Otitis externa caused by Malassezia slooffiae complicated with mastoiditis: A case report.

Herein, we report a case of otitis externa caused by Malassezia slooffiae complicated with mastoiditis. A 70-year-old male complained of fever and severe otorrhea from left external auditory canal 2 months after undergoing a craniotomy to remove a hematoma. He had right-sided paralysis and undertook bed rest. Brain computed tomography revealed continuous fluid accumulation in the left mastoid air cells and middle ear from left external auditory canal in addition to leukocytosis and increased C-reactive protein level. The tympanic membrane was severely swelling. These results indicated the presence of otitis media and mastoiditis. Otorrhea culture showed large amounts of M. slooffiae. The administration of liposomal amphotericin B (L-AMB), the irrigation of external auditory canal with normal saline, and the application of topical ketoconazole ointment were started. The administration of L-AMB for 8 weeks and voriconazole, which was switched from L-AMB, for 4 weeks ameliorated his infection and he was transferred to another hospital to receive rehabilitation. From these results and his clinical course, the diagnosis of otitis externa caused by Malassezia slooffiae complicated with mastoiditis was made. And the possibility of the contamination by M. slooffiae was very low. Clinicians should be aware that M.slooffiae can provoke otological infections since M. slooffiae is the most common Malassezia sp. in external auditory canal.

Development of an efficient Agrobacterium-mediated transformation method and its application in tryptophan pathway modification in Catharanthus roseus.

The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l-1 kanamycin and 20 mg l-1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.

Plant-based production and characterization of a promising Fc-fusion protein against microgravity-induced bone density loss.

Microgravity-induced bone loss is a main obstacle for long term space missions as it is difficult to maintain bone mass when loading stimuli is reduced. With a typical bone mineral density loss of 1.5% per month of microgravity exposure, the chances for osteoporosis and fractures may endanger astronauts' health. Parathyroid Hormone or PTH (1-34) is an FDA approved treatment for osteoporosis, and may reverse microgravity-induced bone loss. However, PTH proteins requires refrigeration, daily subcutaneous injection, and have a short shelf-life, limiting its use in a resource-limited environment, like space. In this study, PTH was produced in an Fc-fusion form via transient expression in plants, to improve the circulatory half-life which reduces dosing frequency and to simplify purification if needed. Plant-based expression is well-suited for space medicine application given its low resource consumption and short expression timeline. The PTH-Fc accumulation profile in plant was established with a peak expression on day 5 post infiltration of 373 ± 59 mg/kg leaf fresh weight. Once the PTH-Fc was purified, the amino acid sequence and the binding affinity to its target, PTH 1 receptor (PTH1R), was determined utilizing biolayer interferometry (BLI). The binding affinity between PTH-Fc and PTH1R was 2.30 × 10-6 M, similar to the affinity between PTH (1-34) and PTH1R (2.31 × 10-6 M). Its function was also confirmed in a cell-based receptor stimulation assay, where PTH-Fc was able to stimulate the PTH1R producing cyclic adenosine monophosphate (cAMP) with an EC50 of (8.54 ± 0.12) x 10-9 M, comparable to the EC50 from the PTH (1-34) of 1.49 × 10-8 M. These results suggest that plant recombinant PTH-Fc exhibits a similar binding affinity and potency in a PTH1R activation assay compared to PTH. Furthermore, it can be produced rapidly at high levels with minimal resources and reagents, making it ideal for production in low resource environments such as space.

Characterization of newly developed pepper cultivars (Capsicum chinense) 'Dieta0011-0301', 'Dieta0011-0602', 'Dieta0041-0401', and 'Dieta0041-0601' containing high capsinoid concentrations and a strong fruity aroma.

Capsaicinoids are responsible for the pungent flavor of peppers (Capsicum sp.). The cultivar CH-19 Sweet is a non-pungent pepper mutant that biosynthesizes the low-pungent capsaicinoid analogs, capsinoids. Capsinoids possess important pharmaceutical properties. However, capsinoid concentrations are very low in CH-19 Sweet, and Capsicum cultivars with high content capsinoids are desirable for industrial applications of capsinoids. Habanero, Bhut Jolokia, and Infinity are species of Capsicum chinense, and have strong pungency and intense fruity flavors. In the present study, we report new cultivars with high concentrations of capsinoids (more than ten-fold higher than in CH-19 Sweet), and showed that these cultivars (Dieta0011-0301 and Dieta0011-0602 from Bhut Jolokia, Dieta0041-0401 and Dieta0041-0601 from Infinity) are of nutritional and medicinal value and have fruity aromas. We also obtained a vanilla bean flavor, vanillyl alcohol, and vanillyl ethyl ether from capsinoids in the fruit of these cultivars following the addition of ethanol at room temperature. ABBREVIATIONS: p-AMT: putative aminotransferase; C. annuum: Capsicum annuum; C. chinense: Capsicum chinense; dCAPS: derived Cleaved Amplified Polymorphic Sequences.

Virus-induced gene silencing in chili pepper by apple latent spherical virus vector.

Apple latent spherical virus (ALSV) can infect a variety of crops, usually without inducing symptoms. Partial gene sequences can be introduced into ALSV vectors for the induction of virus-induced gene silencing (VIGS). These features are beneficial for the estimation of gene functions in plants, with relatively concise experimental manipulations. Given that the infectability of chili peppers (Capsicum spp.) by ALSV was unknown, an ALSV infectivity test was performed on the highly pungent Capsicum chinense cultivar 'Habanero'. The chili pepper plants were not infected after rub-inoculation with a crude homogenate of ALSV-infected Chenopodium quinoa leaves, whereas inoculating them with a concentrated ALSV virus preparation caused an infection. Inoculation with an ALSV RNA preparation by gold particle bombardment resulted in high infection rates (about 90%). The infection was systemic and the infected plants were symptomless. For the induction of VIGS, 201-nucleotide fragments of the putative aminotransferase (pAMT) gene were introduced into the ALSV vector. These ALSV vectors infected 80-90% of RNA-inoculated chili pepper seedlings. Expression of pAMT-mRNA was repressed in the placenta of immature fruit of infected plants. The silencing of pAMT in the infected plants caused a substantial decrease in capsaicin content and a concomitant moderate accumulation of the non-pungent bioactive metabolite capsiate in these plants. These results showed that ALSV could be used to study gene functions by VIGS and to enhance capsiate accumulation in chili pepper through genetic modification.

Both negative and positive G1 cell cycle regulators undergo proteasome-dependent degradation during sucrose starvation in Arabidopsis.

The proteasome pathway regulates many aspects of biological processes in plants, such as plant hormone signaling, light responses, the circadian clock and regulation of cell division. Key cell-cycle regulatory proteins including B-type cyclins, Cdc6, cyclin-dependent kinase inhibitors and E2Fc undergo proteasome-dependent degradation. We used the proteasome inhibitor MG132 to show that proteolysis of Arabidopsis RETINOBLASTOMA-RELATED 1 (AtRBR1) and three E2Fs is mediated by the proteasome pathway during sucrose starvation in Arabidopsis suspension MM2d cells. We found previously that estrogen-inducible RNAi-mediated downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Degradation of not only negative (AtRBR1 and E2Fc) but also positive (E2Fa and E2Fb) cell cycle regulators after sucrose starvation may be required for arrest in G1 phase, when cells integrate a variety of nutritional, hormonal and developmental signals to decide whether or not to commit to entry into the cell cycle.

Arabidopsis G1 cell cycle proteins undergo proteasome-dependent degradation during sucrose starvation.

Although sucrose availability is crucial for commitment to plant cell division during G1 phase, it has remained uncertain how protein levels of core cell cycle genes are regulated. We found that Arabidopsis retinoblastoma-related protein1 (AtRBR1) and three E2F proteins were degraded under limited sucrose conditions, while protein abundance increased in response to treatment with the proteasome inhibitor MG132. We conclude that Arabidopsis key cell cycle proteins are degraded in a proteasome-dependent manner during sucrose starvation in Arabidopsis suspension MM2d cells.

Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 is involved in G1 phase cell cycle arrest caused by sucrose starvation.

Although sucrose availability is crucial for commitment to plant cell division during G1 phase by controlling the expression of D-type cyclins, it has remained unclear how these factors mediate entry into the cell cycle. Here we show that Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 (AtRBR1) is involved in G1-phase cell cycle arrest caused by sucrose starvation. We generated estrogen-inducible AtRBR1 RNA interference (RNAi) Arabidopsis suspension MM2d cells, and found that downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Synchronization experiments confirmed that downregulation of AtRBR1 leads to a prolonged G2 phase and delayed activation of G2/M marker genes. Downregulation of AtRBR1 also stimulated the activation of E2F-regulated genes when these genes were repressed in the uninduced cells under the limited sucrose conditions. We conclude that AtRBR1 is a key effector for the ability of sucrose to modulate progression from G1 phase.