Detection of Leishmania RNA virus in Leishmania parasites.

BACKGROUND Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. METHODOLOGY/PRINCIPAL FINDINGS This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. CONCLUSIONS/SIGNIFICANCE We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

XENOESTROGEN-SPECIFIC MECHANISMS OF DEVELOPMENTAL REPROGRAMMING CORRELATE WITH GENE EXPRESSION AND TUMOR DEVELOPMENT

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. We have shown that neonatal exposure to the xenoestrogen diethylstilbestrol (DES) can developmentally reprogram the reproductive tract in genetically susceptible Eker rats giving rise to complete penetrance of uterine leiomyoma. Based on this, we hypothesized that xenoestrogens, including genistein (GEN) and bisphenol A (BPA), reprogram estrogen-responsive gene expression in the myometrium and promote the development of uterine leiomyoma. We proposed the mechanism that is responsible for the developmental reprogramming of gene expression was through estrogen (E2)/ xenoestrogen inducedrapid ER signaling, which modifies the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) via activation of the PI3K/AKT pathway. We further hypothesized that there is a xenostrogen-specific effect on this pathway altering patterns of histone modification, DNA methylation and gene expression. In addition to our novel finding that E2/DES-induced phosphorylation of EZH2 by AKT reduces the levels of H3K27me3 in vitro and in vivo, this work demonstrates in vivo that a brief neonatal exposure to GEN, in contrast to BPA, activates the PI3K/AKT pathway to regulate EZH2 and decreases H3K27me3 levels in the neonatal uterus. Given that H3K27me3 is a repressive mark that has been shown to result in DNA methylation and gene silencing we investigated the methylation of developmentally reprogrammed genes. In support of this evidence, we show that neonatal DES exposure in comparison to VEH, leads to hypomethylation of the promoter of a developmentally reprogrammed gene, Gria2, that become hyper-responsive to estrogen in the adult myometrium indicating vi that DES exposure alter gene expression via chromatin remodeling and loss of DNA methylation. In the adult uterus, GEN and BPA exposure developmentally reprogrammed expression of estrogen-responsive genes in a manner opposite of one another, correlating with our previous data. Furthermore, the ability of GEN and BPA to developmental reprogram gene expression correlated with tumor incidence and multiplicity. These data show that xenoestrogens have unique effects on the activation of non-genomic signaling in the developing uterus that promotes epigenetic and genetic alterations, which are predictive of developmental reprogramming and correlate with their ability to modulate hormone-dependent tumor development.

A reverse crystal Engineering approach

The enormous impact of crystal engineering in modern solid state chemistry takes advantage from the connection between a typical basic science field and the word engineering. Regrettably, the engineering aspect of organic or metal organic crystalline materials are limited, so far, to descriptive structural features, sometime entangled with topological aspects, but only rarely with true material design. This should include not only the fabrication and structural description at micro- and nano-scopic level of the solids, but also a proper reverse engineering, a fundamental discipline for engineers. Translated into scientific language, the reverse crystal engineering refers to a dedicated and accurate analysis of how the building blocks contribute to generate a given material property. This would enable a more appropriate design of new crystalline material. We propose here the application of reverse crystal engineering to optical properties of organic and metal organic framework structures, applying the distributed atomic polarizability approach that we have extensively investigated in the past few years[1,2].

Calculation of crystal optical properties from molecular electron density

We have recently developed a method to obtain distributed atomic polarizabilities adopting a partitioning of the molecular electron density (for example, the Quantum Theory of Atoms in Molecules, [1]), calculated with or without an applied electric field. The procedure [2] allows to obtained atomic polarizability tensors, which are perfectly exportable, because quite representative of an atom in a given functional group. Among the many applications of this idea, the calculation of crystal susceptibility is easily available, either from a rough estimation (the polarizability of the isolated molecule is used) or from a more precise estimation (the polarizability of a molecule embedded in a cluster representing the first coordination sphere is used). Lorentz factor is applied to include the long range effect of packing, which is enhancing the molecular polarizability. Simple properties like linear refractive index or the gyration tensor can be calculated at relatively low costs and with good precision. This approach is particularly useful within the field of crystal engineering of organic/organometallic materials, because it would allow a relatively easy prediction of a property as a function of the packing, thus allowing "reverse crystal engineering". Examples of some amino acid crystals and salts of amino acids [3] will be illustrated, together with other crystallographic or non-crystallographic applications. For example, the induction and dispersion energies of intermolecular interactions could be calculated with superior precision (allowing anisotropic van der Waals interactions). This could allow revision of some commonly misunderstood intermolecular interactions, like the halogen bonding (see for example the recent remarks by Stone or Gilli [4]). Moreover, the chemical reactivity of coordination complexes could be reinvestigated, by coupling the conventional analysis of the electrostatic potential (useful only in the circumstances of hard nucleophilic/electrophilic interaction) with the distributed atomic polarizability. The enhanced reactivity of coordinated organic ligands would be better appreciated. [1] R. F. W. Bader, Atoms in Molecules: A Quantum Theory. Oxford Univ. Press, 1990. [2] A. Krawczuk-Pantula, D. Pérez, K. Stadnicka, P. Macchi, Trans. Amer. Cryst. Ass. 2011, 1-25 [3] A. S. Chimpri1, M. Gryl, L. H.R. Dos Santos1, A. Krawczuk, P. Macchi Crystal Growth & Design, in the press. [4] a) A. J. Stone, J. Am. Chem. Soc. 2013, 135, 7005−7009; b) V. Bertolasi, P. Gilli, G. Gilli Crystal Growth & Design, 2013, 12, 4758-4770.

FUNCTION OF ZNF668 IN CANCER DEVELOPMENT

Human cancer develops as a result of accumulation of mutations in oncogenes and tumor suppressor genes. Zinc finger protein 668 (ZNF668) has recently been identified and validated as one of the highly mutated genes in breast cancer, but its function is entirely unknown. Here, we report two major functions of ZNF668 in cancer development. (1) ZNF668 functions as a tumor suppressor by regulating p53 protein stability and function. We demonstrate that ZNF668 is a nucleolar protein that physically interacts with both MDM2 and p53. By binding to MDM2, ZNF668 regulates MDM2 autoubiquitination and prevents MDM2-mediated p53 ubiquitination and degradation; ZNF668 deficiency impairs DNA damage-induced p53 stabilization. Notably, ZNF668 effectively suppresses breast cancer cell proliferation and transformation in vitro and tumorigenicity in vivo. Consistently, ZNF668 knockdown readily transforms normal mammary epithelial cells. Together, our studies identify ZNF668 as a novel breast tumor suppressor gene that acts at least in part by regulating the stability and function of p53. (2) ZNF668 functions as a DNA repair protein by regulating histone acetylation. DNA repair proteins need to access the chromatin by chromatin modification or remodeling to use DNA template within chromatin. Dynamic posttranslational modifications of histones are critical for cells to relax chromatin in DNA repair. However, the precise underlying mechanism mediating enzymes responsible for these modifications and their recruitment to DNA lesions remains poorly understood. We observed ZNF668 depletion causes impaired chromatin relaxation as a result of impaired DNA-damage induced histone H2AX hyper-acetylation. This results in the decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after DNA damage, albeit with the presence of a functional ATM/ATR dependent DNA-damage signaling cascade. Importantly, the impaired loading of repair proteins and the defect in DNA repair in ZNF668-deficient cells can be counteracted by chromatin relaxation, indicating that the DNA-repair defect that was observed in the absence of ZNF668 is due to impeded chromatin accessibility at sites of DNA breaks. Our findings therefore identify ZNF668 as a key molecule that links chromatin relaxation with response to DNA damage in the control of DNA repair.

Middle and Late Pleistocene humid periods recorded in palaeolake deposits of the Nafud desert, Saudi Arabia

Present climate in the Nafud desert of northern Saudi Arabia is hyper-arid and moisture brought by north-westerly winds scarcely reaches the region. The existence of abundant palaeolake sediments provides evidence for a considerably wetter climate in the past. However, the existing chronological framework of these deposits is solely based on radiocarbon dating of questionable reliability, due to potential post-depositional contamination with younger 14C. By using luminescence dating, we show that the lake deposits were not formed between 40 and 20 ka as suggested previously, but approximately ca 410 ka, 320 ka, 200 ka, 125 ka, and 100 ka ago. All of these humid phases are in good agreement with those recorded in lake sediments and speleothems from southern Arabia. Surprisingly, no Holocene lake deposits were identified. Geological characteristics of the deposits and diatom analysis suggest that a single, perennial lake covered the entire south-western Nafud ca 320 ka ago. In contrast, lakes of the 200 ka, 125 ka, and 100 ka humid intervals were smaller and restricted to interdune depressions of a pre-existing dune relief. The concurrent occurrence of humid phases in the Nafud, southern Arabia and the eastern Mediterranean suggests that moisture in northern Arabia originated either from the Mediterranean due to more frequent frontal depression systems or from stronger Indian monsoon circulation, respectively. However, based on previously published climate model simulations and palaecolimate evidence from central Arabia and the Negev desert, we argue that humid climate conditions in the Nafud were probably caused by a stronger African monsoon and a distinct change in zonal atmospheric circulation.

Diagnosis and Treatment of Venous Malformations Consensus Document of the International Union of Phlebology (IUP): updated 2013

Venous malformations (VMs) are the most common vascular developmental anomalies (birth defects) . These defects are caused by developmental arrest of the venous system during various stages of embryogenesis. VMs remain a difficult diagnostic and therapeutic challenge due to the wide range of clinical presentations, unpredictable clinical course, erratic response to the treatment with high recurrence/persistence rates, high morbidity following non-specific conventional treatment, and confusing terminology. The Consensus Panel reviewed the recent scientific literature up to the year 2013 to update a previous IUP Consensus (2009) on the same subject. ISSVA Classification with special merits for the differentiation between the congenital vascular malformation (CVM) and vascular tumors was reinforced with an additional review on syndrome-based classification. A "modified" Hamburg classification was adopted to emphasize the importance of extratruncular vs. truncular sub-types of VMs. This incorporated the embryological origin, morphological differences, unique characteristics, prognosis and recurrence rates of VMs based on this embryological classification. The definition and classification of VMs were strengthened with the addition of angiographic data that determines the hemodynamic characteristics, the anatomical pattern of draining veins and hence the risk of complication following sclerotherapy. The hemolymphatic malformations, a combined condition incorporating LMs and other CVMs, were illustrated as a separate topic to differentiate from isolated VMs and to rectify the existing confusion with name-based eponyms such as Klippel-Trenaunay syndrome. Contemporary concepts on VMs were updated with new data including genetic findings linked to the etiology of CVMs and chronic cerebrospinal venous insufficiency. Besides, newly established information on coagulopathy including the role of D-Dimer was thoroughly reviewed to provide guidelines on investigations and anticoagulation therapy in the management of VMs. Congenital vascular bone syndrome resulting in angio-osteo-hyper/hypotrophy and (lateral) marginal vein was separately reviewed. Background data on arterio-venous malformations was included to differentiate this anomaly from syndrome-based VMs. For the treatment, a new section on laser therapy and also a practical guideline for follow up assessment were added to strengthen the management principle of the multidisciplinary approach. All other therapeutic modalities were thoroughly updated to accommodate a changing concept through the years.

Characterization of Phosphorylation- and RNA-Dependent UPF1 Interactors by Quantitative Proteomics

Human up-frameshift 1 (UPF1) is an ATP-dependent RNA helicase and phosphoprotein implicated in several biological processes but is best known for its key function in nonsense-mediated mRNA decay (NMD). Here we employed a combination of stable isotope labeling of amino acids in cell culture experiments to determine by quantitative proteomics UPF1 interactors. We used this approach to distinguish between RNA-mediated and protein-mediated UPF1 interactors and to determine proteins that preferentially bind the hypo- or the hyper-phosphorylated form of UPF1. Confirming and expanding previous studies, we identified the eukaryotic initiation factor 3 (eIF3) as a prominent protein-mediated interactor of UPF1. However, unlike previously reported, eIF3 binds to UPF1 independently of UPF1’s phosphorylation state. Furthermore, our data revealed many nucleus-associated RNA-binding proteins that preferentially associate with hyper-phosphorylated UPF1 in an RNase-sensitive manner, suggesting that UPF1 gets recruited to mRNA and becomes phosphorylated before being exported to the cytoplasm as part of the mRNP.

Feasibility of post mortem cardiac proton density weighted fast field echo imaging in two cases of sudden death

OBJECTIVE The aim of this work is to investigate and compare cardiac proton density (PD) weighted fast field echo (FFE) post-mortem magnetic resonance (PMMR) imaging with standard cardiac PMMR imaging (T1-weighted and T2-weighted turbo spin-echo (TSE)), postmortem CT (PMCT) as well as autopsy. MATERIALS AND METHODS Two human cadavers sequentially underwent cardiac PMCT and PMMR imaging (PD-weighted FFE, T1-weighted and T2-weighted TSE) and autopsy. The cardiac PMMR images were compared to each other as well as to PMCT and autopsy findings. RESULTS For the first case, cardiac PMMR exhibited a focal region of low signal in PD-weighted FFE and T2-weighted TSE images, surrounded by a signal intense rim in the T2-weighted images. T1-weighted TSE and PMCT did not appear to identify any focal abnormality. Macroscopic inspection identified a blood clot; histology confirmed this to be a thrombus with an adhering myocardial infarction. In the second case, a myocardial rupture with heart tamponade was identified in all PMMR images, located at the anterior wall of the left ventricle; PMCT excluded additional ruptures. In PD-weighted FFE and T2-weighted TSE images, it occurred hypo-intense, while resulting in small clustered hyper-intense spots in T1-weighted TSE. Autopsy confirmed the PMMR and PMCT findings. CONCLUSIONS Presented initial results have shown PD-weighted FFE to be a valuable imaging sequence in addition to traditional T2-weighted TSE imaging for blood clots and myocardial haemorrhage with clearer contrast between affected and healthy myocardium.

Detecting Functional Hubs of Ictogenic Networks

Quantitative EEG (qEEG) has modified our understanding of epileptic seizures, shifting our view from the traditionally accepted hyper-synchrony paradigm toward more complex models based on re-organization of functional networks. However, qEEG measurements are so far rarely considered during the clinical decision-making process. To better understand the dynamics of intracranial EEG signals, we examine a functional network derived from the quantification of information flow between intracranial EEG signals. Using transfer entropy, we analyzed 198 seizures from 27 patients undergoing pre-surgical evaluation for pharmaco-resistant epilepsy. During each seizure we considered for each network the in-, out- and total "hubs", defined respectively as the time and the EEG channels with the maximal incoming, outgoing or total (bidirectional) information flow. In the majority of cases we found that the hubs occur around the middle of seizures, and interestingly not at the beginning or end, where the most dramatic EEG signal changes are found by visual inspection. For the patients who then underwent surgery, good postoperative clinical outcome was on average associated with a higher percentage of out- or total-hubs located in the resected area (for out-hubs p = 0.01, for total-hubs p = 0.04). The location of in-hubs showed no clear predictive value. We conclude that the study of functional networks based on qEEG measurements may help to identify brain areas that are critical for seizure generation and are thus potential targets for focused therapeutic interventions.

A new Bayesian approach to the reconstruction of spectral functions

We present a novel approach for the reconstruction of spectra from Euclidean correlator data that makes close contact to modern Bayesian concepts. It is based upon an axiomatically justified dimensionless prior distribution, which in the case of constant prior function m(ω) only imprints smoothness on the reconstructed spectrum. In addition we are able to analytically integrate out the only relevant overall hyper-parameter α in the prior, removing the necessity for Gaussian approximations found e.g. in the Maximum Entropy Method. Using a quasi-Newton minimizer and high-precision arithmetic, we are then able to find the unique global extremum of P[ρ|D] in the full Nω » Nτ dimensional search space. The method actually yields gradually improving reconstruction results if the quality of the supplied input data increases, without introducing artificial peak structures, often encountered in the MEM. To support these statements we present mock data analyses for the case of zero width delta peaks and more realistic scenarios, based on the perturbative Euclidean Wilson Loop as well as the Wilson Line correlator in Coulomb gauge.

Double seronegative myasthenia gravis with antiphospholipid syndrome: a case report

INTRODUCTION Myasthenia gravis is an autoimmune disease characterized by fluctuating muscle weakness. It is often associated with other autoimmune disorders, such as thyroid disease, rheumatoid arthritis, systemic lupus erythematosus, and antiphospholipid syndrome. Many aspects of autoimmune diseases are not completely understood, particularly when they occur in association, which suggests a common pathogenetic mechanism. CASE PRESENTATION We report a case of a 42-year-old Caucasian woman with antiphospholipid syndrome, in whom myasthenia gravis developed years later. She tested negative for both antibodies against the acetylcholine receptor and against muscle-specific receptor tyrosine-kinase, but had typical decremental responses at the repetitive nerve stimulation testing, so that a generalized myasthenia gravis was diagnosed. Her thromboplastin time and activated partial thromboplastin time were high, anticardiolipin and anti-β2 glycoprotein-I antibodies were slightly elevated, as a manifestation of the antiphospholipid syndrome. She had a good clinical response when treated with a combination of pyridostigmine, prednisone and azathioprine. CONCLUSIONS Many patients with myasthenia gravis test positive for a large variety of auto-antibodies, testifying of an immune dysregulation, and some display mild T-cell lymphopenia associated with hypergammaglobulinemia and B-cell hyper-reactivity. Both of these mechanisms could explain the occurrence of another autoimmune condition, such as antiphospholipid syndrome, but further studies are necessary to shed light on this matter.Clinicians should be aware that patients with an autoimmune diagnosis such as antiphospholipid syndrome who develop signs and neurological symptoms suggestive of myasthenia gravis are at risk and should prompt an emergent evaluation by a specialist.

Epithelial NAIPs protect against colonic tumorigenesis.

NLR family apoptosis inhibitory proteins (NAIPs) belong to both the Nod-like receptor (NLR) and the inhibitor of apoptosis (IAP) families. NAIPs are known to form an inflammasome with NLRC4, but other in vivo functions remain unexplored. Using mice deficient for all NAIP paralogs (Naip1-6(Δ/Δ)), we show that NAIPs are key regulators of colorectal tumorigenesis. Naip1-6(Δ/Δ) mice developed increased colorectal tumors, in an epithelial-intrinsic manner, in a model of colitis-associated cancer. Increased tumorigenesis, however, was not driven by an exacerbated inflammatory response. Instead, Naip1-6(Δ/Δ) mice were protected from severe colitis and displayed increased antiapoptotic and proliferation-related gene expression. Naip1-6(Δ/Δ) mice also displayed increased tumorigenesis in an inflammation-independent model of colorectal cancer. Moreover, Naip1-6(Δ/Δ) mice, but not Nlrc4-null mice, displayed hyper-activation of STAT3 and failed to activate p53 18 h after carcinogen exposure. This suggests that NAIPs protect against tumor initiation in the colon by promoting the removal of carcinogen-elicited epithelium, likely in a NLRC4 inflammasome-independent manner. Collectively, we demonstrate a novel epithelial-intrinsic function of NAIPs in protecting the colonic epithelium against tumorigenesis.

Phosphate disorders: hyperphosphatemia or pseudohyperphosphatemia?

We report the case of a 79 year old woman presenting with progressive confusion and drowsiness. Renal insufficiency with hyperkalemia as well as hypercalcemia and severe hyperphosphatemia were diagnosed. Renal insufficiency improved with treatment. However, hyperphosphatemia persisted without apparent explanation. We discuss possible causes of hyper- and pseudohyperphosphatemia. Specifically, phosphate analysis may be disturbed by the paraproteins in patients with multiple myeloma, resulting in pseudohyperphosphatemia. We review the standard laboratory phosphate measurement and the mechanisms of interference with paraproteins.

Do we need antiplatelet therapy in thrombocytosis? Pro. Diagnostic and pathophysiologic considerations for a treatment choice.

Thrombocytosis (defined as platelets >450 x 10(9)/l) has several aetiologies. After having excluded spurious thrombocytosis (e. g., due to microspherocytes, schistocytes, cryoglobulins, or bacteria), the differential diagnosis of true thrombocytosis encompasses secondary causes (as diverse as inflammation, infection, malignancy, iron deficiency, or asplenia), primary hereditary (rare forms of familial thrombocytosis) and primary acquired entities (either in the context of a myelodysplastic syndrome or more frequently a myeloproliferative neoplasia). This manuscript addresses the following aspects: 1) diagnostic approach to thrombocytosis; 2) various mechanisms leading to a high platelet count; 3) potential of some of these mechanisms to modulate platelet function, producing hyper-reactive platelets and thus exerting a direct impact on the thrombotic risk; 4) indication of anti-thrombotic treatment in patients with thrombocytosis. There is a single prospective randomized clinical trial showing the benefit of acetyl-salicylic acid in polycythaemia vera. For other types of primary thrombocytosis and for secondary forms, treatment decisions have to be individualized according to the patient thrombotic and bleeding risks, taking into account the mechanism causing thrombocytosis. This manuscript discusses experimental and clinical data suggesting that besides patients with essential thrombocythaemia and other forms of primary thrombocytosis also those with thrombocytosis in the context of chronic inflammation, malignancy, or exposure to high altitude might benefit from anti-platelet treatment.