Defective AMPA-mediated synaptic transmission and morphology in human neurons with hemizygous SHANK3 deletion engrafted in mouse prefrontal cortex.

Genetic abnormalities in synaptic proteins are common in individuals with autism; however, our understanding of the cellular and molecular mechanisms disrupted by these abnormalities is limited. SHANK3 is a postsynaptic scaffolding protein of excitatory synapses that has been found mutated or deleted in most patients with 22q13 deletion syndrome and about 2% of individuals with idiopathic autism and intellectual disability. Here, we generated CRISPR/Cas9-engineered human pluripotent stem cells (PSCs) with complete hemizygous SHANK3 deletion (SHANK3+/-), which is the most common genetic abnormality in patients, and investigated the synaptic and morphological properties of SHANK3-deficient PSC-derived cortical neurons engrafted in the mouse prefrontal cortex. We show that human PSC-derived neurons integrate into the mouse cortex by acquiring appropriate cortical layer identities and by receiving and sending anatomical projections from/to multiple different brain regions. We also demonstrate that SHANK3-deficient human neurons have reduced AMPA-, but not NMDA- or GABA-mediated synaptic transmission and exhibit impaired dendritic arbors and spines, as compared to isogenic control neurons co-engrafted in the same brain region. Together, this study reveals specific synaptic and morphological deficits caused by SHANK3 hemizygosity in human cortical neurons at different developmental stages under physiological conditions and validates the use of co-engrafted control and mutant human neurons as a new platform for studying connectivity deficits in genetic neurodevelopmental disorders associated with autism.

New insights into pradimicin biosynthesis revealed by two O-methyltransferases.

Pradimicins are a group of antiviral and antifungal natural products from Actinomadura hibisca. Two putative O-methyltransferase genes, pdmF and pdmT, are present in the pradimicin biosynthetic gene cluster. However, there is only one methoxy group (11-OCH3) in pradimicins. Through heterologous expression and in vitro reactions with various substrates, PdmF was characterized as the C-11 O-methyltransferase with a relatively broad substrate specificity. To probe the role of PdmT in pradimicin biosynthesis, the corresponding gene was disrupted through homologous recombination, leading to the production of pradimicinone II. This enzyme was then expressed in Escherichia coli with an N-terminal His6 tag and purified by Ni-NTA chromatography. Reaction of pradimicinone II with PdmT generated 7-O-methylpradimicinone II, confirming that this enzyme is a C-7 O-methyltransferase. Characterization of PdmT suggests a novel pathway that leads to the "flip" of 7-OH to C-14 in pradimicin biosynthesis.

Three enzymes involved in the N-methylation and incorporation of the pradimicin sugar moieties.

Pradimicins are antifungal and antiviral natural products from Actinomadura hibisca P157-2. The sugar moieties play a critical role in the biological activities of these compounds. There are two glycosyltransferase genes in the pradimicin biosynthetic gene cluster, pdmS and pdmQ, which are putatively responsible for the introduction of the sugar moieties during pradimicin biosynthesis. In this study, we disrupted these two genes using a double crossover approach. Disruption of pdmS led to the production of pradimicinone I, the aglycon of pradimicin A, which confirmed that PdmS is the O-glycosyltransferase responsible for the first glycosylation step and attaching the 4',6'-dideoxy-4'-amino-d-galactose or 4',6'-dideoxy-4'-methylamino-d-galactose moiety to the 5-OH. Disruption of pdmQ resulted in the production of pradimicin B, indicating that this enzyme is the second glycosyltransferase that introduces the d-xylose moiety to the 3'-OH of the first sugar moiety. Insertion of an integrative plasmid before pdmO might have interfered with the dedicated promoter, yielding a mutant that produces pradimicin C as the major metabolite, which suggested that PdmO is the enzyme that specifically methylates the 4'-NH2 of the 4',6'-dideoxy-4'-amino-d-galactose moiety. Functional characterization of these sugar-decorating and -incorporating enzymes thus facilitates the understanding of the pradimicin biosynthetic pathway.

Synergistic actions of tailoring enzymes in pradimicin biosynthesis.

Three key tailoring enzymes in pradimicin biosynthesis: PdmJ, PdmW, and PdmN, were investigated. PdmW was characterized as the C-6 hydroxylase by structural characterization of the corresponding product, 6-hydroxy-G-2A. The efficiencies of the C-5 and C-6 hydroxylations, catalyzed respectively by PdmJ and PdmW, were low when they were expressed individually with the early biosynthetic enzymes that form G-2A. When these two cytochrome P450 enzymes were co-expressed, a dihydroxylated product, 5,6-dihydroxy-G-2A, was efficiently produced, indicating that these two enzymes work synergistically in pradimicin biosynthesis. Heterologously expressed PdmN in Streptomyces coelicolor CH999 converted G-2A to JX137a by ligating a unit of D-alanine to the carboxyl group. PdmN has relaxed substrate specificity toward both amino acid donors and acceptors. Through combinatorial biosynthesis, a series of new pradimicin analogues were produced.

Epidemiología de las dermatomicosis en 30 años de estudio en el Instituto de Medicina Tropical Daniel A. Carrión, Universidad Nacional Mayor de San Marcos, Lima, Perú / Epidemiology of dermatomycoses in 30 years of study at Daniel A. Carrion Institute of Tropical Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru

Objetivos: Determinar la evolución epidemiológica de las dermatomicosis en pacientes de consultorio externo durante el periodo 1976-2005. Diseño: Estudio descriptivo, retrospectivo y analítico. Lugar: Instituto de Medicina Tropical æDaniel Alcides CarriónÆ, Universidad Nacional Mayor de San Marcos, Lima, Perú. Participantes: Pacientes positivos a dermatomicosis. Intervenciones: Se revisó las historias clínicas de 7 185 (55,3 por ciento) casos positivos a dermatomicosis. El instrumento de investigación empleado fue la ficha de levantamiento de información. Principales medidas de resultados: Agente etiológico, estación del año, sexo, edad y forma clínica. Resultados: El estudio demostró que los más afectados fueron del grupo etario de 16 a 30 años (42,7 por ciento) y sexo femenino (52,1 por ciento). La dermatomicosis más frecuente fue la onicomicosis (43,6 por ciento). Los agentes patógenos de mayor prevalencia fueron Trichophyton rubrum (33,2 por ciento), Cándida albicans (15,3 por ciento), Cándida no albicans (11,8 por ciento), Trichophyton mentagrophytes (9,4 por ciento), Malassezia spp (9,1 por ciento) y las infecciones mixtas (7,2 por ciento). Las micosis de cuero cabelludo muestran continuo aumento durante todo el estudio. El dermatofito Epidermophyton floccosum fue aislado por última vez en la década del 90. A partir de 1995 ha aumentado la prevalencia de Cándida no albicans y se encontró como especie re-emergente a la levadura Cándida tropicalis. Conclusiones: Entre los años 1976 y 2005 hubo importantes variaciones epidemiológicas en relación a las formas clínicas y a la etiología de las dermatomicosis...

Objectives: To determine dermatomycoses epidemiological evolution in outpatients during the period 1976-2005. Design: Descriptive, retrospective, and analytical study. Setting: Daniel Alcides Carrion Institute of Tropical Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru. Participants: Patients positive to dermatomycoses. Interventions: Medical records of 7 185 (55.3 per cent) dermatomycoses-positive patients were reviewed. Main outcome measures: Etiologic agent, season, gender, age, and clinical forms. Results: Females (52.1 per cent) and the 16 to 30 year-old group (42.7 per cent) were the most affected. Most frequent dermatomycoses was onychomycosis (43.6 per cent). Most prevalent pathogens were Trichophyton rubrum (33.2 per cent), Candida albicans (15.3 per cent), Candida non albicans (11.8 per cent), Trichophyton mentagrophytes (9.4 per cent), Malassezia spp. (9.1 per cent), and mixed infections (7.2 per cent). The fungal scalp infection showed steady increase during the period studied. Epidermophyton floccosum dermatophyte was isolated for the last time in the 1990s. Since 1995 prevalence of Candida non albicans has increased and Candida tropicalis yeast species are re-emerging. Conclusions: Epidemiological changes in dermatomycoses clinical forms and etiology were found between 1976 and 2005...

A key cytochrome P450 hydroxylase in pradimicin biosynthesis.

Pradimicins A-C (1-3) are a group of antifungal and antiviral polyketides from Actinomadura hibisca. The sugar moieties in pradimicins are required for their biological activities. Consequently, the 5-OH that is used for glycosylation plays a critical role in pradimicin biosynthesis. A cytochrome P450 monooxygenase gene, pdmJ, was amplified from the genomic DNA of A. hibisca and expressed in Escherichia coli BL21(DE3). PdmJ introduced a hydroxyl group to G-2A (4), a key pradimicin biosynthetic intermediate, at C-5 to form JX134 (5). A d-Ala-containing pradimicin analog, JX137a (6) was tested as an alternative substrate, but no product was detected by LC-MS, indicating that PdmJ has strict substrate specificity. Kinetic studies revealed a typical substrate inhibition of PdmJ activity. The optimal substrate concentration for the highest velocity is 115µM under the test conditions. Moreover, the conversion rate of 4 to 5 was reduced by the presence of 6, likely due to competitive inhibition. Coexpression of PdmJ and a glucose 1-dehydrogenase in E. coli BL21(DE3) provides an efficient method to produce the important intermediate 5 from 4.