The reliability of cryoSEM for visualizing gas embolisms in xylem vessels of intact, functioning roots is examined and discussed. The possibility that these embolisms form as a result of freezing water columns under tension is discounted by a double‐freeze experiment. Two regions of the same root, one frozen under tension, the other isolated from the tension by the first freeze, had the same percentage of embolisms, as did also long pieces of root frozen simultaneously along their length. The reliability of energy‐dispersive X‐ray analysis to measure xylem sap concentration in situ in frozen tissue was established by measurement of KCl standard solution frozen on stubs, and within xylem vessels. Solute heterogeneity within the vessels varied with freezing procedure; deep‐freeze > LN2 > cryopliers > liquid ethane, but only the deep‐freeze method gave unsatisfactory estimates of concentration for the standard solution. It is concluded that cryoanalytical SEM is useful for direct observation of gas and liquid‐filled compartments, and for solute analyses at depth within intact plant organs.

BackgroundRetinitis pigmentosa (RP) is a blinding genetic disorder that is caused by the death of photoreceptors in the outer nuclear layer of the retina. To date, 39 different genetic loci have been associated with the disease, and 28 mutated genes have been identified. Despite the complexity of the underlying genetic basis for RP, the final common pathway is photoreceptor cell death via apoptosis.Methodology/Principal FindingsIn this study, P23H and S334ter rhodopsin transgenic rat models of RP were used to test the neuroprotective effects of anti-apoptotic gene therapy. Adeno-associated viruses (AAV) carrying the X-linked inhibitor of apoptosis (XIAP) or green fluorescent protein (GFP) were delivered subretinally into the eye of transgenic rat pups. Histological and functional measures were used to assess neuroprotection. XIAP is known to block apoptosis by inhibiting the action of caspases-3, -7 and -9. The results show that XIAP gene therapy provides long-term neuroprotection of photoreceptors at both structural and functional levels.Conclusions/SignificanceOur gene therapy strategy targets the apoptotic cascade, which is the final common pathway in all forms of retinitis pigmentosa. This strategy holds great promise for the treatment of RP, as it allows for the broad protection of photoreceptors, regardless of the initial disease causing mutation.

We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin+ cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP+ embryonic cones and postnatal Nrl−/− ‘cods’ into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.

The results suggest that XIAP is protective against this potent chemotoxic agent and holds promise as a therapeutic agent in gene therapy approaches to treating retinitis pigmentosa.

The vessel lumenal surface has a dual nature, namely a mosaic of hydrophilic and hydrophobic patches at the micrometre scale, with hydrophilic predominating. A key role is shown, for the first time, of overarching borders of pits in determining the dual nature of the surface. In gas-filled (embolized) vessels they are hydrophobic. When wetted by sap (vessels refilling or full) they are hydrophilic. A hypothesis is proposed to explain the switch between the two states.