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Dysregulated arousal often accompanies neurodevelopmental disorders such as attention deficit hyperactivity disorder and autism spectrum disorder. Recently, we have found that adolescent homozygous Brattleboro (Hom) rats, which contain a mutation in the arginine vasopressin (AVP) gene, exhibit lower behavioral arousal than their heterozygous (Het) littermates in the open field test. This hypoaroused phenotype could be due to loss of AVP in magnocellular cells that supply AVP to the peripheral circulation and project to limbic structures or parvocellular cells that regulate the stress axis and other central targets. Alternatively, hypoarousal could be a side effect of diabetes insipidus - polydipsia and polyuria seen in Hom rats due to loss of AVP facilitation of water reabsorption in the kidney. We developed a viral-rescue approach to "cure" magnocellular AVP cells of their Brattleboro mutation. Infusion of a recombinant adeno-associated virus (rAAV) containing a functional Avp gene and promoter (rAAV-AVP) rescued AVP within magnocellular cells and fiber projections of the paraventricular nucleus of the hypothalamus (PVN) of male and female adolescent Hom rats. Furthermore, water intake was markedly reduced, ameliorating the symptoms of diabetes insipidus. In contrast, open field activity was unaffected. These findings indicate that the hyporaoused phenotype of adolescent Hom rats is not due to the loss of AVP function in magnocellular cells or a side effect of diabetes insipidus, but favors the hypothesis that central, parvocellular AVP mechanisms underlie the regulation of arousal during adolescence.

Among mammalian soft tissues, articular cartilage is particularly interesting because it can endure a lifetime of daily mechanical loading despite having minimal regenerative capacity. This remarkable resilience may be due to the depth-dependent mechanical properties, which have been shown to localize strain and energy dissipation. This paradigm proposes that these properties arise from the depth-dependent collagen fiber orientation. Nevertheless, this structure-function relationship has not yet been quantified. Here, we use confocal elastography, quantitative polarized light microscopy, and Fourier-transform infrared imaging to make same-sample measurements of the depth-dependent shear modulus, collagen fiber organization, and extracellular matrix concentration in neonatal bovine articular cartilage. We find weak correlations between the shear modulus |G(∗)| and both the collagen fiber orientation and polarization. We find a much stronger correlation between |G(∗)| and the concentration of collagen fibers. Interestingly, very small changes in collagen volume fraction vc lead to orders-of-magnitude changes in the modulus with |G(∗)| scaling as (vc - v0)(ξ). Such dependencies are observed in the rheology of other biopolymer networks whose structure exhibits rigidity percolation phase transitions. Along these lines, we propose that the collagen network in articular cartilage is near a percolation threshold that gives rise to these large mechanical variations and localization of strain at the tissue's surface., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel(r)) and Escherichia coli aspartate-semialdehyde dehydrogenase (asd(Ec)), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn7-derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ, gfp, and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-Deltabla-Tel(r)-FRT efficiently transposed within Burkholderia cenocepacia, Burkholderia thailandensis, B. pseudomallei, and B. mallei. We then utilized the mini-Tn7-Tel(r)-based transposon vector (mini-Tn7-Tel(r)-betBA) and a transposase-containing helper plasmid (pTNS3-asd(Ec)) to complement the B. thailandensis DeltabetBA mutation. Next, one of the FRT-lacZ fusion vectors (pFRT1-lacZ-Tel(r)) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-oriT-asd(Ec)) to construct the B. thailandensis DeltabetBA::FRT-lacZ-Tel(r) reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing beta-galactosidase assays on the B. thailandensis DeltabetBA::FRT-lacZ-Tel(r) fusion strain. Finally, we engineered B. thailandensis DeltabetBA::FRT-gfp-Tel(r) and DeltabetBA::FRT-lux-Tel(r) fusion strains by utilizing fusion vectors pFRT1-gfp-Tel(r) and pFRT1-lux-Tel(r), respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.

Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the alpha-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Tel(r)) and trimethoprim (Tp(r)) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Tel(r)-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tp(r)), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Tel(r)) and pFLP-AB5 (Tp(r)). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the Delta betBA::Tel(r)-FRT and Delta betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.