extracts were electrophoresed under reducing conditions on SDS-PAGE minigels and electroblotted onto PVDF membrane (Amersham Pharmacia Biotech). Bound antibody was visualized by chemiluminescence . Lipid peroxidation in mitochondrial fractions was determined using an OxiSelect™ thiobarbituric acid reactive substances assay Kit (TBARS) . Methionine oxidation was detected using αMetO antibodies .Cellular fractionation into cytoplasmic and mitochondrial fractions was performed essentially as described by Gregg et al. 2.5d-galactopyranoside (ONPG) hydrolyzed per minute per microgram of total protein (U). Values shown are the means of at least three determinations. The gsh1 mutant was grown in minimal SCD media in the presence or absence of 1 mM GSH.For the determination of β-galactosidase activity, transformants were assayed essentially as described previously 2.6Experiments were performed according to 2.7p-value less than 0.05.Data are presented as mean values±SD. Statistical analysis was performed by one-way ANOVA and results were considered statistically significant with a 33.1M1L). The Glr1M1L mutant and wild-type Glr1 were expressed as the sole copies of Glr1 in glr1 mutant cells. Cells were fractionated and Glr1 detected using western blot analysis  to compare the redox potentials in different intracellular compartments glr1 mutant as would be expected due to the lack of Glr1 . These data indicate that the presence of Glr1 improves the growth recovery of a gsh1 mutant, presumably acting to recycle limiting amounts of oxidized GSSG. The addition of 0.1 μM GSH only partially restored the growth of the gsh1 glr1 mutant strain containing the Glr1M1L mutant  results in an increase in total glutathione levels which is thought to act as a compensatory mechanism in response to the increased redox load trx1 trx2) and found that OxD values were increased by approximately two-fold for both the cytoplasmic and mitochondrial roGFP2 fluorescent probes under fermentative or respiratory conditions  or expression of the Glr1M1L mutant did not alter the redox state of Trx3. Taken together, these data indicate that in contrast to the cytoplasmic redox systems, the redox states of mitochondrial glutathione and Trx3 are maintained independently and the mitochondrial GSSG:2GSH redox couple glutathione is unaffected by the loss of the mitochondrial thioredoxin.Loss of 3.5GLR1 and TRX3 to test whether there is a requirement for Trx3 when the GSSG:2GSH redox couple becomes oxidized. In agreement with previous observations glr1 GLR1 with glr1 trx3 GLR1). However, loss of Trx3 exacerbated the sensitivity of the glr1 mutant containing empty vector or Glr1M1L to both hydrogen peroxide and diamide stress. This suggests that Trx3 can act as a back-up antioxidant to promote oxidant tolerance, under conditions where mitochondrial glutathione becomes oxidized.Although a complete mitochondrial thioredoxin system comprising a thioredoxin (Trx3) and a thioredoxin reductase (Trr2) has been identified in yeast glr1 and trx3 mutant strains to confirm whether Trx3 has an antioxidant function. All amino acids are potential targets for oxidation, but methionine residues are particularly sensitive forming methionine sulphoxide (MetO) in cells glr1 and trx3 mutants. We found that the levels of mitochondrial MetO are comparable in wild-type, glr1 and trx3 mutants. In contrast, mitochondrial MetO concentrations were elevated in the glr1 trx3 double mutant  which is active as a peroxidase and can protect cells against hydrogen peroxide stress e mutant B. Malonde mutant C. These 3.6GLR1 resulted in a modest decrease in viability during stationary phase, although maximal CLS (~16 days) was unaffected relative to the CLS of wild-type and trx3 mutant strains  to a more oxidized state glr1 mutant strains glr1 mutant has a much greater percentage of oxidized glutathione in both the cytosol and the mitochondria compared with a wild-type strain. Hence, there appears to be sufficient GSH available in a glr1 mutant to maintain iron sulphur cluster assembly, which is proposed to only require trace amounts gsh1 Glr1M1L mutant compared with a gsh1 GLR1 mutant. This emphasizes the particular importance of the mitochondrial GSSG:2GSH redox couple for cell growth and oxidant tolerance, since the cytoplasmic GSSG:2GSH redox couple is maintained at wild-type levels in the Glr1M1L mutant.Previous studies have emphasized the role of glutathione in iron metabolism M1L mutant suggesting that similar cytosolic redox environments are maintained in these strains. The fact that the mitochondrial roGFP2 probe becomes more oxidized in a mutant lacking mitochondrial Glr1 suggests that the cytosolic pool of glutathione is insufficient to protect mitochondria against oxidation following exposure to an exogenous oxidant. It is unclear whether oxidant sensitivity and mitochondrial glutathione oxidation in a glr1 mutant is caused by loss of an antioxidant activity which is normally mediated by glutathione. For example, mitochondrial glutaredoxin (Grx2) and peroxiredoxin (Prx1) require glutathione for their antioxidant activities glr1 mutant might arise due to a general loss of mitochondrial thiol redox regulation, and non-specific oxidation of reactive cysteine residues in mitochondrial proteins.Transport of hydrogen peroxide across cell membranes can be facilitated by transporters such as aquaporins, but once inside cells it should be freely diffusible trx1 trx2 mutant, whereas there is no significant alteration in a trx3 mutant. It is surprising that the mitochondrial glutathione pool is oxidized in a trx1 trx2 mutant since previous studies using GFP-based redox sensors have suggested that the glutathione pools in the cytosol and mitochondrial matrix are not linked glr1 mutant does not affect the redox state of cytoplasmic thioredoxins , and thioredoxin oxidation is only detected in response to severe glutathione depletion Extensive overlaps have been reported between the yeast thioredoxin and glutathione systems TRX3 does not alter the redox state of the cytosolic or mitochondrial roGFP2 fluorescent probes suggesting that loss of the mitochondrial thioredoxin does not impose a redox load on the glutathione system. Mitochondrial thioredoxin is however, known to be more reliant on the glutathione redox couple. Unlike cytoplasmic thioredoxins, the redox state of mitochondrial Trx3 is buffered by the GSSG:2GSH redox couple under oxidative stress conditions trr2), but is accumulated in a trr2 glr1 double mutant under both fermentative and respiratory growth conditions GLR1, since the oxidant sensitivity of a glr1 mutant is exacerbated in the absence of TRX3 and markers of oxidant damage including MetO and MDA accumulate in a glr1 trx3 double mutant. We therefore suggest that mitochondrial Trx3 plays an ancillary or back-up role for mitochondrial glutathione, perhaps under stress or growth conditions where mitochondrial glutathione is oxidized.Loss of mitochondrial GLR1 or TRX3 alone does not significantly alter chronological lifespan. However, in a glr1 trx3 double mutant longevity is significantly reduced. The role of Trx3 in CLS appears to require a complete mitochondrial thioredoxin system since a similar defect in CLS is observed in a glr1 trr2 mutant. This is the first time a thioredoxin system-dependent phenotype has been described for Trx3 since the only other reported phenotypes have been detected in a trr2 mutant and not a trx3 mutant It was originally reported that Trx3 is the physiological electron donor for mitochondrial Prx1, which is unexpected since 1-Cys Prx’s are not thought to form a disulphide which could act as a substrate for thioredoxin 5Thiol redox regulation plays a long-recognized role in the response of cells to oxidative stress conditions. Our current data emphasize the importance of compartmentalized redox regulation when cells are subjected to oxidative stress conditions. Whilst cytosolic glutathione represents the first major pool of thiols which would be a target of oxidation in response to exposure to an exogenous oxidant, it is the mitochondrial glutathione pool which is crucial for oxidant tolerance.


The images for Figs 7, 9 and 10 appear incorrectly in the published article. The figure captions are correct. Please see the correct Figs


Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.SRY (Sex Determining Region Y)-Box 4 or  This is a rare event within the mammalian genome but is common in the plant.•The data provides a modified method for brain cell fixation and immobilisation on glass slides for effective RNA-FISH analysis.•Sox4 natural antisense transcripts, known as Sox4ot1 and Sox4ot2 in the production of Sox4_sir3 in vitro.Comparison of two different •Sox4 gene locus allows clear, concise and easy visualisation of various features defined in the region by using Artemis software.Compilation of all the information within the 11.1Sox4 gene locus. The Sox4 gene locus is featured by multiple overlapping sense and natural antisense transcripts (NATs) In silico data mining and mapping were also carried out to enrich the features within the locus and the detailed information is summarized in a GenBank file format as Supplementary GenBank File. A snapshot of the annotated Sox4 gene locus visualized using Artemis Genome Browser and Annotation Tool www.ensembl.org), Sox4ot1, Sox4ot2, Sox4_sir3, untranslated regions, coding region and exons/introns.The data reported here consists of information related to the The most important information within the Supplementary GenBank File is the mapped FANTOM Paired-End Ditags (PET) sequences. Twelve pairs of PET sequences were mapped to the locus indicating the presence of 6 different NATs. These NATs were named PET1-6 with 4 of them were successfully cloned and further analysed in Ling et al. 1.2Sox4 sense was generally diffused all over the cytoplasm whereas Sox4 NATs were depicted as aggregates within the cytoplasm. Whenever Sox4 NATs aggregates were observed, Sox4 sense aggregates were found at the same loci within the cytoplasm.The data article also describes the results for RNA-FISH experiments performed on cells isolated from different regions of the mouse brain . All celSox4, RNA FISH was performed on cells obtained from P1.5 olfactory bulbs using probes against the Hmbs housekeeping gene  to quantitatively estimate the intensity of bands in Western blotting experiments. Pixels from each band from two independent experiments were calculated by using a fixed rectangular selection approach (T-test (2-tailed) was used to compare PET/pcDNA3 and control groups for any significant differences but none of the p-values were lesser than 0.05.We used ImageJ software  Sox4 gene locus  and PET6 (NAT that overlaps Sox4_sir3 origin site). The overexpression of PET3 and PET6 both did not alter the level of sense transcript expression. As expected, the expression of the Sox4 NAT at region overlapped by PET3 and PET6 were significantly upregulated . Amplicons were analysed using agarose gel electrophoresis to estimate the size of PET6. The analysis showed that there were 2 forms of PET6 NATs, one is unspliced and the other one is spliced . Sanger mall RNA .1.7in situ hybridization experiments are appropriately controlled to avoid misinterpretation of noisy signals. Locked Nucleic Acid (LNA)-in situ hybridization (ISH) for small RNA is usually controlled with a scramble probe, a mutated antisense probe or a sense probe. As the control for Sox4_sir3 LNA-ISH reported in Ling et al. Sox4_sir3 probe  to determine the relative levels for various Sox4 sense and NATs expression. All RT-qPCR data presented here were conforming to the criteria described elsewhere


Area at risk measurements often rely on T2 weighted images, but subtle differences in T2 may be overlooked with this method. Quantitative T2 mapping may bring us beyond some of the technical limitations associated with T2-weighted images . We hypothesize that T2 quantification can detect differences between salvaged and infarcted myocardium within the AAR in a reperfused model of acute myocardial infarction.Dogs underwent 2 hours of coronary occlusion followed by 4 or 48 hours of reperfusion before imaging. CMR imaging was performed at 1.5T (Siemens) using native T2 mapping, T2-prepared SSFP imaging, and late gadolinium enhancement (LGE). One midventricular slice was chosen per animal for analysis. LGE images were used to define infarcted, salvaged, and remote myocardial ROIs. Another ROI of both the infarcted and salvaged areas was defined as the AAR. Data were analyzed with ANOVA and Bonferroni correction for multiple testing. A p value < 0.05 was considered significant.22 animals were imaged after 4 n = 11) or 48 hours (n = 11) of reperfusion. The signal intensity of the AAR was significantly greater than the remote myocardium on T2-prepared SSFP images, both at 4 hours  of reperfusion and 48 hours . This was also the case with T2 quantification of the AAR compared to remote at 4 hours and 48 hours . Dividing the AAR into infarcted and salvaged myocardium demonstrated that the T2 of salvaged myocardium was significantly longer than remote myocardium at both 4 and 48 hours of reperfusion. The T2 of infarcted myocardium was also longer than remote myocardium (see Figures 1 or 48 hT2 mapping techniques quantitatively differentiated sub-regions within the AAR during the first days of reperfusion. The T2 of salvaged myocardium was significantly higher than remote myocardium after both 4 and 48 hours of reperfusion, though the magnitude of the difference was greater at 4 hours. T2 mapping was also able to distinguish salvaged from infarcted myocardium.Funded by the Intramural Research Program of the National Heart, Lung, and Blood Institute of The National Institutes of Health.


Experiments have revealed that membrane proteins can form two-dimensional clusters with regular translational and orientational protein arrangements, which may allow cells to modulate protein function. However, the physical mechanisms yielding supramolecular organization and collective function of membrane proteins remain largely unknown. Here we show that bilayer-mediated elastic interactions between membrane proteins can yield regular and distinctive lattice architectures of protein clusters, and may provide a link between lattice architecture and lattice function. Using the mechanosensitive channel of large conductance (MscL) as a model system, we obtain relations between the shape of MscL and the supramolecular architecture of MscL lattices. We predict that the tetrameric and pentameric MscL symmetries observed in previous structural studies yield distinct lattice architectures of MscL clusters and that, in turn, these distinct MscL lattice architectures yield distinct lattice activation barriers. Our results suggest general physical mechanisms linking protein symmetry, the lattice architecture of membrane protein clusters, and the collective function of membrane protein lattices. Superresolution light microscopy and electron cryo-tomography have revealed12357810111314161718202122232425262728293031323334353637383940414243In this Article we study the most favorable (minimum-energy) lattice architectures, and corresponding modulation of protein function, due to bilayer-mediated elastic interactions between mechanosensitive membrane proteins. A diverse range of integral membrane proteins have been shown to be mechanosensitive2020in vitro145253in vivo, and the physiological significance of tetrameric MscL is a matter of debate14155253in vivo52in vivo14145253in vitro experiments, or varying the temperature. In this Article we take the available MscL structures as our starting point, and consider the lattice architectures and collective functions of clusters of both tetrameric and pentameric MscL, as well as mixtures of tetrameric and pentameric MscL.MscL switches from a closed to an open state with increasing membrane tension204951In vitro and in vivo studies have suggested that bilayer-mediated interactions stabilize large clusters of hundreds of MscL5555Bilayer-mediated protein clustering may be driven by curvature deformations212223242526272829303132333132333435362438394041424320435558a is one-half the hydrophobic thickness of the unperturbed lipid bilayer, and, for generality, we considerτ to u as well as to area changes. Experiments roughly yield kBT/nm2 20where the thickness deformation field Mycobacterium tuberculosisBased on structural data48R captures the size of MscL, ε is the amplitude of angular undulations, and ω denotes the orientation of MscL with respect to the x-axis. The observed structure of closed pentameric MscLStaphylococcus aureusin polar coordinates, where u along the bilayer-protein interface in the closed and open states of MscL, where the unperturbed bilayer half-thickness E. coli20436368On the basis of structural data on MscL, the hydrophobic thickness of MscL in the closed and open states has been estimatedWhile the anisotropic thickness deformations due to a few proteins can be obtained by minimizing equation 6543U as a summation over elements,Following the standard finite element discretization procedure, we rewrite the variation of the energy in equation where the element stiffness matrix D is a block diagonal matrix with the lipid bilayer parameters as coefficients. The strain-displacement transformation matrix B combines the DKT shape functions H with the linear triangular shape functions M:are integrated over the local coordinates H are given by Batoz et al.M can be found in standard finite element textbooks—see, e.g., ref. 71Explicit forms of the DKT shape functions d is the center-to-center distance between the two MscL and d row of the interaction energy array by rounding d. For fast evaluation of hard-core steric constraints, we constructed an analogous array for the minimum allowed distances To confirm our predictions of the minimum-energy lattice architectures of tetrameric and pentameric MscL we carried out Monte Carlo simulations with simulated annealing of pair interaction potentials796 Monte Carlo steps, and decreased the temperature linearly starting from around In our simulated annealing Monte Carlo simulations, a single Monte Carlo step consists of one displacement and one rotation trial per MscL on average. We used a unit displacement edom see , we firsd, which are greater than d. For fixed protein shape and orientation, thickness-mediated interactions are most favorable for the smallest value of d allowed by steric constraints on lipid size, d, non-pairwise contributions to thickness-mediated interactions can be For curvature- and fluctuation-mediated interactions it has been suggested232532334359d, and the close-packed hexagonal lattice with Thickness-mediated MscL clustering was studied before4320634849u normal to their bilayer boundaries .


Haematological malignancies are associated with treatment releated morbidity and mortality. Intensive chemotherapy and haematopoetic stem cell transplantation has increased treatment releated complications. Transferring a severely ill patients to the intensive care unit for life support is often a difficult decision.To detect risk factors effecting haematological malignancy patients mortality after admission to the medical intensive care unit.This study was performed prospectively in the medical intensive care unit of Erciyes Universty Hospital. History, physical exam and laboratory findings on admisssion,and therapeutic interventions during ICU stay were recorded. The study end point was ICU mortality.32 (%60) of the 53 patients included into this study were male. The average age of the patients was 49 ± 19 years. The hematological diagnosis of the patients were as follows; 19 were AML, 12 were MM, 7 were Non-Hodgin Lymphoma and 6were ALL. The most common reasons for ICU admissions were respiratory failure (%57) and septic shock (%17). The mean time delay starting from deteriation to ICU was 6 hours (range 1-48). APACHE-2 score was 26 ± 8 and the early warning system score was 8 (range 1-14). APACHE-2 score and time delay for ICU admission of nonsurvivors were higher . Serum total cortisol levels were lower in survivors compared to nonsurvivors (p= 0.023). ICU mortality rate was 60%.The mortality rate of haematological malignancy patients rate was high. The time delay for ICU admission and APACHE-2 score were important risk factors for ICU mortality. These patients should be admitted to the ICU as soon as possible when vital signs are detoriated.


C9orf72 is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) acting through a loss of function mechanism due to haploinsufficiency of C9orf72 or a gain of function mediated by aggregates of bidirectionally transcribed HRE-RNAs translated into di-peptide repeat (DPR) proteins. To fully understand regulation of C9orf72 expression we surveyed the C9orf72 locus using Cap Analysis of Gene Expression sequence data (CAGEseq). We observed C9orf72 was generally lowly expressed with the exception of a subset of myeloid cells, particularly CD14+ monocytes that showed up to seven fold higher expression as compared to central nervous system (CNS) and other tissues. The expression profile at the C9orf72 locus showed a complex architecture with differential expression of the transcription start sites (TSSs) for the annotated C9orf72 transcripts between myeloid and CNS tissues suggesting cell and/or tissue specific functions. We further detected novel TSSs in both the sense and antisense strand at the C9orf72 locus and confirmed their existence in brain tissues and CD14+ monocytes. Interestingly, our experiments showed a consistent decrease of C9orf72 coding transcripts not only in brain tissue and monocytes from C9orf72-HRE patients, but also in brains from MAPT and GRN mutation carriers together with an increase in antisense transcripts suggesting these could play a role in regulation of C9orf72. We found that the non-HRE related expression changes cannot be explained by promoter methylation but by the presence of the C9orf72-HRE risk haplotype and unknown functional interactions between C9orf72, MAPT and GRN.A non-coding hexanucleotide repeat expansion (HRE) in The online version of this article (doi:10.1186/s40478-016-0306-7) contains supplementary material, which is available to authorized users. C9orf72 gene as the major cause for chromosome 9-linked ALS and FTD with or without concomitant motor neuron disease 3 probe (sense foci), or [GGGGCC]3 probe (antisense foci) (Exiqon) as previously described [CD14+ monocytes from three escribed . Brieflyescribed  and usedhttps://www.r-project.org/).All statistical analyses, correlations and plots were performed using the free software environment R . All tissue requests received at the NBB are reviewed by NBB's scientific committee and all material and data collected by the NBB are obtained on the basis of written informed consent. Procedures, information and consent forms of the NBB have been approved by the Medical Ethics Committee of the VU Medical Centre. The use of human brain samples for the work leading to the generation of the CAGEseq dataset 3 was approved by the NIH Office for Human Subjects Research as indicated in C. Blauwendraat, under review in another journal. Controls and disease blood donors gave informal consent and study approval was obtained by the Ethics Committee of the University of Tübingen.C9orf72 locus and identify possible new transcripts on the sense and antisense strands that contribute to C9orf72 expression, we used publically available CAGEseq expression data generated in the context of the FANTOM5 project [C9orf72 is expressed. We observed C9orf72 was expressed above 1 tpm in the sense strand in 83.3 % of the samples and in the antisense strand in 22.1 % of the samples. Particularly, C9orf72 expression was very high in CD14+ monocytes, eosinophils, and neutrophils, a subset of myeloid cells involved in innate and adaptive immunity, from now on referred to as myeloid-high , or lymphoid-derived cells like T and B cells were much lower , NM_18325 (Transcript 2) and NM_1256054 (Transcript 3)  Fig. . The C9oile Fig.  shows thC9orf72 locus: a TSS on the reverse strand downstream of exons 1A and 1B of the annotated transcripts  with the IPO7 gene encoding for importin 7, a nucleocytoplasmic transport protein [The distinct modes of expression of  protein  when conC9orf72 expression between myeloid cells and CNS, we investigated whether this finding was also true across distinct CNS regions. In addition to the FANTOM5 collection that contains data for a range of CNS regions in a limited numbers of adult donors, we analyzed an additional CAGEseq brain dataset consisting of 49 control libraries generated in our laboratory, with data from seven CNS regions from seven control donors .We next investigated roarrays . To confC9orf72 expression in microglia. Therefore we isolated RNA from commercially available microglia from human fetal brain tissue. Digital PCR experiments  and therefore we excluded it from further analysis. In agreement with our CAGEseq data we observed an increase for AS1 in the C9orf72-HRE patients and AS3 TSSs in the C9orf72-HRE and GRN patients as compared to controls .We then investigated whether and to which extent the newly detected ols Fig. . HoweverC9orf72 expression is also reduced in non-HRE FTD patients suggests that the reduction of C9orf72 expression is not entirely dependent on the repeat expansion. Sequence analysis of the GC-rich low-complexity sequence (LCS) adjacent to the HRE did not reveal any DNA variants, including the 10 bp deletion observed in up to 25 % of C9orf72-HRE patients [C9orf72 expression; therefore we explored other potential causes. One possible explanation might be that the observed C9orf72 decrease in GRN and MAPT cases is a consequence of the neurodegeneration process. We therefore performed a series of qPCR experiments on cDNA from brain RNA of patients with different neurodegenerative disorders including sporadic FTD, AD, ALS, PSP, PD, HD and MS patients   (data not shown). On average, we observed a three fold increase in the expression of S1 + 3a and S1 + S3b TSSs in the individuals homozygous for the risk haplotype as compared to the individuals carrying the non-risk haplotype and a 1.2 and 1.5 decrease in the expression of S2a and S2b TSSs, respectively.Our eQTL analysis shows that the risk haplotype is associated with higher expression of the ly) Fig.  and to lNext we determined by genotyping which samples used in our qPCR experiments on brain samples were carrying the risk haplotype. We identified five individuals homozygous and 34 heterozygous for the risk haplotype and 29 individuals non-carriers of the risk haplotype Table .C9orf72 transcripts expression levels determined by qPCR with the concordance to the risk haplotype. In our qPCR experiments we could measure the effect of the risk haplotype C9orf72 transcript 1 and 3 in separate assays differently than with CAGEseq as C9orf72 transcript 1 and transcript 3 share the same TSS. We observed a significant association of the risk haplotype with a higher expression of C9orf72 transcript 3 in agreement with our CAGEseq eQTL findings  but not with transcript 1, with the influence of the risk haplotype on C9orf72 transcript 3 clearly shown in Fig. C9orf72 transcript 2 probably because of the small effect and sample size.We then correlated C9orf72 expression in brain tissue, but it does not fully explain the observed C9orf72 reduction in MAPT and GRN patients as it was also detected in cases not carrying the risk allele. Therefore additional mechanisms must exist to explain the observed C9orf72 decrease.Taken together these results suggest that the risk haplotype affects C9orf72 promoter region are associated with down-regulation of C9orf72 mRNA expression in HRE patients [C9orf72 promoter in FTD cases with MAPT and GRN mutations has not been yet investigated.Several studies have shown that hypermethylation and H3 and H4 lysine trimethylation of the patients , 48, 53,C9orf72-HRE cases, two samples were strongly hypermethylated and overall C9orf72 promoter methylation levels were higher in C9orf72 patients as compared to controls, MAPT and GRN mutations carriers  while no significant differences were observed between MAPT cases versus controls and GRN cases versus controls, ruling out a direct influence of C9orf72 methylation level on C9orf72 expression in these patients. We then investigated whether the risk haplotype would influence the methylation level of the C9orf72 promoter in the MAPT and GRN mutation carriers. We did not observe a significant association between the risk haplotype, in the homozygous or heterozygous state and C9orf72 promoter methylation level (data not shown). Our results therefore suggest that C9orf72 promoter methylation level by itself or in combination with the risk haplotype does not account for the C9orf72 reduction observed in MAPT and GRN cases.We therefore performed an HhaI methylation-sensitive digestion assay combined with qPCR as described by Russ and colleagues . Among tC9orf72 reduction observed in MAPT and GRN mutation carriers could also imply a functional interaction within the molecular pathways disrupted by the C9orf72, MAPT and GRN mutations respectively. In this respect the decrease in MAPT transcripts containing exon 10 observed by Prudencio et al in RNAseq data from C9orf72-HRE patients is interesting [C9orf72 in MAPT patients, which would be consistent with a functional interaction.The C9orf72 transcripts 2 and 3, MAPT and GRN. qPCR experiments on cDNAs from the transduced cells showed a clear influence on expression of C9orf72 transcripts 2 and 3 after knock down of MAPT and GRN separately compared to scrambled controls  M17 human neuroblastoma cells with lentiviral constructs expressing shRNAs targeting 9orf72 expression using CAGEseq data across the FANTOM5 collection showed that C9orf72 is highly expressed in myeloid cells, particularly in CD14+ monocytes, in agreement with previous findings based on microarrays [Our analysis of global Croarrays .C9orf72 in CD14+ monocytes (up to seven fold increase) as compared to brain  than the percentage reported for peripheral blood leukocytes (7 %) [C9orf72 is rather similar to that in the CNS [C9orf72 is much more abundant in CD14+ monocytes.Overall the percentage of RNA foci for the antisense es (7 %)  and for es (7 %) , while t the CNS , 47, 55,C9orf72 expression by using CAGEseq data reveals new and interesting features for the C9orf72 promoter and gene locus. A major strength of CAGEseq lies in its ability to distinguish closely spaced TSS usage preference that is often tissue or cell specific. Indeed we observed several novel TSSs at the C9orf72 locus with distinct modes of expression in a subset of myeloid cells and CNS suggesting they have a cell and/or tissue specific function. Interestingly, expression levels of S1 + S3b TSS for C9orf72 transcripts 1 and 3 show a high correlation with the IPO7 gene encoding for the importin 7 protein, a member of the β-karyopherin family proteins that mediate nuclear import of ribosomal proteins and export of ribosomal subunits necessary for ribosome biogenesis [C9orf72-IPO7 correlation reflects a functional interaction, but it has been shown that C9orf72 protein isoforms localize to the nuclear membrane in healthy neurons and they interact with components of the nuclear pore complex [C9orf72 transcripts 1 and 3 in the CNS might be physiologically involved in nuclear import and export that would be affected by a decrease in their expression. This potential detrimental effect could be exacerbated in C9orf72-HRE patients by the presence of HRE-containing transcripts, as indicated by several studies highlighting defects in nucleocytoplasmic trafficking as results of HRE expression [Our analysis on global ogenesis . Current complex . It is tpression , 11, 57.C9orf72 functions in CNS, particularly when considering therapeutic efforts aiming at simply decreasing C9orf72 expression to ameliorate RNA foci and DPR proteins aggregates formation.More research is therefore warranted in characterizing C9orf72 expression in C9orf72-HRE patients but also in MAPT and GRN mutations carriers pointing to additional regulatory mechanisms besides the HRE-mediated C9orf72 repression. On control brains we observed an association of the C9orf72-HRE risk haplotype with an increase in expression of C9orf72 TSS for transcript 1 and 3 and decrease in TSS for transcript 2. The association of C9orf72 expression with SNPs in the risk haplotype has been already reported in monocytes but never in brain [C9orf72 transcripts 1 and 3 and we replicated the association of the risk haplotype with the increase of C9orf72 transcript 3 but not with transcript 1 or 2, likely because of the small effect and sample size. At first glance the increased expression of transcript 3 determined by the risk haplotype appears to be in contradiction with the CAGEseq and qPCR expression data showing general reduction of transcript 3 in the C9orf72-HRE and FTD cases with MAPT and GRN mutations compared with controls. However the increase in expression of C9orf72 transcript 3 in individuals carrying the risk haplotype is small. Additionally, approximately half of the GRN and MAPT samples do not carry the risk haplotype.Our CAGEseq data and qPCR results showed a consistent decrease in in brain , 59. In C9orf72 transcript 3 might have consequences in C9orf72-HRE patients. The HRE can be transcribed in the pre-mRNA of abortive and/or mature C9orf72 transcripts 1 and 3 that would accumulate in RNA foci [C9orf72-HRE cases might intensify RNA foci formation although not necessarily DPR proteins aggregation as it has been suggested RAN translation might require transcripts containing the first entire intron as template [Albeit small, the increase in expression of RNA foci , 60. In template .C9orf72 transcript 3 expression are unknown. The C9orf72-HRE occurs on a risk haplotype covering 110 kb region between MOB3B and C9orf72. The HRE is highly polymorphic and prone to expansion particularly in the context of the risk haplotype [C9orf72-HRE patients [GRN and MAPT mutation carriers used in this study.The molecular reasons underlying the association of the risk haplotype with the increase in patients , 52; howC9orf72 expression. We gathered convincing evidence for the existence of three distinct antisense transcripts head-to-head to C9orf72 that are highly expressed in myeloid cells and to a lower extent in brain tissues in C9orf72-HRE cases as well as in individual without the repeat expansion. Several C9orf72 antisense transcripts have been already detected in intron 1b [C9orf72-AS2 and AS3 transcripts are adjacent but downstream the HRE and they might be involved in C9orf72 regulation.Therefore, additional mechanisms must play a role in regulating ntron 1b , 15 and C9orf72 regulation mainly in the context of the G-rich HRE that would mediate RNA-DNA hybrid formation (R-loops) facilitating RNA polymerase II pausing before efficient termination causing transcriptional stalling and nucleolar stress [C9orf72 sense transcripts and antisense transcript AS3 in brain tissues from C9orf72-HRE patients and therefore it might be relevant to investigate whether the novel antisense transcripts hamper or amplify the HRE antisense effects. In addition increasing evidence shows that antisense transcripts act at nearly every level of gene regulation  and even function simultaneously at multiple steps. It has been shown that antisense expression can regulate transcription by affecting DNA methylation. Several studies, including our own, show that hypermethylation of the CpG-island at the 5′ end of the repeat contributes to C9orf72 repression in a high of C9orf72-HRE carriers, yet down-regulation of C9orf72 is more prevalent and shown among FTD sporadic patients [MAPT and GRN mutation carriers (this study). Very recently, co-localization of para-nucleolar DPR proteins inclusions with heterochomatin and H3K9me2 [C9orf72 antisense AS3 affects histone modifications at the C9orf72 locus not only in C9orf72-HRE but also in MAPT and GRN patients.Gene regulation by antisense transcription is rather intriguing. Antisense transcripts are transcribed from the opposite strand of protein-coding genes. This genomic arrangement immediately suggests that the sense and antisense transcripts might act on each other and increasing evidence corroborates such an hypothesis . Antisenr stress . We obsepatients  and MAPT H3K9me2 , a marke H3K9me2 . In lighC9orf72 expression in myeloid cells and the changes in C9orf72 expression in CD14+ monocytes after exposure to microbial pathogens suggest that C9orf72 might be involved in immune-related processes, in agreement also with the recent findings that in mice is required for normal function of myeloid cells [C9orf72 might play a role in the initial steps of autophagy as suggested by its interaction with FIP200, a component of the ULK1-ATG13 autophagosome initiation complex [The robust id cells . Moreove complex .C9orf72 role in autophagy, neurons differentiated from patients-derived iPSC, are more sensitive to autophagy inhibitors and presented higher p62 levels suggesting autophagy might be compromised [Consistent with a possible promised .The half-life of monocytes in blood is relatively short: approximately 1 day in mice and 3 days in human. Once monocytes are stimulated by an inflammatory response they activate pro-survival pathways, migrate to tissues where they mediate direct antimicrobial activity by releasing tumor necrosis factor, chemokines, engage in phagocytosis and differentiate to macrophages through molecular mechanisms that involve autophagy .C9orf72 in the autophagy process it is tempting to speculate that reduced C9orf72 expression might impair autophagy affecting monocyte-macrophage differentiation and thereby hamper host inflammatory responses. Although our findings will need to be replicated in a larger samples size including age-matched controls, our qPCR results on CD14+ monocytes from C9orf7-HRE carriers and from patients with clinical diagnosis of FTD and ALS are in line with this hypothesis. Currently we do not have evidence that monocytes in these patients are impaired but we show that they carry a very high antisense RNA foci burden.While waiting for further research to clarify the exact role of C9orf72 expression. We showed that the decrease in C9orf72 expression is a widespread phenomenon in FTD pathogenesis suggesting C9orf72 plays a more general role in neurodegeneration. Moreover the C9orf72 TSSs profile in CNS and myeloid cells suggests distinct C9orf72 transcripts have tissue specific function and they call attention to a potential C9orf72 role in immune response.In conclusion, our findings strongly imply that several mechanisms acting independently from the HRE, or in a concerted manner, contribute to regulate


Solanum lycopersicum) transcription factor SlZFP2 fine tunes ABA biosynthesis during fruit development through direct suppression of ABA biosynthetic genes and it also regulates fruit ripening through transcriptional suppression of the ripening regulator CNR. This indicates that SlZFP2 likely modulates the cross-talk between ABA and ethylene in regulation of fruit development and ripening in tomato. Gene expression analysis using ABA deficient mutants sit and flc as well as the SlZFP2 RNAi lines of high fruit ABA production showed that ethylene biosynthetic genes LeACS1A, LeACS1 and LeACO1 were positively regulated by ABA during early fruit growth. We reason that ABA promotes basal ethylene biosynthesis in system 1 during fruit growth and likely plays a minor role in ripening regulation after the onset of ripening process.The stress hormone ABA not only regulates stress response, but is also required for plant development and growth. Some evidences indicate that ABA plays a pivotal role in the ripening process of non climacteric as well as climacteric fruits. In a recent study, we showed that the tomato ( SlZFP2Solanum lycopersicum zinc finger protein 2HA-SlZFP2an expression cassette of hemagglutinin-SlZFP2 fusion proteinACS1-aminocyclopropane-1-carboxylic acid synthaseACOACC oxidaseCNRCOLORLESS NON-RIPENINGNOTNOTABILISSITSITIENSFLCFLACCASlAO1Solanum lycopersicum aldehyde oxidase 1ABAabscisic aciddpadays post anthesisRNAiRNA interferenceFPKMFragments Per Kilobase of transcript per Million mapped reads.SlZFP2, encoding a single C2H2 zinc finger protein, in tomato fruit development.HA-SlZFP2 under 35S promoter repressed ABA biosynthesis in leaves and fruits, whereas silencing its expression increased ABA production in young fruits at 5 and 10 dpa. We also revealed that SlZFP2 regulates fruit ripening through transcriptional repression of the ripening regulator CNR. Thus, the SlZFP2 pathway likely modulates crosstalk between ABA biosynthesis and the regulatory network of fruit ripening in tomato.ABA is well known for its roles in seed maturation and germination, in addition to its pivotal roles in stress response.NOT, FLC, SIT and SlAO1. Since SlZFP2 is mainly expressed during fruit development, it likely plays an important role in maintenance of the dynamic ABA production post pollination. Indeed, SlZFP2 expression negatively correlates with ABA level during fruit development, for example, SlZFP2 expression was relatively low in anthesis ovaries and 20 dpa fruits when high ABA production was observed . Their expression was not impacted at the onset of ripening process by overexpression or RNAi-mediated repression of SlZFP2. Thus, the gene expression analysis suggests that elevated or repressed ABA biosynthesis by manipulating SlZFP2 expression has little impact on ethylene production at the onset of ripening process.However, the action of LeACS1A, LeACS6, LeACO1, 3 and 4 are responsible for the basal ethylene production.SlZFP2 does not directly regulate the induction of ethylene biosynthesis in system 2. However, we found LeACO3 and LeACO4 expression was increased significantly in the 2 dpa fruits of the representative SlZFP2 RNAi line 207 through transcriptome analysis by RNA-seq. The other ethylene biosynthetic genes LeACS1A, LeACS2 and LeACO1, although the LeACS1A and LeACS2 were expressed at low levels, were also expressed at higher levels in the young fruits (SlZFP2 may regulate ethylene biosynthesis in system 1. Thus, the problematic fruit set observed in these SlZFP2 RNAi lines can be explained by elevated ethylene biosynthesis. Our observation is consistent with early studies that ABA promotes flower and fruit abscission through ethylene biosynthesis.SlZFP2 RNAi fruits, SlZFP2 likely regulates ethylene biosynthesis during early fruit growth through ABA pathway. To test the possibility, we analyzed the expression of ethylene biosynthetic genes in ABA deficient mutants sit and flc. We found that LeACO1 was downregulated in both the 5 and 10 dpa fruits of the 2 mutants; LeACS6 expression was also repressed at 5 dpa (Ethylene is the predominant plant hormone regulating climacteric-fruit ripening. In tomato, two systems of ethylene biosynthesis have been proposed, which basal ethylene production is maintained in system 1 during fruit growth and later its production is increased drastically in system 2 during ripening.SlZFP2 plays at least two roles in regulation of fruit development and ripening (SlZFP2, likely induced by high ABA at anthesis, represses ABA biosynthesis after anthesis, and in turn the decrease in ABA level limits ethylene production during fruit set and early fruit growth. Fine-tuning ABA biosynthesis likely helps to maintain ethylene production at its basal level in system 1 for normal fruit growth. Second, SlZFP2 also prevents CNR expression before the onset of ripening process. However, it remains to be determined whether or not the ABA biosynthesis regulated by SlZFP2 interconnects with the CNR-mediated ripening regulation.Collectively,


Background: During March 2009 a novelInfluenza A virus emerged in Mexico. We describe the clinical picture of the pandemicInfluenza A (H1N1) Influenza in cancer patients during the 2009 influenza season.Methods: Twelve centers participated in a multicenter retrospective observational study of cancer patients with confirmed infection with the 2009 H1N1Influenza A virus (influenza-like illness or pneumonia plus positive PCR for the 2009 H1N1Influenza A virus  in respiratory secretions). Clinical data were obtained by retrospective chart review and analyzed. Results: From May to August 2009, data of 65 patients were collected. Median age was 51 years, 57 % of the patients were female. Most patients (47) had onco-hematological cancers and 18 had solid tumors. Cancer treatment mainly consisted of chemotherapy (46), or stem cell transplantation (SCT) (16). Only 19 of 64 patients had received the 2009 seasonal Influenza vaccine. Clinical presentation included pneumonia (43) and upper respiratory tract infection (22). Forty five of 58 ambulatory patients were admitted. Mechanical ventilation was required in 12 patients (18%). Treatment included oseltamivir monotherapy or in combination with amantadine for a median of 7 days. The global 30-day mortality rate was 18%. All 12 deaths were among the non-vaccinated patients. No deaths were observed among the 19 vaccinated patients. Oxygen saturation <96% at presentation was a predictor of mortality .Conclusions: In our cancer patient population, the pandemic 2009 Influenza A (H1N1) virus was associated with high incidence of pneumonia (66%), and 30-day mortality (18.5%). Saturation <96% was significantly associated with death. No deaths were observed among vaccinated patients. Influenza with a mortality rate of up to 28%1. Non-transplant cancer patients can also have a high mortality rate of up to 38%2, being higher in patients with lung, hematological and colorectal cancer, in patients that develop lower respiratory tract infections, and in patients with other co-morbid conditions. In Argentina, seasonal Influenza in onco-hematological patients is associated to a 12% incidence of pneumonia and to a 5% of 30-day mortality3.Seasonal influenza is a known cause of morbidity and mortality among cancer and transplant patients. During influenza season, 20 to 30% of stem cell transplant SCT recipients with respiratory symptoms can test positive forInfluenza A virus, later known as 2009pandemic influenza A (H1N1), emerged in Mexico. The new strain initially spread among travelers to the USA and Canada, and subsequently infected people worldwide4. Clinical presentations ranged from mild symptoms to severe cases that lead to pneumonia and respiratory failure–related deaths.In March 2009 a novelInfluenza A (H1N1) in Argentina were reported in May 2009, in travelers returning from Mexico and the USA. From May to December 2009 there were 11931 cases of confirmedInfluenza A H1N1 in Argentina, 617 deaths, and over 90% of the circulating respiratory viruses in adults were the novelInfluenza A H1N15.The first cases of pandemic16. In these studies, the incidence of pneumonia ranges from 2011 to 52%8, while the reported mortality rate ranges from 0–10%16 to as high as 21–31%15.Data from different studies on the impact of this new virus in the adult cancer and SCT population are somewhat contradictory. Many studies from different countries were reportedInfluenza A H1N1 continued to circulate .During the 2013 winter season, pandemicpandemic H1N1Influenza during the 2009 influenza season, in patients with cancer and SCT in two cities of Argentina.In this study, we examined the effects and severity ofThis is a multicenter retrospective observational study that included 12 medical centers. From May to August 2009, cancer and SCT patients older than 16 years, who presented a confirmed influenza infection by real-time PCR were included.pandemic Influenza A H1N1 virus were performed on nasopharyngeal swabs or bronchoalveolar lavage samples when available. Either of two PCR protocols were used for detection of thepandemic Influenza A H1N1 virus depending on test availability: the Real-time ready InfluenzaA/H1N1DetectionSet® Version June 2009  and the PCR protocol used by the WHO .The following data were obtained anonymously: underlying illness, type and date of SCT, whether patients were or were not receiving immunosuppressive treatment, at the time of the influenza diseases, immunization for seasonal influenza, clinical presentation (influenza like or pneumonia), laboratory and radiology results, anti-viral treatment, and outcome. In addition, data on the time between the onset of symptoms and the initiation of antiviral therapy, need for ventilation support, and presence of co-infections were also collected. Hypoxemia was defined as an oxygen saturation value lower than 96%. We diagnosed lymphopenia when the absolute lymphocyte count was less than 1000/µL. The RT-PCR tests forCategorical variables are shown as percentages and they are compared with the χ²-distribution test or Fisher test. Numeric variables such as median and range are compared with the Wilcoxon test. The association between baseline variables and events is presented as OR with the 95% CI. In all cases, statistical significance was assumed at a value of p=<0.05.From May to August 2009, 12 centers sent data of 65 cancer patients with 2009 H1N1 virus disease confirmed by positive PCR in BAL (3) or nasopharyngeal wash (62). The median age of the patients was 51 years (range 17 to 81), and 57% were female. The majority of patients (47) had onco-hematological cancer (72%) and 18 (28%) had solid tumors. Cancer treatment included chemotherapy (46), SCT (16), no treatment (2) and surgery (1). History of 2009 seasonal influenza vaccination was present in 19/64 patients (30%). No patient had received influenza chemoprophylaxis. The median time of patients follow up from the onset of symptoms was 61 days, range 5 to 259.Data on overall clinical presentation and outcome are shown in17.Patients started treatment at a median of two days from onset of symptoms (range 0 to 45 days). Sixty eight percent (43/63) of patients started treatment within the 48h after the onset of symptoms. Some patients received combined antiviral treatment because of the potential circulation of seasonal Influenza A H1N1 known to be resistant to oseltamivirMost patients acquired the infection in the community  while 7 (11%) of infections were acquired in the hospital setting despite the implementation of adequate standard precautions and isolation measures during this outbreak. Detailed descriptions of the outcome of patients with community acquired (CAPIA) and nosocomially-acquired (NAPIA) pandemic Influenza A H1N1 infection patients are described inMost  of outpatients required hospital admission. Reasons for patient admission included mainly oxygen desaturation, but, in many cases, patients were admitted because of their severe state of immune suppression and the lack of information about this emergent virus, especially when the patient’s social environment prevented him/her from easy access to medical care.Outpatients who presented with upper respiratory tract symptoms (URTI), had the most benign course since the majority  resolved their infections with antiviral therapy in the outpatient setting, and, among the 8 (42%) who were admitted, none of them required ICU admission or developed signs of pneumonia. The 30-day mortality among CAPIA URTI was 0.Outpatients who presented with pneumonia had a more severe course since almost all of them  were admitted, 15/39 (38%) required ICU, 11/39 (28%) required mechanical ventilation (MV), and the 30-day mortality in this group was of 23% (9/39). The worst prognosis in this group was seen among those who presented with pneumonia and desaturation (25), leading to an admission rate of 100% , and a 30-day mortality of 36%.Patients, who developed NAPIA, belonged to 3 different centers and started having symptoms at median of 20 days after admission (range 2 to 33). This group had the poorest prognosis since the 30-day mortality rate was 43% (3/7).One of three (33%) NAPIA URTI progressed to pneumonia, while none of the 19 patients with CAPIA URTI did. Therefore, the overall progression from URTI to pneumonia was of 4.5% (1/22).The 30-day mortality according to the clinical presentation and setting is best described inAcinetobacter baumanii, and GNR that was only seen in direct examination), 2 pneumonias  and 1 meningitis (Ps. aeruginosa). The median time from the onset of symptoms to the development of a bacterial complication was 11 days, range 0–34. Bacterial complications developed only among patients who presented with pneumonia  by the pandemicInfluenza A H1N1. No bacterial complication developed among the 22 patients who presented with URTI.Bacterial complications were documented in 6 (9%) patients and included 3 bacteremias  and bleeding (2) (lung and brain). Most patients presented more than one complication.Non-infectious complications developed in 14 (22%) patients. They included: renal failure (5), respiratory failure (5), shock (3), hypokalemia (3), nonbacterial infections (3)  at onset of symptoms and the delay in treatment were not associated to death or mechanical ventilation. By univariate analysis lack of history of vaccination, and the following baseline characteristics: pneumonia, oxygen saturation <96%, and lymphocyte count <800 cells/μL, were associated to 30-day mortality and mechanical ventilation. By multivariate analysis only lack of history of vaccination (OR did not apply because none died in the vaccinated group) and baseline oxygen saturation <96%  were associated to mechanical ventilation and death. There might be a bias regarding the apparent benefit of vaccination because in cancer patients, immunization is usually advised when the period of major immunosuppression has finished.Baseline characteristics, clinical presentation, treatment and outcome of 65 cancer or SCT patientsClick here for additional data file.Copyright: © 2015 Dignani MC et al.2015Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Influenza A H1N1 virus in 65 cancer patients from 12 institutions located in two cities of Argentina. Eleven percent of these infections were nosocomially acquired. Overall we found a high rate of pneumonia (66%) and mortality (18%). The clinical course was less severe in those who presented with an URTI in the outpatient setting in contrast to those who presented with pneumonia and desaturation especially in the hospital setting. We also found that the best predictors of death were oxygen desaturation at presentation and lack of vaccination against seasonal Influenza.Our study shows the clinical course of the infection by the 2009 pandemic20 and it is also higher than the incidence of lower respiratory tract infections (LRTI) caused by the 2009 pandemicInfluenza A H1N1 in the hospitalized general population (40–44%)21, in solid organ transplant recipients (23%)16, in HCT recipients (21–56%)22 and in patients with hematological malignancies (48%)23. Only one small study that includes 15 confirmed cases of 2009 pandemicInfluenza A H1N1 infection in onco-hematological patients reports a higher incidence of pneumonia (87%)15.The incidence of pneumonia we found is higher than the one reported with seasonal influenza in cancer patients (5–44%)Adenovirus, Influenza,Parainfluenza, and RSV (5%)3. However, it is similar to the mortality rate reported in hematological patients with pneumonia byInfluenza,Parainfluenza,Picornavirus and RSV at a US institution (15%)24.The 30-day mortality rate we show in this study is more than three times the one observed in Argentina in the same patient population when looking at infections by other respiratory viruses such as20. The pandemicInfluenza A H1N1 virus is an independent risk factor for progression to LRTI  and hypoxemia  compared with seasonalinfluenza virus in HCT recipients1. In addition, immunosuppression was a main risk factor for early mortality among 337 Argentinean patients admitted to ICU with influenza like illness and respiratory failure that required mechanical ventilation. Mortality was highly associated with refractory hypoxemia25. These data explain our high mortality rate observed among the 17 patients who were admitted to ICU  or among the 12 patients who developed respiratory failure . These values are comparable to those reported in the same type of population22, but are higher than those reported in other populations, which ranged from 0–24%29. Indeed, the overall mortality rate observed among Argentinean patients admitted to ICU and requiring mechanical ventilation was 46%25. To further support the high mortality of patients with pandemic 2009 Influenza pneumonia, we identified hypoxemia at onset of symptoms as an independent predictor of mortality.It is well known that seasonal influenza-induced pneumonia is independently associated with mortality after HCT 30, and profound lymphopenia (<100 cell/µL) was reported as a significant risk factor for requirement of mechanical ventilation and death in HCT recipients infected with seasonal influenza virus30. In our study, having fewer than 800 lymphocytes/µL at presentation was a predictor for the need for mechanical ventilation and death in a univariate but not in a multivariate analysis. We did not analyze a lower value such as <100 of lymphocytes due to the small number of patients included with this value.Lymphocytopenia has been described as a risk factor for progression from upper to lower viral respiratory tract infection in cancer patientsIt is noteworthy that co-infections or bacterial complications developed in less than 15% of patients.30 and hypoxemia30 when instituted early after onset of symptoms. It was reported that delaying therapy in cancer patients with the pandemicInfluenza A H1N1 virus infection was significantly associated with death16. Early initiation of antiviral therapy in these patients may attenuate the severity of disease27. In our series, antiviral therapy was started early after a median of two days after the onset of symptoms, with a range from 0–45 days. We did not find any correlation between days from onset of symptoms to therapy or diagnosis to therapy by univariate or multivariate analysis.Neuraminidase inhibitor therapy appears to be effective in preventing progression to LRTI31. In our series half of the outpatients with URTI remained as such, while the other half was admitted but did not require ICU. There is a possibility that most of the admitted patients could have been managed as outpatients as well.It is known that patients with URTI can be treated as outpatients and can recover completely from their infection20.The global progression from URTI to pneumonia in our study was of 4.5% (1/22). This single patient had a nosocomial infection and died 21 days later with sepsis, respiratory failure and neutropenia. This is according to reports of progression to LRTI that may occur even after one week of symptoms22. Higher doses could also be considered in a setting of profound lymphocytopenia30. All our patients received standard dose of oseltamivir (75 mg po twice a day) for a minimum of 10 days based on data on slower viral clearance30.In contrast, patients with LRTI required hospitalization with a high number of them requiring admission to ICU for ventilation support. The dismal outcome seen in these patients despite treatment with oseltamivir probably indicates that this high-risk group needs to be treated differently from patients with isolated URTI. Some authors have suggested an initial treatment with high dose of oseltamivir and/or combination therapy approaches in the case of respiratory failure32 and pandemic 2009 Influenza A H1N1 infection33 can develop even in the setting of appropriate infection control measures. Seven (11%) of our patients had hospital-acquired influenza. Three of them (43%) died. This mortality rate is higher than the previously reported 13–27%, however, the number of patients in our report is too small to make any conclusion.Nosocomial outbreaks of seasonal34. In our study all deaths occurred among the non-vaccinated patients, while there were no deaths among the vaccinated patients. Individuals vaccinated against seasonal Influenza A or with previous seasonal influenza infection may benefit from preexisting cross-reactive memory CD4+ T cells and CD8+ T cells reducing their susceptibilityto Influenza A H1N1 infection or explaining, at least in part, the unexpected mild illness in the community39. Whether the trivalent seasonal Influenza vaccine is protective against the pandemicInfluenza A H1N1 virus in cancer patients is still a matter of debate38.Seasonal influenza vaccination is recommended yearly for all patients with cancer and HSCT recipientsInfluenza A H1N1 infection with a high incidence of hospitalization, severe pneumonia, ICU admission, mechanical ventilation, and 30-day mortality. In our series hypoxemia and lack of vaccination with seasonal trivalent influenza vaccine were predictors of mechanical ventilation requirement and death. A larger study is needed to evaluate the possibility of cross protection with the seasonal influenza vaccination.In conclusion, we report a series of cancer patients with the pandemicThe data referenced by this article are under copyright with the following copyright statement: Copyright: © 2015 Dignani MC et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).F1000Research: Dataset 1. Baseline characteristics, clinical presentation, treatment and outcome of 65 cancer or SCT patients doi:http://dx.doi.org/10.5256/f1000research.5251.d10027640Ethical committee approval was not required in Argentina at the time of the study. This is an interesting article that shows the impact of pandemic 2009 influenza A (H1N1) in a highly vulnerable population for influenza complications such as immunocompromised oncohaematological patients.Did you observe any difference in time elapsed since the initiation of symptoms between those with community acquired pandemic 2009 influenza A (H1N1) who presented with upper respiratory tract infections and those who presented with pneumonia? Previous vaccination against seasonal influenza showed that some degree of preexisting immunity to pandemic 2009 influenza A (H1N1) strains exists, especially among adults aged >60 years. Have you found any difference in progression from UTRI to pneumonia or to death between those older or younger than 69 years of age?The paper is well written and I would like to make some comments or questions to the authors:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Question 1:Patients with community acquired 2009 Influenza A (H1N1) who presented with URTI (N=19) had symptoms for a median of 2 days (range 1-11). Patients who presented with pneumonia (N=39) had symptoms for a median  3 days (range 1-20).There was a trend towards a longer period with symptoms in patients who presented with pneumonia.Question 2:Patients older than 60 years old who had been vaccinated against seasonal Influenza  (N=10) had 60% rate of pneumonia and 0 mortality at day 30 of treatment. Patients with the same age that had not been vaccinated (N=4) had 100% rate of pneumonia and 25% mortality. Patients younger than 60 years old that had been vaccinated (N=9) had 55% rate of pneumonia and 0 mortality at day 30 of treatment. Patients with the same age that had not been vaccinated (N=31) had 68% rate of pneumonia and 26% mortality. Vaccination against seasonal Influenza seemed to have some degree of protection against death in all age groups. Does the report include all patients fulfilling the inclusion criteria at the 12 hospitals?Why was the cut-off for lymphocytopenia chosen as <800? Most previous studies have chosen a lower cut-off.One of several reports on outcome of the H1N1 pandemic in 2009. A couple of issues:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Sirt1 gene that abolishes SIRT1 catalytic activity. Tumor latency was reduced in animals fed a high fat diet but this effect was not dependent on SIRT1 activity. Resveratrol had little effect on tumor formation except in animals heterozygous for the mutant Sirt1 gene. We conclude that the effects of these dietary interventions on tumorigenesis are not mediated by modulation of SIRT1 catalytic activity.The protein deacetylase SIRT1 is involved in the regulation of a large number of cellular processes that are thought to be required for cancer initiation and progression. Both SIRT1 activity and tumorigenesis can be influenced by dietary fat and polyphenolics. We set out to determine whether dietary modulations of tumorigenesis are mediated by SIRT1 catalytic functions. We introduced a mammary gland tumor-inducing transgene, MMTV-PyMT, into stocks of mice bearing a H355Y point mutation in the  The NAD+ produced in this manner may function as a cofactor for the SIRT1 enzyme The rapid proliferation of cancer cells is associated with a metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, a phenomenon known as the Warburg effect Sirt1 may have the properties of a tumor suppressor gene and several studies have suggested that this may be the case Sirt1 is an oncogene Sirt1 has no effect on oncogenesis In mammals, caloric restriction (CR) can delay the onset of various ageing-related diseases including cancer Sirt1 alleles or contain mutations in the Sirt1 gene, suggesting a role as a non-classical tumor suppressor Sirt1 genes. We report below that both dietary modifications modestly affected the onset of tumorigenesis but were not dependent on SIRT1 activity.A relatively scant amount of clinical data has suggested an oncogenic role for SIRT1 in breast cancer Sirt1Y/+), mice on a mixed 129sv/CD1 background and carrying the tm2McbySirt1 allele. This allele harbours a missense mutation in the Sirt1 gene (H355Y) and encodes a catalytically inactive protein Sirt1 alleles was performed as previously described Male FVB/N-Tg(MMTV-PyMT)634Mul/J mice , were a generous gift from Dr. Bill Muller Guidelines for the Care and Use of Animals established by the Canadian Council on Animal Care with protocols approved by the Animal Care Committee of the University of Ottawa, Ottawa, Ontario, Canada.Animals received food and water ad libitum. Our previous work has shown that the caloric intake is similar between mice of all Sirt1 genotypes 2 asphyxiation. Tumours were removed, weighed and fixed in 10% neutral buffered formalin. Lungs were examined for macroscopically visible metastatic nodules prior to fixation in formalin.Animals were euthanized via COP-values are two-sided. Analysis was performed using Graphpad Prism statistical software .The probability of significant differences was determined by analysis of variance (ANOVA), employing the Kruskal-Wallis test with the Dunn's multiple comparison test. Survival and time-to-detection curves were compared using the LogRank test. Correlation was tested using the Spearman rank test. Data is expressed as mean±SEM (standard error of the mean) and Sirt1 gene  and studied the emergence of mammary tumors in +/+Sirt1, +/YSirt1, and Y/YSirt1 females. Animals were monitored weekly via digital palpation for the presence of any palpable mass in the mammary glands. It should be noted that no compensatory increase in the mRNA levels of other sirtuin family members, Sirt2-7, was observed in the Sirt1Y/Y animals  consisting of 60% calories from fat or a standard rodent diet (6% fat) from time of weaning until they had reached humane endpoint, generally due to significant tumor burden. HFD decreased tumor latency in both Y/Y mice . The eff reduced . The HFDY/+ mice .Sirt1Y/Y mice weighed 20–30% less than their Sirt1Y/+ and Sirt1+/+ counterparts (Sirt1Y/Y mice gained weight faster than their Sirt1+/+ and Sirt1Y/+ littermates. By 70 days of age (approximately 7 weeks on HFD) Sirt1Y/Y mice weighed an average of 10–20% more than the Sirt1+/+ and Sirt1Y/+ mice  and animals on the low phytoestrogen control diet (r2 = 0.48) (There was no significant difference in tumor burden for any of the her diet . Tumor b = 0.48) .Sirt1Y/Y mice were less likely to have macroscopically visible metastatic lung nodules than Sirt1+/+ or Sirt1+/Y mice  has yet to be investigated.In all of the studies described here as well as in those reported previously Figure S1Y/Y mice.There is no compensatory increase in expression of other sirtuin family members in tissues of Sirt1 Sirt1-7 mRNA levels measured in A) liver (n = 1 per group) or B) brain (n = 2 per group) of Sirt1+/+ and Sirt1Y/Y mice. Values are expressed as the mean ± SEM of the threshold cycle (CT).(TIF)Click here for additional data file.Figure S2Weight gain over time.Sirt1+/+, Sirt1Y/+, and Sirt1Y/Y  mice were weighed once weekly and the weights represented here range from one week after commencing the specified diet (approximately 28 days of age) until the final week at which all animals in each group were still alive (70 days of age). A) standard rodent diet. B) high fat diet. C) low phytoestrogen diet. D) resveratrol containing diet. N = 10 for all groups.(TIF)Click here for additional data file.File S1Materials and Methods: Quantitative Polymerase Chain Reaction (QPCR). Measurement of Sirt1 mRNA levels via QPCR.(DOCX)Click here for additional data file.


Bipolar disorder is a highly heritable neuropsychiatric disorder affecting nearly 2.5% of the population. Prior genetic studies identified a panel of common and rare single-nucleotide polymorphisms associated with the disease that map to the first intron of the PDE10A gene. RNA sequencing of striatal brain tissue from bipolar and healthy control subjects identified a novel transcript of PDE10A, named PDE10A19, that codes for a PDE10A isoform with a unique N terminus. Genomic sequences that can encode the novel N terminus were conserved in other primates but not rodents. The RNA transcript was expressed at equal or greater levels in the human striatum compared with the two annotated transcripts, PDE10A1 and PDE10A2. The PDE10A19 transcript was detected in polysomal fractions; western blotting experiments confirmed that the RNA transcript is translated into protein. Immunocytochemistry studies using transfected mouse striatal and cortical neurons demonstrated that the PDE10A19 protein distributes to the cytosol, like PDE10A1, and unlike PDE10A2, which is associated with plasma membranes. Immunoprecipitation and immunocytochemical experiments revealed that the PDE10A19 isoform interacts physically with PDE10A2 and, when expressed at elevated levels, interferes with the plasma membrane localization of PDE10A2. These studies illustrate the complexity of PDE10A gene expression in the human brain and highlight the need to unravel the gene's complex and complete coding capabilities along with its transcriptional and translational regulation to guide the development of therapeutic agents that target the protein for the treatment of neuropsychiatric illness. Individuals exhibiting mania experience periods of markedly elated moods, are easily distracted and tend to engage in impulsive and high-risk behaviors.11 We recently queried potential BD-associated loci using a ‘molecular pathway approach,' performing a case–control study focusing on genes that comprise the cAMP pathway,12 prompted in part by numerous reports showing disrupted cAMP signaling in BD.16 Our results identified a series of common and rare single-nucleotide polymorphisms within the first large intron of the PDE10A gene as associated with BD, which generated great interest given the prominent expression of this enzyme in striatal cells,20 the role of the striatum in coordinating and integrating cortical and dopaminergic inputs23 and the promise of PDE10A as a pharmacological target for brain disorders including schizophrenia and BD.24Genome-wide and case–control association studies have identified numerous susceptibility genes for BD with variants exhibiting modest effect sizes.GMP-stimulated PDEs, Anabaenaadenylyl cyclases and Escherichia coli transcription factor Fhla) domains and hydrolyze both cAMP and cGMP, but have a higher affinity for cAMP.25 The PDE10A gene is currently known to be comprised of 23 exons26 and maps to chromosome 6q26,18 a region previously associated with BD.27 The known isoforms of PDE10A, PDE10A1  were obtained from the Human Brain and Spinal Fluid Resource Center . The samples were de-identified of all the information other than clinical diagnosis, sex, age at death and postmortem interval . For RNAThe 5′ RNA ligase-mediated rapid amplification of complementary DNA (cDNA) ends (RLM-RACE) studies were performed with HC putamen and a primer that bound within exon 4. The resulting 5′ DNA fragments were cloned and sequenced. To determine whether PDE10A transcripts were alternately spliced between exons 4–22, total RNA was treated with DNAseI and used for cDNA synthesis primed by random hexamers. PCR primer pairs  were the32 RNA isolation, cDNA synthesis and real-time PCR were performed as described above.Polysomes were isolated from four HC striatal tissue specimens, obtained from the Harvard Brain Tissue Resource Center , a part of the NIH NeuroBioBank , via ultracentrifugation on sucrose gradients.Custom anti-peptide antibodies were generated for each PDE10A isoform by an external contractor. The Scripps Research Institute IACUC approved the production of these antibodies. Full-length PDE10A transcripts with 3′-HA or Flag epitope tags were constructed  and usedThe PDE10A19-HA or PDE10A2-HA enzyme was affinity purified from transfected HEK293 cell lysates using the HA-tagged Protein Purification Kit  according to the manufacturer's instructions. Enzymes from four affinity purifications were pooled and desalted by gel filtration to remove excess phosphate from the samples. PDE10A activity was measured within the linear phase of the enzyme's activity over a 15-min time course using the Cyclic Nucleotide Phosphodiesterase Assay Kit  according to the manufacturer's instructions.33 DIV14–18 primary neurons were fixed, incubated with anti-HA Alexa 488-conjugated monoclonal antibody and/or anti-peptide antibodies  and imaged.34 Immunoreactivity intensity profiles across neurites were computed and normalized for neurite diameter differences and background intensity. The images were analyzed using Image J and statistical analyses performed using Excel and/or Prism.Primary neuronal cultures from 0- to 1-day-old C57BL/6J pups were prepared for immunocytochemistry as described.de novo RNA transcript assembly for each of the samples. From this de novo alignment, the annotated PDE10A2 transcript (NM_001130690) was detected in all of the HC samples, determined by obtaining reads specific to exon A1/2.1 and spliced to exon 2  were found linked to the annotated exons 2, 3 and 4 (RNA sequencing (RNAseq) was performed on total RNA isolated separately from the putamen and caudate nucleus of postmortem striatal brain tissue from four individuals diagnosed with BD and four individuals with no reported symptoms of any neuropsychiatric disorder (HCs). On average, 20 million reads were obtained from each sample  and aligo exon 2 , while to exon 2 . Surpriso exon 2 .35, 36 T 3 and 4 . A human 3 and 4 . A TATA We generated and sequenced overlapping RT-PCR (PCR with reverse transcription) amplicons spanning PDE10A annotated exons 4–22 to ascertain whether any additional RNA transcripts might be produced by alternative splicing across this region. These efforts failed to reveal any major splice variants that arise from the coding region for these exons . A smallThus, our combined RNAseq and 5′ RLM-RACE data indicate the existence of a novel first exon for PDE10A, a heretofore unknown but predicted protein isoform of the human PDE10A gene, named PDE10A19, and a promoter for the PDE10A gene residing upstream of the novel first exon which may confer different regulatory properties on PDE10A19 expression compared with other PDE10A RNA transcripts. The data further indicate that the N terminus of PDE10A19 is unique but comes into frame with the other PDE10A isoforms at exon 2. Therefore, it is predicted to contain the same GAF binding and catalytic domains found in the PDE10A2 and PDE10A1 isoforms .We queried online protein sequence databases and found that the green monkey and the crab-eating macaque both have predicted proteins that are highly homologous to PDE10A19 . We alsode novo assembly analysis (P=0.0482) and PDE10A2 (P=0.0150) transcripts in the putamen when the data for all the eight subjects were pooled and compared (P=0.0087) and PDE10A1 (P=0.0010), while no significant difference was found in the abundance estimate between the PDE10A2 and PDE10A1 transcripts. We also examined the expression level of the three transcripts between the HC and BD subjects. The PDE10A2 transcript was found to be expressed fivefold less abundantly in the BD putamen samples compared with the HC from the RNAseq data (P=0.0039).To estimate the abundance of the PDE10A19 transcript relative to PDE10A1 and PDE10A2, we counted the number of RNAseq reads residing within the novel exon of the transcript and normalized these reads for exon size and number of reads that mapped to the upstream PDE10A genomic region used for the analysis . The PDEcompared . The PDEWe isolated polysomes from postmortem HC striatal tissue to determine whether the three different mRNAs were loaded into protein synthesis machinery. Tissue samples were lysed in the presence of the translational inhibitor cycloheximide and the cytosolic supernatant was separated on a sucrose gradient via ultracentrifugation. The relative transcript abundances in gradient fractions were measured by quantitative RT-PCR. Messenger RNA for all the three PDE10A isoforms, PDE10A2, PDE10A1 and PDE10A19, was detected in both low molecular weight (free RNA) and high molecular weight (containing polysomes) fractions, suggesting their association with polysomes in striatal neurons .We generated anti-peptide antibodies to the unique N termini of the three different PDE10A isoforms to assay their expression in both human tissue and transfected cells. We tested the anti-peptide antibodies using an indirect ELISA assay to determine how the anti-peptide antibodies bound to their specific antigenic peptide . This asWe tested this hypothesis using IP experiments. Cell lysates expressing the HA-tagged PDE10A19 isoform were immunoprecipitated with anti-PDE10A19 anti-peptide antibody, anti-PDE10A2 anti-peptide antibody, anti-HA antibody and normal rabbit IgG. These IP reactions were fractionated and probed with the anti-HA antibody . This prTo test whether the PDE10A19 protein is expressed in human brain, we performed IP experiments using human striatal tissue. A commercially available PDE10A antibody that targets the conserved carboxyl terminus of PDE10A was used to IP PDE10A proteins from HC 3589 and the immunoprecipitates were probed with the PDE10A19 anti-peptide antibody. As shown in m, respectively.To confirm that PDE10A19 hydrolyzed both cAMP and cGMP and exhibited enzymatic properties similar to other PDE10A isoforms, HA-tagged PDE10A19 and HA-tagged PDE10A2 enzymes were affinity purified from transfected HEK293 cell lysates for use in cyclic nucleotide phosphodiesterase assays. 30 None of the three isoforms were expressed in the nucleus.  that mapped to regions of the genome currently annotated as intronic. Others have reported that up to 64% of the total RNAseq reads map to non-exonic regions of the genome using human brain tissue,38 unlike other tissues.39 Thus, our data along with others indicate that the complexity of gene expression in the human brain is greatly underappreciated. This dictates a strategy that most, if not all, neurogenetic studies that focus on individual genes should begin with a complete structural characterization of the gene of interest to reduce the possibility of false conclusions.Our prior case–control study of 29 different genes comprising the cAMP signaling pathway identified seven common and eight rare nucleotide variants associated with BD that mapped to a 23 kb intronic region of the PDE10A gene.The strategy applied here to the PDE10A gene revealed unknown complexity in RNA transcripts and the spectrum of protein isoforms encoded by them. The 5′ RLM-RACE experiments using HC putamen tissue confirmed the existence of the PDE10A19 transcript and argued that it includes a unique transcriptional start site and regulatory region, since multiple and independent cDNA clones of PDE10A19 terminated in the identical 5′ nucleotide. The expression of PDE10A19 transcripts was measured at levels equal to or higher than the two annotated forms of PDE10A, PDE10A1 and PDE10A2. Thus, the impact of PDE10A19 on the physiology of human striatal neurons cannot be trivial. This critical fact now needs to underlie all studies of how cAMP signaling influences the physiological state of medium spiny neurons.40 As rodents do not express the PDE10A19 isoform, mouse physiology and behavioral studies become difficult to perform. However, the primate-specific presence of PDE10A19 is consistent with the thought that neuropsychiatric disorders like BD and schizophrenia are human-specific diseases with human-specific etiology.A very intriguing observation made was that the genomic sequences encoding the novel N terminus of PDE10A19 is conserved only among primates, and not rodents. This is in contrast to PDE10A1 and PDE10A2, which are conserved isoforms in both primates and rodents. This suggests that the PDE10A19 isoform evolved after the separation of primates from rodents. Species expression differences of PDE isoforms are not unusual; in fact, in the case of the PDE4B4 isoform, while humans and rats have relatively conserved genomic sequences for this isoform, only rats express this protein due to the stop codons positioned in every reading frame of the human nucleotide sequence.30 Therefore, this isoform may be primarily responsible for submembranous PDE activity, including the PDE10A activity at the membranes of dendritic spines. Our data argue that the three human PDE10A isoforms are non-nuclear, in contrast to some studies of rat non-striatal neurons where PDE10A was primarily localized to the nucleus.41 In addition, we show here that PDE10A19 and PDE10A2 interact; and that PDE10A19, when expressed at high levels, can redirect PDE10A2 from a membrane-bound position to a cytosolic one. Thus, the level of expression of PDE10A19 under normal circumstances is predicted to influence the ratio of membrane/cytosolic PDE activity. It also seems possible that mis-expression of PDE10A19 in a diseased state would dysregulate the normal trafficking of PDE10A2, rendering it unable to associate with the plasma membrane and unable to control cAMP signaling at the synapse and/or across cellular membranes. One function of PDE10A activity is to inhibit striatal output by reducing medium spiny neuron excitability.42 If this function is primarily because of PDE10A2 membrane localization, then mis-expression of PDE10A19 would be predicted to redirect PDE10A2 to the cytosol, reducing membrane-associated PDE activity, and increasing the excitable state. This idea that a lack of PDE10A enzyme could cause cAMP signaling disruptions has also been suggested as a cause of the motor and psychiatric deficits seen in Huntington's Disease.44 Competitive inhibitors of PDE10A have been proposed as therapeutic targets to ameliorate psychosis.45 However, if the molecular pathology of psychotic disorders arises from a failure of PDE10A2 to regulate excitability at the membrane, due to mis-expression of PDE10A19, such therapies may be ineffective.Besides providing cytosolic PDE activity, PDE10A19 may modulate cAMP signaling by influencing the subcellular localization of its binding partners. PDE10A2 is only one of the three isoforms investigated in this study that appears capable of interacting with the plasma membrane.47 This multimerization occurs through the GAF-B binding domain of the protein,48 and as this region is conserved between the PDE10A19 and PDE10A2 isoforms, it stands to reason that these two isoforms may interact as a heterodimer. The dimerization of PDE10A2 with PDE10A19 may in some way protect or expose the N-terminal antigenic site of PDE10A2.In addition, we discovered that our PDE10A2-specific anti-peptide antibody failed to recognize PDE10A2 protein when expressed alone, but did so when PDE10A19 was expressed. This lack of recognition can be explained by at least two hypotheses: (1) the PDE10A2 anti-peptide antibody fails to recognize the PDE10A2 N terminus because the N-terminal sequences are cleaved from the protein posttranslationally or (2) PDE10A2 palmitoylation at Cys-11 ref.  or phospOur data reveal that the landscape of PDE10A gene expression and protein function in the striatum is much more complex than originally thought. Our studies identify a novel and reasonably abundant form of PDE10A along with its own promoter. We also show that the expression of this isoform influences the subcellular localization of other isoforms, specifically PDE10A2. The cell biological knowledge of this important family of enzymes is critical for understanding the enzyme's role in contributing to neurospsychiatric diseases such as BD. Will the large intronic region found to be associated with BD consist of regulatory elements responsible for controlling the expression of different PDE10A isoforms? As discussed above, disturbances to this balance of transcript/protein abundance could dysregulate cAMP signaling, perhaps in specific subcellular compartments, and alter behavior. Thus, more studies targeted at understanding how these various isoforms are transcriptionally and translationally regulated within the human striatum are warranted.