Transformation of Kalanchoe pinnata by Agrobacterium tumefaciens with ZsGreen1

Plant Cell, Tissue and Organ Culture (PCTOC) - Screening tests in which 30 species of succulent plants were inoculated with A136 or C58 strains of Agrobacterium tumefaciens led to selecting genus...     

Mimicry of bioactive peptides via non-natural,sequence-specific peptidomimetic oligomers

Non-natural, sequence-specific peptidomimetic oligomers are being designed to mimic bioactive peptides, with potential therapeutic application. Cationic, facially amphipathic helical beta-peptide oligomers have been developed as magainin mimetics. Non-natural mimics of HIV-Tat protein, lung surfactant proteins, collagen, and somatostatin are also being developed. Pseudo-tertiary structure in beta-peptides and peptoids may herald the creation of entirely artificial proteins.     

The interaction between early life epilepsy and autistic-like behavioral consequences: a role for the mammalian target of rapamycin (mTOR) pathway

Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR) modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1) signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236), as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308) and phospho-ERK (Thr202/Tyr204). Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures.     

The HOXB4 homeoprotein promotes the ex vivo enrichment of functional human embryonic stem cell-derived NK cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34(+)CD45RA(+) precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells.     

Field determination of denitrification in water-logged forest soils

Abstract Denitrification rates were measured as N₂O production in two water-logged forest soils at monthly intervals. The effect of acetylene inhibition and the addition of nitrate, glucose, acetate and celloboise in field incubations was examined.
N₂O release from the two soils was very low, 26 mg N₂m⁻²y⁻¹ in ash and 178 mg N₂ in alder. In acetylene inhibited incubations N₂O production was higher, 296 and 486 mg N₂m⁻² y⁻¹ in ash and alder respectively. After addition of nitrate and C-sources to a 10 mM concentration, denitrification rates increased to 5–15 times higher values.
The denitrification rates below 4°C were low and most N₂O was produced in late spring and summer.
The highest rate of denitrification during a 50 h incubation experiment occurred between 3 and 23 h.     

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-Invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient.     

NMEGylation: a novel modification to enhance the bioavailability of therapeutic peptides

We have evaluated "NMEGylation"--the covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain--as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic polyethylene glycol-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as nonfouling surface coatings and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated before this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, "C20," as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end, we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C-terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affected peptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.     

Biomimetic N-terminal alkylation of peptoid analogues of surfactant protein C

Surfactant protein C (SP-C) is a hydrophobic lipopeptide that is critical for lung function, in part because it physically catalyzes the formation of surface-associated surfactant reservoirs. Many of SP-C's key biophysical properties derive from its highly stable and hydrophobic α-helix. However, SP-C's posttranslational modification with N-terminal palmitoyl chains also seems to be quite important. We created a new (to our knowledge) class of variants of a synthetic, biomimetic family of peptide mimics (peptoids) that allow us to study the functional effects of biomimetic N-terminal alkylation in vitro. Mimics were designed to emulate the amphipathic patterning, helicity, and hydrophobicity of SP-C, and to include no, one, or two vicinal amide-linked, N-terminal octadecyl chains (providing a reach equivalent to that of natural palmitoyl chains). Pulsating bubble surfactometry and Langmuir-Wilhelmy surface balance studies showed that alkylation improved biomimetic surface activities, yielding lower film compressibility and lower maximum dynamic surface tensions. Atomic force microscopy studies indicated that alkyl chains bind to and retain segregated interfacial surfactant phases at low surface tensions by inducing 3D structural transitions in the monolayer's fluid-like phase, forming surfactant-associated reservoirs. Peptoid-based SP-C mimics are easily produced and purified, and offer much higher chemical and secondary structure stability than polypeptide-based mimics. In surfactant replacements intended for medical use, synthetic SP mimics reduce the odds of pathogen contamination, which may facilitate the wider use of surfactant treatment of respiratory disorders and diseases.     

A fluorescence polarization assay using an engineered human respiratory syncytial virus F protein as a direct screening platform

Human respiratory syncytial virus (hRSV) typically affects newborns and young children. Even though it can cause severe and, in some cases, lifelong respiratory infections, there are currently no Food and Drug Administration (FDA)-approved therapeutics that control this virus. The hRSV F protein facilitates viral fusion, a critical extracellular event that can be targeted for therapeutic intervention by disrupting the assembly of a postfusion 6-helix bundle (6HB) within the hRSV F protein. Here we report the development of a fluorescence polarization (FP) assay using an engineered hRSV F protein 5-helix bundle (5HB). We generated the 5HB and validated its ability to form a 6HB in an FP assay. To test the potential of 5HB as a screening tool, we then investigated a series of truncated peptides derived from the “missing” sixth helix. Using this FP-based 5HB system, we have successfully demonstrated that short peptides can prevent 6HB formation and serve as potential hRSV fusion inhibitors. We anticipate that this new 5HB system will provide an effective tool to identify and study potential antivirals to control hRSV infection.