Zinc-finger and helix-loop-helix transcription factors regulate Purkinje neuron neurogenesis and cerebellar corticogenesis

Many regulatory genes have been pinpointed as orchestrators of cerebellar development, from the onset of neurogenesis to the patterning of the adult cerebellar cortex, with a special reference to the development of cerebellar Purkinje cells (PCs). PCs provide the sole output from cerebellar cortical circuits, where each PC integrates myriads of presynaptic inputs, both inhibitory and excitatory. In the murine cerebellar primordium PCs are generated from a pool of ventricular zone progenitors facing the fourth ventricle between embryonic day (E) 10.5 and 13.5. This progenitor pool expands in the ventricular zone (VZ) through symmetric cell division until E10.5, when a gradual switch to asymmetric cell division occurs, regulated by Notch1 (Lutolf et al., 2002) and its interactor (Masserdotti et al., 2010), the Zn-finger TF Zfp423 (Alcaraz et al., 2006; Warming et al., 2006; Croci et al., submitted). Zfp423 was recently implicated in Joubert syndrome and cerebellar vermis hypoplasia (Chaki et al., 2012). Allelic mutations of Zfp423 produce distinct alterations in PC development (Croci et al., submitted). PCs arise from a pool of progenitors positive for the basic-helix-loop-helix transcription factors (TFs) neurogenin (Ngn) 1 and 2 (Zordan et al., 2008; Lundell et al., 2009). Ngn2 regulates cell cycle progression and dendritic arbor generation in PC precursors (Florio et al., 2012). PCs also express HLH transcription factors of the Olf/EBF family. In Ebf2 -/- mutants, PC migration and survival are affected (Croci et al., 2006). Neonatal PC death is due to local downregulation of Igf1 gene expression (Croci et al., 2011). Finally, EBF2 regulates cortical patterning in the adult cerebellum, regulating its subdivision into alternate parasagittal stripes of distinct PC subtypes. Indeed, EBF2 is required to repress the zebrin II+ phenotype in postnatal PCs (Croci et al., 2006; Chung et al., 2008)

Domain-specific regulation of cerebellar morphogenesis by Zfp423 / ZNF423, a gene implicated in Joubert syndrome and cerebellar vermis hypoplasia

The Zfp423 gene encodes a 30-Zn-finger transcription factor that acts as a scaffold for the assembly of complex transcriptional and cellular machineries regulating neural development. While null Zfp423 mutants feature a sharp decrease in the total number of cerebellar Purkinje cells (PCs), the underlying mechanisms remain unclear. Mutations of the human homolog ZNF423 have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), associated with other signs of classical ciliopathy outside the central nervous system. To further characterize the role of ZFP423 in cerebellar neurogenesis, we have performed morphological, cellular and molecular studies on two mutant mouse lines carrying allelic in-frame deletions of Zfp423. While both lines exhibit cerebellar hypoplasia, considerable differences are observed between the two mutants, with respect to neural progenitor differentiation, cell survival and morphogenesis. The results of this in vivo and in vitro structure-function analysis point to domain- and context-specific roles played by ZFP423 in different aspects of cerebellar development, and contribute to our understanding of its role as a disease / modifier gene in JS, CVH and other ciliopathies

High-sensitivity visualisation of contaminants in heparin samples by spectral filtering of H-1 NMR spectra

A novel application of two-dimensional correlation analysis has been employed to filter H-1 NMR heparin spectra distinguishing acceptable natural variation and the presence of foreign species. Analysis of contaminated heparin samples, compared to a dataset of accepted heparin samples using two-dimensional correlation spectroscopic analysis of their 1-dimensional H-1 NMR spectra, allowed the spectral features of contaminants to be recovered with high sensitivity, without having to resort to more complicated NMR experiments. Contaminants, which exhibited features distinct from those of heparin and those with features normally hidden within the spectral mass of heparin could be distinguished readily. A heparin sample which had been pre-mixed with a known contaminant, oversulfated chondroitin sulfate (OSCS), was tested against the heparin reference library. It was possible to recover the 1 H NMR spectrum of the OSCS component through difference 2D-COS power spectrum analysis of as little as 0.25% (w/w) with ease, and of 2% (w/w) for more challenging contaminants, whose NMR signals fell under those of heparin. the approach shows great promise for the quality control of heparin and provides the basis for greatly improved regulatory control for the analysis of heparin, as well as other intrinsically heterogeneous and varied products.Wellcome TrustRoyal SocietyBBSRCFinlambardia SPA 'Fondo per la promozione di Accordi Istituzionali'Univ Liverpool, Sch Biol Sci, Liverpool L69 3BX, Merseyside, EnglandIst Ric Chim & Biochim G Ronzoni, I-20133 Milan, ItalyUNIFESP Universidade Federal de São Paulo, Dept Bioquim, Disciplina Biol Mol, BR-04044020 São Paulo, BrazilKeele Univ, Inst Sci & Technol Med, Keele ST5 5BG, Staffs, EnglandNatl Inst Biol Stand & Controls, Potters Bar EN6 3QG, Herts, EnglandUNIFESP Universidade Federal de São Paulo, Dept Bioquim, Disciplina Biol Mol, BR-04044020 São Paulo, BrazilWeb of Scienc

Unravelling Structural Information from Complex Mixtures Utilizing Correlation Spectroscopy Applied to HSQC Spectra

The first use of <i>statistical</i> correlation spectroscopy to extract chemical information from 2D-HSQC spectra, termed HSQC correlation spectroscopy (<b>HSQCcos</b>), is reported. <b>HSQCcos</b> is illustrated using heparin, a heterogeneous polysaccharide, whose diverse composition causes signals in HSQC spectra to disperse. <b>HSQCcos</b> has been used to probe the chain modifications that cause this effect and reveals hitherto unreported structural details. An interesting finding was that the signal for position 2 of trisulfated glucosamine [N-, 3-O-, and 6-O-sulfated] (<b>A</b>*) is bifurcated, owing to the presence of <b>A</b>* residues in both the “normal” antithrombin binding site and also at the nonreducing end of the molecule, which is reported in intact heparin for the first time. The method was also applied to investigating the environment around other rare sequences/disaccharides, suggesting that the disaccharide; 2-O-sulfated iduronic acid linked to 6-O-sulfated N-glucosamine, which contains a free amine at position 2, is adjacent to the heparin linkage region. <b>HSQCcos</b> can extract chemically related signals from information-rich spectra obtained from complex mixtures such as heparin

Structure-Based Design of a B Cell Antigen from <i>B. pseudomallei</i>

<i>Burkholderia pseudomallei</i> is the etiological agent of melioidosis, a severe endemic disease in South-East Asia, causing septicemia and organ failure with high mortality rates. Current treatments and diagnostic approaches are largely ineffective. The development of new diagnostic tools and vaccines toward effective therapeutic opportunities against <i>B. pseudomallei</i> is therefore an urgent priority. In the framework of a multidisciplinary project tackling melioidosis through reverse and structural vaccinology, BPSL1050 was identified as a candidate for immunodiagnostic and vaccine development based on its reactivity against the sera of melioidosis patients. We determined its NMR solution structure and dynamics, and by novel computational methods we predicted immunogenic epitopes that once synthesized were able to elicit the production of antibodies inducing the agglutination of the bacterium and recognizing both BPSL1050 and <i>B. pseudomallei</i> crude extracts. Overall, these results hold promise for novel chemical biology approaches in the discovery of new diagnostic and prophylactic tools against melioidosis

Benchmarking postoperative outcomes after open liver surgery for cirrhotic patients with hepatocellular carcinoma in a national cohort

Background: Benchmark analysis for open liver surgery for cirrhotic patients with hepatocellular carcinoma (HCC) is still undefined. Methods: Patients were identified from the Italian national registry HE.RC.O.LE.S. The Achievable Benchmark of Care(ABC) method was employed to identify the benchmarks. The outcomes assessed were the rate of complications, major comorbidities, post-operative ascites(POA), post-hepatectomy liver failure(PHLF), 90-day mortality, rate of R0 and the length of stay. Benchmarking was stratified for surgical complexity(CP1, CP2 and CP3). Results: A total of 978 of 2698 patients fulfilled the inclusion criteria. 431(44.1%) patients were treated with CP1 procedures, 239(24.4%) with CP2 and 308(31.5%) with CP3 procedures. Patients submitted to CP1 had a worse underlying liver function, while the tumor burden was more severe in CP3 cases. The ABC for complications(13.1%, 19.2% and 28.1% for CP1, CP2 and CP3 respectively), major complications(7.6%, 11.1%, 12.5%) and 90-day mortality (0%, 3.3%, 3.6%) increased with the surgical difficulty, but not POA (4.4%, 3.3% and 2.6% respectively) and PHLF (0% for all groups). Conclusions: We propose benchmarks for open liver resections in HCC cirrhotic patients, stratified for surgical complexity. The difference between the benchmark values and the results obtained during everyday practice reflects the room for potential growth, with the aim to encourage constant improvement among liver surgeons