Deniz Yüksel

Mutations in CLN7/MFSD8 are a common cause of variant late-infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs), the most common neurodegenerative disorders of childhood, are characterized by the accumulation of autofluorescent storage material mainly in neurons. Although clinically rather uniform, variant late-infantile onset NCL (vLINCL) is genetically heterogeneous with four major underlying genes identified so far. We evaluated the genetic background underlying vLINCL in 119 patients, and specifically analysed the recently reported CLN7/MFSD8 gene for mutations in 80 patients. Clinical data were collected from the CLN7/MFSD8 mutation positive patients. Eight novel CLN7/MFSD8 mutations and seven novel mutations in the CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8 genes were identified in patients of various ethnic origins. A significant group of Roma patients originating from the former Czechoslovakia was shown to bear the c.881C>A (p.Thr294Lys) mutation in CLN7/MFSD8, possibly due to a founder effect. With one exception, the CLN7/MFSD8 mutation positive patients present a phenotype indistinguishable from the other vLINCL forms. In one patient with an in-frame amino acid substitution mutation in CLN7/MFSD8, the disease onset was later and the disease course less aggressive than in variant late-infantile NCL. Our findings raise the total number of CLN7/MFSD8 mutations to 14 with the majority of families having private mutations. Our study confirms that CLN7/MFSD8 defects are not restricted to the Turkish population, as initially anticipated, but are a relatively common cause of NCL in different populations. CLN7/MFSD8 should be considered a diagnostic alternative not only in variant late-infantile but also later onset NCL forms with a more protracted disease course. A significant number of NCL patients in Turkey exist, in which the underlying genetic defect remains to be determined.

Nogo-A is a negative regulator of CNS angiogenesis

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A–specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti–Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

The effect of the baby-friendly hospital initiative on long-term breast feeding

The aim of this study was to evaluate the effects of ‘baby‐friendly hospital initiative’ (BFHI) on breast feeding.In the four consecutive months after BFHI in Gazi University Hospital (November 2002–February 2003), breast feeding status until the second year of life in 297 babies, born in the same hospital was compared with the values of 258 babies born before BFHI (November 2001–February 2002).The exclusive breast feeding rate in the first 6 months was higher in the babies born after BFHI. Cox regression analysis revealed that BFHI increases the duration of breast feeding 1.5 times. At the end of the second year, cumulative rate of breast feeding was higher in the group after‐BFHI (p = 0.0036).The rate of breast feeding was increased by BFHI implementation.

Peptide Tertiary Structure Nucleation by Side-Chain Crosslinking with Metal Complexation and Double “Click” Cycloaddition

Stabilized helical peptides show potential as therapeutics capable of modulating protein function through enhanced helix-protein binding.[1] Helix turn stabilization may be accomplished though covalent sidechain crosslinking on one face of the helix, in i and i+4 or i+7 positions,[2–4] or replacement of a main chain hydrogen bond with a covalent linker.[3, 5, 6] We report herein a new helix-nucleating[7] i and i+4 crosslinking strategy based on copper catalyzed azide-alkyne [3+2] “click” cycloaddition[8] and demonstrate the ability of this method and metal complexation to restore coiled-coil dimerization in a folding-incompetent sequence crippled by 2 helix-breaking glycine residues. Elegant applications of azide-alkyne cycloaddition chemistry in peptide conjugation[9] and structure stabilization[10] are known in the literature; this design strategy complements known intramolecular methods for structure nucleation such as ring closing metathesis[2, 5, 11] (RCM) and allows convergent installation of functional groups pendant to the bis-alkyne linker that could be used to modulate peptide binding, targeting, and membrane permeability. We studied 21 residue sequences derived from the GCN4 leucine zipper[12] in which the central heptad contains glycine residues in the c and e helix positions and crosslinking residues (X) in the b and f (i and i+4) positions, leaving the hydrophobic core residues in the a and d positions intact as isoleucine and leucine, respectively (Figure 1). Metal complexation with i and i+4 X=His residues is known to induce monomeric helix folding,[4, 13] though it has not been previously demonstrated to restore structure in peptides containing 2 glycine residues. We postulated that the bis-triazole product of a double [3+2] cycloaddition between i and i+4 azidoalanine[14] (Az) residues and a bis alkyne could yield a non-labile covalent linker isosteric with i and i+4 His-His metal complex (Figure 1A). We prepared peptides 1 and 2 using standard Fmoc solid phase synthesis which may be crosslinked in the central heptad by Ni2+ complexation with histidine and bis-alkyne cyclization with Az, respectively (Figure 1B). Indeed, the circular dichroism (CD) spectrum of 1 exhibits a saturable increase in helicity upon treatment with NiCl2, which may be reversed with EDTA treatment (Figure 3). While the free peptide is a completely unfolded monomer, the nickel-complexed peptide melts cooperatively with a Tm of 46 °C, and is found by analytical ultracentrifugation (AUC) to be dimeric (Figure 1D), indicating the ability of sidechain crosslinking to restore tertiary structure even in sequences containing 2 centrally placed glycine residues. Figure 1 (A) Strategies for helix structure nucleation with i and i+4 crosslinking using metal complexation (top) or double click cycloaddition (bottom). aDi-azidoalanine peptide was treated in aqueous buffer with bis alkynes 6–9, sodium ascorbate, CuSO ... Figure 3 (A) Nickel dependent folding of 1, from 0 NiCl2 (ο) to 100 μM NiCl2 (•), with intermediate concentrations shown in black. (B) Binding isotherms of NiCl2 to peptide 1 (•) and peptide 9a (ο), fit to a 1:1 binding ... We sought to use copper catalyzed “click” chemistry to similarly induce secondary[15] and tertiary structure by linking azidoalanines in peptide 2 with bis alkyne linkers 6–9. These intermolecular cyclizations were successful with both resin-bound protected peptide in DMF using CuI/DIEA and in aqueous buffer using a copper (I) catalyst[16] (Figure 1A). Click adducts were subjected to Staudinger reduction conditions with PPh3 (on resin) or TCEP (in aqueous solution) to confirm ring closure; while starting material 2 reacted with phosphine to yield reduction and other products, all adducts remained unchanged, consistent with conversion of azide to triazole. Additionally, MS-MS peptide fragmentation patterns were indicative of cyclic “double-click” products (Figure 2). Fragmentation of the bisalkyne treated diazido peptides yielded all possible peptide bond mass fragments except for those putatively joined by a bis-triazole linker, which should not fragment under these conditions. The identical experiment with the click adduct to a monoazide peptide 3, in which the C-terminal Az residue was replaced with serine yielded all possible peptide mass fragments. Notably, the observed ratio of ion intensities of the 4+ parent ion relative to the common mass fragment y1 is much higher in the spectrum of cycloadduct of 2 (Figure 2B) relative to the cycloadduct of 3. This is consistent with double cycloaddition to 2 that results in the coalescence of ion intensities of 4 possible distinct mass fragments into a single, more intense peak. Figure 2 Representative MS-MS data indicating fragmentation at the peptide bonds of the adduct of linker 6 to (A) peptide 3 and (B) peptide 2 (6a). Arrows indicate diagnostic C-terminal y-fragment ions, N-terminal b-fragment ions and g-parent ions (4+). Interestingly, though linkers were used in large excess on-resin, the bis-adduct was never observed and diazide 2 could be near quantitatively converted on resin to the double cycloadduct (determined as described above), suggesting that the second click reaction to close the ring is much faster than the first, despite the 23 or 24-atom ring closure required. Similarly efficient peptide ring closures have been observed using RCM, suggesting that the peptide backbone may greatly reduce the entropic cost of cyclization through templating effects or hindered amide bond rotations.[2, 11] Cyclization in aqueous buffer also yielded desired product in acceptable (40–50%) isolated yield, though bis-adducts were observed when linker was used in moderate excess; the more rigid unsaturated linkers 7 and 8 gave more bis-adducts relative to linkers 6 and 9, which have single bonds connecting the 2 alkynes. The functional effect of ring-closure via double cycloaddition was obtained by comparing the folding properties of monoazide peptides in which one AzAla residue was replaced by serine. These negative control peptides are also readily coupled with 6–9, but remain unfolded under all conditions, like diazido starting material 2. (Figure 3). Commercially available 1,5-hexadiyne (linker 6) induced the most significant change in helicity upon double cycloaddition, nucleating helical structure in the central heptad with sufficient efficiency to restore dimerization to the folding-crippled 2. Like the nickel complex of 1, the 1,5-hexadiyne adduct peptide (6a) melts cooperatively (Tm=40°C) but dimerizes independently of nickel concentration (Figure 3). Linkers 7–8 were used to probe the steric requirements of side chain constraint. When the ethyl spacer of 1,5-hexadiyne 6 is replaced by the rigid 2 carbon linker of the ortho-diethynylbenzoic acid linker 7, the folded state is not favored. The meta-diethynylbenzoic acid 8 and di-propargylated glycine linker, 9 both extend the chain by 1 atom, with a rigid unsaturated linker and more flexible saturated linker, respectively. The cycloadducts of diazidopeptide 2 with 7 (7a) and 9 (9a) exhibit random coil signatures while 8a is insoluble in aqueous solvent. Interestingly, treatment of 9a with NiCl2 increases the helicity of the system, suggesting the formation of a new nickel binding site, presumably composed of the triazoles, tertiary amine, and carboxylate from the ligated linker. Nickel concentration dependence of ellipticity indicated tighter nickel binding by adduct 9a (Kd=10−6 M) than di-histidine peptide 1 (Kd=10−4 M), consistent with a more organized binding site in cycloadduct relative to the open chain peptide 1. Indeed, the double cycloadduct of propargyl alcohol with 2 installs the two triazole moieties in a non-cyclic product and this peptide remains unfolded even in the presence of nickel chloride, indicating the importance of cyclization (Supporting Information). However, AUC measurements indicated a monomer-dimer equilibrium for 9a under all conditions, suggesting that metal complexation does not completely restore dimerization. This result stands in constrast to the enhanced helicity and dimerization found upon crosslinking with linker 6 to obtain 6a. It is possible that the additional atom in linker 9 actually prevents the formation of a helical turn, and metal binding induces a contraction of the cyclic binding site that allows partial, but incomplete folding and dimerization. Weak dimerization and partially helical signature in 9a suggests that, similar to the parent “uncrippled” coiled-coils, helix folding and oligomerization in these systems is coupled.[17] Peptides 4 and 5 are dimerization-inhibited by charged residues at the dimerization interface or scrambled coiled-coil sequences, respectively. These soluble peptides (X=Az or His) retained random coil CD signatures upon metal complexation with NiCl2 and were insoluble upon cycloaddition, consistent with coupled folding and assembly; this is likely accentuated by the presence of 2 helix-breaking glycines in the sequence. We have thus demonstrated a new methodology for helix structural nucleation using double azide-alkyne [3+2] cycloadditions to form i and i+4 constrained peptide sequences. This chemistry is sufficiently robust to allow intermolecular cyclization with few sideproducts, setting the stage for convergent functionalization and structure nucleation. This technology may be useful as a means of synthesizing new building blocks for peptide-based materials[18] or preparing stabilized helical peptides capable of binding protein interfaces and altering biological function. These studies are currently underway in our lab.

Model system for cell adhesion mediated by weak carbohydrate-carbohydrate interactions.

The multivalent carbohydrate-carbohydrate interaction between membrane-anchored epitopes derived from the marine sponge Microciona prolifera has been explored by colloidal probe microscopy. An in situ coupling of sulfated and non-sulfated disaccharides to membrane-coated surfaces was employed to mimic native cell-cell contacts.The dynamic strength of the homomeric self-association was measured as a function of calcium ions and loading rate. A deterministic model was used to estimate the basic energy landscape and number of participating bonds in the contact zone.

Prevalence of some risk factors in children with epilepsy compared to their controls

AIM The goal of this case-control study was to identify the significance of certain risk factors for epilepsy in Turkey. METHOD A total of 805 cases, aged 1-16 years, followed-up for epilepsy at the Pediatric Neurology Department and a control group consisting of 846 age-matched cases without epilepsy were included in the study. The risk factors examined were gender, neurological impairment, febrile convulsion, head trauma, central nervous system infections, parental consanguinity, family history of epilepsy, prenatal and natal risk and newborn jaundice. Data regarding the investigated epilepsy risk factors were obtained through a questionnaire via personal interviews and the medical records and were assessed using univariate and multivariate analysis. RESULT Univariate analysis showed an increased risk for epilepsy with a history of atypical febrile seizure (21.97-fold), severe and moderate head injury (27.76- and 7.09-fold respectively), CNS infection (4.76-fold), history of epilepsy in first-, second- or third-degree relatives (6.42-, 3.09- and 2.66-fold, respectively), presence of maternal hypertension (4.31-fold), an apgar score < or =6 at any time (7.78-fold) and neonatal jaundice (3.12-fold). Abnormal neurological signs increased the epilepsy risk 5.92 times in univariate analysis and 30.26 times in multivariate analysis. CONCLUSION The most important risk factors for epilepsy in this study were neurological impairment, history of atypical febrile seizures, severe head injury and a low apgar score. Other important risk factors were moderate head trauma and a history of epilepsy in the family.

Devic's neuromyelitis optica in an infant case.

Devics neuromyelitis optica was orginally described as an acute severe monophasic syndrome characterised by myelitis and optic neuritis. The mean age at onset was reported to be around 40 years, with a wide range. However, Devics neuromyelitis optica has also been seen in children. Prognosis of the syndrome was poor, and no satisfactory treatment was known. This article reports a 23-month-old boy with acute myelitis and optic neuritis who was diagnosed with Devics neuromyelitis optica. The response of the patient to therapy was poor, and he developed severe sequelae.

Tau proteins in the cerebrospinal fluid of patients with subacute sclerosing panencephalitis

Neurodegenerative diseases characterized by cytoskeletal deformation and neurofibrillary tangles are associated with altered levels of tau and related proteins in cerebrospinal fluid (CSF). Neuronal or glial fibrillary tangles have been shown in 20% of subacute sclerosing panencephalitis (SSPE) patients. We therefore investigated CSF samples from 60 newly diagnosed SSPE and 31 neurological control patients for total tau (t-tau), phosphorylated tau (p-tau), and S100-B levels by ELISA. There was no difference between patient and control groups in t-tau and S100-B levels. p-Tau was lower in the SSPE group (p=0.009). Past history of measles infection, measles immunization status, latent period between measles and onset of SSPE, duration of symptoms, frequency of myoclonia, neurological deficit index, stage and progression rate of the disease, CSF glucose levels and cell counts, CSF and serum measles IgG titer, distribution of lesions on brain magnetic resonance imaging were not related to t-tau, p-tau and S100-B levels. Mental status and age were negatively correlated with t-tau, and male gender and EEG abnormalities were associated with higher t-tau levels. The levels of tau proteins in our patients suggest there is no, or only scarce and immature, neurofibrillary tangle formation in SSPE. Autopsy studies showing neurofibrillary tangles might have examined older patients with longer disease and more parenchymal involvement.

SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis

Background: Fibronectin matrix assembly is mediated by cell traction at the RGD loop of 10FNIII, which is predicted to unravel β-strands A and B. Results: Sequence SLLISWD from strand B initiates fibronectin multimerization. Conclusion: SLLISWD mediates cell-mediated fibronectin fibril assembly. Significance: This matrix motif provides insight into physiological fibronectin fibrillogenesis with utility in initiating matrix assembly for tissue repair. Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B β-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid “multimerization sequence” (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and β-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated β-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.

Prothrombotic risk factors in children with hemiplegic cerebral palsy

Background: The purpose of the present paper was to investigate the prevalence of prothrombotic risk factors associated with hemiplegic cerebral palsy (CP).