Identification of the Arabidopsis dry2/sqe1-5 mutant reveals a central role for sterols in drought tolerance and regulation of reactive oxygen species.

Squalene epoxidase enzymes catalyse the conversion of squalene into 2,3-oxidosqualene, the precursor of cyclic triterpenoids. Here we report that the Arabidopsis drought hypersensitive/squalene epoxidase 1-5 (dry2/sqe1-5) mutant, identified by its extreme hypersensitivity to drought stress, has altered stomatal responses and root defects because of a point mutation in the SQUALENE EPOXIDASE 1 (SQE1) gene. GC-MS analysis indicated that the dry2/sqe1-5 mutant has altered sterol composition in roots but wild-type sterol composition in shoots, indicating an essential role for SQE1 in root sterol biosynthesis. Importantly, the stomatal and root defects of the dry2/sqe1-5 mutant are associated with altered production of reactive oxygen species. As RHD2 NADPH oxidase is de-localized in dry2/sqe1-5 root hairs, we propose that sterols play an essential role in the localization of NADPH oxidases required for regulation of reactive oxygen species, stomatal responses and drought tolerance.

Arabidopsis 3-hydroxy-3-methylglutaryl-CoA reductase is regulated at the post-translational level in response to alterations of the sphingolipid and the sterol biosynthetic pathways.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR, EC 1.1.1.34) catalyzes the major rate-limiting step in the mevalonate (MVA) pathway for isoprenoid biosynthesis. Its activity is regulated at different levels, from transcriptional to post-translational. Treatment of Arabidopsis thaliana plants with myriocin, a specific inhibitor of serine palmitoyltransferase (SPT), the first enzyme of sphingolipid biosynthesis, resulted in a concomitant reduction of both HMGR activity and the sterol content, which reveals regulatory cross-talk between these two lipid biosynthesis pathways. Myriocin-induced down-regulation of HMGR activity is exerted at the post-translational level, like the regulatory response of HMGR to enhancement or depletion of the flux through the sterol pathway.

Anther development in tribe Epidendreae: orchids with contrasting pollination syndromes.

BACKGROUND: Epidendreae is one of the most diverse tribes among the orchids with remarkable variation in life form, floral morphology and pollination syndromes. Its circumscription was recently revised and subtribes Agrostophyllinae and Calypsoinae were transferred into this tribe. One of the principal floral characters utilized in classification of orchids is the incumbency or bending of the column. This study records and compares late stages of anther, column and lip development, and discusses anther characters in fifteen representative taxa of five of the six subtribes in Epidendreae with respect to classification and pollination biology. METHODS: A series of late floral stages were sampled and fixed for examination under scanning electron microscope. RESULTS: Anther incumbency or bending in this group varies from 90° to almost 180°. Incumbency in the late stages of development is reached in Bletiinae, Ponerinae, Pleurothallidinae and Laeliinae whereas incumbency is reached early in its development in Corallorhiza and Govenia of Calypsoinae. DISCUSSION: Our observations indicate that the position of Chysis in subtribe Bletiinae needs revision based on differences in a number floral, and in particular of anther characters; and that Coelia only shares the early anther incumbency with Calypsoinae members, but not the rest of floral and anther characters. Anatomical characters such as crystals around the actinocytic stomata on the anther cap and sugar crystals in Laeliinae; lack of rostellum in Bletiinae; coalescent anther with the column, lack of trichomes and papillae on lip keels, and underdeveloped rostellum in Chysis; a mechanism by which the anther cap comes off (it is joined with the grooved lip by a claw) in Isochilus are all related to pollination syndromes and reproductive biology.

Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.

In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI.

Highly rapid amplification-free and quantitative DNA imaging assay.

There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a 'sample-in-result-out' mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 µL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.