Wnt-controlled sphingolipids modulate Anthrax Toxin Receptor palmitoylation to regulate oriented mitosis in zebrafish.

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis.

SWAN-Venule: An Optimized MRI Technique to Detect the Central Vein Sign in MS Plaques.

BACKGROUND AND PURPOSE: Multiple sclerosis lesions develop around small veins that are radiologically described as the so-called central vein sign. With 7T MR imaging and magnetic susceptibility-based sequences, the central vein sign has been observed in 80%-100% of MS lesions in patients' brains. However, a lower proportion ∼50% has been reported at 3T using susceptibility-weighted angiography (SWAN). Our aim was to assess a modified version of SWAN optimized at 3T for sensitive detection of the central vein sign. MATERIALS AND METHODS: Thirty subjects with MS were scanned on a 3T clinical MR imaging system. 3D T2-weighted FLAIR and optimized 3D SWAN called SWAN-venule, were acquired after injection of a gadolinium-based contrast agent. Patients showing >3 focal white matter lesions were included. The central vein sign was recorded by 2 trained raters on SWAN-venule images in the supratentorial brain. RESULTS: Twenty patients showing >3 white matter lesions were included. A total of 380 white matter lesions (135 periventricular, 144 deep white matter, and 101 juxtacortical) seen on both FLAIR and SWAN-venule images were analyzed. Overall, the central vein sign was detected in 86% of the white matter lesions (periventricular, 89%; deep white matter, 95%; and juxtacortical, 78%). CONCLUSIONS: The SWAN-venule technique is an optimized MR imaging sequence for highly sensitive detection of the central vein sign in MS brain lesions. This work will facilitate the validation and integration of the central vein sign to increase the diagnostic certainty of MS and further prevent misdiagnosis in clinical practice.

Disease activity impacts disability progression in primary progressive multiple sclerosis.

BACKGROUND: Although solid information on the natural history of primary progressive multiple sclerosis (PPMS) is available, evidence regarding impact of disease activity on PPMS progression remains controversial. OBJECTIVE: To describe the clinical characteristics, presence or absence of MRI activity, and natural history of a PPMS cohort from two referral centers in Argentina and assess whether clinical and/or radiological disease activity correlated with disability worsening. METHODS: Retrospective study conducted at two MS clinics in Buenos Aires, Argentina, through comparative analysis of patients with and without evidence of disease activity. RESULTS: Clinical and/or radiologic activity was presented in 56 (31%) of 178 patients. When stratified by age at onset, we found that for every 10 years of increase in age at onset, risk of reaching EDSS scores of 4 and 6 increased by 26% and 31%, respectively (EDSS 4: HR 1.26, CI 95%: 1.06-1.50; EDSS 6: HR 1.31, CI 95%: 1.06-1.62). Patients who presented clinical exacerbations reached EDSS scores of 6, 7 and 8 faster than those without associated exacerbations (p = 0.009, p = 0.016 and p = 0.001, respectively). Likewise, patients who presented gadolinium-enhancing lesions during the course of disease reached EDSS scores of 7 earlier (p = 0.002). CONCLUSION: Older age at onset and presence of clinical and/or radiological disease activity correlated with accelerated disability progression in this cohort of PPMS patients.

Multiple sclerosis and environmental factors: the role of vitamin D, parasites, and Epstein-Barr virus infection.

Pathogenic mechanisms underlying multiple sclerosis development have yet to be clearly identified, but considerable evidence indicates that autoimmunity plays an important role in the etiology of the disease. It is generally accepted that autoimmune diseases like MS arise from complex interactions between genetic susceptibility and environmental factors. Although environmental factors unequivocally influencing MS development have yet to be established, accumulating evidence singles out several candidates, including sunlight-UV exposure or vitamin D deficiency, viral infections, hygiene, and cigarette smoking. Vitamin D deficiency has been associated with different autoimmune diseases. Several investigations indicate 125 (OH)2 vitamin D plays a critical role in shaping T-cell response and inducing T cells with immunosuppressive properties. Likewise, helminth infections represent another potential environmental factor exerting immunomodulatory properties. Both epidemiological and experimental data provide evidence to support autoimmune down-regulation secondary to parasite infections in patients with MS, through regulatory T- and B-cell action, with effects extending beyond simple response to an infectious agent. Finally, different epidemiological studies have demonstrated that Epstein-Barr virus infection confers added risk of developing MS. Proposed mechanisms responsible for this association include activation and expansion of self-reactive T and B cells, lower threshold for self-tolerance breakdown, and enhanced autoreactive B-cell survival, all to be discussed in this review. Understanding environmental factors influencing propensity to MS will lead to new and more effective approaches to prevent and treat the disease.

Anthrax toxin receptor 2a controls mitotic spindle positioning.

Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis.

Fractal and nonlinear changes in the long-term baseline fluctuations of fetal heart rate.

The interpretation of heart rate patterns obtained by fetal monitoring relies on the definition of a baseline, which is considered as the running average heart rate in the absence of external stimuli during periods of fetal rest. We present a study along gestation of the baseline's fluctuations, in relation to fractal and nonlinear properties, to assess these fluctuations according with time-varying attracting levels introduced by maturing regulatory mechanisms. A low-risk pregnancy was studied weekly from the 17th to 38th week of gestation during long-term recording sessions at night (>6 h). Fetal averaged pulse rate samples and corresponding baseline series were obtained from raw abdominal ECG ambulatory data. The fractal properties of these series were evaluated by applying detrended fluctuation analysis. The baseline series were also explored to evaluate nonlinear properties and time ordering by applying the scaling magnitude and sign analyses. Our main findings are that the baseline shows fractal and even nonlinear anticorrelated fluctuations. This condition was specially the case before mid-gestation, as revealed by α values near to unit, yet becoming significantly more complex after 30 weeks of gestation as indicated by α(mag) values >0.5. The structured (i.e. not random) fluctuations and particular nonlinear changes that we found thus suggest that the baseline provides on itself information concerning the functional integration of cardiac regulatory mechanisms.

The missing link: implementation of morphogenetic growth control on the cellular and molecular level.

In the wing imaginal disc of Drosophila melanogaster, the morphogen Dpp controls growth, probably in an instructive manner. Many models for growth control by Dpp have been proposed and have been extensively discussed elsewhere. In this review, we speculate on how instructive growth control could provide a link between Dpp signaling and cell growth and/or cell cycle progression and so implement morphogenetic growth control on the cellular and molecular levels.

Dynamics of Dpp signaling and proliferation control.

Morphogens, such as Decapentaplegic (Dpp) in the fly imaginal discs, form graded concentration profiles that control patterning and growth of developing organs. In the imaginal discs, proliferative growth is homogeneous in space, posing the conundrum of how morphogen concentration gradients could control position-independent growth. To understand the mechanism of proliferation control by the Dpp gradient, we quantified Dpp concentration and signaling levels during wing disc growth. Both Dpp concentration and signaling gradients scale with tissue size during development. On average, cells divide when Dpp signaling levels have increased by 50%. Our observations are consistent with a growth control mechanism based on temporal changes of cellular morphogen signaling levels. For a scaling gradient, this mechanism generates position-independent growth rates.

Criteria and outcome of limb salvage surgery.

When sufficient margins of resection surrounding the tumor can be achieved, limb salvage surgery, as opposed to amputation, has become the standard of care in treating patients with bone and soft tissue sarcoma of the extremities. Currently, 90-95% of patients with primary malignant bone and soft-tissue tumors involving the extremities can be treated safely with wide resection and limb salvage surgery with a low risk of recurrence and the same disease-free survival rate as amputative surgery. However, discussions persist regarding the indications and criteria, and whether limb salvage provides superior functional results and quality of life for cancer patients. In this study we aimed to review and update the current criteria, indications and contraindications of limb salvage surgery and discuss its role in the quality of life of cancer patients.

Microfluidic based contactless dielectrophoretic device: Modeling and analysis.

While there have been many attempts at patterning cells onto substrates, a reliable method for trapping cell clusters and forming cell arrays in a predefined geometry remains to be demonstrated. We intend to develop a multielectrode array platform to initially trap cells via dielectrophoresis (DEP) and to later measure their electrical activity. As a first step toward that objective, here we present an interdigitated microfabricated comb structure. We designed an optimal insulation layer via finite element modeling for maximum dielectrophoretic field strength in solution and minimal cell damage. The microfabricated structure was combined with a microfluidic channel to vertically constrain cell position. With the objective of capturing cells onto the substrate, we here show that there is an optimal thickness of dielectric which limits electrolysis in solution and still allows for sufficient dielectrophoretic force on the cells to pull them onto the surface.

Provocative test with propofol: experience in patients with cerebral arteriovenous malformations who underwent neuroendovascular procedures.

BACKGROUND AND PURPOSE: Provocative testing before AVM embolization has been shown to be a predictor of a successful endovascular treatment without neurologic deficits. Propofol has been used previously as an alternative agent in Wada testing with adequate results. The purpose of this study was to show our experience with the use of propofol as a safe and effective alternative to barbiturate provocative testing in AVM embolization procedures. MATERIALS AND METHODS: A series of 20 patients, undergoing 38 embolization sessions, was treated for cerebral AVMs between November 2007 and February 2009 by endovascular methods. All patients were treated under conscious sedation. Pre-embolization neurologic assessment was performed with provocative testing by using propofol at 7-mg doses by an intra-arterial route after microcathether placement in or near the AVM nidus. RESULTS: Among these 20 patients, 3 developed transient neurologic deficits after provocative testing, precluding initial or further embolization. One of the patients passing the provocative test developed slight paresis as a result of embolization with n-BCA, resulting in a PPV of 97%. CONCLUSIONS: Propofol use during provocative testing in AVM embolization procedures represents an effective alternative to barbiturate testing and can have a positive impact in improving safety under sedation.

Quantification of growth asymmetries in developing epithelia.

Many developmental processes of multicellular organisms involve the patterning and growth of two-dimensional tissues, so called epithelia. We have quantified the growth of the wing imaginal disk, which is the precursor of the adult wing, of the fruit fly Drosophila melanogaster. We find that growth follows a simple rule with exponentially decreasing area growth rate. Anisotropies of growth can be precisely determined by comparing experimental results to a continuum theory. Growth anisotropies are to good approximation constant in space and time. They are weak in wild-type wing disks but threefold increased in GFP-Dpp disks in which the morphogen Dpp is overexpressed. Our findings indicate that morphogens such as Dpp control tissue shape via oriented cell divisions that generate anisotropic growth.

Repeatability of heart rate variability in congenital hypothyroidism as analysed by detrended fluctuation analysis.

The analysis of heart rate fluctuations, or heart rate variability (HRV), may be applied to explore children's neurodevelopment. However, previous studies have reported poor reliability (repeatability) of HRV measures in children at rest and during light exercise. Whether the reliability can be improved by controlling variables such as physical activity, breathing rate and tidal volume, or by selecting non-conventional techniques for analysing the data remains as an open question. We evaluated the short-term repeatability of RR-interval data from medicated children with congenital hypothyroidism (CH). The alpha(1) exponents, obtained by detrended fluctuation analysis (DFA), from the data of 21 children collected at two different sessions were compared. Elapsed days between sessions were 59 +/- 33, and data were obtained during 10 min, trying to restrict the children's activity while being seated. We found statistical agreement between the means of alpha(1) exponents for each session (p = 0.94) and no bias with a low-coefficient variation (9.1%); an intraclass correlation coefficient ri = 0.48 ([0.14 0.72], 95% confidence interval) was also estimated. These findings, which were compared with results obtained by conventional time and frequency techniques, indicate the existence of agreement between the alpha(1) exponents obtained at each session, thereby providing support concerning the repeatability of HRV data as analysed by DFA in children with congenital hypothyroidism. Of particular interest was also the agreement found by using the central frequency of the high-frequency band and the parameter pNN20, both showing better or similar ri than alpha(1) (0.77 [0.57 0.89] and 0.51 [0.17 0.74], respectively), yet considerably better repeatability than other conventional time and frequency parameters.

Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division.

Endocytosis has a crucial role during Notch signalling after the asymmetric division of fly sensory organ precursors (SOPs): directional signalling is mediated by differential endocytosis of the ligand Delta and the Notch effector Sanpodo in one of the SOP daughters, pIIb. Here we show a new mechanism of directional signalling on the basis of the trafficking of Delta and Notch molecules already internalized in the SOP and subsequently targeted to the other daughter cell, pIIa. Internalized Delta and Notch traffic to an endosome marked by the protein Sara. During SOP mitosis, Sara endosomes containing Notch and Delta move to the central spindle and then to pIIa. Subsequently, in pIIa (but not in pIIb) Notch appears cleaved in Sara endosomes in a gamma-secretase- and Delta internalization-dependent manner, indicating that the release of the intracellular Notch tail to activate Notch target genes has occurred. We thus uncover a new mechanism to bias signalling even before asymmetric endocytosis of Sanpodo and Delta takes place in the daughter cells: already during SOP mitosis, asymmetric targeting of Delta and Notch-containing Sara endosomes will increase Notch signalling in pIIa and decrease it in pIIb.

Association between polymorphisms of folate-metabolizing enzymes and risk of prostate cancer.

Polymorphisms of the genes 5'-10'-methylenetetrahydrofolate reductase (MTHFR, 677CT and 1298AC), methionine synthase (MTR, 2756AC) and methionine synthase reductase (MTRR, 66AC) provoke variations in enzyme activity, which can lead to alterations in the metabolism of folates and in the synthesis of S-adenosyl-methionine (SAM), the most active methyl donor in the body. This could play an important role in carcinogenesis through the degree of DNA methylation and of nucleotide synthesis. In the present study, four polymorphisms were studied, two of the methylenetetrahydrofolate reductase gene, and the other two of methionine synthase and methionine synthase reductase. Our aim was to study the association between prostate carcinoma susceptibility and these polymorphisms. A hospital-based case-control study was conducted in 182 patients (mean age: 70.7+/-7.29 years) with histologically confirmed prostate carcinoma and in 205 control subjects (mean age: 70.3+/-7.82 years) diagnosed with benign prostatic hyperplasia (BPH). Genomic DNA was extracted from peripheral leukocytes. Comparison of the MTHFR CT and TT genotypes in patients and the controls revealed significant differences (0.57 vs 0.38) (OR: 2.19, 95% CI: 1.46-3.30) and (0.06 vs 0.15) (OR: 0.36, 95% CI: 0.17-0.73), respectively. No statistically significant differences were found between patients and controls with respect to the MTHFR 1298AC, the MTR 2756AC and the MTRR 66AC polymorphisms. However, among the patients, the MTR 2756 allele C was related to a high Gleason score. We conclude that the polymorphism MTHFR C677T is clearly related to prostatic carcinogenesis, on the contrary to the other polymorphisms studied, although the MTR 2756 allele C acts as a factor of tumor aggressiveness, this being found in tumors with high carcinogenic potential.

Genotypes of the C677T and A1298C polymorphisms of the MTHFR gene as a cause of human spontaneous embryo loss.

BACKGROUND Polymorphisms C677T and A1298C of the MTHFR gene have been implicated in fetal viability. In this study, we determined the allele and genotype frequencies of these polymorphisms in different populations, including spontaneous abortion (SA) fetal tissues, with the objective of evaluating their impact on fetal viability. METHODS 342 samples of fetal tissues, selected from SA occurring during the 1980s, 230 samples from subjects born in the 1980s and a third set of samples from 204 subjects born in the 1950s, were genotyped by using TaqMan probes. RESULTS The wild CC genotype of the C677T polymorphism showed a strong protective effect against abortion (0.03 in SA versus 0.47 in 1950s and 0.43 in 1980s) (P < 0.0001). Genotypes of three mutations in the combinations of polymorphisms for C677T and A1298C showed a very low frequency in the living population; however, the three mutations genotypes were over expressed in the SA group (0.02 in 1950s; 0.03 in 1980s and 0.17 in SA) (P < 0.0001). Samples with four mutations (n = 2) were found only in the SA group. CONCLUSIONS There is no linkage disequilibrium between C667T and A1298C polymorphisms. Fetal viability is directly related to the CC genotype as a protector while the three and four mutation MTHFR genotypes appear to be a determinant on fetal non-viability and SA.

Morphogen transport in epithelia.

We present a general theoretical framework to discuss mechanisms of morphogen transport and gradient formation in a cell layer. Trafficking events on the cellular scale lead to transport on larger scales. We discuss in particular the case of transcytosis where morphogens undergo repeated rounds of internalization into cells and recycling. Based on a description on the cellular scale, we derive effective nonlinear transport equations in one and two dimensions which are valid on larger scales. We derive analytic expressions for the concentration dependence of the effective diffusion coefficient and the effective degradation rate. We discuss the effects of a directional bias on morphogen transport and those of the coupling of the morphogen and receptor kinetics. Furthermore, we discuss general properties of cellular transport processes such as the robustness of gradients and relate our results to recent experiments on the morphogen Decapentaplegic (Dpp) that acts in the wing disk of the fruit fly Drosophila.

Robust formation of morphogen gradients.

We discuss the formation of graded morphogen profiles in a cell layer by nonlinear transport phenomena, important for patterning developing organisms. We focus on a process termed transcytosis, where morphogen transport results from the binding of ligands to receptors on the cell surface, incorporation into the cell, and subsequent externalization. Starting from a microscopic model, we derive effective transport equations. We show that, in contrast to morphogen transport by extracellular diffusion, transcytosis leads to robust ligand profiles which are insensitive to the rate of ligand production.

Differential transcriptional expresión of the polymorphic myxovirus resistance protein A in response to interferon-alpha treatment.

Levels of myxovirus resistance protein A (MxA) mRNA were studied for a single nucleotide polymorphism in the promoter region at nucleotide position -88 of the gene to identify individual-specific responses to interferon (IFN)-alpha2 that might predict responsiveness to IFN-alpha therapy. We quantified MxA expression by reverse transcription and real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMC) in vitro, induced by IFN-alpha2, from 22 healthy donors, in relation with G/T polymorphism located in the promoter of the MxA. MxA mRNA was significantly upregulated in all subjects (mean of 53-fold) in response to IFN-alpha2 in vitro (P < 0.01). Comparison of the inducibility of MxA mRNA expression in relation with G/T polymorphism showed a 4.26-fold higher induction of MxA mRNA levels in PBMC from carriers of the mutant allele (GT or TT) than homozygotes with the wild-type allele (GG) (P < 0.001). We propose that expression of the IFN-inducible MxA is affected by a single nucleotide polymorphism in the MxA promoter which can identify an individual response to IFN-alpha2.