Results: We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images.
Conclusions: Our protocol should be adaptable for monitoring activation for other plant small GTPases and protein-protein interactions for other FRET sensors in various plant cells.
METHODS: This is a 5-year retrospective audit on patients who underwent phototherapy between 2011 and 2015.
RESULTS: There were 892 patients, M:F=1.08:1, aged from 4- 88 years, with a median age of 38.8 years who underwent phototherapy. Majority (58.9%) had skin phototype IV, followed by type III (37.7%) and type II (0.7%). There were 697(78.1%) who underwent NBUVB, 136 (15.2%) had topical PUVA, 22(2.5%) had oral PUVA, 12(1.4%) had UVA1 and 23(2.6%) had NBUVB with topical or oral PUVA/UVA1 at different time periods. The indications were psoriasis (46.6%), vitiligo (26.7%), atopic eczema (9.8%), pityriasis lichenoides chronica (5.3%), mycosis fungoides (3.9%), lichen planus (2.5%), nodular prurigo (2.2%), scleroderma (1.2%), alopecia areata (0.7%) and others. The median number of session received were 27 (range 1-252) for NBUVB, 30 (range 1-330) for topical PUVA, 30 (range 3-190) for oral PUVA and 24.5 (range 2-161) for UVA1. The acute adverse effects experienced by patients were erythema (18%), pruritus (16.3%), warmth (3.3%), blister formation (3.1%), cutaneous pain (2.4%), and xerosis (0.8%), skin swelling (0.7%) and phototoxicity (0.2%).
CONCLUSION: Narrow-band UVB was the most frequently prescribed phototherapy modality in our center. The most common indication for phototherapy in our setting was psoriasis. Acute adverse events occurred in a third of patients, although these side effects were mild.
METHODS: We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients.
RESULTS: The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed.
CONCLUSION: We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.