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For floral induction in adult citrus, low temperature is one of the most important environmental factors. FLOWERING LOCUS C (FLC) plays a very important role in low-temperature-induced Arabidopsis flowering by repressed FLC expression under exposure to prolonged low-temperature conditions. However, little is known about the FLC regulation mechanism in perennial woody plants such as citrus. In this study, the functions of citrus FLC homolog (PtFLC) were investigated by ectopic expression in Arabidopsis. Transcription factor of homeodomain leucine zipper I (HD-ZIP I) as an upstream regulator of PtFLC was identified by yeast one-hybrid screen to regulate its transcription. The HD-ZIP I transcription factor was highly homologous to Arabidopsis ATHB13 and thus was named PtHB13. Ectopically expressed PtHB13 inhibited flowering in transgenic Arabidopsis. Furthermore, the expression of PtFLC and PtHB13 showed a seasonal change during the floral induction period and was also affected by low temperature. Thus, we propose that PtHB13 binds to PtFLC promoter to regulate its activity during the citrus floral induction process.

For flowering plants, flowering transition is induced in response to endogenous and extrinsic environmental signals. During this process, the members of the APETALA family from MADS-box transcription factors play an important role. Here, to better understand the molecular mechanism of APETALA1 (AP1) in the regulation of citrus flowering, an APETALA1-like homologue gene (PtAP1) was isolated from an early flowering trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf) and normal trifoliate orange. The results indicated that the sequence of PtAP1 was not any differences in two genotypes of trifoliate orange. Sequence alignment and phylogenetic analysis revealed that the PtAP1 fell in the euAP1 clade. Specific spatial and/or temporal patterns of PtAP1 were investigated in precocious trifoliate orange by real-time PCR. The results showed that PtAP1 was detected in almost all reproductive tissues, but only faint expression in vegetative tissues. Moreover, the expression pattern of PtAP1 was closely correlated with floral induction and flowering at phase change stage. Transcription of PtAP1 in Arabidopsis ap1-1 mutant showed partially rescued AP1 function. Over-expression of PtAP1 in wild-type Arabidopsis resulted in early flowering and changed architecture. About 1KB promoter sequence of PtAP1 was obtained by chromosome walking from two genotypes of trifoliate orange, there was also not nucleotide difference in the two-promoter sequence. Meanwhile, a number of putative cis-regulatory elements were predicted in PtAP1 promoter by bioinformatics analysis. To determine the expression of PtAP1 promoter, GUS reporter gene was transformed into wild-type Arabidopsis under the control of PtAP1 promoter. The results revealed that the PtAP1 promoter had strong activity and showed developmental phase and tissue specificity, which might be ideal for citrus genetic improvement. In addition, a yeast one-hybrid assay indicated that PtAP1 may be interacted with some flowering proteins, which have not yet been functionally characterized in citrus. These findings suggested that PtAP1 may play an important role during the early flowering of precocious trifoliate orange.

Keloids is a fibroproliferative disease. The incidence of keloids among Asians has not been thoroughly studied. The objective of this study is to determine the incidence of keloids in Taiwan, which mainly consists of ethnic Chinese. Furthermore, we want to determine the comorbidity rate of other fibrosis-related diseases among keloid patients. This study was based on the National Health Insurance Research Database, which contains the data of 1 million randomly selected patients. Multivariate logistic regression analyses were employed to estimate the relative odds of keloids as a function of fibrosis-related diseases. The annual keloid incidence rate in Taiwan was 0.15 % for the general population. With a 1.33 ratio, women outnumbered men. Women with uterine leiomyoma have a 2.25-fold greater risk of keloids, compared with women without leiomyoma. We concluded that keloid incidence in Taiwan is approximately 0.15 %. Women with leiomyoma have a greater risk of keloids, this implicates that both diseases share a common etiopathological pathway.

The transition from vegetative to reproductive growth in perennial woody plants does not occur until after several years of repeated seasonal changes and alternative growth. To better understand the molecular basis of flowering regulation in citrus, a MADS-box gene was isolated from trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.). Sequence alignment and phylogenetic analysis showed that the MADS-box gene is more closely related to the homologs of the AGAMOUS-LIKE 24 (AGL24) lineage than to any of the other MADS-box lineages known from Arabidopsis; it is named PtAGL24. Expression analysis indicated that PtAGL24 was widely expressed in the most organs of trifoliate orange, with the higher expression in mature flowers discovered by real-time PCR. Ectopic expression of PtAGL24 in wild-type Arabidopsis promoted early flowering and caused morphological changes in class I transgenic Arabidopsis. Yeast two-hybrid assay revealed that PtAGL24 interacted with Arabidopsis AtAGL24 and other partners of AtAGL24, suggesting that the abnormal morphology of PtAGL24 overexpression in transgenic Arabidopsis was likely due to the inappropriate interactions between exogenous and endogenous proteins. Also, PtAGL24 interacted with SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (PtSOC1) and APETALA1 (PtAP1) of citrus. These results suggest that PtAGL24 may play an important role in the process of floral transition but may have diverse functions in citrus development.

Most of what we know about the molecular genetics of flowering time regulation comes from studies in the model plants. However, little is known about the regulation of flowering transition in perennial species or in species with particular growth habits compared with model plants. Here comparative transcriptome and proteome profiling of spring shoots was performed on an early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata) and its wild-type. A total of 19,215 read-mapped genes were observed in two genotypes by RNA-Seq. Of these, 1450 and 1159 genes were specifically observed in the mutant and wild-type libraries, respectively. There were 355 genes that were expressed differently in the two genotypes. A total of 1664 proteins were identified by the iTRAQ technique, and transcript and protein profiles were parallel across the time course for 50% of the comparisons made, but divergent patterns were also observed, indicative of post-transcriptional events. In addition, a global survey of messenger RNA splicing events identified 16,343 splice junctions among 12,688 genes and showed that alternative 3' splice is the most prevalent form of alternative splicing. We further identify 5698 novel transcribed regions that are not overlapping with annotated citrus transcriptome in two genotypes. Understanding of the regulation of flowering transition in citrus can help in the development of new genetic or management strategies to improve fruit production.

Summary Background Diabetic foot ulcers (DFUs) superimposed by infection and ischaemia may result in amputation without prompt and adequate management. We investigated whether the diabetic foot ulcer treatment programme (DFUTP) involving immediate debridement within 12 h, flap coverage and/or revascularisation improved the outcome of patients with infected DFUs. Method Between 2006 and 2009, we randomly enrolled 350 patients in the DFUTP group and compared them with control patients (the non-DFUTP group, n = 386) in Taiwan. Inclusion criteria consisted of infected diabetic foot ulcers with or without ischaemia. The risk factors, dynamics and outcome of amputation and re-amputation were analysed in terms of patient demographics, glycaemic control and infection. Result The results of logistic regression analyses indicated that risk factors of amputation in both groups were HbA1c (odds ratio (OR) = 1.63, 95% confidence interval (CI) 1.31–2.02) and C reactive protein (OR = 1.12, 95% CI 1.01–1.24). The DFUTP group showed a lower amputation rate than the non-DFUTP group ( p = 0.001). The association between the amputation and University of Texas (UT) classification was not statistically significant. The Kaplan–Meier estimate showed that the time to complete recovery of the sugar level in the DFUTP group was faster than in the non-DFUTP group ( p = 0.001). For patients at stage D, the hospital stay in the non-DFUTP group was longer than in the DFUTP group ( p = 0.014). Conclusion The DFUTP provides an effective treatment programme for decreasing the amputation rate with infected DFUs. Immediate debridement and flap reconstruction decrease the amputation and re-amputation rate respectively.

To isolate differentially expressed genes during the juvenile-to-adult phase transition of an early-flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata), suppression subtractive hybridization was performed. In total, 463 cDNA clones chosen by differential screening of 1,920 clones were sequenced and 178 differentially expressed genes were identified, among which 41 sequences did not match any known nucleotide sequence. Analysis of expression profiles of the differentially expressed genes through hybridization on customized chips revealed their expression change was associated with the phase transition from juvenile to adult in the mutant. Open reading frames of nine selected genes were successfully determined by rapid amplification of cDNA ends. Expression analysis of these genes by real-time RT-PCR showed that transcript levels of several genes were associated with floral induction and inflorescence development. Among these genes, HM596718, a sequence sharing a high degree of similarity with Arabidopsis EARLY FLOWERING 5 (AtELF5) was discovered. Real-time PCR and in situ hybridization indicated its expression pattern was closely correlated with floral induction and flowering of the mutant. Ectopic expression of the gene in Arabidopsis caused early flowering; however, its functional characterization is different than the role of AtELF5 observed in Arabidopsis. A yeast two-hybrid assay indicated that PtELF5 significantly interacted with DUF1336 domain of a hypothetical protein, which has not yet been functionally characterized in woody plants. These findings suggest that PtELF5 may be a novel gene that plays an important role during the early flowering of precocious trifoliate orange.

Obstructive sleep apnea (OSA) is a major concern in modern medicine; however, it is difficult to diagnose. Screening questionnaires such as the Berlin questionnaire, Rome questionnaire, and BASH'IM score are used to identify patients with OSA. However, the sensitivity and specificity of these tools are not satisfactory. We aim to introduce an artificial intelligence method to screen moderate to severe OSA patients (apnea-hypopnea index ≧15).One hundred twenty patients were asked to complete a newly developed questionnaire before undergoing an overnight polysomnography (PSG) study. One hundred ten validated questionnaires were enrolled in this study. Genetic algorithm (GA) was used to build the five best models based on these questionnaires. The same data were analyzed with logistic regression (LR) for comparison.The sensitivity of the GA models varied from 81.8% to 88.0%, with a specificity of 95% to 97%. On the other hand, the sensitivity and specificity of the LR model were 55.6% and 57.9%, respectively.GA provides a good solution to build models for screening moderate to severe OSA patients, who require PSG evaluation and medical intervention. The questionnaire did not require any special biochemistry data and was easily self-administered. The sensitivity and specificity of the GA models are satisfactory and may improve when more patients are recruited.

MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes.

This paper formally presents the resident physician scheduling problem, which is one of the most important scheduling problems in hospital. The resident physician scheduling problem is characterized as satisfying the fair schedule constraint, the physician specification constraint and the safe schedule constraint simultaneously. To minimize the penalties from violating the constraints, this study adopts the evolutionary approach to propose a genetic algorithm for solving the problems. In addition the well-known genetic operators, this study proposed a new mutation operator called dynamic mutation for solving the resident physician scheduling problem. The experimental results show that the proposed algorithm performs well in searching optimal schedules.

Background After several years in the juvenile phase, trees undergo flowering transition to become mature (florally competent) trees. This transition depends on the balanced expression of a complex network of genes that is regulated by both endogenous and environmental factors. However, relatively little is known about the molecular processes regulating flowering transition in woody plants compared with herbaceous plants. Results Comparative transcript profiling of spring shoots after self-pruning was performed on a spontaneously early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata) with a short juvenile phase and the wild-type (WT) tree by using massively parallel signature sequencing (MPSS). A total of 16,564,500 and 16,235,952 high quality reads were obtained for the WT and the mutant (MT), respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed genes in the MT (31,468) was larger than in the WT (29,864), suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts revealed that 2735 genes had more than twofold expression difference in the MT compared with the WT. In addition, we identified 110 citrus flowering-time genes homologous with known elements of flowering-time pathways through sequencing and bioinformatics analysis. These genes are highly conserved in citrus and other species, suggesting that the functions of the related proteins in controlling reproductive development may be conserved as well. Conclusion Our results provide a foundation for comparative gene expression studies between WT and precocious trifoliate orange. Additionally, a number of candidate genes required for the early flowering process of precocious trifoliate orange were identified. These results provide new insight into the molecular processes regulating flowering time in citrus.