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Background : fMRI of mental phenomena is quite difficult to perform because lack of patient's cooperation or because the symptoms are stable. In some exceptional cases, however, fMRI and DTI are capable to provide insights on the anatomy of organic hallucinations. Methods : In this report we describe a 14-year-old boy with a left fronto-dorsal tumor who experienced chronic complex brief, frequent and repetitive complex visual and auditory hallucinations. His clinical picture included multiple and severe social and mood problems. During a presurgical fMRI mapping the patient complained of having the visual and auditory hallucinations. A block-design FMRI paradigm was obtained from the event timecourse. Deterministic DTI of the brain was obtained seeding the lesion as ROI. The patient underwent surgery and electrocorticography of the lesional area. Results : The fMRI of the hallucinations showed activation in the left inferior frontal gyrus (IFG) and the peri-lesional area. The tractography of the tumor revealed structural aberrant connectivity to occipital and temporal areas in addition to the expected connectivity with the IFG via the aslant fasciculus and homotopic contralateral areas. Intraoperative EEG demonstrated epileptic discharges in the tumor and neighboring areas. After resection, the patient's hallucinations stopped completely. He regained his normal social life and recover his normal mood. He remained asymptomatic for 90 days. Afterwards, hallucinations reappeared but with less intensity. Conclusions : To our knowledge, this is the first reported case of combined functional and structural connectivity imaging demonstrating brain regions participating in a network involved in the generation of complex auditory and visual hallucinations.

Introduction and Aims: Traumatic dental injuries (TDI), alcohol and illicit drugs consumption are highly-prevalent public health concerns. The aim of this study was to evaluate the association between alcohol and/or illicit drugs consumption and the presence of TDI., Design and Methods: This review was performed in accordance with MOOSE and PRISMA guidelines. Electronic searches were performed to found observational studies that investigated subjects that consumed alcohol and/or illicit drugs associated with TDI. Data extraction and a risk of bias assessment were performed. Three meta-analyses (MA) were performed to evaluate the association between TDI and alcohol consumption (1 st MA), subgrouping the studies into alcohol intake and binge-drinking, alcohol-related problems (2 nd MA), and drugs (3 rd MA). The odds ratio (OR) were calculated (p ≤ 0.05) for all analyses. The evidence was certainty-tested using the GRADE approach., Results: Twelve articles met the eligibility criteria. Nine studies were methodologically sound, while three had some confounding factors. In the pooled MA, an association between alcohol consumption and TDI (OR 1.57[1.33,1.85],p = 0.00001) was determined with moderate certainty of evidence, confirmed in the subgroups of alcohol intake (OR 1.49[1.17,1.90],p = 0.001) and binge-drinking (OR 1.68[1.30,2.18],p = 0.0001). No associations were found between TDI and risk for alcohol-related problems and drug use (OR 0.75[0.50,1.14],p = 0.18, and OR 1.20[0.82,1.75],p = 0.27, respectively), with both analyses having a very low certainty of evidence., Discussion and Conclusions: There is moderate evidence concerning the association between alcohol use and TDI. Despite the absence of an association between illicit drugs use, alcohol-related problems and the presence of TDI, these results should be viewed with caution, since they had very low-certainly-evidence., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined., Results: Here we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask., Conclusion: Our protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.

Objectives: To evaluate the desensitizing effect of nano-hydroxyapatite (n-HAP) on dentine hypersensitivity (DH)., Sources: Seven electronic databases were searched on April 27, 2018., Study Selection: Randomized clinical trials (RCTs) were included based on the PICO strategy: Participants - Humans with DH; Intervention - n-HAP-containing desensitizing; Comparison -n-HAP-free treatments or placebo/negative control; and Outcomes - relief of DH. The risk of bias was classified by the Cochrane guidelines. Five meta-analyses were performed to evaluate the efficacy of n-HAP with regard to pain assessment stimuli (primary outcome); comparison of n-HAP with other treatments or placebo/negative control, and effectiveness of at-home and in-office n-HAP use (secondary outcomes). The quality of the evidence was evaluated using the GRADE., Data: Six RCTs with 4 weeks of follow-up were included in the meta-analysis. For the primary outcome, n-HAP showed a better desensitizing effect for evaporative stimuli (SMD -1.09 [-1.24, -0.94], p < 0.00001) and tactile stimuli (SMD -0.93 [-1.42, -0.43]) than other treatments (p = 0.0002). However, there was no difference between n-HAP and other treatments for the cold stimuli (SMD -0.17 [-0.81, 0.48], p = 0.61). In an overall analysis, n-HAP-containing treatment showing the most significant desensitizing effect (SMD -0.93 [-1.19, -0.68], p < 0.00001) with a high quality of evidence for pooled results. In the secondary outcomes, n-HAP showed the best effect in the overall analysis (p < 0.05) with moderate quality evidence., Conclusions: The n-HAP-containing treatment showed better clinical performance than other treatments for DH relief. However, long-term follow-up RCTs are required in the future before definitive recommendations can be made., Clinical Significance: Dentin hypersensitivity is a common global condition and its multifactorial etiology has led to the development of several treatments. The n-HAP-containing treatment showed greater DH relief when compared to other desensitizing agents, placebo or negative control., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Drug delivery to the brain represents a challenge, especially in the therapy of central nervous system malignancies. Simvastatin (SVT), as with other statins, has shown potential anticancer properties that are difficult to exploit in the central nervous system (CNS). In the present work the physico⁻chemical, mucoadhesive, and permeability-enhancing properties of simvastatin-loaded poly-ε-caprolactone nanocapsules coated with chitosan for nose-to-brain administration were investigated. Lipid-core nanocapsules coated with chitosan (LNC chit ) of different molecular weight (MW) were prepared by a novel one-pot technique, and characterized for particle size, surface charge, particle number density, morphology, drug encapsulation efficiency, interaction between surface nanocapsules with mucin, drug release, and permeability across two nasal mucosa models. Results show that all formulations presented adequate particle sizes (below 220 nm), positive surface charge, narrow droplet size distribution (PDI < 0.2), and high encapsulation efficiency. Nanocapsules presented controlled drug release and mucoadhesive properties that are dependent on the MW of the coating chitosan. The results of permeation across the RPMI 2650 human nasal cell line evidenced that LNC chit increased the permeation of SVT. In particular, the amount of SVT that permeated after 4 hr for nanocapsules coated with low-MW chitosan, high-MW chitosan, and control SVT was 13.9 ± 0.8 μg, 9.2 ± 1.2 µg, and 1.4 ± 0.2 µg, respectively. These results were confirmed by SVT ex vivo permeation across rabbit nasal mucosa. This study highlighted the suitability of LNC chit as a promising strategy for the administration of simvastatin for a nose-to-brain approach for the therapy of brain tumors.

Energy efficiency is crucial in the design of battery-powered end devices, such as smart sensors for the Internet of Things applications. Wireless communication between these distributed smart devices consumes significant energy, and even more when data need to reach several kilometers in distance. Low-power and long-range communication technologies such as LoRaWAN are becoming popular in IoT applications. However, LoRaWAN has drawbacks in terms of (i) data latency; (ii) limited control over the end devices by the gateway; and (iii) high rate of packet collisions in a dense network. To overcome these drawbacks, we present an energy-efficient network architecture and a high-efficiency on-demand time-division multiple access (TDMA) communication protocol for IoT improving both the energy efficiency and the latency of standard LoRa networks. We combine the capabilities of short-range wake-up radios to achieve ultra-low power states and asynchronous communication together with the long-range connectivity of LoRa. The proposed approach still works with the standard LoRa protocol, but improves performance with an on-demand TDMA. Thanks to the proposed network and protocol, we achieve a packet delivery ratio of 100% by eliminating the possibility of packet collisions. The network also achieves a round-trip latency on the order of milliseconds with sensing devices dissipating less than 46 mJ when active and 1.83 μ W during periods of inactivity and can last up to three years on a 1200-mAh lithium polymer battery.

Cell line-based proteomics studies are susceptible to intrinsic biological variation that contributes to increasing false positive claims; most of the methods that follow these changes offer a limited understanding of the biological system. We applied a quantitative proteomic strategy (iTRAQ) to detect intrinsic protein variation across SH-SY5Y cell culture replicates. More than 95% of the quantified proteins presented a coefficient of variation (CV) < 20% between biological replicates and the variable proteins, which included cytoskeleton, cytoplasmic and housekeeping proteins, are widely reported in proteomic studies. We recommend this approach as an additional quality control before starting any proteomic experiment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Catheters are extensively used for coronary angiographies. These devices are long, thin, and flexible tubes of variable diameters that are made of the following polymer compounds: polyamide, polyurethane and Teflon. The objectives of this study are to identify changes that occur in the polymeric structure of the catheters, including an analysis of microcracks, fissures, cuts, perforations, roughness, and porosity in the internal and external layers after the first, second, third, and seventh reprocessing., Methods: Five catheters of the same brand and manufacturer were selected for each analysis, except for the new and seventh catheters, which had only one sample. The catheters that we analyzed included new catheters, catheter reprocessed once (1st), catheters reprocessed twice (2nd), catheters reprocessed three times (3rd), and catheters reprocessed up to seven times (7th). Samples were cut into small pieces and the scanning electron microscope and infrared spectroscopy were used for analysis., Results: Scanning electron microscopy (SEM) of reprocessed catheters revealed rougher surface with porosity, stainless steel wires, peeling of the inner layers, micropores, small holes, disordered cracks, cracks, grooves, microcracks, granular appearance, dots whitish, diffuse microcracks, desquamation, diffuse recesses, depressions, protrusions and deep crevices. Infrared spectroscopy suggested a possible change in the chemical structures of the polymer. There was a directly proportional relationship between the reprocessing number and the carbonyl ratio., Conclusions: The combined SEM and infrared analyses reveal that reprocessing damages the integrity of coronary angiography catheters. Therefore, the reprocessing of such catheters is not recommended.

In this work, we will test the hypothesis that the connectivity of language areas in normal children is asymmetric between the hemispheres. Intrahemispheric region of interest (ROI)-to-ROI connectivity was assessed in 40 normal right-handed children. Asymmetries were assessed (1) between the hemispheres (global language connectivity); (2) between Brodmann areas (BAs) pairs (pairwise connectivity); and (3) between two homotopic BA (Global BA connectivity). Sixteen BAs were selected: 6, 7, 9, 19, 21, 22, 37, 38, 39, 40, 41, 42, 44, 45, 46, and 47. T scores for connectivity of each BA pair were ascertained using the MATLAB toolbox CONN. Lateralization index (LI) scores based on T-values were obtained. Only LIs with 2SD above the mean were considered as significant. Comparisons between T-value groups (per side and per BA) were performed utilizing double-sided T-tests. Null hypothesis was rejected for p < 0.05. There was not a statistical difference between global left and right connectivity strength (p = 0.40). There was significant pairwise connectivity asymmetry for the following pairs: BA7-BA44 (LI = 0.662); BA21-BA42 (LI = -0.616); BA21-BA40 (LI = -0.595); BA38-BA44 (LI = 0.470); BA39-BA44 (LI = -0.903); and BA42-BA47 (LI = -0.445). Language-related brain connectivity asymmetries have been demonstrated in a group of children and young adolescents. Two pairs related to Broca's area were left dominant (BA44-BA38 and BA44-BA7) and four pairs right dominant (BA42-BA47, BA39-BA44, BA21-BA40, and BA21-BA42).

Plant secondary metabolites play important ecological and evolutionary roles, most notably in the deterrence of natural enemies. The classical theory explaining the evolution of plant chemical diversity is that new defences arise through a pairwise co-evolutionary arms race between plants and their specialized natural enemies. However, plant species are bombarded by dozens of different herbivore taxa from disparate phylogenetic lineages that span a wide range of feeding strategies and have distinctive physiological constraints that interact differently with particular plant metabolites. How do plant defence chemicals evolve under such multiple and potentially contrasting selective pressures imposed by diverse herbivore communities? To tackle this question, we exhaustively characterized the chemical diversity and insect herbivore fauna from 31 sympatric species of Amazonian Protieae (Burseraceae) trees. Using a combination of phylogenetic, metabolomic and statistical learning tools, we show that secondary metabolites that were associated with repelling herbivores (1) were more frequent across the Protieae phylogeny and (2) were found in average higher abundance than other compounds. Our findings suggest that generalist herbivores can play an important role in shaping plant chemical diversity and support the hypothesis that chemical diversity can also arise from the cumulative outcome of multiple diffuse interactions.

Wireless sensor nodes are traditionally powered by individual batteries, and a significant effort has been devoted to maximizing the lifetime of these devices. However, as the batteries can only store a finite amount of energy, the network is still doomed to die, and changing the batteries is not always possible. A promising solution is to enable each node to harvest energy directly in its environment, using individual energy harvesters. Moreover, novel ultra-low power wake-up receivers, which allow continuous listening of the channel with negligible power consumption, are emerging. These devices enable asynchronous communication, further reducing the power consumption related to communication, which is typically one the most energy-consuming tasks in wireless sensor networks. Energy harvesting and wake-up receivers can be combined to significantly increase the energy efficiency of sensor networks. In this paper, we propose an energy manager for energy harvesting wireless sensor nodes and an asynchronous medium access control protocol, which exploits ultra-low power wake-up receivers. The two components are designed to work together and especially to fit the stringent constraints of wireless sensor nodes. The proposed approach has been implemented on a real hardware platform and tested in the field. Experimental results demonstrate the benefits of the proposed approach in terms of energy efficiency, power consumption and throughput, which can be up to more than two-times higher compared to traditional schemes., Competing Interests: The authors declare no conflict of interest.

The transport properties of zigzag graphene nanoribbons (zGNRs) were studied by density functional theory (DFT) in conjunction with Green's function analysis. In particular, spin transport through a zGNR (12,0) device was investigated under the constraint of ferromagnetic coordination of the ribbon edges. Several configurations with two vacant sites in the edge and the bulk region of the zGNR device were derived from this system. For all structures, magnetocurrent ratios (MCRs) were recorded as a function of the bias as well as the amount of strain applied longitudinally to the devices. ZGNR devices with vacancies in the edge regime turn out to exhibit perfect spin-filter activity for well-defined choices of the strain and the bias, carrying completely polarized minority spin currents. In the alternative structure, characterized by vacancies in the bulk regime, spin currents with majority orientation prevail. With respect to both the sign and the size, the MCR is seen to depend sensitively on the device parameters, i.e., the vacancy locations, the bias, and the amount of strain. These results are interpreted in terms of density-of-states distributions, transmission spectra, and transmission operator eigenstates.

Background: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive., Objective: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators., Methods: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation., Results: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced., Conclusion: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Intensive exploitation of base metal deposits in the Sulcis-Iglesiente district (Sardinia, Italy), lasted from the 1850s to the 1990s, determined a high environmental impact on the coastal area, but the effects on marine environment have never been investigated. A marine sediment core, dated with 14 C, was characterized for grain size, chemical and mineralogical composition, in order to reconstruct the sedimentary history of the area and to assess the environmental impact of mining. The comparison of chemical and mineralogical characteristics of recent sediments with those of pre-industrial age allowed discriminating the real anthropogenic impact from the natural metal enrichment. The correspondence, in the upper core, of anthropogenic trace metal enrichment with the presence of mine waste minerals is attributed to the exploiting over industrial scale; the still high metal enrichment in sediment surface levels suggests a still existing impact due to mine dumps and tailings weathering., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background and Objective: To evaluate the efficacy of combined bevacizumab-triamcinolone intravitreal injection in the treatment of diabetic macular edema (DME) compared to monotherapy., Patients and Methods: At eight clinical sites, 111 patients with DME were randomly assigned to receive an intravitreal injection of bevacizumab (Avastin; Genentech, South San Francisco, CA), triamcinolone (Ophthalmos Pharmaceutical Industry, São Paulo-SP, Brazil), or their combination. The primary outcome was visual acuity (VA) at 6 months' follow-up., Results: The average number of injections was 3.2 in the bevacizumab group, 2.4 in the combined group, and 2.1 in the triamcinolone group. All groups presented with improvements in VA (P < .001); however, no differences between groups were observed (P = .436). Mean reduction in central retinal thickness was statistically different only between the triamcinolone and bevacizumab groups (P < .015)., Conclusion: Mono- or combination therapy was effective for DME treatment. No synergistic effects were observed; however, triamcinolone alone or a drug combination may reduce the number of injections required when compared to bevacizumab alone. [Ophthalmic Surg Lasers Imaging Retina. 2017;48:734-740.]., (Copyright 2017, SLACK Incorporated.)

SH-SY5Y neuroblastoma cells are susceptible to differentiation using retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), providing a model of neuronal differentiation. We compared SH-SY5Y cells proteome before and after RA/BDNF treatment using iTRAQ and phosphopeptide enrichment strategies. We identified 5587 proteins, 366 of them with differential abundance. Differentiated cells expressed proteins related to neuronal development, and, undifferentiated cells expressed proteins involved in cell proliferation. Interactive network covered focal adhesion, cytoskeleton dynamics and neurodegenerative diseases processes and regulation of mitogen-activated protein kinase-related signaling pathways; key proteins involved in those processes might be explored as markers for neuronal differentiation.

A large-scale proteomic approach was devised to advance the understanding of venom composition. Bothrops jararaca venom was fractionated by OFFGEL followed by chromatography, generating peptidic and proteic fractions. The latter was submitted to trypsin digestion. Both fractions were separately analyzed by reversed-phase nanochromatography coupled to high resolution mass spectrometry. This strategy allowed deeper and joint characterizations of the peptidome and proteome (proteopeptidome) of this venom. Our results lead to the identification of 46 protein classes (with several uniquely assigned proteins per class) comprising eight high-abundance bona fide venom components, and 38 additional classes in smaller quantities. This last category included previously described B. jararaca venom proteins, common Elapidae venom constituents (cobra venom factor and three-finger toxin), and proteins typically encountered in lysosomes, cellular membranes and blood plasma. Furthermore, this report is the most complete snake venom peptidome described so far, both in number of peptides and in variety of unique proteins that could have originated them. It is hypothesized that such diversity could enclose cryptides, whose bioactivities would contribute to envenomation in yet undetermined ways. Finally, we propose that the broad range screening of B. jararaca peptidome will facilitate the discovery of bioactive molecules, eventually leading to valuable therapeutical agents., Biological Significance: Our proteopeptidomic strategy yielded unprecedented insights into the remarkable diversity of B. jararaca venom composition, both at the peptide and protein levels. These results bring a substantial contribution to the actual pursuit of large-scale protein-level assignment in snake venomics. The detection of typical elapidic venom components, in a Viperidae venom, reinforces our view that the use of this approach (hand-in-hand with transcriptomic and genomic data) for venom proteomic analysis, at the specimen-level, can greatly contribute for venom toxin evolution studies. Furthermore, data were generated in support of a previous hypothesis that venom gland secretory vesicles are specialized forms of lysosomes. Two testable hypotheses also emerge from the results of this work. The first is that a nucleobindin-2-derived protein could lead to prey disorientation during envenomation, aiding in its capture by the snake. The other being that the venom's peptidome might contain a population of cryptides, whose biological activities could lead to the development of new therapeutical agents., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This paper presents a robotic team suited for bottom sediment sampling and retrieval in mudflats, targeting environmental monitoring tasks. The robotic team encompasses a four-wheel-steering ground vehicle, equipped with a drilling tool designed to be able to retain wet soil, and a multi-rotor aerial vehicle for dynamic aerial imagery acquisition. On-demand aerial imagery, properly fused on an aerial mosaic, is used by remote human operators for specifying the robotic mission and supervising its execution. This is crucial for the success of an environmental monitoring study, as often it depends on human expertise to ensure the statistical significance and accuracy of the sampling procedures. Although the literature is rich on environmental monitoring sampling procedures, in mudflats, there is a gap as regards including robotic elements. This paper closes this gap by also proposing a preliminary experimental protocol tailored to exploit the capabilities offered by the robotic system. Field trials in the south bank of the river Tagus' estuary show the ability of the robotic system to successfully extract and transport bottom sediment samples for offline analysis. The results also show the efficiency of the extraction and the benefits when compared to (conventional) human-based sampling., Competing Interests: The authors declare no conflict of interest.

Proinflammatory cytokines are critical mediators that control Mycobacterium tuberculosis (Mtb) growth during active tuberculosis (ATB). To further inhibit bacterial proliferation in diseased individuals, drug inhibitors of cell wall synthesis such as isoniazid (INH) are employed. However, whether INH presents an indirect effect on bacterial growth by regulating host cytokines during ATB is not well known. To examine this hypothesis, we used an in vitro human granuloma system generated with primary leukocytes from healthy donors adapted to model ATB. Intense Mtb proliferation in cell cultures was associated with monocyte/macrophage activation and secretion of IL-1β and TNF. Treatment with INH significantly reduced Mtb survival, but altered neither T-cell-mediated Mtb killing, nor production of IL-1β and TNF. However, blockade of both IL-1R1 and TNF signaling rescued INH-induced killing, suggesting synergistic roles of these cytokines in mediating control of Mtb proliferation. Additionally, mycobacterial killing by INH was highly dependent upon drug activation by the pathogen catalase-peroxidase KatG and involved a host PI3K-dependent pathway. Finally, experiments using coinfected (KatG-mutated and H37Rv strains) cells suggested that active INH does not directly enhance host-mediated killing of Mtb. Our results thus indicate that Mtb-stimulated host IL-1 and TNF have potential roles in TB chemotherapy., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The study of benthic foraminifera in sediment cores provides the opportunity to recognize environmental changes, including those due to the anthropogenic impact. The integration of these data with chemical-physical parameters provides a comprehensive quality assessment. This research was applied to a sediment core collected in the Augusta bay, where a very large commercial and military harbor and one of the largest petrochemical poles in Europe are present. Inside the petrochemical area also operated, from 1958 to 2003, a chlor-alkali plant with mercury cell technology which caused anthropic contamination of surrounding land and marine areas. The sediment core was collected in front of this plant and characterized for grain size and pollutants directly associated to chlor-alkali activity, such as mercury (Hg), barium (Ba), polychlorobiphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). Composition of foraminiferal assemblages and faunal parameters such as specific diversity, faunal density, abundance of abnormal specimens, and foraminiferal size were investigated as potential indicators of environmental status. Statistical analysis indicated a main common origin for Hg, Ba, and PCBs and the influence of pollutants on species distribution and faunal diversity and density. Exceptionally high Hg concentrations (63-680 mg/kg d.w.) were recorded in the whole core, where the geochronological study attributed the most contaminated levels to the period of maximum activity of the chlor-alkali plant, while a decrease of contamination was recorded after the stop of the activity. Distinct foraminiferal assemblages identified different ecozones along the core, which suggested decreasing anthropogenic impact from the bottom to the top.

Wireless sensor nodes have a limited power budget, though they are often expected to be functional in the field once deployed for extended periods of time. Therefore, minimization of energy consumption and energy harvesting technology in Wireless Sensor Networks (WSN) are key tools for maximizing network lifetime, and achieving self-sustainability. This paper proposes an energy aware Adaptive Sampling Algorithm (ASA) for WSN with power hungry sensors and harvesting capabilities, an energy management technique that can be implemented on any WSN platform with enough processing power to execute the proposed algorithm. An existing state-of-the-art ASA developed for wireless sensor networks with power hungry sensors is optimized and enhanced to adapt the sampling frequency according to the available energy of the node. The proposed algorithm is evaluated using two in-field testbeds that are supplied by two different energy harvesting sources (solar and wind). Simulation and comparison between the state-of-the-art ASA and the proposed energy aware ASA (EASA) in terms of energy durability are carried out using in-field measured harvested energy (using both wind and solar sources) and power hungry sensors (ultrasonic wind sensor and gas sensors). The simulation results demonstrate that using ASA in combination with an energy aware function on the nodes can drastically increase the lifetime of a WSN node and enable self-sustainability. In fact, the proposed EASA in conjunction with energy harvesting capability can lead towards perpetual WSN operation and significantly outperform the state-of-the-art ASA.

MicroRNAs are important posttranscriptional regulators of gene expression, which have been shown to fine-tune innate immune responses downstream of pattern recognition receptor (PRR) signaling. This study identifies miR-650 as a novel PRR-responsive microRNA that is downregulated upon stimulation of primary human monocyte-derived dendritic cells (MDDCs) with a variety of different microbe-associated molecular patterns. A comprehensive target search combining in silico analysis, transcriptional profiling, and reporter assays reveals that miR-650 regulates several well-known interferon-stimulated genes, including IFIT2 and MXA. In particular, downregulation of miR-650 in influenza A infected MDDCs enhances the expression of MxA and may therefore contribute to the establishment of an antiviral state. Together these findings reveal a novel link between miR-650 and the innate immune response in human MDDCs., (© 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: This study evaluated the effects of cattle removal on the Culicidae mosquito community structure in a tropical dry forest in Brazil., Methods: Culicidae were collected during dry and wet seasons in cattle presence and absence between August 2008 and October 2010 and assessed using multivariate statistical models., Results: Cattle removal did not significantly alter Culicidae species richness and abundance. However, alterations were noted in Culicidae community composition., Conclusions: This is the first study to evaluate the impact of cattle removal on Culicidae community structure in Brazil and demonstrates the importance of assessing ecological parameters such as community species composition.

Myeloid differentiation 88 (MyD88) is the key signaling adapter of Toll-like and interleukin-1 receptors. Recurrent lymphoma-associated mutations, particularly Leu265Pro (L265P), within the MyD88 Toll/interleukin-1 receptor (TIR) domain sustain lymphoma cell survival due to constitutive nuclear factor κB signaling. We found that mutated TIR domains displayed an intrinsic propensity for augmented oligomerization and spontaneous formation of cytosolic Myddosome aggregates in lymphoma cell lines, mimicking the effect of dimerized TIR domains. Blocking of MyD88 oligomerization induced apoptosis. The L265P TIR domain can recruit the endogenous wild-type MyD88 for oligomer formation and hyperactivity. Molecular dynamics simulations and analysis of additional mutations suggest that constitutive activity is caused by allosteric oligomerization., (© 2014 by The American Society of Hematology.)

Peptide spectrum matching is the current gold standard for protein identification via mass-spectrometry-based proteomics. Peptide spectrum matching compares experimental mass spectra against theoretical spectra generated from a protein sequence database to perform identification, but protein sequences not present in a database cannot be identified unless their sequences are in part conserved. The alternative approach, de novo sequencing, can make it possible to infer a peptide sequence directly from a mass spectrum, but interpreting long lists of peptide sequences resulting from large-scale experiments is not trivial. With this as motivation, PepExplorer was developed to use rigorous pattern recognition to assemble a list of homologue proteins using de novo sequencing data coupled to sequence alignment to allow biological interpretation of the data. PepExplorer can read the output of various widely adopted de novo sequencing tools and converge to a list of proteins with a global false-discovery rate. To this end, it employs a radial basis function neural network that considers precursor charge states, de novo sequencing scores, peptide lengths, and alignment scores to select similar protein candidates, from a target-decoy database, usually obtained from phylogenetically related species. Alignments are performed using a modified Smith-Waterman algorithm tailored for the task at hand. We verified the effectiveness of our approach using a reference set of identifications generated by ProLuCID when searching for Pyrococcus furiosus mass spectra on the corresponding NCBI RefSeq database. We then modified the sequence database by swapping amino acids until ProLuCID was no longer capable of identifying any proteins. By searching the mass spectra using PepExplorer on the modified database, we were able to recover most of the identifications at a 1% false-discovery rate. Finally, we employed PepExplorer to disclose a comprehensive proteomic assessment of the Bothrops jararaca plasma, a known biological source of natural inhibitors of snake toxins. PepExplorer is integrated into the PatternLab for Proteomics environment, which makes available various tools for downstream data analysis, including resources for quantitative and differential proteomics., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Snake venom metalloproteinases (SVMPs) are zinc-dependent enzymes responsible for most symptoms of human envenoming. Like matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAM) proteins, SVMPs are synthesized as zymogens, and enzyme activation is regulated by hydrolysis of their prodomain, but the processing of SVMPs is still unclear. In this study, we attempted to identify the presence of prodomain in different compartments of snake venom glands as zymogens or in the free form to elucidate some mechanism involved in SVMP activation. Using antibodies obtained by immunization with a recombinant prodomain, bands of zymogen molecular mass and prodomain peptides were detected mostly in gland extracts all along the venom production cycle and in the venom collected from the lumen at the peak of venom production. Prodomain was detected in secretory cells mostly in the secretory vesicles near the Golgi. We hypothesize that the processing of SVMPs starts within secretory vesicles and continues in the lumen of the venom gland just after enzyme secretion and involves different steps compared to ADAMs and MMPs but can be used as a model for studying the relevance of peptides resulting from prodomain processing and degradation for controlling the activity of metalloproteinases.

Purpose: To present the development and initial experience of a novel colored perfluorocarbon liquid (PFCL) in vitreoretinal surgery., Methods: This was an experimental laboratory study and prospective human interventional study. F6H8 (Fluoron GmbH) was colored by adding 0.3 g/L blue anthraquinone dye. Subsequently, 20% colored F6H8 was prepared by mixing with perfluorooctane or perfluorodecalin (Fluoron GmbH). The novel product is not yet FDA approved for human application. In the laboratory, the colored PFCL was covered with 1) uncolored PFCL, 2) BSS, and 3) silicone oil. Cell toxicity was evaluated in L929 mouse fibroblasts using a growth inhibition assay. Porcine ex vivo eyes were evaluated after vitrectomy followed by intravitreal and subretinal colored PFCL infusion. A pilot, prospective, noncomparative interventional study was conducted in patients with retinal detachment with proliferative vitreoretinopathy (PVR)., Results: The density of the colored PFLC mixture was 1.664 g/cm for perfluorooctane and 1.802 g/cm for perfluorodecalin. There was no relevant cell growth inhibition with any concentration of colored PFCL tested. Experiments in pigs revealed that infusion of the colored PFCL caused neither staining of the internal limiting membrane nor intravitreal residual droplets. In the prospective study, 9 eyes (75%) underwent surgery for rhegmatogenous retinal detachment with at least grade C PVR. The colored PFCL enabled retinal break examination and detection of residual intravitreal droplets in all surgeries. There was no case of separation or leakage of the dye from the PFCL solution that could have caused unwanted staining of the vitreous or epiretinal surface., Conclusion: The colored PFCL enabled intraoperative maneuvers such as endolaser use. In addition, removal of the colored PFCL was easily achieved at the end of surgery.

The present work aims at characterizing T. harzianum secretome when the fungus is grown in synthetic medium supplemented with one of the four substrates: glucose, cellulose, xylan, and sugarcane bagasse (SB). The characterization was done by enzymatic assays and proteomic analysis using 2-DE/MALDI-TOF and gel-free shotgun LC-MS/MS. The results showed that SB induced the highest cellulolytic and xylanolytic activities when compared with the other substrates, while remarkable differences in terms of number and distribution of protein spots in 2-DE gels were also observed among the samples. Additionally, treatment of the secretomes with PNGase F revealed that most spot trails in 2-DE gels corresponded to N-glycosylated proteoforms. The LC-MS/MS analysis of the samples identified 626 different protein groups, including carbohydrate-active enzymes and accessory, noncatalytic, and cell-wall-associated proteins. Although the SB-induced secretome displayed the highest cellulolytic and xylanolytic activities, it did not correspond to a higher proteome complexity because CM-cellulose-induced secretome was significantly more diverse. Among the identified proteins, 73% were exclusive to one condition, while only 5% were present in all samples. Therefore, this study disclosed the variation of T. harzianum secretome in response to different substrates and revealed the diversity of the fungus enzymatic toolbox.

Defining the proteomes encoded by each chromosome and characterizing proteins related to human illnesses are among the goals of the Chromosome-centric Human Proteome Project (C-HPP) and the Biology and Disease-driven HPP. Following these objectives, we investigated the proteomes of the human anterior temporal lobe (ATL) and corpus callosum (CC) collected post-mortem from eight subjects. Using a label-free GeLC-MS/MS approach, we identified 2454 proteins in the ATL and 1887 in the CC through roughly 7500 and 5500 peptides, respectively. Considering that the ATL is a gray-matter region while the CC is a white-matter region, they presented proteomes specific to their functions. Besides, 38 proteins were found to be differentially expressed between the two regions. Furthermore, the proteome data sets were classified according to their chromosomal origin, and five proteins were evidenced at the MS level for the first time. We identified 70 proteins of the chromosome 15 - one of them for the first time by MS - which were submitted to an in silico pathway analysis. These revealed branch point proteins associated with Prader-Willi and Angelman syndromes and dyskeratosis congenita, which are chromosome-15-associated diseases. Data presented here can be a useful for brain disorder studies as well as for contributing to the C-HPP initiative. Our data are publicly available as resource data to C-HPP participant groups at http://yoda.iq.ufrj.br/Daniel/chpp2013. Additionally, the mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD000547 for the corpus callosum and PXD000548 for the anterior temporal lobe.

The brain activation associated with the Spinning Dancer Illusion, a cognitive visual illusion, is not entirely known. Inferences from other study modalities point to the involvement of the dorso-parieto-occipital areas in the spontaneous switchings of perception in other bistable non-kinetic illusions. fMRI is a mature technique used to investigate the brain responses associated with mental changes. Resting-state fMRI is a novel technique that may help ascertain the effects of spontaneous brain changes in the top-down regulation of visual perception. The purpose of this report is to describe the brain activation associated with the subjective illusory changes of perception of a kinetic bistable stimulus. We hypothesize that there is a relationship between the perception phases with the very slow cortical spontaneous fluctuations, recently described. A single normal subject who was trained to produce voluntarily perception phase switches underwent a series of fMRI studies whose blocks were either defined post-hoc or accordingly with a predefined timeline to assess spontaneous and voluntarily evoked visual perception switches, respectively. Correlation of findings with resting-state fMRI and independent component analysis of the task series was sought. Phases of the rotation direction were found associated with right parietal activity. Independent component analysis of the task series and their comparison with basal resting-state components suggest that this activity is related to one of the very slow spontaneous brain fluctuations. The spontaneous fluctuations of the cortical activity may explain the subjective changes in perception of direction of the Spinning Dancer Illusion. This observation is a proof-of-principle, suggesting that the spontaneous brain oscillations may influence top-down sensory regulation.

Affinity purification of protein complexes and identification of co-purified proteins by mass spectrometry is a powerful method to discover novel protein-protein interactions. Application of this method to the study of biological systems often requires the ability to process a large number of samples. Hence, there is great need to generate proteomic workflows compatible with large-scale studies. The major goal of this protocol is to present a fast, reliable, and scalable method to characterize protein complexes by mass spectrometry to overcome the limitations of conventional geLC-MS/MS or MudPIT protocols. This method was successfully employed for the discovery and characterization of novel protein complexes in cultured yeast, mammalian cells, and mice.

In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled inline to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 proteins were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in PPDB, SUBA, or PlProt databases while homologues for 13 proteins were not found in any of the three plastid proteins databases but were marked as plastidial by at least one of the three prediction programs used. Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in the plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, our study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level.

Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

Biosurfactants are a class of functional molecules produced and secreted by microorganisms, which play important roles in cell physiology such as flagellum-dependent or -independent bacterial spreading, cell signaling, and biofilm formation. They are amphipathic compounds and comprise a variety of chemical structures, including rhamnolipids, typically produced by Pseudomonas spp. and also reported within other bacterial genera. The present study is focused on Burkholderia kururiensis KP23(T), a trichloroethylene (TCE)-degrading, N-fixing, and plant growth-promoting bacterium. Herein, we describe the production of rhamnolipids by B. kururiensis, and its characterization by LTQ-Orbitrap Hybrid Mass Spectrometry, a powerful tool that allowed efficient identification of molecular subpopulations, due to its high selectivity, mass accuracy, and resolving power. The population of rhamnolipids produced by B. kururiensis revealed molecular species commonly observed in Pseudomonas spp. and/or Burkholderia spp. In addition, this strain was used as a platform for expression of two Pseudomonas aeruginosa biosynthetic enzymes: RhlA, which directly utilizes β-hydroxydecanoyl-ACP intermediates in fatty acid synthesis to generate the HAA, and RhlB, the rhamnosyltransferase 1, which catalyzes the transfer of dTDP-L-rhamnose to β-hydroxy fatty acids in the biosynthesis of rhamnolipids. We show that rhamnolipid production by the engineered B. kururiensis was increased over 600 % when compared to the wild type. Structural analyses demonstrated a molecular population composed mainly of monorhamnolipids, as opposed to wild-type B. kururiensis and P. aeruginosa in which dirhamnolipids are predominant. We conclude that B. kururiensis is a promising biosurfactant-producing organism, with great potential for environmental and biotechnological applications due to its non-pathogenic characteristics and efficiency as a platform for metabolic engineering and production of tailor-made biosurfactants.

Background: Aedes albopictus is a vector for several fatal arboviruses in tropical and sub-tropical regions of the world. The midgut of the mosquito is the first barrier that pathogens must overcome to establish infection and represents one of the main immunologically active sites of the insect. Nevertheless, little is known about the proteins involved in the defense against pathogens, and even in the processing of food, and the detoxification of metabolites. The identification of proteins exclusively expressed in the midgut is the first step in understanding the complex physiology of this tissue and can provide insight into the mechanisms of pathogen-vector interaction. However, identification of the locally expressed proteins presents a challenge because the Ae. albopictus genome has not been sequenced., Methods: In this study, two-dimensional electrophoresis (2DE) was combined with liquid chromatography in line with tandem mass spectrometry (LC-MS/MS) and data mining to identify the major proteins in the midgut of sugar-fed Ae. albopictus females., Results: Fifty-six proteins were identified by sequence similarity to entries from the Ae. aegypti genome. In addition, two hypothetical proteins were experimentally confirmed. According to the gene ontology analysis, the identified proteins were classified into 16 clusters of biological processes. Use of the STRING database to investigate protein functional associations revealed five functional networks among the identified proteins, including a network for carbohydrate and amino acid metabolism, a group associated with ATP production and a network of proteins that interact during detoxification of toxic free radicals, among others. This analysis allowed the assignment of a potential role for proteins with unknown function based on their functional association with other characterized proteins., Conclusion: Our findings represent the first proteome map of the Ae. albopictus midgut and denotes the first steps towards the description of a comprehensive proteome map of this vector. In addition, the data contributes to the functional annotation of Aedes spp. genomes using mass spectrometry-based proteomics data combined with complementary gene prediction methods.

In the present work, we report the synthesis of the novel esters of indomethacin (IDMC) and an ester of reduced IDMC. For this purpose, IDMC is covalently bound by using a spacer chain to the pyrrole (Py) in the 3-position. The innovative pyrrole-indomethacin (3-Py-IDMC) derivates show no cytotoxic effects in primary calvarial osteoblasts. The designed IDMC derivates have been studied because they could be injected locally as a component of polymeric micro-particles. In fact, the new 3-Py-IDMC derivatives will assure their further polymerization since the 2- and 5-monomer positions are free., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Plant cell wall-degrading enzymes produced by microorganisms possess important biotechnological applications, including biofuel production. Some anaerobic bacteria are able to produce multienzymatic complexes called cellulosomes while filamentous fungi normally secrete individual hydrolytic enzymes that act synergistically for polysaccharide degradation. Here, we present evidence that the fungus Trichoderma harzianum, cultivated in medium containing the agricultural residue sugarcane bagasse, is able to secrete multienzymatic complexes. The T. harzianum secretome was firstly analyzed by 1D-BN (blue native)-PAGE that revealed several putative complexes. The three most intense 1D-BN-PAGE bands, named complexes [I], [II], and [III], were subsequently subjected to tricine SDS-PAGE that demonstrated that they were composed of smaller subunits. Zymographic assays were performed using 1D-BN-PAGE and 2D-BN/BN-PAGE demonstrating that the complexes bore cellulolytic and xylanolytic activities. The complexes [I], [II], and [III] were then trypsin digested and analyzed separately by LC-MS/MS that revealed their protein composition. Since T. harzianum has an unsequenced genome, a homology-driven proteomics approach provided a higher number of identified proteins than a conventional peptide-spectrum matching strategy. The results indicate that the complexes are formed by cellulolytic and hemicellulolytic enzymes and other proteins such as chitinase, cutinase, and swollenin, which may act synergistically to degrade plant cell wall components., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Our current knowledge in biology has been mostly derived from studying model organisms and cell lines in which only a small fraction of all described species have been extensively studied. Although these model organisms are amenable to genetic manipulations, this blinds researchers to the true variability of life. Groundbreaking discoveries are often achieved by analyzing "noncanonical" species; for example, the characterization of Taq polymerase from Thermus aquaticus ultimately led to a revolution in the field of molecular biology. Brazil possesses a rich biodiversity and a considerable fraction of Brazilian groups use current proteomic techniques to explore this natural treasure-trove. However, in our opinion, much more than the widely adopted peptide spectrum match approach is required to explore this rich "proteomosphere." Here, we provide a critical overview of the available strategies for the analysis of proteomic data from "noncanonical" biological samples (e.g. proteins from unsequenced genomes or genomes with high levels of polymorphisms), and demonstrate some limitations of existing approaches for large-scale protein identification and quantitation. An understanding of the premises behind these computational tools is necessary to properly deal with their limitations and draw accurate conclusions., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

TP53 (tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short interfering RNA library. We identify a variety of TP53 synthetic interactions unmasking the complex connections of p53 to cellular physiology and growth control. Molecular dissection of the TP53 synthetic interaction with UNRIP indicates an enhanced dependency of TP53-negative cells on small nucleolar ribonucleoprotein (snoRNP) assembly. This dependency is mediated by the snoRNP chaperone gene NOLC1 (also known as NOPP140), which we identify as a physiological p53 target gene. This unanticipated function of TP53 in snoRNP assembly highlights the potential of RNAi-mediated synthetic interaction screens to dissect molecular pathways of tumour suppressor genes.

Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes, oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed., (Published by Elsevier Ltd.)

DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair., Competing Interests: The authors have declared that no competing interests exist.

Efficient chromosome segregation during mitosis relies on the coordinated activity of molecular motors with proteins that regulate kinetochore attachments to dynamic spindle microtubules [1]. CLASPs are conserved kinetochore- and microtubule-associated proteins encoded by two paralog genes, clasp1 and clasp2, and have been previously implicated in the regulation of kinetochore microtubule dynamics [2-4]. However, it remains unknown how CLASPs work in concert with other proteins to form a functional kinetochore microtubule interface. Here we have identified mitotic interactors of human CLASP1 via a proteomic approach. Among these, the microtubule plus-end-directed motor CENP-E [5] was found to form a complex with CLASP1 that colocalizes to multiple structures of the mitotic apparatus in human cells. We found that CENP-E recruits both CLASP1 and CLASP2 to kinetochores independently of its motor activity or the presence of microtubules. Depletion of CLASPs or CENP-E by RNA interference in human cells causes a significant and comparable reduction of kinetochore microtubule poleward flux and turnover rates and rescues spindle bipolarity in Kif2a-depleted cells. We conclude that CENP-E integrates two critical functions that are important for accurate chromosome movement and spindle architecture: one relying directly on its motor activity, and the other involving the targeting of key microtubule regulators to kinetochores.

Defense responses of Fagus sylvatica seedlings elicited by infection with the root pathogen Phytophthora citricola and root or leaf wounding were compared at local and systemic levels in differential display experiments using two-dimensional gel electrophoresis followed by homology-driven mass spectrometric identification of proteins. A total of 68 protein spots were identified representing 51 protein functions related to protein synthesis and processing, energy, primary and secondary metabolism, as well as signal transduction, stress and defense. Changes in the abundance of root and leaf proteins partly overlapped between plant responses to the different stressors. The response to pathogen infection was rather late, weak and unspecific and accompanied by adjustments of the energy and primary metabolism which suggested either a lack of recognition or a suppression of host's defense reaction by the invading pathogen. The response to wounding involved changes in the basal metabolism as well as activation of defense mechanisms. Both types of changes were largely specific to the wounded organ. Similarities between the defense mechanisms activated by root infection and root wounding were also observed.

Proliferation of mammalian cells requires the coordinated function of many proteins to accurately divide a cell into two daughter cells. Several RNAi screens have identified previously uncharacterised genes that are implicated in mammalian cell division. The molecular function for these genes needs to be investigated to place them into pathways. Phenotypic profiling is a useful method to assign putative functions to uncharacterised genes. Here, we show that the analysis of protein localisation is useful to refine a phenotypic profile. We show the utility of this approach by defining a function of the previously uncharacterised gene C13orf3 during cell division. C13orf3 localises to centrosomes, the mitotic spindle, kinetochores, spindle midzone, and the cleavage furrow during cell division and is specifically phosphorylated during mitosis. Furthermore, C13orf3 is required for centrosome integrity and anaphase onset. Depletion by RNAi leads to mitotic arrest in metaphase with an activation of the spindle assembly checkpoint and loss of sister chromatid cohesion. Proteomic analyses identify C13orf3 (Ska3) as a new component of the Ska complex and show a direct interaction with a regulatory subunit of the protein phosphatase PP2A. All together, these data identify C13orf3 as an important factor for metaphase to anaphase progression and highlight the potential of combined RNAi screening and protein localisation analyses.

Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.

Araucaria angustifolia is the only native conifer of economic importance in the Brazilian Atlantic Rainforest. Due to a clear-cutting form of exploitation this species has received the status of vulnerable. The aim of this work was to investigate and characterize changes in protein expression profile during seed development of this endangered species. For this, the proteome of developing seeds was characterized by 2-DE and LC-MS/MS. Ninety six proteins were confidently identified and classified according to their biological function and expression profile. Overaccumulated proteins in early seed development indicated a higher control on oxidative stress metabolism during this phase. In contrast, highly expressed proteins in late stages revealed an active metabolism, leading to carbon assimilation and storage compounds accumulation. Comprehensive protein expression profiles and identification of overaccumulated proteins provide new insights into the process of embryogenesis in this recalcitrant species. Considerations on the improvement and control of somatic embryogenesis through medium manipulation and protein markers screening using data generated are also discussed.

Homology-driven proteomics aims at exploring the proteomes of organisms with unsequenced genomes that, despite rapid genomic sequencing progress, still represent the overwhelming majority of species in the biosphere. Methodologies have been developed to enable automated LC-MS/MS identifications of unknown proteins, which rely on the sequence similarity between the fragmented peptides and reference database sequences from phylogenetically related species. However, because full sequences of matched proteins are not available and matching specificity is reduced, estimating protein abundances should become the obligatory element of homology-driven proteomics pipelines to circumvent the interpretation bias towards proteins from evolutionary conserved families.