A pilot open-label trial of minocycline in patients with autism and regressive features.

BACKGROUND: Minocycline is a tetracycline derivative that readily crosses the blood brain barrier and appears to have beneficial effects on neuroinflammation, microglial activation and neuroprotection in a variety of neurological disorders. Both microglial activation and neuroinflammation have been reported to be associated with autism. The study was designed to evaluate the effects of minocycline treatment on markers of neuroinflammation and autism symptomatology in children with autism and a history of developmental regression. METHODS: Eleven children were enrolled in an open-label trial of six months of minocycline (1.4 mg/kg). Ten children completed the trial. Behavioral measures were collected and cerebrospinal fluid (CSF), serum and plasma were obtained before and at the end of minocycline treatment and were analyzed for markers of neuroinflammation. RESULTS: Clinical improvements were negligible. The laboratory assays demonstrated significant changes in the expression profile of the truncated form of brain derived neurotrophic factor (BDNF) (P = 0.042) and hepatic growth factor (HGF) (P = 0.028) in CSF. In serum, the ratio of the truncated BDNF form and α-2 macroglobulin (α-2 M), was also significantly lower (P = 0.028) while the mature BDNF/α-2 M ratio revealed no difference following treatment. Only the chemokine CXCL8 (IL-8) was significantly different (P = 0.047) in serum while no significant changes were observed in CSF or serum in chemokines such as CCL2 (MCP-1) or cytokines such as TNF-α, CD40L, IL-6, IFN-γ and IL-1ß when pre- and post-treatment levels of these proteins were compared. No significant pre- and post-treatment changes were seen in the profiles of plasma metalloproteinases, putative targets of the effects of minocycline. CONCLUSIONS: Changes in the pre- and post-treatment profiles of BDNF in CSF and blood, HGF in CSF and CXCL8 (IL-8) in serum, suggest that minocycline may have effects in the CNS by modulating the production of neurotrophic growth factors. However, in this small group of children, no clinical improvements were observed during or after the six months of minocycline administration. TRIAL REGISTRATION: NCT00409747.

Transplastomics in Arabidopsis: progress toward developing an efficient method.

Protocols developed for plastome engineering in Nicotiana tabacum rely on biolistic delivery of the transforming DNA to chloroplasts in intact leaf tissue; integration of the foreign DNA into the plastid genome by homologous recombination via flanking plastid DNA (ptDNA) targeting regions; and gradual dilution of non-transformed ptDNA during cultivation in vitro. Plastid transformation in Arabidopsis was obtained by combining the tobacco leaf transformation protocol with Arabidopsis-specific tissue culture and plant regeneration protocols. Because the leaf cells in Arabidopsis are polyploid, this protocol yielded sterile plants. Meristematic cells in a shoot apex or cells of a developing embryo are diploid. Therefore, we developed a regulated embryogenic root culture system that will generate diploid tissue for plastid transformation. This embryogenic culture system is created by steroid-inducible expression of the BABY BOOM transcription factor. Plastid transformation in Arabidopsis will enable the probing of plastid gene function, and the characterization of posttranscriptional mechanisms of gene regulation and the regulatory interactions of plastid and nuclear genes.

A guide to choosing vectors for transformation of the plastid genome of higher plants.

Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3'rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3''-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3''-adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs.

Exceptional paternal inheritance of plastids in Arabidopsis suggests that low-frequency leakage of plastids via pollen may be universal in plants.

Plastid DNA is absent in pollen or sperm cells of Arabidopsis thaliana. Accordingly, plastids and mitochondria, in a standard genetic cross, are transmitted to the seed progeny by the maternal parent only. Our objective was to test whether paternal plastids are transmitted by pollen as an exception. The maternal parent in our cross was a nuclear male sterile (ms1-1/ms1-1), spectinomycin-sensitive Ler plant. It was fertilized with pollen of a male fertile RLD-Spc1 plant carrying a plastid-encoded spectinomycin resistance mutation. Seedlings with paternal plastids were selected by spectinomycin resistance encoded in the paternal plastid DNA. Our data, in general, support maternal inheritance of plastids in A. thaliana. However, we report that paternal plastids are transmitted to the seed progeny in Arabidopsis at a low (3.9 x 10(-5)) frequency. This observation extends previous reports in Antirrhinum majus, Epilobium hirsutum, Nicotiana tabacum, Petunia hybrida, and the cereal crop Setaria italica to a cruciferous species suggesting that low-frequency paternal leakage of plastids via pollen may be universal in plants previously thought to exhibit strict maternal plastid inheritance. The genetic tools employed here will facilitate testing the effect of Arabidopsis nuclear mutations on plastid inheritance and allow for the design of mutant screens to identify nuclear genes controlling plastid inheritance.

DNA markers define plastid haplotypes in Arabidopsis thaliana.

To identify genetic markers in the Arabidopsis thaliana plastid genome (ptDNA), we amplified and sequenced the rpl2-psbA and rbcL-accD regions in 26 ecotypes. The two regions contained eight polymorphic sites including five insertions and/or deletions (indels) involving changes in the length of A or T mononucleotide repeats and three base substitutions. The 27 alleles defined 15 plastid haplotypes, providing a practical set of ptDNA markers for the Columbia, Landsberg erecta and Wassilewskija ecotypes that are commonly used in genetic studies and also for the C24 and RLD ecotypes that are the most amenable for cell culture manipulations.

A novel approach to plastid transformation utilizes the phiC31 phage integrase.

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.

Identification of functional lox sites in the plastid genome.

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.