Colonization study of gfp-tagged Achromobacter marplatensis strain in sugar beet

This study details the introduction of a gfp marker into an endophytic bacterial strain (Achromobacter marplatensis strain 17, isolated from sugar beet) to monitor its colonization of sugar beet (Beta. vulgaris L.). Stability of the plasmid encoding the gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under nonselective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated sugar beet plants that grew for 10 or 20 days. gfp-Expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the Achromobacter marplatensis 17:gfp strain in plants 20 days after inoculation, even in the absence of selective pressure, suggests that it is good colonizer. These results also suggest that this strain could be a useful tool for the delivery of enzymes or other proteins into plants. In addition, the study highlights that sugar beet plants can be used effectively for detailed in vitro studies on the interactions between A. marplatensis strain 17 and its host, particularly if a gfp-tagged strain of the pathogen is used.     

Omega-3 fatty acids concentrate production by enzyme-catalyzed ethanolysis of supercritical CO<Subscript>2</Subscript> extracted oyster oil

The separation of oil by a suitable technique from the Pacific oyster muscle is important for the utilization of the oil as a ω-3 polyunsaturated fatty acids (ω-3 PUFAs) source and production of bio-functional peptides/ oligosaccharides from oil-free residue. This study was conducted to prepare ω-3 PUFAs concentrate from supercritical carbon dioxide (SC-CO₂) extracted Pacific oyster oil by enzyme-catalyzed ethanolysis reactions. SC-CO₂ extractions were done at different temperatures and pressures to optimize suitable extraction conditions and extracted oils were compared with Soxhlet (n-hexane) extracted oil to evaluate the yield and quality. Oil extracted by SC-CO₂ at optimized conditions was used for ethanolysis reaction catalyzed by immobilized sn-1,3 specific lipases, namely Novozymes-435, Lipozyme TLIM, and Lipozyme RMIM to produce 2-monoacylglycerols (2-MAG) rich in ω-3 PUFAs. The optimum temperature and pressure for SC-CO₂ extractions of oyster oil was 50°C and 30 MPa. In this condition, the yield of oil was 5.96% and the acid, peroxide, free fatty acid, and p-anisidine values were 4.49 mg KOH/g, 4.72 meq/kg, 3.42%, and 10.03, respectively. The ω-3 PUFAs content significantly increased in 2-MAG obtained from Novozymes 435, Lipozyme TLIM, and Lipozyme RMIM to 43.03 ± 0.36, 45.95 ± 0.29, and 40.50 ± 0.77%, respectively (p < 0.05). A thin layer chromatography (TLC) analysis confirmed the production and separation of 2-MAG in the ethanolysis process. The ratio of total ω-3 to ω-6 fatty acids was almost twice in 2-MAG of SC-CO₂ extracted oyster oil. SC-CO₂ extracted Pacific oyster oil can be used for sn-1,3 specific lipases catalyzed ethanolysis to produce ω-3 PUFAs rich in 2-MAG.     

TRIAD3/RNF216 mutations associated with Gordon Holmes syndrome lead to synaptic and cognitive impairments via Arc misregulation

Multiple loss‐of‐function mutations in TRIAD3 (a.k.a. RNF216) have recently been identified in patients suffering from Gordon Holmes syndrome (GHS), characterized by cognitive decline, dementia, and movement disorders. TRIAD3A is an E3 ubiquitin ligase that recognizes and facilitates the ubiquitination of its target for degradation by the ubiquitin‐proteasome system (UPS). Here, we demonstrate that two of these missense substitutions in TRIAD3 (R660C and R694C) could not regulate the degradation of their neuronal target, activity‐regulated cytoskeletal‐associated protein (Arc/Arg 3.1), whose expression is critical for synaptic plasticity and memory. The synaptic deficits due to the loss of endogenous TRIAD3A could not be rescued by TRIAD3A harboring GHS‐associated missense mutations. Moreover, we demonstrate that the loss of endogenous TRIAD3A in the mouse hippocampal CA1 region led to deficits in spatial learning and memory. Finally, we show that these missense mutations abolished the interaction of TRIAD3A with Arc, disrupting Arc ubiquitination, and consequently Arc degradation. Our current findings of Arc misregulation by TRIAD3A variants suggest that loss‐of‐function mutations in TRIAD3A may contribute to dementia observed in patients with GHS driven by dysfunctional UPS components, leading to cognitive impairments through the synaptic protein Arc.     

Primary identifications and palynological observations of Perilla in China

Unresolved controversies concerning classification of the monotypic genus Perilla L. have hindered the complete understanding and subsequent sustainable use of these vital food, oil and medicinal plants to their full potential. We attempted to use scanning electron microscopy to obtain palynological evidence from Perilla plants of 21 samples from seven provinces in China as a potential extra attribute in classification. The findings showed that pollen grains from plants of 11 samples were oblate, while those of the other 10 were suboblate in shape, and there were no any type of prolate pollen grains being observed. Pollen grains of all the samples had diverse exine ornamentations. Based on whether having continuous tecta on the ornamentations, all of the pollen grains derived from the 21 samples were classified into two categories, fourteen of them with irregular reticulates, seven with continuous tecta with no perforations. None of the samples were bireticulates. The ornamentation pattern and size of pollen grains jointly provided evidence that it is appropriate to classify the genus Perilla into five varieties of one species. Furthermore, by comparison, it is concluded that shapes and exine ornamentations of Perilla are unique among those of the seven genera already investigated in the subfamily Lamioideae. Using these pollen features, Perilla could be easily distinguished from two other subtribes (Menthinae Briq. and Thyminae Briq.) in the same tribe, supporting the view that Perilla and other four related genera were divided into one subtribe (Perillinae).     

A PCR-RFLP-based method to distinguish sable (<Emphasis Type="Italic">Martes zibellina</Emphasis>) and pine marten (<Emphasis Type="Italic">Martes martes</Emphasis>)

Species identification is an important issue in conservation and a particular focus for wildlife forensics. Molecular biological methods retain a unique power to differentiate between difficult samples that lack other identifiable characteristics. The pine marten (Martes martes) and sable (Martes zibellina) are closely related species with very similar pelage characteristics and are often difficult to distinguish from each other. The sable, however, in contrast to the pine marten, remains an endangered and protected animal in China with both hunting and fur trade strictly prohibited for this species. Here, we present a polymerase chain reaction-based restriction fragment length polymorphism method for distinguishing the two species. We sequenced a 638-bp fragment of cytochrome b gene in 39 sables, 68 pine martens, and 10 stone martens and identified all variable nucleotides. A new primer pair was subsequently designed to amplify a 316-bp fragment containing restriction sites of enzyme BseG I and BamH I that are different among martens. When the fragment was cut using BseG I, the resulting restriction pattern was identical in the sable and pine marten, but differed from all other martens. When cut using BamH I, the fragment generated two diagnostic fragments in the sable which could distinguish them from pine martens. This method was valid for all haplotypes of sable and pine marten thus far identified and has high potentially applicability for the identification of the two species.