Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation.Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity , and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation (“buried topsoils”), resulting from a decrease in fungal abundance compared to recent (“unburied”) topsoils.  It has been predicted that due to the strong increase in mean annual surface air temperature (up to 8°C), 25% of the arctic permafrost could thaw until the end of this century IPCC, . IncreasThe diversity of microbial communities in permafrost-affected soils, their functional potential and the coupling of SOM properties and microbial activity have been increasingly recognized and addressed in several studies in Siberia  at three sites in northeastern Greenland. We aimed to (1) explore the microbial diversity and community composition, (2) determine potential activities of oxidative and hydrolytic extracellular enzymes involved in the degradation of organic matter, and (3) assess possible correlations between enzyme activities and shifts in community composition. We hypothesized that different sampling sites and/or soil horizons harbor distinct communities and that changes in community structure are reflected in the enzymatic and thus the degradation potential of these communities. We were particularly interested in the distribution patterns of both fungal and bacterial decomposers  that have previously been identified as critical components in the delayed decomposition of organic matter in cryoturbated soils . Site 2 was a wet fen dominated by mosses and grasses . Two to three replicate plots that were distributed within an area of approximately 50 × 50 m were sampled at each sampling site  and kept cold until further processing. Samples for the analyses of soil properties, microbial biomass and potential enzyme activities were stored in closed polyethylene bags at 4°C until analyzed.Soils were sampled in close vicinity to the Zackenberg Research Station, northeast Greenland . The climate is characterized by a mean annual air temperature of around −10°C and an annual precipitation of about 150 mm. Minimum air temperatures are below −40°C, and sub-surface temperatures (5 cm below the surface) are below −18° for about 4 months per year . Microbial C and N were calculated as the difference in C and N concentrations between fumigated and non-fumigated samples.Soil water content was estimated by drying the soil over night at 60°C and reweighing the samples. Total organic carbon (TOC) and total nitrogen (TN) contents were determined in dried (60°C) and ground samples with an EA IRMS system . Microbial C and N were determined using chloroform-fumigation extraction  were measured according to Kaiser et al.  using milater. Approximately 0.5 g of soil was added to a FastPrep™ Lysis Matrix E tube . Hexadecyltrimethylammonium bromide (CTAB) extraction buffer containing 5% CTAB  and 0.5 mL phenol-chloroform-isoamylalcohol (25:24:1) was added and shaken in a FastPrep Instrument  at speed 5–6 for 45 s. After bead beating, the samples were extracted with chloroform and precipitated in a PEG 6000/1.6 M NaCl solution. Pellets were washed with 70% ethanol and re-suspended in molecular biology grade water. Nucleic acids were further purified using the CleanAll DNA/RNA Clean-up and Concentration Micro Kit . Total DNA was quantified using SybrGreen  of a known amount of purified PCR product obtained from genomic Escherichia coli DNA and Natrialba magadii DNA by using the SSU gene-specific primers 8F/1492R and 21F/1492R, respectively  of a known amount of purified PCR product obtained from genomic Fusarium oxysporum DNA by using the SSU gene-specific primers nu-SSU-0817-5′ and nu-SSU1196-3′ . Amplicons were sequenced on the Illumina MiSeq platform .Nucleic acid extration and bacterial and archaeal SSU rRNA amplification were performed according to standardized protocols of the Earth Microbiome Project  was used to summarize data on soil properties and microbial biomass. Data were standardized (zero mean and unit standard deviation), and the number of dimensions were estimated by cross-validation and missing values were imputed using the package missMDA , and a wet fen site , and from four different soil horizons at each of these sites: organic and mineral topsoil , mineral subsoil , buried topsoil , and the permafrost layer . Topsoil horizons exhibited a higher moisture, TOC and TN content, as well as higher microbial biomass  compared to buried topsoils, mineral subsoils and permafrost samples . Significant differences in FB ratio were found between O and A topsoil horizons as well as between O and J horizons . Archaeal gene copies followed the bacterial distribution pattern and were highest in topsoil horizons and buried topsoils. They were two to three orders of magnitude lower in mineral subsoils and permafrost samples. The fraction of archaeal SSU rRNA genes proportionally increased with depth being lowest in O and A horizons  and highest in permafrost samples (0.4%).On average, bacterial SSU rRNA gene abundances per gram dry soil were highest in topsoil samples and buried topsoils and were two to four orders of magnitude lower in mineral subsoils and permafrost samples Figure . In contGeneral observations as described above were also apparent for the individual sampling sites, including highest bacterial, archaeal and fungal abundances in topsoils and buried topsoils, and lower abundances in subsoils and permafrost samples Figure . While dP-values or insignificant differences, Figure Similar patterns in bacterial, archaeal and fungal SSU rRNA gene distributions were found when soil organic carbon (OC) was taken into account Figure . Howeverhttp://www.microbio.me/emp/, study no. 1034). Sequencing was successful for 20 of these samples , Acidobacteria, Actinobacteria, Verrucomicrobia, Bacteroidetes, and Firmicutes accounting for ~84% of all sequences , and 38 other rare phyla (<0.5% each) were identified. Rare phyla included archaeal taxa that represented 0.3% of all sequence reads. The majority of archaeal reads was assigned to the classes Methanomicrobia and Methanobacteria within the Euryarchaeota comprising 39.8% of all archaeal reads. For site 2, sequence reads assigned to archaeal taxa comprised 0.9% of all reads obtained from site 2 samples, whereas they only accounted for 0.05 and 0.01% at sites 1 and 3, respectively  were subjected to Illumina tag sequencing of the V4 SSU rRNA gene region  = 0.001) and soil horizon  = 0.007), but not for the soil pit factor  = 0.097). No interactions between factors  were apparent . PCoA based on unweighted UniFrac distances was used to visualize clusters of similar communities according to sampling site, soil horizon and soil pit  = 0.001), followed by soil horizons  = 0.002), and soil pits  = 0.025). Topsoil samples from sites 1 and 3 formed a distinct cluster that also included two out of four of the buried soil samples from these sites (samples A4 and G4), but excluded topsoil and buried topsoil samples from site 2 . Samples from buried topsoils at sites 1 and 3 were highly variable in species richness and diversity resulting in statistically insignificant differences between horizons . Potential NAG activity was similarly high in topsoil and buried topsoil samples and significantly lower in mineral subsoils and permafrost samples. Potential oxidative enzyme activities were highest in samples from topsoils (O) and buried topsoils . Potential POX and PER activities were significantly higher in active layer samples from site 2 compared to the corresponding soil samples from sites 1 and 3  were highest in topsoil samples (O and/or A), followed by buried topsoils, mineral subsoils and permafrost samples Figure . This dis Figure . HoweverWhen potential enzyme activities were calculated per gram organic carbon (OC), differences between horizons became smaller and less significant for hydrolytic enzymes Figure Actinobacteria  and the Bacteroidetes , as well as putatively anaerobic members of the Firmicutes (Clostridia), the Chloroflexi , and methanogenic Euryarchaeota (Methanomicrobia) were positively correlated with potential POX and PER activities  and type II , and the recently described Methylacidiphilae within the phylum Verrucomicrobia .Sequences matching putative anaerobic taxa  were highly abundant in the wet fen site accounting for up to 38% of all sequences in the O horizon Figure . While tia Table . Type I 2 and CH4  in buried topsoils was affiliated with the family Intrasporangiaceae (Actinobacteria). Its abundance was particularly high in buried topsoils from site 2 . Although this OTU was also present in 90% of all other soil samples, it only accounted for an average of 1.7% to the total community in non-buried topsoils, mineral subsoils and permafrost samples. Members of the Intrasporangiaceae also constituted a major fraction of the prokaryotic community in mineral subsoils and buried topsoils from the Siberian Arctic, where they accounted for up to 30 and 47% of the total community, respectively , where fungal degradation activities are restricted  dominated in the topsoil horizons, whereas Methanosarcinales dominated in mineral subsoils. The latter are able to generate methane from a broad range of substrates including acetate, H2/CO2 and methylated compounds . This depth-related succession from a hydrogenotrophic to a metabolically more flexible methanogenic community has been found in other permafrost environments, such as arctic wetlands and peat soils, and has been attributed to advantages in the competition for substrates and their independence from syntrophic interactions . Together with few other studies  determining the dominance of one or the other MOB type, the balance between methane oxidation and methanogenesis and the ecological implications for arctic environments need further research.In line with previous reports from various cold environments  and stable oxidative enzyme activities with depth supported the hypothesis that bacteria might resume the role of fungi in the degradation of phenolic compounds such as lignin.The collective results presented here shed light on the highly diverse microbial communities in permafrost-affected soils in Northeast Greenland, their enzymatic degradation potential and the distribution of functional guilds involved in the anaerobic degradation of SOM . Site-specific differences were apparent between the typical tundra sites and the wet fen site, the latter being a potential hot spot for degradation activities. In addition, shifts in community composition between unburied and buried topsoils (decrease in fungal abundance and a predominance of Antje Gittel, Jiří Bárta, Tim Urich and Andreas Richter designed the research presented in this manuscript. Antje Gittel directed and performed the molecular work and sequence data analyses on prokaryotic communities. Jiří Bárta and Iva Kohoutová performed the quantification of fungal communities. Petr Čapek contributed with data on microbial biomass. Jörg Schnecker and Birgit Wild determined enzyme activity potentials and soil geochemical properties. Samples were collected by Christina Kaiser and Andreas Richter. Antje Gittel performed all statistical analyses. Antje Gittel, Jiří Bárta and Tim Urich wrote the manuscript with input from Christina Kaiser, Jörg Schnecker, Birgit Wild, Vigdis L. Torsvik, and Andreas Richter.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our data showed that UUO induced renal fibrosis and combined with the activation of ERK signaling, the upregulation of fibronectin, collagen I, and transforming growth factor-β (TGF-β). The administration of metformin inhibited the activation of ERK signaling and attenuated the production of extracellular matrix (ECM) proteins and collagen deposition in the obstructed kidneys. In cultured renal fibroblasts, Ang II increased the expression of fibronectin and collagen I and also activated ERK signaling and TGF-β in a time-dependent manner. Pretreatment of the cells with metformin blocked Ang II–induced ERK signaling activation and ECM overproduction. Our results show that metformin prevents renal fibrosis, possibly through the inhibition of ERK signaling, and may be a novel strategy for the treatment of renal fibrosis.Renal fibrosis is the final common pathway of chronic kidney disease (CKD), and no effective medication is available clinically for managing its progression. Metformin was initially developed as an anti-diabetic drug and recently gained attention for its potential in the treatment of other diseases. In this study, we investigated its effects on renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO)  Renal fibrosis is a common feature of all forms of chronic kidney disease (CKD), regardless of the initial cause of the disease ,2,3. It Renal fibrosis can be initiated by many pathological factors, including toxicity, ischemia, pathogenic microorganism infections, an inherited defect, or endocrine and immunological diseases . When inet al. have demonstrated that the anti-fibrotic effect of metformin in renal fibrosis in vivo [Metformin  is a big in vivo .in vivo and in vitro. Unilateral ureteral obstruction (UUO) is a well-characterized animal model of renal fibrosis [In this study, we investigated the effects of metformin on renal fibrosis both fibrosis ,20. Our in vivo. The Western blot results showed that the metformin inhibited UUO-induced upregulation of fibronectin and collagen I protein expression in the obstructed kidney  is associated with various deleterious effects, including vasoconstriction, inflammation, hypertrophy, and ECM protein synthesis ,24,25,26To examine the effect of metformin in renal fibrosis, metformin was administered to UUO mice. As shown by our results, metformin treatment in the UUO mice dramatically attenuated the expression of ECM proteins, including fibronectin and collagen I. The upregulation of TGF-β induced by UUO was abolished by metformin administration. Moreover, ERK phosphorylation was reduced by metformin, indicating that metformin attenuated the activation of ERK signaling. Taken together, these results suggested that metformin attenuated renal fibrosis in UUO mice, accompanied by a decrease in TGF-β and inhibition of the associated ERK signaling pathway.in vitro experiments were performed in renal fibroblast NRK-49F cells. The level of ERK phosphorylation was elevated significantly after Ang II treatment, indicating that Ang II induced the activation of ERK signaling. Furthermore, treatment with Ang II induced significant profibrotic effects including the upregulation of TGF-β and excessive production of ECM proteins. Metformin treatment reversed the Ang II–induced ECM overproduction. Moreover, it also abolished the Ang II–induced upregulation of TGF-β, and the activation of ERK signaling. Taken together, these results suggest that metformin could be effective in reversing the profibrotic responses in renal fibroblasts.Resident renal fibroblasts are the most important source of scar-producing myofibroblasts. The activation and proliferation of resident fibroblasts in the kidney result in the enhanced production and excessive deposition of ECM proteins. To confirm the effect of metformin in renal fibrosis, Dulbecco’s modified Eagle’s medium (DMEM), recombinant human angiotensin II, metformin, and PD98059 were purchased from Sigma-Aldrich . Bovine calf serum (BCS) was obtained from Gibco . The BCA Protein Assay Kit was from Shenergy Biocolor BioScience and Technology . The anti-fibronectin antibody was obtained from Sigma-Aldrich , the anti-collagen I antibody and anti-ATIR antibody from Abcam , the anti-TGF-β antibody from Santa Cruz Biotechnologies, Inc. , the anti-phospho-ERK and anti-ERK antibodies from Cell Signaling Technology , and the anti-GAPDH antibody and an enhanced chemiluminescence (ECL) detection kit and hydrogen peroxide assay kits from Beyotime Institute of Biotechnology . The Ang II ELISA kit was from Westang Biotech . Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore . The proteinase inhibitor and phosphatase inhibitor were obtained from Roche . All other chemicals and reagents used were of analytical grade.Male C57BL/6J mice (20–25 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . Mice were anesthetized with 5% chloral hydrate via intraperitoneal injection. The left ureter was visualized by a flank incision and ligated with 4–0 silk. The sham group underwent the same surgery apart from the left ureter ligation. Over the course of the study, six mice were included in each group and were euthanized three or seven days after surgery. Mice that underwent UUO surgery were randomly divided into three groups: UUO with vehicle , UUO with metformin (200 mg/kg body weight per day) and UUO with PD98059 (150 mg/kg body weight per day) administered by gavage for seven days. The kidneys were then harvested. All animal experiments were performed according to the Criteria of the Medical Laboratory Animal Administrative Committee of Shanghai and the Guide for Care and Use of Laboratory Animals of Fudan University, and the protocols were approved by the Ethics Committee for Experimental Research, Shanghai Medical College, Fudan University.2–95% air at 37 °C. They were deprived of serum for 24 h when they reached approximately 80% confluence. The cells were pretreated with 1 mM metformin, 0.1 μM PD98059, or vehicle for 1 h followed by the administration of 1 μM Ang II.Normal rat kidney fibroblasts (NRK-49F) were purchased from the Institute of Biochemistry and Cell Biology . Cells were cultured in DMEM containing 10% BCS, in 5% COProtein was isolated from the homogenized frozen kidneys or cell lysate homogenates. Protein concentrations were examined using a BCA Protein Assay Kit according to the manufacturer’s instructions. A standardized amount (30 μg per sample) of total protein was loaded and separated by electrophoresis on a 10% SDS-PAGE gel and then transferred to PVDF membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature with gentle shaking. The membranes were then incubated with the primary antibodies overnight at 4 °C . The membranes were then washed three times with Tris-buffered saline (TBS) with 0.1% Tween and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature. After another three washes with TBS/Tween, the hybridizing bands were developed using the ECL detection kit according to the manufacturer’s instructions. Protein bands were visualized by the GE chemiluminescence system for the required time. Bands were normalized using GAPDH or ERK.The concentration of Ang II was measured using commercially available enzyme immunoassay kits . The procedures were performed according to the manufacturer’s instructions. The Ang II concentration was calculated from the standard curve constructed with standard solutions.The kidneys were fixed in 10% neutral buffered formalin, and then embedded in paraffin. Sections were collected and prepared. Slides of paraffinized tissue sections were deparaffinized, rehydrated, and washed in distilled water. Sections were stained with Masson’s trichrome. Histological changes were observed at 200× optical magnification.post hoc analysis using Tukey’s multiple comparison test. The differences between two groups were compared using Student’s t test. p < 0.05 was considered to indicate a statistically significant difference between mean values.Data are presented as means ± SEM with the statistical analysis performed using one-way analysis of variance with in vitro and in vivo, and that these effects were associated with the downregulation of TGF-β and the inhibition of the ERK signaling pathway. Given its high safety and cheapness, we propose that metformin could potentially be used clinically to treat renal fibrosis.In summary, our study showed that metformin attenuated renal fibrosis effectively both


Physcomitrella patens, we provide microscopic evidence for the existence of mitochondria-ER interactions in plants and their correlation with mitochondrial constriction and fission. We further investigate a previously identified protein of unknown function (MELL1), and show that it modulates the amount of mitochondrial association to the ER, as well as mitochondrial shape and number.Whereas contact sites between mitochondria and the ER have been in the focus of animal and fungal research for several years, the importance of this organellar interface and the molecular effectors are largely unknown for plants. This work gives an introduction into known evolutionary differences of molecular effectors of mitochondrial dynamics and interactions between animals, fungi, and plants. Using the model plant  These GTPases contain two C-terminal transmembrane domains and mediate tethering of neighboring organelles and outer membrane fusion  protein from Although evidence for links between plant mitochondrial form, function, and dynamics exist, the molecular identity of interaction sites is mostly unclear and modulators and effectors known from animal systems, such as Bcl2-like proteins, mitofusins, PINK, and Parkin Logan,  are lackWe recently identified a plant protein with LEA (Late Embryogenesis Abundant) and LysM domains and a conspicuous subcellular localization to ER and mitochondria (MELL1), which influences mitochondrial shape , a third of the mitochondria showed an elongated shape with clear constriction sites  which is required for the localization of DRP3A to plant mitochondria  fission  as N-terminal targeting signal . To assemble this vector, PTA2 5′ homologous region  and PTA2 3′ homologous region  were amplified from genomic DNA, joined with two template PCR , and codons for the ER retention signal KDEL added to the C-terminus . This construct was introduced in the pAct5_PTA2 via restriction and ligation using SalI and Ecl136II. Subsequently, the signal peptide from moss aspartic protease  Bruch & Schimp. Gransden strain  were prepared and transfected as described previously  was co-transfected in a ratio of 3:1 with the construct for homologous recombination. For transient transfection, pAct5_PTA2 vectors containing organelle marker constructs and MELL1 overexpression vector , or diode lasers  using three separate tracks. Fluorescence was detected for chlorophyll from 670–756 nm , for GFP from 505–550 nm , for mtEOSred from 575–615 nm . Pinhole was set to 1 AU for Cerulean channel and section thickness adjusted accordingly in all other channels. Pixel dwell was 1.61 μs. Images were taken using 4 averages and 256 × 256 pixel for time series (~7 s per time point) and using 16 averages and 512 × 512 pixel for snaps. The zoom factor was adjusted to guarantee 1.5-2x overimaging of pixels, as recommended for deconvolution . Bleaching Figure  settingshttp://icy.bioimageanalysis.org; de Chaumont et al., http://www.graphpad.com/quickcalcs/), using two-tailed T-test. Bar graphs show mean and standard deviation.Confocal images were all deconvolved prior to subsequent analyses using Huygens Remote Manager . Co-localization analysis was performed in Huygens using Mander's coefficient (Manders et al., SM and RR planned and designed the research. SM performed experiments and analyzed data. SM and RR wrote the manuscript. All authors discussed data and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cerebellopontine angle (CPA) and cerebellomedullary fissure (CMF) tumors are rare in children and information is scarce in the literature. This retrospective study reports their histological distribution and tumor origin, and describes surgical resections and post-operative outcome based upon the authors’ consecutive personal series.Clinical data of infants and children 16 years old or younger of age treated from 2001 to 2012 by a single surgeon was retrospectively reviewed. All had histologically verified CPA/CMF tumors and underwent radical tumor resection through craniotomy except for two children who had a stereotactic biopsy for malignant tumors ). Tumors’ pathological distributions, tumors’ origin, surgical approaches, and patients’ outcome were reviewed.There were 44 infants and children with the age at diagnosis ranging from 11 weeks to 16 years; 32 were predominantly in the CPA and/or CMF whereas 12 showed an extension to the fourth ventricle. Pathology showed 14 ependymomas, 12 benign gliomas , 4 atypical teratoid rhabdoid tumors (ATRTs), 4 epidermoids, 3 primitive neuroectodermal tumors (PNETs), 3 meningiomas, 3 nerve sheath tumors, and 1 glioblastoma. The anatomical site of tumor origin was the lateral recess of the fourth ventricle in 13 patients, the ventral cerebellar hemisphere in 8, the cerebellar peduncle in 7, and the brain stem in 6. Others were from embryonal nest, cranial nerve, or meninges. For 42 tumor resections, 38 were approached through a posterior fossa craniotomy and 4 through a temporal craniotomy and transtentorial approach. At tumor resection, 26 had a gross total or near total resection, 12 subtotal resection, and 4 partial resection. There were no mortalities. The most significant morbidity was ninth and tenth nerve palsy; 15 patients had unilateral vocal cord palsy or dysphagia. Of these, nine were treated with nasogastric (NG) feeding tube, five with a combination of gastrostomy (G-tube) and tracheotomy, and one with G-tube. All had successful removal of NG feeding from 1 month to 2 years (average 6 months). The tracheostomy and G-tube were removed between 4 months and 2 years (average 14 months) in all.A plethora of tumor types occur in childhood at the CPA/CMF and our review indicated 50 % were benign in histology. High rates of lower cranial nerve morbidity were experienced but their dysfunctions were often recovered or compensated in 2 years. However, one should be cognizant of these complications and conduct resection with appropriate surgical approach, intraoperative monitoring, and surgical microscope. The majority of posterior fossa tumors in children develop in the midline including the fourth ventricle, cerebellar vermis and brain stem, or in the cerebellar hemispheres. However, the cerebellopontine angle (CPA) is a rare site and less than 10 % of posterior fossa tumors in childhood were located in CPA [The CPA is a subarachnoid space located in the ventral surface of the brainstem and medial cerebellar hemisphere, laterally bordered by the superior and inferior limbs of the cerebellopontine fissure (CPF). The CPF is formed by the ventrolateral wings of the quadrangular lobule (superiorly) and simple lobule (inferiorly) folding around the middle cerebellar peduncle. The superior and inferior limbs of the CPF meet at the lateral corner of the CPA, lateral to the flocculus, and extend laterally to the horizontal fissure of the cerebellum. Inferiorly, the inferior limb of the CPF extends to the cerebellomedullary fissure (CMF), which is bordered by the medulla oblongata and the medial wall of the biventer lobule, forming the lateral cerebellomedullary cistern. Medially at the CPA, the lateral recess of the fourth ventricle opens to the CPA through the foramen of Luschka. The inferior cerebellar peduncle forms the ventral and rostral wall of the lateral recess. In the CPA and CMF, where respective cerebrospinal fluid (CSF) cisterns are present, multiple cranial nerves from the 5th to 12th nerve are present. At the foramen of Luschka, fourth ventricular choroid plexus protrudes into the CPA cistern. Also present are the arterial branches of vertebrobasilar system including posterior inferior cerebellar and anterior inferior cerebellar arteries.A plethora of tumors can occur in the CPA and CMF, which derive from neuroglial tissues, cranial nerve sheath, meninges, and embryonic remnants . Thus, tIn this communication, the authors retrospectively reviewed a series of pediatric CPA and CMF tumors focusing on the histological distribution, their tumor origin, surgical approach and techniques, and their outcomes.This series is composed of the senior author’s (TT) personal pediatric surgical cases of primary tumors located in the CPA and/or CMF which were diagnosed and treated between 2001 and 2012. All patients had their tumor histology verified. Excluded are those with intrinsic expansion of the brain stem or cerebellum that is confined by the pia. Among tumors that affect both the fourth ventricle and the CPA/CMF, only those with the cisternal portion larger than the fourth ventricle portion are included. Vestibular schwannomas confined within the internal auditory canal(s) are excluded. Primary bony tumors arising from the skull base are also excluded.There were 44 infants and children in this series. There were 22 females and 22 males. The age at diagnosis ranged from 11 weeks to 16 years. All were evaluated with CT and MRI preoperatively.Of locations of these tumors, 32 were predominantly in the CPA and/or CMF whereas 12 showed a dumbbell-shaped extension into the fourth ventricle. Pathology of all tumors was verified: 14 ependymomas, 12 gliomas , 4 atypical teratoid rhabdoid tumors (ATRTs), 4 epidermoids, 3 primitive neuroectodermal tumors (PNETs), 3 meningiomas, 3 nerve sheath tumors, and 1 glioblastoma. The presumed origin of the tumor correlating with the pathology is shown in Table At diagnosis, 23 patients were noted to have hydrocephalus. The most common presentation was headaches or irritability/lethargy. Gait disturbances among the children and developmental arrest or regression among infants were common among hydrocephalic patients. Of 44 patients, 15 were found to have cranial nerve dysfunctions preoperatively; hearing loss in 4, dysphagia/dysphonia in 5, facial weakness in 2, facial pain in 2, and sleep apnea and shoulder droop in 1 patient each.Of 23 cases with pre-operative hydrocephalus, 3 had a shunt placed elsewhere and all others had external ventricular drainage at the time of craniotomy for tumor resection. All patients had surgical resection through 42 craniotomies and 2 stereotactic biopsies (PNET and glioblastoma). Of 42 craniotomies, 38 patients had a posterior fossa (PF) craniotomy and 4 patients had temporal craniotomy with subtemporal transtentorial approach to the CPA. All were resected with surgical microscope and neurophysiological monitoring. For posterior fossa craniotomy among 38 cases, 36 patients were in the prone position, with 26 patients receiving a “hockey-stick” incision Fig.  with a cThere were no deaths related to surgical procedures in our series. However, surgical morbidity rates were high: 17 patients had unilateral vocal cord palsy or dysphagia; 2 were noted preoperatively. Of these, postoperatively, 10 were treated with nasogastric (NG) feeding tube, 5 with a combination of gastrostomy (G-tube) and tracheotomy, and 1 with G-tube. All had successful removal of NG feeding from 1 month to 2 years (average 6 months). The tracheostomy and G-tube were removed between 4 months and 2 years (average 14 months) in all. Two had fifth nerve and one had sixth nerve dysfunctions. Two patients with lateral medulla oblongata tumor had a trigeminal trophic syndrome, which occurred 6–15 months postoperatively. Dense hemiparesis occurred in two patients (one persisted) and increased cerebellar ataxia occurred in two others.During the follow-up period up to 15 years, 13 patients were known to be dead. They were 3 of 4 ATRTs, 8 out of 12 ependymomas, and 1 each of PNET and glioblastoma. Both groups of the patients with benign astrocytoma and those with schwannoma, meningioma, and epidermoid are all alive whereas overall survival rate of those with ependymoma and ATRT/PNET/glioblastoma was 42.8 and 37.5 %, respectively  in the CPA with no obvious point of origin from the cerebellum or brain stem . HoweverThirty-two of our patients were predominantly in the CPA and/or CMF cisterns whereas 12 showed a dumbbell-shaped extension between the fourth ventricle and the CPA. Tumors, particularly those predominantly in the CMF, may look like they are growing into the fourth ventricle cavity, but they may be displacing the fourth ventricle contralaterally without internal invasion. Tsai et al. classified 29 pediatric CPA tumors into three categories: those exclusively confined in the CPA (8 patients), those with tumors predominantly within CPA 16 patients), and those arising from the vicinity and growing into CPA (5 patients) [ patientsIn the CPA and CMF, a plethora of tumors occurs, but they are rare during childhood, only representing 1 to 3 % of pediatric brain tumors. According to Phi et al., the CPA and CMF location consists of only 10 % of posterior fossa tumors  to more According to Phi et al., of 26 children with CPA tumors, 18 (69 %) were malignant and 8 were benign . Of theiPosterior fossa ependymoma is the most common histology. They originate from ependymal rests of the fourth ventricle. Some take their origin within the lateral recess of the fourth ventricle and grow both laterally into the CPA and medially into the fourth ventricle . When thJPAs originate often from cerebellar peduncle or lateral brain stem into the CPA/CMF. It may be difficult to distinguish the tumor origin between the brainstem and cerebellar peduncle due to the lack of anatomical borders between them . The patAccording to Rorke and Biegel, 52 % of ATRTs occur in the posterior fossa and they stated that the tendency to arise in the CPA with invasion of the surrounding structures is a distinct feature of this tumor . The celVestibular schwannomas are very prevalent in adult CPA tumors. However, some report higher incidence of vestibular schwannomas in pediatric CPA; 60 % of pediatric CPA and IAC tumors according to Holman et al. , and 86 CPA epidermoid tumors are considered to be third most common after vestibular schwannoma and meningioma in adults. Of four epidermoids in this series, three cysts contained thick viscid contents whereas another had a typical pearly whitish appearance.Tumors of the CPA and CMF often displace the brainstem and cerebellar hemisphere and may cause hydrocephalus. According to Zuccaro and Sosa, about 33 % of patients presented with hydrocephalus . In our A retro-sigmoid approach has been practiced most commonly for the CPA tumors . This apAlthough Phi et al. did not disclose the complication details in their report, they state that complication rates for malignant CPA tumors were much higher than the same tumors located in the midline location . MalignaInjury to the spinal trigeminal tract during the resection of the tumor from the lateral medulla oblongata is likely the cause of trigeminal trophic syndrome occurred in two of patients. Its occurrence in the pediatric population is rare .The review of our personal series of surgically treated pediatric CPA/CMF tumors shows a plethora of histology types; 50 % are malignant including ependymoma, ATRT, PNET, and glioblastoma, whereas another 50 % were benign consisting of JPA, epidermoid, schwannoma, and meningioma. Common adult tumors seen in adulthood such as schwannoma and meningiomas are uncommon. The caveat of this finding, however, is that this series only included the senior author’s surgical cases. None of intra-canalicular or bilateral vestibular schwannomas was included in this study as they were treated conservatively.Resecting CPA/CMF tumors remains difficult in children. One should choose appropriate surgical approaches for maximum exposure of the posterior fossa structures and minimum brain retraction. A hockey stick scalp incision with a bilateral posterior fossa craniotomy going far lateral on one side provides wide access to the CPA/CMF and cervical subarachnoid space. However, surgical resections carry high rates of surgical complications, particularly involving the lower cranial nerve deficits. Most of the new neurologic deficits caused from surgery resolves or compensates over time. None-the-less, in order to minimize complications, one should pay meticulous attention during tumor resection from the CPA/CMF. The use of intraoperative neurophysiological monitoring and the surgical microscope is important in order to reduce these complications.


To describe Ebola cases in the district Ebola management centre of in Kailahun, a remote rural district of Sierra Leone, in terms of geographic origin, patient and hospitalisation characteristics, treatment outcomes and time from symptom onset to admission.Data of all Ebola cases from June 23rd to October 5th 2014 were reviewed. Ebola was confirmed by reverse-transcriptase-polymerase-chain-reaction assay.P = 0.05). The median community infectivity time was 6.5 days for both general population and health workers (P = 0.4).Of 489 confirmed cases , 166 (34%) originated outside Kailahun district. Twenty-eight (6%) were health workers: 2 doctors, 11 nurses, 2 laboratory technicians, 7 community health workers and 6 other cadres. More than 50% of patients had fever, headache, abdominal pain, diarrhoea/vomiting. An unusual feature was cough in 40%. Unexplained bleeding was reported in 5%. Outcomes for the 489 confirmed cases were 227 (47%) discharges, 259 (53%) deaths and 3 transfers. Case fatality in health workers (68%) was higher than other occupations (52%, One in three admitted cases originated outside Kailahun district due to limited national access to Ebola management centres – complicating contact tracing, safe burial and disinfection measures. The comparatively high case fatality among health workers requires attention. The community infectivity time needs to be reduced to prevent continued transmission. Ebola virus disease (EVD), also called Ebola haemorrhagic fever or simply Ebola, is a disease of humans and other primates caused by viruses of the family filoviridae , 2. It wThe incubation period lasts 2–21 days and pathogenesis involves immune suppression and a systemic inflammatory response that causes impairment of the vascular, coagulation and immune systems, leading to multiorgan failure , 4, contOn 8 August 2014 WHO declared the West African Ebola epidemic in Guinea , LiberiaAlthough WHO has analysed the epidemiologic characteristics of the outbreak using multicountry data from West Africa,  no analyThis observational study in November 2014 included all patients who were enrolled consecutively at arrival at the Ebola management centre between 23rd June and 5th October 2014. Follow-up was censured on 10th November 2014.2. It lies in the north-east of Sierra Leone and is bordered by Liberia to the east and Guinea to the north.Sierra Leone has an estimated population of six million and despite decades of mining of diamonds, titanium, bauxite and gold, 70% of its people live in poverty . The 199The district health management team of the Ministry of Health and Sanitation is responsible for overall coordination of Ebola control activities and partners. Partners include the International Federation of the Red Cross , Save the Children (contact tracing), WHO , the World Food Programme (providing food for households under quarantine), the National Microbiological Laboratory, Winnipeg, Canada (laboratory diagnosis of Ebola) and MSF . A national Ebola call centre receives alerts and despatches teams to investigate and implement control activities. This includes investigating alerts of suspect cases and deaths in the community. At the time of this report, only three dedicated ambulances were available in the whole district for patient transfers to the Ebola management centre.Confirmed and suspected Ebola cases from Kailahun and those referred from neighbouring districts were admitted to the Ebola management centre, which progressively increased its bed capacity from 72 to 94 beds. The set-up and functioning of the centre has been previously described . In briePatients arrive by ambulances and are assessed in a triage area. Their clinical signs are then recorded, and they are admitted to the suspect or probable area of the centre depending on their case classification . All casSupportive care is provided until the PCR turns negative. Those with two negative PCR tests for Ebola are discharged to seek care from the general health services. All cases receive a systematic course of antimalarials, a broad-spectrum antibiotic and symptomatic care for fever, diarrhoea and vomiting.Treatment outcomes were standardised and documented as recovered ; death after being admitted; abandoned ; transferred (transferred to another facility). A patient admitted as an Ebola suspect but found negative on repeated PCRs was classified as a non-case.An epidemiologist gathered data from patient files daily and encoded them into a password-protected database used for the analysis. Information on contacts was sourced from the district health office. Treatment outcomes for the period June 23rd to October 5th 2014 were censured on 10th November 2014.P ≤ 0.05, and 95% confidence intervals (CIs) were used. Data analysis was performed using STATA 11 software .The cumulative incidence of death was estimated and expressed graphically using the Kaplan–Meier method. The number of days from onset of symptoms to admission at the Ebola management centre was considered the community infectivity time. Differences between groups were compared using chi-square and Wilcoxon Rank-sum test. The level of significance was set at The study used data collected during surveillance and response activities for Ebola at district level and stripped of patient identifiers. Informed consent was not applicable. The study satisfied the MSF Ethics Review Board criteria for studies using routinely collected data ; the Ebola interventions were approved by the Ministry of Health, Sierra Leone.FigureFigureOf 489 confirmed cases, 250 (51%) were male. The median age was 28 years . Only 3 individuals had eaten bush meat, while 76 (16%) had attended a funeral in the previous 21 days. The great majority (401) of cases were thus likely related to direct contact with body fluids of infected individuals at home or in the community.Twenty-eight health workers were admitted with Ebola (19 from Kailahun district and 12 from other districts) comprising 11 nurses, 7 community health workers, 2 doctors, 2 laboratory technicians, an ambulance driver, a vaccinator, a national staff working for an international organization a contact tracer, a counsellor and a surveillance officer.TableOf the 489 admitted Ebola cases, one outcome was unrecorded in the database. Of the remaining 488, 227  were discharged, 259 died , and 3 (1%) were transferred.FigureP = 0.05).Of the 28 health workers admitted, 19 (68%) died . Case fatality in health workers  was higher than in other occupations  (P = 0.4). Median CIT was 4 days  for cases from Kailahun district and 8 days  for those from other districts (P < 0.001).In addition to 489 confirmed Ebola cases, there were 36 probable cases – 19 deaths on ambulance arrival and 17 unexplained community deaths. The number of contacts for all 525 cases was estimated at 5250, of whom 1923 (37%) were identified. The median community infectivity time (CIT) was 6.5 days (IQR 3–8 days) for both general population and health workers , myocarditis, pericardial effusions, encephalitis or other reasons may be to blame. Research into the cause of late mortality is important and should be pursued. There might also have been associated comorbidity (e.g. HIV) that we were unaware of.Finally, of serious concern is that 28 health workers were affected and suffered a higher case fatality than others. Surprisingly, health workers did not present earlier than the general population, which is worrying as symptomatic health workers are at high risk of both acquiring and transmitting infection. In a country that had only 0.2 doctors and 1.7 nurses per 10 000 inhabitants before the epidemic, additional staff losses are devastating and will have long-term repercussions for an already weak, crippled health system , 14. MeaFocused immediate and long-term attention on a national scale is needed to ensure infection control and protection for the remaining health workers. Curbing the population spread of Ebola plus new vaccines and treatments would provide a safety net for health workers and need to be fast-tracked . CompensDespite severe limitations, commendable efforts have been made towards trying to control the Ebola outbreak in a rural district of Sierra Leone. The recovery rate of almost 50% in difficult circumstances is positive, but balanced by a significant loss of healthcare workers, woeful inadequacies of transport and case-finding and difficult access to the Ebola management centre. Urgent and sustained action by national and international partners is necessary to support Ebola management centres in all districts, solve operational problems and contain the epidemic.


While many sex differences in structure and function of the mammalian brain have been described, the molecular correlates of these differences are not broadly known. Also unknown is how sex differences at the protein level are perturbed by mutations that lead to intellectual disability (ID). Down syndrome (DS) is the most common genetic cause of ID and is due to trisomy of human chromosome 21 (Hsa21) and the resulting increased expression of Hsa21-encoded genes. The Dp(10)1Yey mouse model (Dp10) of DS is trisomic for orthologs of 39 Hsa21 protein-coding genes that map to mouse chromosome 10 (Mmu10), including four genes with known sex differences in functional properties. How these genes contribute to the DS cognitive phenotype is not known.Using reverse phase protein arrays, levels of ~100 proteins/protein modifications were measured in the hippocampus, cerebellum, and cortex of female and male controls and their trisomic Dp10 littermates. Proteins were chosen for their known roles in learning/memory and synaptic plasticity and include components of the MAPK, MTOR, and apoptosis pathways, immediate early genes, and subunits of ionotropic glutamate receptors. Protein levels were compared between genotypes, sexes, and brain regions using a three-level mixed effects model and the Benjamini-Hochberg correction for multiple testing.In control mice, levels of approximately one half of the proteins differ significantly between females and males in at least one brain region; in the hippocampus alone, levels of 40 % of the proteins are significantly higher in females. Trisomy of the Mmu10 segment differentially affects female and male profiles, perturbing protein levels most in the cerebellum of female Dp10 and most in the hippocampus of male Dp10. Cortex is minimally affected by sex and genotype. Diverse pathways and processes are implicated in both sex and genotype differences.The extensive sex differences in control mice in levels of proteins involved in learning/memory illustrate the molecular complexity underlying sex differences in normal neurological processes. The sex-specific abnormalities in the Dp10 suggest the possibility of sex-specific phenotypic features in DS and reinforce the need to use female as well as male mice, in particular in preclinical evaluations of drug responses.The online version of this article (doi:10.1186/s13293-015-0043-9) contains supplementary material, which is available to authorized users. Sex differences in brain function and dysfunction are well documented , 2. Sex Sex differences are attributed at least in part to molecular events that occur during development and throughout postnatal life in the regulation and levels of sex hormones and their receptors , 9. Sex Intellectual disability (ID) affects 1–3 % of the population worldwide . For ID DS is difficult to model well in mouse, not only because it is a contiguous gene syndrome with candidate genes spanning the length of Hsa21q but also because orthologs of Hsa21 genes are distributed on segments of three mouse chromosomes: the telomeric region of mouse chromosome 16 (Mmu16) and internal segments of mouse chromosome 17 (Mmu17) and mouse chromosome 10 (Mmu10) . Many paOverexpression at the RNA level of ADAR2, S100B, and TRPM2 has been demonstrated in the Dp10 mice . This waTo investigate sex differences in molecular features that may underlie normal LM and perturbations that may contribute to impaired LM in DS, we describe here expression levels of ~100 proteins in the hippocampus, cortex, and cerebellum of cohorts of male and female control mice and their age- and sex-matched trisomic littermates from the Dp10 line. The proteins include components of the MAPK, MTOR, and apoptosis signaling pathways, immediate early gene (IEG) proteins, subunits of ionotropic glutamate receptors, and additional proteins involved in synaptic plasticity and/or known to be mutated in subsets of patients with ID or in mouse mutants showing LM deficits or abnormal in patients with AD or mouse models of AD. We show that, in the hippocampus of control mice, levels of almost half of these proteins differ between male and female mice and that in each case, the level is higher in females than in males. In contrast, sex influences on levels of the same proteins are minimal in the cortex and cerebellum. Trisomy-associated perturbations are also sex-specific, with hippocampus most affected in male Dp10 and cerebellum most affected in female Dp10. The observation that females and males differ in their baseline profiles of proteins critical to learning and memory suggests that molecular responses to the stimulation of learning will differ. The data also suggest that Hsa21 genes with orthologs on Mmu10 may influence, positively or negatively, cognitive features in people with DS, and that the molecular basis of these features, and their modulation by pharmacological treatment, will differ between males and females.The Dp10)1Yey mice , 46, ori01Yey micDNA was prepared from a 1-mm tail snip by lysis in 50 nM NaOH at 98 °C for 1 h, followed by neutralization with 1 M Tris Base (pH 8.0). Lysates were stored at −20 °C until use. Mice were genotyped by standard PCR using the following primer pairs: control gene reverse primer 5-′CTAGGCCACAGAATTGAAAGATCT- 3′ and forward primer 5′GTAGGTGGAAATTCTAGCATCATCC 3′; trisomic gene forward primer 5′GGCGAACGTGGCGAGAAA 3′ and reverse primer 5′CCTGCTGCCAAGCCATCAG 3′.To preserve protein phosphorylation, mice were sacrificed by cervical dislocation without anesthetic. The whole brain was removed, immediately snap frozen in liquid nitrogen, and stored at −80 °C. For lysate preparation, the brains were removed from the freezer and, without thawing, rapidly heated to 95 °C under vacuum in the Stabilizor T1  as described previously (48). The cortex, hippocampus, and cerebellum were dissected out, weighed, placed in 10 volumes of IEF buffer  and homogenized by sonication with three bursts 5 s long in a Branson Sonic Power Co. . Lysates were centrifuged to remove debris, and the protein concentration of the cleared supernatant was determined using the 660 nM Protein Assay Kit (Pierce); all sample protein concentrations were 9–11 mg/ml. Information for each mouse, age, littermates, and tissue weight is provided in Additional file Proteins screened for expression level are listed in Additional file Each sample lysate was prepared in 5 dilutions, neat plus 4 serial dilutions with a 0.8 dilution factor, and 1 buffer control, in a 384-well V-shaped ABgene plate . Samples were printed, in triplicate, onto nitrocellulose-coated glass slides  using an Aushon BioSystems 2470 Arrayer  with 185-μm pins and a single touch. The arrays were produced in two major print runs and slides were stored at 4 °C until further use.Procedures for array screening have been described previously . Brieflyp value <0.05 with a false discovery rate (FDR) of 5 % was considered for overall statistical significance across the entirety of the hypotheses. Results of all comparisons carried out for the three brain regions are provided in Additional file Signals on each slide were quantified using Scan Array Express software  where the antibody signal intensity for each spot was normalized to the corresponding SyproRuby signal. Details of quantification and review of data quality and reproducibility were as described previously , 49, 50.p < 0.05 were inspected and correlations with artifactually high r values  were eliminated. All data analysis was carried out using SAS® version 9.3 .For correlation analysis, data were reduced to one observation per mouse. Protein values for each brain region of each individual of each sex/genotype were used to compute Spearman correlation coefficients. Graphs for data from protein pairs with correlation coefficients greater than 0.8 with http://www.ebi.ac.uk/intact/), HPRD , and BioGRID  databases. Subsets of primary and secondary interactions for sex hormone receptors and proteins screened by RPPA were retained for networks in Fig. Protein interaction partners of each protein encoded in the Dp10 trisomic segment for each of the proteins measured by RPPA and for proteins encoded on the X chromosome that escape X inactivation –13 were The goals of the protein measurements were first to assess sex differences in control mice and then to determine how trisomy of the Hsa21 syntenic region on Mmu10 influences both sex-dependent and sex-independent protein profiles. A total of ~100 proteins/protein modifications were screened in whole tissue lysates from the hippocampus, cortex, and cerebellum of ~8-month-old mice. Four pairwise comparisons were carried out for each brain region: (i) protein levels in control females were compared to those in control males to determine sex differences normally present in the inbred C57BL/6JEi background, (ii) levels in trisomic females were compared to those in trisomic males to determine if and how trisomy alters normal sex differences, (iii) levels in trisomic males were compared to those in control males, and (iv) levels in trisomic females were compared to those in control females, to determine sex-independent and sex-specific perturbations caused by trisomy. Proteins measured included 18 components of the MAP kinase pathway and 14 from the MTOR pathway, 4 immediate early gene proteins, subunits of ionotropic glutamate receptors, and a number of proteins associated with AD. Fourteen proteins encoded by Hsa21, 4 of which are trisomic in the Dp10, were also measured. Proteins were chosen because of their specific individual importance, or the importance of the pathways in which they function, to LM or synaptic plasticity or because they have been shown to be abnormal in brains from people or mouse models of DS, ID, or AD. Proteins include those used in previous studies of the Tc1 mouse model of DS and of the Ts65Dn with and without memantine treatment and exposure to CFC , 49. TheFigure Figure It is evident from Fig. Figure Figure As shown in Table Levels of 14 Hsa21 orthologs were measured. Genes encoding three, S100B, PRMT2, and ADAR2, are trisomic in the Dp10 mice. As shown in Fig. Proteins encoded by four Hsa21 genes that are not trisomic in the Dp10 were also affected by sex and/or trisomy with brain region specificities Fig. . In the Consistent with the overall modest sex and trisomy effects on protein expression in cortex, perturbation of Hsa21 protein levels were slight. Levels of S100B were elevated in trisomy . Unique to cortex, however, sex comparisons showed significantly higher levels in Dp10 females than Dp10 males of the Hsa21 orthologs TIAM1, CBS, and RRP1  were significantly increased; of these, AKT and pAKT were increased by 66 and 45 %, respectively, and pMTOR and pS6 each by ~30 %. As a result, levels of 12 of 13 components of the MTOR pathway were higher in the cerebella of female Dp10 mice than male; only AKT shows similar levels in male and female trisomics. Thus, the picture of perturbations in MTOR is strongly sex, genotype, and brain region-specific.In the hippocampus, 14 components of the MTOR pathway were measured, including 7 phosphoproteins. Results are shown in Fig. In comparison to MTOR, perturbations in the MAPK pathway are minimal. In the hippocampus, four components differed between male and female control mice, pERK, pELK, RSK, and pRSK, and only pBRAF and pERK showed sex differences in Dp10 mice. Only 5 of 14 components of the classical MAPK pathway were perturbed, all increased, in male Dp10, and only one was altered in female Dp10. Data are shown in Additional file We previously reported in the hippocampus of male Dp10 mice  measuremData from the cerebellum again present a very different picture. Levels of only four of the 26 AD-related proteins, CDK5, IL1B, NR1 and NR2B, differed between sexes in controls, while the majority, 18 of 26, differed between sexes in trisomic mice. Levels of nine proteins were altered in male Dp10, and 16 were altered in female Dp10. Perturbations in the levels of five proteins, CDK5, pGSK3BY216, NR1, P35/25, and pNUMB, were of the same magnitude in both male and female trisomics, but opposite in direction, decreased in males and increased in females relative to their respective controls. These data are shown in Additional file Compared with hippocampus and cerebellum, protein levels in the cortex showed few sex differences and genotype perturbations. However, as shown in Fig. We compared protein levels between brain regions in controls and asked how these relationships changed, or not, with trisomy. The bar graphs in Fig. Figure Figure Figure In Dp10 females, most components of the MTOR pathway were increased in the cerebellum, but not perturbed in the hippocampus, relative to female controls, and this is reflected in the magnitudes of the bar graphs in Fig. Results of a similar analysis of MAPK components are shown in Additional file In prior work, correlations among functionally related proteins were noted in the hippocampus of Tc1 and Ts65Dn mice , 49. In Levels of ~100 proteins/protein modifications were measured in the hippocampus, cerebellum, and cortex of cohorts of adult female and male mice. Cohorts included mice trisomic for the Mmu10 region syntenic with Hsa21 and their littermate controls. Analysis uncovered both sex and genotype, brain region-specific differences in protein expression. In control mice, levels of half the proteins differed between females and males in at least one brain region, and in the hippocampus alone, expression levels of 41 proteins were significantly higher in females than those in males. Levels of only nine proteins showed sex differences in the cerebellum in controls. Trisomy differentially affected protein levels in females and males. In female trisomic mice, levels of a total of 69 proteins differed from female controls; 62 were increased in the cerebellum, while only 18 were perturbed in the hippocampus. In contrast, in male Dp10 mice, while levels of a total of 68 proteins were perturbed in at least one brain region, 49 were elevated in the hippocampus and 21 were altered in the cerebellum; the majority decreased relative to male controls. In both control and trisomic mice, cortex showed the fewest sex differences: levels of only one protein differed in control mice, and 13 and 8 were altered in female and male Dp10, respectively. Because of the sex specificities of the trisomy-induced perturbations, male and female Dp10 mice differed in levels of 15 proteins in hippocampus, 70 in the cerebellum, and 23 in the cortex, a very different profile from their littermate controls.The number of sexually dimorphic proteins is of interest given that the entire protein set was selected from those shown by mutational analysis to function in brain development, ID or LM, to be components of pathways relevant to these processes or to show abnormalities in brains of patients or mouse models of DS or AD , 47. IndIn both controls and trisomic mice, proteins showing sex differences are diverse in their functional classifications. They include subsets of the components of the MTOR, MAPK, and apoptosis pathways, in control mice, several AMPA receptor subunits, and in trisomic mice, NMDA receptor subunits. The cerebellum of trisomic mice shows the most dramatic sex differences, with the magnitudes of the differences, averaging >30 %, greater than in those in other genotype/brain regions. These are due to the sensitivity of female mice to trisomy of the Dp10 segment, where 62 proteins differ from control females, compared to only 21 in male Dp10.Sex differences were also seen in levels of proteins with previously reported abnormalities in brains of patients with AD or mouse models . Ten of A comprehensive study of sex differences in gene expression at the RNA level found that 14 % of mRNAs expressed in the brain differed in levels between females and males . This coSeveral proteins assayed here have been shown individually to contribute to sex-specific phenotypic features, at least when mutated. For example, in an AD-related study, when a FYN kinase null mutant was crossed with the AD “triple transgenic” model , male offspring were delayed in the development of Aβ pathology and spatial learning deficits, while female offspring showed no such temporal protection, i.e., decreased levels of FYN were protective only in males . AnotherSex differences are also relevant to molecular responses to drug treatments. The effects of fluoxetine were examined in rats that had been exposed to chronic stress. When initially evaluated in the forced swim test, stressed male rats showed increased immobility while stressed females showed increased hyperactivity. In both sexes, stress response behavior was normalized by fluoxetine . Levels Sex hormones, X chromosome genes that escape inactivation, and environmental effects all may contribute to the sexually dimorphic patterns of protein expression observed here in control mice. That these patterns are different in Dp10 mice suggests that trisomic Mmu10 genes impact one or more of these normal processes. Based on the known functions of Mmu10 trisomic genes, candidates for perturbations of sex differences in expression can be proposed . Figure Knockouts of two genes trisomic in the Dp10 have been shown to be associated with sex-specific phenotypes. TRPM2, a calcium-permeable cation channel primarily activated by intracellular adenosine-diphosphate ribose (ADPR) , 70, shoNot included in Fig. In humans, although it has been consistently shown that ~15 % of X chromosome genes escape inactivation, the consequences for protein levels have not been well characterized. The number and identity of the genes expressed from both X chromosomes, and the resulting mRNA levels, varies among individuals and between tissues/cell types –78. In mEnvironmental conditions can also influence gene expression, potentially contributing to sex or genotype differences. General environmental conditions are controlled, e.g., all mice are exposed to the same level and frequency of noise, light/dark cycle, and access to food and water. In addition, all mice were sacrificed between noon and 2 p.m. to control for normal circadian variations in protein expression. However, if there are sex or genotype differences in sensitivity to any of these features, some  part of the proteome would be affected. Behavioral studies are necessary to determine if the Dp10, males or females, are differentially affected by environmental conditions of noise or handling or if they exhibit altered sleep/wake or feeding patterns.An additional environmental influence on the proteome arises from housing conditions and potential effects of social hierarchy. A dominant male would have higher levels of androgens than a submissive male, with downstream consequences for some protein expression. All mice here were housed with their littermates of the same sex. Because we did not ascertain the effects of trisomy on the propensity for dominance, there are three possible scenarios: (i) control and Dp10 males are equally likely to be the dominant animal, (ii) controls will always be dominant over a Dp10, or (iii) a Dp10 will always be dominant over a control. Given the number of each genotype in each of the seven litters  Dp10 . These d


Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is a vector of the phloem-limited bacterium ‘Candidatus Liberibacter solanacearum’ (Lso), the putative causal agent of zebra chip disease of potato. Little is known about how potato psyllid transmits Lso to potato. We used electrical penetration graph (EPG) technology to compare stylet probing behaviors and efficiency of Lso transmission of three haplotypes of potato psyllid . All haplotypes exhibited the full suite of stylet behaviors identified in previous studies with this psyllid, including intercellular penetration and secretion of the stylet pathway, xylem ingestion, and phloem activities, the latter comprising salivation and ingestion. The three haplotypes exhibited similar frequency and duration of probing behaviors, with the exception of salivation into phloem, which was of higher duration by psyllids of the Western haplotype. We manipulated how long psyllids were allowed access to potato  to examine the relationship between phloem activities and Lso transmission. Between 25 and 30% of psyllids reached and salivated into phloem at an IAP of 1 hr, increasing to almost 80% of psyllids as IAP was increased to 24 h. Probability of Lso-transmission was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids which salivated into the phloem failed to transmit Lso. Logistic regression showed that probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit Lso, for unknown reasons. Information from these studies increases our understanding of Lso transmission by potato psyllid, and demonstrates the value of EPG technology in exploring questions of vector efficiency.The potato psyllid,  Candidatus Liberibacter solanacearum’ (Lso), transmitted by the potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) [Zebra chip disease of potato is thought to be caused by the phloem-limited bacterium ‘iozidae) –4. Zebraiozidae) . While ziozidae) , it was iozidae) –7.Little is known about the mechanisms by which the potato psyllid transmits Lso to potato and other solanaceous species . Plant dThe relationship between feeding behaviors and Lso transmission may be complicated further by the discovery of genetic variants, or haplotypes, of potato psyllid –10. ThreAn innovative approach known as Electrical Penetration Graph (EPG) technology –20 is a This study used EPG technology to compare stylet probing behavior among psyllids of the Central, Western, and Northwestern haplotypes –10, and Our second objective was to examine the relationship between stylet behaviors and probability of Lso transmission to potatoes. We determined whether probability of transmission is predicted by the amount of time a psyllid spends salivating into phloem tissue . ThLso-free and Lso-infected potato psyllid colonies of three haplotypes were established at the USDA-ARS facility in Wapato, WA , with insects collected from Weslaco, Texas  , Albany, California  (Western haplotype), and Prosser, Washington  (Northwestern haplotype). No collection or import permit was required by Texas, California, or Washington; potato psyllid is not an endangered or protected species. The colonies were started with isofemale lines and were maintained on Atlantic potatoes under laboratory conditions of 25±1°C, 40±5% RH, and a photoperiod of 16:8 (L: D) h. Haplotype of psyllids was confirmed using high resolution melting analysis [Certified disease-free potatoes (var. Atlantic) grown from seed tubers obtained from CSS Farms Inc.  were used for this study. This variety was selected due to its high susceptibility to zebra chip , 33–34. 9 Ohms (Ω), and DC substrate voltage.EPG system wiring and recordings were conducted as described by Pearson et al. . PsyllidWe first verified that our methods produced waveform results for all three haplotypes similar to those obtained by Pearson et al.  for psylPearson et al.  reportedOnce we had validated our EPG methods, we then examined the relationship between duration of stylet behaviors and transmission of Lso to potato. We compared the EPG waveforms of psyllids that successfully transmitted Lso and waveforms of infected psyllids that had failed to transmit Lso. We limited our analyses to an examination of how variation in the E1 waveform affected probability of transmission, as both Pearson et al.  and SandPsyllids of each of the three haplotypes were collected from Lso-infected colonies and starved for 6 h at 4°C prior to EPG hook-up . Each teEach plant from the EPG portion of the assays was then monitored for development of zebra chip symptoms. Because symptoms associated with zebra chip develop more readily in field-grown plants than in potted plants, all plants were transplanted into small field cages located out-of-doors . TransplThe presence or absence of Lso in psyllid and plant samples was determined using PCR to amplify a region of 16S rDNA. DNA from psyllids was extracted using the cetyltrimethlyammonium bromide (CTAB) method of Zhang et al. , whereasThe average duration and number of events of intercellular stylet penetration/pathway phase (C); xylem ingestion (G); initial contact with phloem tissue (D), salivation into phloem sieve elements (E1), and phloem sap ingestion (E2) probing events were compared among psyllid haplotypes using the GLIMMIX procedure of SAS 9.3 . Data weWe examined the interactive effects of haplotype and IAP on probability of plant infection and on probability that the psyllid had accessed phloem tissues as a 3 x 8 (haplotype x IAP) factorial model in GLIMMIX, specifying the DIST = BINARY option. Lastly, we used logistic regression to examine the relationship between duration of E1  events and probability of successful transmission of Lso. The analyses were done using PROC LOGISTIC . HaplotyWaveforms produced by infected psyllids of all three haplotypes were similar in appearance to those shown by Pearson et al.  for uninThe EPG waveforms showed that the first probe was initiated on average within a few minutes (< 10 min) following onset of the assay . The mea7, 467 = 7.7, P < 0.0001) but not by haplotype  or the haplotype x IAP interaction . Profile contrasts were extracted to compare mean probabilities between adjacent levels of the IAP factor, and showed that there was a significant increase between the 2 h and 3 h levels of IAP in probability that a psyllid had fed in phloem tissues . No other contrasts were significant, indicating that probabilities were statistically flat between 1 h and 2 h IAP, and between 3 h and 24 h IAP , again with a statistically significant increase in probabilities only between the 2 and 3 h IAP levels . Haplotype effects were non-significant , as was the haplotype x IAP interaction .Probability of plant infection was consistently lower across all IAP levels than probability that the psyllid had fed in phloem tissues, indicating that a percentage of infected psyllids which reached phloem tissues nonetheless failed to induce disease in the potato plant . Mean prThe observation that probability of Lso-transmission  was cons2 = 0.9, df = 2, P = 0.65; haplotype × duration, χ2 = 2.4, P = 0.31; longest E1 duration: haplotype, χ2 = 0.6, df = 2, P = 0.75; haplotype × duration, χ2 = 1.2, P = 0.55), so the final logistic models were fitted without either effect , and showed that probability of plant infection increased with increasing duration of E1 events. Transmission of Lso occurred in some instances even following a very short interval of salivation, shown by the large numbers of psyllids at short durations which nonetheless induced plant disease (the “YES” psyllids in the scatter plots of both panels). However, there were also a number of infected psyllids which failed to produce disease in plants despite extremely long intervals of E1 behavior, as shown by the occurrence of long-duration psyllids labeled “NO” in the scatter plots  to estimate what minimum duration of E1 can lead to Lso-transmission. We limited these data to psyllids that exhibited a single E1 event over the course of an assay, and that exhibited an E1 duration shorter than 30 min . Eight pThe present study is the first to compare feeding behavior and Lso transmission among different haplotypes of potato psyllid. Results showed that the shape and form of the EPG waveforms  produced by Lso-infected psyllids of the three haplotypes were similar in appearance to waveforms described by Pearson et al.  Figs 2–, for uniWe manipulated the amount of time psyllids were allowed access to the host substrate (IAP) to determine the effects of this variable on probability of Lso-transmission, and in duration and frequency of phloem feeding events. Transmission rates were not statistically different among haplotypes across a range of IAP levels between 1 h and 24 h . Rates oWe also examined the relationship between IAP and phloem feeding. Particular attention was paid to the time the psyllids spent salivating into the phloem tissue. The phloem salivation (E1) and ingestion (E2) phases  are consOne unexpected result from our IAP assays was the observation that probability of salivation into phloem tissues during an assay was higher than the probability that the assayed psyllid had also transmitted the Lso pathogen, irrespective of IAP compare . That isResults of this study further demonstrate the value of electrical penetration graph technology for understanding feeding behaviors of Hemiptera and how those behaviors lead to transmission of plant pathogens –31, 26. Lastly, this study provides some insight into the potential importance of the different haplotypes as vectors of Lso within the PNW growing region. While zebra chip was first reported in Mexico in 1994 and in Texas in 2000 , the disS1 TableTotal duration: durations summed across multiple E1 events for each psyllid; longest duration: longest E1 event for each psyllid.(XLSX)Click here for additional data file.