Perinatal loss of galactosylceramidase in both oligodendrocytes and microglia is crucial for the pathogenesis of Krabbe disease in mice.

Lysosomal galactosylceramidase (GALC) is expressed in all brain cells, including oligodendrocytes (OLs), microglia, and astrocytes, although the cell-specific function of GALC is largely unknown. Mutations in GALC cause Krabbe disease (KD), a fatal neurological lysosomal disorder that usually affects infants. To study how Galc ablation in each glial cell type contributes to Krabbe pathogenesis, we used conditional Galc-floxed mice. Here, we found that OL-specific Galc conditional knockout (CKO) in mice results in a phenotype that includes wasting, psychosine accumulation, and neuroinflammation. Microglia- or astrocyte-specific Galc deletion alone in mice did not show specific phenotypes. Interestingly, mice with CKO of Galc from both OLs and microglia have a more severe neuroinflammation with an increase in globoid cell accumulation than OL-specific CKO alone. Moreover, the enhanced phenotype occurred without additional accumulation of psychosine. Further studies revealed that Galc knockout (Galc-KO) microglia cocultured with Galc-KO OLs elicits globoid cell formation and the overexpression of osteopontin and monocyte chemoattractant protein-1, both proteins that are known to recruit immune cells and promote engulfment of debris and damaged cells. We conclude that OLs are the primary cells that initiate KD with an elevated psychosine level and microglia are required for the progression of neuroinflammation in a psychosine-independent manner.

Disease correction in mucopolysaccharidosis type IIIB mice by intraparenchymal or cisternal delivery of a capsid modified AAV8 codon-optimized NAGLU vector.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene that encodes the protein N-acetyl-glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes leading to progressive central nervous system (CNS) degeneration. Intravenous treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry into the CNS. Additionally, we have demonstrated that AAV8 provides a broader transduction area in the MPS IIIB mouse brain compared with AAV5, 9 or rh10. A triple-capsid mutant (tcm) modification of AAV8 further enhanced GFP reporter expression and distribution. Using the MPS IIIB mouse model, we performed a study using either intracranial six site or intracisterna magna injection of AAVtcm8-codon-optimized (co)-NAGLU using untreated MPS IIIB mice as controls to assess disease correction. Disease correction was evaluated based on enzyme activity, heparan sulfate storage levels, CNS lysosomal signal intensity, coordination, activity level, hearing and survival. Both histologic and enzymatic assessments show that each injection method results in supranormal levels of NAGLU expression in the brain. In this study, we have shown correction of lifespan and auditory deficits, increased CNS NAGLU activity and reduced lysosomal storage levels of heparan sulfate following AAVtcm8-coNAGLU administration and partial correction of NAGLU activity in several peripheral organs in the murine model of MPS IIIB.

Liquid Chromatography-Tandem Mass Spectrometry in Newborn Screening Laboratories.

Tandem mass spectrometry (MS/MS) is the most universal platform currently available for the analysis of enzymatic activities and biomarkers in dried blood spots (DBS) for applications in newborn screening (NBS). Among the MS/MS applications in NBS, the most common is flow-injection analysis (FIA-) MS/MS, where the sample is introduced as a bolus injection into the mass spectrometer without the prior fractionation of analytes. Liquid chromatography combined with MS/MS (LC-MS/MS) has been employed for second-tier tests to reduce the false-positive rate associated with several nonspecific screening markers, beginning two decades ago. More recently, LC-MS/MS has been applied to primary screening for new conditions for which FIA-MS/MS or other methods, including genomic screening, are not yet adequate. In addition to providing a list of the currently used LC-MS/MS-based assays for NBS, the authors share their experience regarding the maintenance requirements of LC-MS/MS vs. FIA-MS/MS systems. The consensus is that the maintenance of LC-MS/MS and FIA-MS/MS instrumentation is similar, and LC-MS/MS has the advantage of allowing for a larger number of diseases to be screened for in a multiplex, cost-effective fashion with a high throughput and an adequate turnaround time.

In Utero Enzyme-Replacement Therapy for Infantile-Onset Pompe's Disease.

Patients with early-onset lysosomal storage diseases are ideal candidates for prenatal therapy because organ damage starts in utero. We report the safety and efficacy results of in utero enzyme-replacement therapy (ERT) in a fetus with CRIM (cross-reactive immunologic material)-negative infantile-onset Pompe's disease. The family history was positive for infantile-onset Pompe's disease with cardiomyopathy in two previously affected deceased siblings. After receiving in utero ERT and standard postnatal therapy, the current patient had normal cardiac and age-appropriate motor function postnatally, was meeting developmental milestones, had normal biomarker levels, and was feeding and growing well at 13 months of age.

A rapid and non-invasive proteomic analysis using DBS and buccal swab for multiplexed second-tier screening of Pompe disease and Mucopolysaccharidosis type I.

PURPOSE: Current newborn screening programs for Pompe disease (PD) and mucopolysaccharidosis type I (MPS I) suffer from a high false positive rate and long turnaround time for clinical follow up. This study aimed to develop a novel proteomics-based assay for rapid and accurate second-tier screening of PD and MPS I. A fast turnaround assay would enable the identification of severe cases who need immediate clinical follow up and treatment. METHODS: We developed an immunocapture coupled with mass spectrometry-based proteomics (Immuno-SRM) assay to quantify GAA and IDUA proteins in dried blood spots (DBS) and buccal swabs. Sensitivity, linearity, reproducibility, and protein concentration range in healthy control samples were determined. Clinical performance was evaluated in known PD and MPS I patients as well as pseudodeficiency and carrier cases. RESULTS: Using three 3.2 mm punches (~13.1 µL of blood) of DBS, the assay showed reproducible and sensitive quantification of GAA and IDUA. Both proteins can also be quantified in buccal swabs with high reproducibility and sensitivity. Infantile onset Pompe disease (IOPD) and severe MPS I cases are readily identifiable due to the absence of GAA and IDUA, respectively. In addition, late onset Pompe disease (LOPD) and attenuated MPS I patients showed much reduced levels of the target protein. By contrast, pseudodeficiency and carrier cases exhibited significant higher target protein levels compared to true patients. CONCLUSION: Direct quantification of endogenous GAA and IDUA peptides in DBS by Immuno-SRM can be used for second-tier screening to rapidly identify severe PD and MPS I patients with a turnaround time of <1 week. Such patients could benefit from immediate clinical follow up and possibly earlier treatment.

Detection of GM1-gangliosidosis in newborn dried blood spots by enzyme activity and biomarker assays using tandem mass spectrometry.

GM1-gangliosidosis is a rare autosomal recessive lysosomal storage disease caused by deficiency of ß-galactosidase (GLB1). Newborn screening (NBS) may be warranted in the near future given the initiation of a number of gene therapy clinical trials. Here, we report a tandem mass spectrometry (MS/MS) enzymatic assay of GLB1 using dried blood spots (DBS), and the demonstration that GLB1 activities in newborn DBS from seven GM1-gangliosidosis patients are well below those measured in random newborn DBS. MS/MS analysis of two glycan biomarkers, dp5 and A2G2, shows high elevation in newborn DBS from GM1-gangliosidosis compared to the levels in the nonaffected reference range.

Tandem Mass Spectrometry Enzyme Assays for Multiplex Detection of 10-Mucopolysaccharidoses in Dried Blood Spots and Fibroblasts.

The mucopolysaccharidoses (MPSs) are a class of inborn errors of metabolism caused by deficiency of each of the enzymes involved in the lysosomal degradation of mucopolysaccharides. Newborn screening panels worldwide have been recently expanded to include one or more MPS disorders, as treatments are available and are most efficacious if initiated early in life. Here we report the first multiplex assay of 10 enzymatic activities in dried blood spots and fibroblast lysates that allow newborn screening and diagnosis of all MPS disorders except the ultrarare MPS-IX. The assay consists of incubation of enzyme-specific substrates with dried blood spot punches or fibroblast lysate followed by quantification of enzymatic products using liquid chromatography-tandem mass spectrometry (LC-MS/MS) together with internal standards. Assay of all MPS enzymes using fluorimetric or other methods has not been possible. The steps of the LC-MS/MS assay are sufficiently simple and rapid to be used in newborn screening and diagnostic laboratories. Assays showed acceptable precision, and enzymatic activities measured in confirmed MPS samples are well below the reference range.

Achieving Congruence among Reference Laboratories for Absolute Abundance Measurement of Analytes for Rare Diseases: Psychosine for Diagnosis and Prognosis of Krabbe Disease.

Measurement of the absolute concentration of the biomarker psychosine in dried blood spots (DBS) is useful for diagnosis and prognosis of Krabbe disease and to support newborn screening of this leukodystrophy. As for assays for more common diseases, it is important to achieve congruence when multiple clinical laboratories provide testing. Four clinical laboratories, one research laboratory, and a commercial vendor collaborated with the goal to achieve congruence in quantitative psychosine measurement in DBS. A set of DBS calibrators was prepared by a single vendor and used in each reference laboratory to make a standard curve of measured psychosine in DBS versus the stated calibrator psychosine level. Congruence between the participating five laboratories was achieved using the psychosine DBS calibrators. This allowed application of disease-specific reference ranges obtained by the reference laboratory with the most extensive data by the other reference laboratories. Congruence between multiple reference laboratories in the measurement of the absolute concentration of biomarkers in DBS (and by extension other samples) is possible by the use of a common set of DBS calibrators.

Detection of Infantile Batten Disease by Tandem Mass Spectrometry Assay of PPT1 Enzyme Activity in Dried Blood Spots.

A new tandem mass spectrometry (MS/MS)-based approach for measurement of the enzymatic activity of palmitoyl protein thioesterase I (PPT1) in dried blood spots (DBS) is presented. Deficiency in this enzyme leads to infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease, CLN1). The assay could distinguish between 80 healthy newborns and three previously diagnosed INCL patients. Unlike the fluorimetric PPT1 assay, the MS/MS assay does not require recombinant ß-glucosidase. Furthermore, the assay could be easily combined with a TPP1 enzyme assay (for CLN2 disease) and can be potentially multiplexed with a large panel of additional lysosomal enzyme assays by MS/MS for newborn screening and postscreening analysis.

Fabrication of pristine Mn2O3 and Ag-Mn2O3 composite thin films by AACVD for photoelectrochemical water splitting.

Pristine Mn2O3 and Ag-Mn2O3 composite thin films have been developed on fluorine doped tin oxide (FTO) coated glass substrates at 450 °C by aerosol assisted chemical vapor deposition (AACVD) using a methanol solution of a 1 : 2 mixture of acetatoargentate(i), Ag(CH3COO), and a newly synthesized manganese complex, [Mn(dmae)2(TFA)4] (1) (dmae = N,N-dimethylaminoethanolate, TFA = trifluoroacetate). The phase purity and stoichiometric composition of the films were investigated by X-ray diffraction (XRD) and Raman spectroscopy techniques. Energy dispersive X-ray (EDX) and X-ray photoelectron spectroscopy (XPS) analyses revealed a Ag to Mn ratio of 1 : 2 and further confirmed the uniform dispersion of Ag nanoparticles into the Mn2O3 structure. Optical studies showed a direct band gap of 2.0 eV for the pristine Mn2O3 film that was lowered to 1.8 eV for Ag-Mn2O3 due to the plasmonic interaction of Ag with Mn2O3. The Ag-Mn2O3 composite film displayed enhanced photocatalytic activity in photoelectrochemical (PEC) water splitting and yielded a photocurrent of 3 mA cm(-2) at 0.7 V versus Ag/AgCl which was 1.6 times higher than a pristine Mn2O3 film alone, under AM 1.5 G illumination (100 mW cm(-2)). The high PEC efficiency is mainly due to the plasmonic effect of Ag nanoparticles, which enhances the visible light absorption, efficient electron-hole separation and high carrier mobility of the Ag-Mn2O3 photoelectrode. The charge carrier density of Ag-Mn2O3 is two times higher than the pristine Mn2O3 as calculated by the Mott-Schottky plot. Based on the PEC studies a mechanism is proposed to elucidate the high activity of Ag-Mn2O3 in PEC water splitting.

Fabrication of CoTiO3-TiO2 composite films from a heterobimetallic single source precursor for electrochemical sensing of dopamine.

Cobalt titanate-titania composite oxide films have been grown on FTO-coated glass substrates using a single-source heterometallic complex [Co2Ti4(µ-O)6(TFA)8(THF)6]·THF () which was obtained in quantitative yield from the reaction of diacetatocobalt(ii) tetrahydrate, tetraisopropoxytitanium(iv), and trifluoroacetic acid from a tetrahydrofuran solution. Physicochemical investigations of complex have been carried out by melting point, FT-IR, thermogravimetric and single-crystal X-ray diffraction analyses. CoTiO3-TiO2 films composed of spherical objects of various sizes have been grown from by aerosol-assisted chemical vapor deposition at different temperatures of 500, 550 and 600 °C. Thin films characterized by XRD, Raman and X-ray photoelectron spectroscopy, scanning electron microscopy, energy-dispersive X-ray analysis have been explored for electrochemical detection of dopamine (DA). The cyclic voltammetry with the CoTiO3-TiO2 electrode showed a DA oxidation peak at +0.215 V while linear sweep voltammetry displayed a detection limit (LoD) of 0.083 µM and a linear concentration range of 20-300 µM for DA. Thus, the CoTiO3-TiO2 electrode is a potential candidate for the sensitive and selective detection of DA.

A new chromanone acid from the stem bark of Calophyllum teysmannii.

A new chromanone acid, namely caloteysmannic acid (1), along with three known compounds, calolongic acid (2), isocalolongic acid (3) and stigmasterol (4) were isolated from the stem bark of Calophyllum teysmannii. All these compounds were evaluated for their cytotoxic and antioxidant activities in the MTT and DPPH assays, respectively. The structure of compound 1 was determined by means of spectroscopic methods including 1D and 2D NMR experiments as well as HR-EIMS spectrometry. The stereochemical assignment of compound 1 was done based on the NMR results and X-ray crystallographic analysis. The preliminary assay results revealed that all the test compounds displayed potent inhibitory activity against HeLa cancer cell line, in particular with compound 1 which exhibited the highest cytotoxic activity comparable to the positive control used, cisplatin. However, no significant antioxidant activity was observed for all the test compounds in the DPPH radical scavenging capacity assay.

Computational studies of a paramagnetic planar dibenzotetraaza[14]annulene Ni(II) complex.

A square-planar Ni(II) dibenzotetraaza[14]annulene complex substituted with two 3,3-dimethylindolenine groups in the meso positions has recently been synthesized and characterized experimentally. In the solid-state, the Ni(II) complex forms linear π-interacting stacks with Ni···Ni separations of 3.448(2) Å. Measurements of the temperature dependence of the magnetic susceptibility revealed a drastic change in the magnetic properties at a temperature of 13 K, indicating a transition from low-to-high spin states. The molecular structures of the free-base ligand, the lowest singlet, and triplet states of the monomer and the dimer of the Ni complex have been studied computationally using density functional theory (DFT) and ab initio correlation levels of theory. In calculations at the second-order Møller-Plesset (MP2) perturbation theory level, a large energy of 260 kcal mol(-1) was obtained for the singlet-triplet splitting, suggesting that an alternative explanation of the observed magnetic properties is needed. The large energy splitting between the singlet and triplet states suggests that the observed change in the magnetism at very low temperatures is due to spin-orbit coupling effects originating from weak interactions between the fine-structure states of the Ni cations in the complex. The lowest electronic excitation energies of the dibenzotetraaza[14]annulene Ni(II) complex calculated at the time-dependent density functional theory (TDDFT) levels are in good agreement with values deduced from the experimental UV-vis spectrum. Calculations at the second-order algebraic-diagrammatic construction (ADC(2)) level on the dimer of the meso-substituted 3,3-dimethylindolenine dibenzotetraaza[14] annulene Ni(II) complex yielded Stokes shifts of 85-100 nm for the lowest excited singlet states. Calculations of the strength of the magnetically induced ring current for the free-base 3,3-dimethylindolenine-substituted dibenzotetraaza[14]annulene show that the annulene ring is very weakly antiaromatic, sustaining a paratropic ring-current strength of only -1.7 nA/T.

Heterometallic Pd(II)-Ni(II) complexes with meso-substituted dibenzotetraaza[14]annulene: double C-H bond activation and formation of a rectangular tetradibenzotetraaza[14]annulene.

Three isomeric 2[Pd(II)-Ni(II)] metal complexes, derived from indoleninyl meso-substituted dibenzotetraaza[14]annulene, were synthesized. The resulting dimers feature Ni···Ni or, alternatively, Ni···π interactions in staggered or slipped cofacial structures. A remarkable insertion of palladium into two different C-H bonds yielded a 4[Pd(II)-Ni(II)] rectangular complex with dimensions of 8.73 × 10.38 Å.

Semiconducting composite oxide Y2CuO4-5CuO thin films for investigation of photoelectrochemical properties.

An octa-nuclear heterobimetallic complex [Y2Cu6Cl0.7(dmae)6(OAc)7.3(OH)4(H2O)2]·3H2O·0.3CH3C6H5 (dmae = dimethylaminoethanoate; OAc = acetato) was synthesized, characterized by melting point analysis, elemental analysis, FT-IR, and single crystal X-ray diffraction analysis and implemented at 600 °C under an oxygen atmosphere for the deposition of Y2CuO4-5CuO composite thin films by aerosol assisted chemical vapor deposition (AACVD). The chemical composition and surface morphology of the deposited thin film have been determined by X-ray diffraction, scanning electron microscopy and energy dispersive X-ray analysis that suggest the formation of impurity-free crystallite mixtures of the Y2CuO4-5CuO composite, with well-defined evenly distributed particles in the size range of 19-24 nm. An optical band gap energy of 1.82 eV was estimated by UV-visible spectrophotometry. PEC studies show that under illumination with a 150 W halogen lamp and at a potential of 0.8 V, a photocurrent density of 9.85 µA cm(-2) was obtained.

Indole-7-carbaldehyde thiosemicarbazone as a flexidentate ligand toward ZnII, CdII, PdII and PtII ions: cytotoxic and apoptosis-inducing properties of the PtII complex.

A new thiosemicarbazone (LH2) derived from indole-7-carbaldehyde was synthesized and reacted with Zn(II), Cd(II), Pd(II) and Pt(II) salts. The reactions with zinc and cadmium salts in 2 : 1 (ligand-metal) molar ratio afforded complexes of the type MX2(LH2)2, (X = Cl, Br or OAc), in which the thiosemicarbazone acts as a neutral S-monodentate ligand. In the presence of potassium hydroxide, the reaction of LH2 with ZnBr2 resulted in deprotonation of the thiosemicarbazone at the hydrazine and indole nitrogens to form Zn(L)(CH3OH). The reaction of LH2 with K2PdCl4 in the presence of triethylamine, afforded Pd(L)(LH2) which contains two thiosemicarbazone ligands: one being dianionic N,N,S-tridentate while the other one is neutral S-monodentate. When PdCl2(PPh3)2 was used as the Pd(II) ion source, Pd(L)(PPh3) was obtained. In a similar manner, the analogous platinum complex, Pt(L)(PPh3), was synthesized. The thiosemicarbazone in the latter two complexes behaves in a dianionic N,N,S-tridentate fashion. The platinum complex was found to have significant cytotoxicity toward four cancer cells lines, namely MDA-MB-231, MCF-7, HT-29, and HCT-116 but not toward the normal liver WRL-68 cell line. The apoptosis-inducing properties of the Pt complex was explored through fluorescence microscopy visualization, DNA fragmentation analysis and propidium iodide flow cytometry.

Ethyl 4-hy-droxy-1-methyl-5-oxo-2-phenyl-pyrrolidine-3-carboxyl-ate 1.25-hydrate.

The asymmetric unit of the title compound, C(14)H(17)NO(4)·1.25H(2)O, consists of four substituted pyrrolidone mol-ecules (two pairs of enanti-omers) and five water mol-ecules. The five-membered rings each have an envelope conformation, with the C atom bonded to the ester group as the flap. The mean planes of the five-membered rings of the four pyrrolidone mol-ecules make dihedral angles of 60.87 (5), 64.45 (5), 62.03 (5) and 65.79 (5)° with respect to the phenyl rings. In the crystal, the pyrrolidone and water mol-ecules are connected through O-H⋯O hydrogen bonds, forming a layer parallel to the ab plane. The two-dimensional network is further stabilized by inter-molecular C-H⋯O hydrogen bonds.

Indolenine meso-substituted dibenzotetraaza[14]annulene and its coordination chemistry toward the transition metal ions Mn(III), Fe(III), Co(II), Ni(II), Cu(II), and Pd(II).

A new dibenzotetraaza[14]annulene bearing two 3,3-dimethylindolenine fragments at the meso positions (LH(2)), has been synthesized through a nontemplate method. X-ray crystallography shows that the whole molecule is planar. The basicity of the indolenine ring permits the macrocycle to be protonated external to the core and form LH(4)(2+)·2Cl(-). Yet another structural modification having strong C-H···π interactions was found in the chloroform solvate of LH(2). The latter two modifications are accompanied by a degree of nonplanar distortion. The antiaromatic core of the macrocycle can accommodate a number of metal ions, Mn(III), Fe(III), Co(II), Ni(II) and Cu(II), to form complexes of [Mn(L)Br], [Mn(L)Cl], [Fe(LH(2))Cl(2)](+)·Cl(-), [Co(L)], [Ni(L)], and [Cu(L)]. In addition, the reaction of LH(2) with the larger Pd(II) ion leads to the formation of [Pd(2)(LH(2))(2)(OAc)(4)] wherein the macrocycle acts as a semiflexible ditopic ligand to coordinate pairs of metal ions via its indolenine N atoms into dinuclear metallocycles. The compounds LH(2), [Co(L)], and [Ni(L)] are isostructural and feature close π-stacking as well as linear chain arrangements in the case of the metal complexes. Variable temperature magnetic susceptibility measurements showed thermally induced paramagnetism in [Ni(L)].

5-Methyl-1H-indole-3-carbaldehyde.

The title mol-ecule, C(10)H(9)NO, is almost planar with an r.m.s. deviation for all non-H atoms of 0.0115 Å. In the crystal, mol-ecules are connected through N-H⋯O hydrogen bonds into chains running along [021]. The chains are further connected via C-H⋯π inter-actions, forming layers in the bc plane.

Cytoprotective effect of benzyl N'-(5-chloro-indol-3-yl-methylidene)-hydrazinecarbodithioate against ethanol-induced gastric mucosal injury in rats.

Indolic compounds have attracted a lot of attention due to their interesting biological properties. The present study was performed to evaluate the subacute toxicity and anti-ulcer activity of BClHC against ethanol-induced gastric ulcers. Experimental animal groups were orally pre-treated with different doses of BClHC (50, 100, 200 and 400 mg/kg) in 10% Tween 20 solution (vehicle). Blank and ulcer control groups were pre-treated with vehicle. The positive group was orally pretreated with 20 mg/kg omeprazole. After one hour, all groups received absolute ethanol (5 mL/kg) to generate gastric mucosal injury except the blank control group which was administered the vehicle solution. After an additional hour, all rats were sacrificed, and the ulcer areas of the gastric walls determined. Grossly, the ulcer control group exhibited severe mucosal injury, whereas pre-treatment with either derivative or omeprazole resulted in significant protection of gastric mucosal injury. Flattening of gastric mucosal folds was also observed in rats pretreated with BClHC. Histological studies of the gastric wall of ulcer control group revealed severe damage of gastric mucosa, along with edema and leucocytes infiltration of the submucosal layer compared to rats pre-treated with either BClHC or omeprazole where there were marked gastric protection along with reduction or absence of edema and leucocytes infiltration of the submucosal layer. Subacute toxicity study with a higher dose of derivative (5 g/kg) did not manifest any toxicological signs in rats. In conclusions, the present finding suggests that benzyl N'-(5-chloroindol-3-ylmethylidene)hydrazinecarbodithioate promotes ulcer protection as ascertained by the comparative decreases in ulcer areas, reduction of edema and leucocytes infiltration of the submucosal layer.