Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein-or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ;2 3 10 5 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.

BackgroundOrchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied.ResultsSeveral molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR). A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI) genes (OnTI1, OnTI2 and OnTI3), which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses.ConclusionsBy combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.

Introduction: Multiple system atrophy (MSA) has detrimental effects on swallowing function. The swallowing function of patients with MSA has not been systematically characterized and the underlying pathophysiological mechanisms of dysphagia remain poorly understood.Objectives: To investigate the characteristics of swallow function in MSA using high-resolution manofluorography (HRMF).Methods: We conducted a retrospective review of twenty-five MSA patients who underwent HRMF from 2016 to 2017. HRMF was utilized on patients with only oral diet (Functional Oral Intake Scale (FOIS) >3). Pharyngoesophageal and proximal esophageal pressure profiles were evaluated and compared to established normative data. The frequency and characteristics of upper esophageal sphincter (UES) and proximal esophageal abnormalities during rest and swallow were calculated.Results: The ages of patient cohort in our study ranged from 48–81 years (median 65 years) with male predominance (68%). We observed a distinct abnormal deglutitive proximal esophageal contraction (ADPEC) in 14 (56% of patients), which appears to reflect a discoordinated response of the striated muscle esophagus. Deficient UES relaxation duration, impaired UES relaxation, hypertensive resting UES pressure and hypotensive resting UES pressure were detected in 8 patients (32%), 3 patients (12%), 1 patient (4%), and 11 patients (44%) respectively.Conclusions: In patients with MSA, abnormal UES resting pressure is common. A discoordinated proximal esophageal pressure response was identified and may be a pathognomonic manometry finding for MSA. These findings may serve as indications of early stage swallowing dysfunction in patients with MSA.

Purpose: Obstructive sleep apnea (OSA) is characterized by recurring hypoxic-apneic events during sleep, and labyrinthine vascular compromise is a pathophysiologic hallmark of idiopathic sudden sensorineural hearing loss (ISSNHL). Some reports have discussed the relationship between OSA and hearing impairment; however, few have examined hearing prognosis in OSA and patients without OSA with ISSNHL. We aimed to investigate clinical manifestations of ISSNHL in patients with OSA, including severity of hearing loss and response to treatment. Patients and Methods: A case-control study was conducted by extracting data from the sleep center and cochlea center databases of the Chang Gung Memorial Hospital. A retrospective chart review was performed to include confirmed adult OSA patients diagnosed with unilateral ISSNHL. Age and sex-matched patients without OSA with ISSNHL were enrolled as controls. Pure-tone average (PTA) thresholds were measured at specific frequencies. Changes in PTA before and after standard treatment with oral prednisolone (1mg/kg/day for 5 days, then tapered) and between participants with OSA and without OSA were compared. Standard treatment was given to all ISSNHL patients. Results: Twenty-eight out of 8500 (0.33%) OSA patients experienced subsequent ISSNHL in 9 years. Patients with OSA (n=28) had poorer high-frequency perception in the unaffected ear than the patients without OSA (n=120), although the difference was not significant. Hearing in the affected ear among patients with OSA was comparable to that patients without OSA at individual frequencies and average, suggesting no difference in hearing loss in the affected ear between the two groups. In terms of high-frequencies (4000 and 8000 Hz) perception, patients with OSA had significantly poorer responses to steroid treatment than patients without OSA. Conclusion: ISSNHL may be one of the auditory complications associated with OSA. Patients with OSA had poorer prednisolone related hearing improvement in high frequencies than patients without OSA. Despite study limitations, OSA-related hypoxia and snoring noise is hazardous to hearing and standard treatments with CPAP is suggested in OSA patients for both holistic and auditory health.

We developed a gene gun method for the transfer of human agouti signalling protein (ASP) cDNA to alter rat skin colour in vivo. Human ASP cDNA was cloned into a modified cytomegalovirus plasmid and delivered to the skin of LongEvans rats by gene gun bombardment. Skin pigmentation, body weight and blood sugar of ASP cDNA-transfected rats were recorded against the control group, which were injected with plasmids encoding for green fluorescent protein. The treated skin showed lighter skin colour after 3 days of ASP gene transfection. This depigmentation effect was most prominent on day 14 and the skin gradually returned to its original pigmentation by day 28. Successful transfection of ASP gene in skin and hair follicles, as well as downregulation of melanocortin-1 receptor (MC1R) and tyrosinase expression upon treatment, was confirmed using immunohistochemistry and Western blot analysis. Body weight and blood sugar in the treated rats did not show statistically significant differences as compared to control groups. These observations demonstrate that gene transfer using the gene gun method can induce high cutaneous ASP production and facilitate a switch from dark to fair colour without systemic pleiotropic effects. Such a colour switch may be that ASP is acting in a paracrine fashion. In addition, this study verifies that ASP exerts its functions by acting as an independent ligand that downregulates the melanocyte MC1R and tyrosinase protein in an in vivo system. Our result offers new, interesting insights about the effect of ASP on pigmentation, providing a novel approach to study the molecular mechanisms underlying skin melanogenesis.