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Summary The Orchidaceae is of economic and ecological importance and constitutes ˜10% of all seed plant species. Here, we report a genome physical map for Cymbidium sinense, a well‐known species belonging to genus Cymbidium that has thousands of natural variation varieties of flower organs, flower and leaf colours and also referred as the King of Fragrance, which make it arose into a unique cultural symbol in China. The high‐quality chromosome‐scale genome assembly was 3.52 Gb in size, 29 638 protein‐coding genes were predicted, and evidence for whole‐genome duplication shared with other orchids was provided. Marked amplification of cytochrome‐ and photosystem‐related genes was observed, which was consistent with the shade tolerance and dark green leaves of C. sinense. Extensive duplication of MADS‐box genes, and the resulting subfunctional and expressional differentiation, was associated with regulation of species‐specific flower traits, including wild‐type and mutant‐type floral patterning, seasonal flowering and ecological adaption. CsSEP4 was originally found to positively regulate gynostemium development. The CsSVP genes and their interaction proteins CsAP1 and CsSOC1 were significantly expanded and involved in the regulation of low‐temperature‐dependent flowering. Important genetic clues to the colourful leaf traits, purple‐black flowers and volatile trait in C. sinense were also found. The results provide new insights into the molecular mechanisms of important phenotypic traits of Cymbidium and its evolution and serve as a powerful platform for future evolutionary studies and molecular breeding of orchids.

Objective To introduce posteromedial corner release with the knee in the figure‐of‐four position versus the conventional position for varus knee arthroplasty. Methods This is a retrospective study. From March 2015 to September 2019, a series of 123 patients (139 knees) with varus knee were randomly and blindly allocated to experimental group (60 patients; 68 knees) and control group (57 patients; 65 knees). Patients in experimental group underwent posteromedial corner release with the knee in the figure‐of‐four position; and patients in control group with the knee in the conventional position. If soft tissue balance was not completely achieved or the medial gap was still tight, an additional loosening technique were used to achieve symmetric medial and lateral space in both groups. Time for soft tissue balancing was defined as the time from the start of the spacer test to the end of the balance test. Length of release was defined as the distance from the osteotomy surface of the tibial plateau to the farthest structures released. The rating system of Hospital for Special Surgery (HSS) knee score was used to evaluate the clinical results. Quantitative variables were described as mean and standard deviation, and compared by one‐way analysis of variance. Results The mean age of experimental group and control group was 70.2 ± 8.7 years and 68.7 ± 6.2 years, respectively (P > 0.05). Preoperatively, the mean HSS score of the groups was 38.2 ± 11.3 and 39.1 ± 10.7, respectively (P > 0.05). The mean varus knee angle was 19.7° ± 9.3° and 19.3° ± 10.7°, respectively (P > 0.05). The mean time for soft tissue balancing was 8.4 ± 3.3 min and 11.3 ± 6.9 min in experimental and control group, respectively (P 0.05). No complications were found during the follow‐up time, and the clinical symptoms were observed to be significantly improved in the patients. Conclusion The posteromedial corner can be released more extensively and thoroughly when the knee is placed in the figure‐of‐four position during varus knee arthroplasty., Application of the figure‐of‐four position to release the soft tissue in the varus knee medial‐posterior corner in total knee arthroplasty. (A) The ankle was taken across the contralateral leg with the knee bent to 90° in the figure‐of‐ four position; (B) To visualize the posteromedial corner structures, the knee anteriorly was dislocated using a Hoffman hook placed behind the tibial plateau. Then, the structures of the posteromedial corner were released. (C, D) The other two patients who got the application of the figure‐of‐four position to release the soft tissue in the varus knee medial‐posterior corner in total knee arthroplasty.

Programmed cell death ligand 1 (PD‐L1), inducing T cell exhaustion to facilitate immune escape of tumor cells, is upregulated by interleukin 6 (IL‐6) in T cell lymphoma and ovarian cancer. The purpose of this study is to investigate the expression of IL‐6 and PD‐L1 in thyroid cancer, and whether IL‐6 regulates PD‐L1 expression. As a result, IL‐6 and PD‐L1 were highly expressed in thyroid cancer tissues. Multivariate logistic analysis showed that tumor size, distant metastasis, and risk stratification were significantly associated with IL‐6 expression (P, IL‐6 and PD‐L1 are highly expressed in thyroid cancer and correlate with disease aggressiveness. IL‐6 activates the MAPK and JAK‐STAT3 signaling pathways in thyroid cancer. In addition, IL‐6 promotes PD‐L1 transcription through the MAPK and JAK‐STAT3 signaling pathways, which function via transcription factors c‐Jun and stat3.

Fine particulate matter (PM2.5) is the primary air pollutant that is able to induce airway injury. Compelling evidence has shown the involvement of IL‐17A in lung injury, while its contribution to PM2.5‐induced lung injury remains largely unknown. Here, we probed into the possible role of IL‐17A in mouse models of PM2.5‐induced lung injury. Mice were instilled with PM2.5 to construct a lung injury model. Flow cytometry was carried out to isolate γδT and Th17 cells. ELISA was adopted to detect the expression of inflammatory factors in the supernatant of lavage fluid. Primary bronchial epithelial cells (mBECs) were extracted, and the expression of TGF signalling pathway‐, autophagy‐ and PI3K/Akt/mTOR signalling pathway‐related proteins in mBECs was detected by immunofluorescence assay and Western blot analysis. The mitochondrial function was also evaluated. PM2.5 aggravated the inflammatory response through enhancing the secretion of IL‐17A by γδT/Th17 cells. Meanwhile, PM2.5 activated the TGF signalling pathway and induced EMT progression in bronchial epithelial cells, thereby contributing to pulmonary fibrosis. Besides, PM2.5 suppressed autophagy of bronchial epithelial cells by up‐regulating IL‐17A, which in turn activated the PI3K/Akt/mTOR signalling pathway. Furthermore, IL‐17A impaired the energy metabolism of airway epithelial cells in the PM2.5‐induced models. This study suggested that PM2.5 could inhibit autophagy of bronchial epithelial cells and promote pulmonary inflammation and fibrosis by inducing the secretion of IL‐17A in γδT and Th17 cells and regulating the PI3K/Akt/mTOR signalling pathway.

Background The role of atopy patch test (APT) in the diagnosis of food allergy (FA) remains largely controversial. In our meta-analysis, we aimed to evaluate the accuracy of APT for diagnosing FA in children. Methods Pubmed, Embase and Cochrane Library were searched for studies regarding the diagnostic value of APT for FA in children compared to oral food challenge (double-blind placebo-controlled food challenge and/or open food challenge). The last search was conducted on November 11, 2017. Two reviewers independently screened relevant studies and assessed the quality by QUADAS-2. Meta-analysis was performed to calculate the pooled sensitivity, specificity, DOR (diagnostic odds ratio), PLR (positive likelihood ratio), NLR (negative likelihood ratio) with their 95% confidence intervals (CIs). Subgroup analyses were conducted according to different food allergens, atopic dermatitis, gastrointestinal symptoms, and age younger than 3 years. Results Forty-one studies were included in the meta-analysis. The pooled sensitivity, specificity, PLR, NLR and DOR were 50.30% (95% CI 48.40%-52.30%), 86.60% (95% CI 85.30%-87.80%), 3.405 (95% CI 2.594-4.470), 0.545 (95% CI 0.469-0.634) and 7.528 (95% CI 5.507-11.206), respectively. However, for children with FA-related gastrointestinal symptoms, the pooled sensitivity and specificity were 57.40% (95% CI 52.10%-62.50%) and 91.50% (95% CI 88.30%-94.10%) respectively. Conclusions Our findings suggest that APT is specific but not sensitive for diagnosing FA in children, especially in children with FA-related gastrointestinal symptoms.

Summary Currently, COVID‐19 has been reported in nearly all countries globally. To date, little is known about the viral shedding duration, clinical course, and treatment efficacy of COVID‐19 near Hubei Province, China. This multicentre, retrospective study was performed in 12 hospitals in Henan and Shaanxi Provinces from January 20 to February 8, 2020. Clinical outcomes were followed up until March 26, 2020. The viral shedding duration, full clinical course, and treatment efficacy were analysed in different subgroups of patients. A total of 149 COVID‐19 patients were enrolled. The median age was 42 years, and 61.1% (91) were males. Of them, 133 (89.3%) had fever, 131 of 144 (91%) had pneumonia, 27 (18.1%) required intensive care unit (ICU) management, 3 (2%) were pregnant, and 3 (2%) died. Two premature newborns were negative for SARS‐CoV‐2. In total, the median SARS‐CoV‐2 shedding period and clinical course were 12 (IQR: 9‐17; mean: 13.4, 95% CI: 12.5, 14.2) and 20 (IQR: 16‐24; mean: 21.2, 95% CI: 20.1, 22.3) days, respectively; and ICU patients had longer median viral shedding periods (21 [17‐24] vs 11 [9‐15]) and clinical courses (30 [22‐33] vs 19 [15.8‐22]) than non‐ICU patients (both p0.05) in real‐life settings. In conclusion, the viral shedding duration and clinical course in Henan and Shaanxi Provinces were shorter than those in Hubei Province, and current antiviral therapies were ineffective for shortening viral shedding duration and clinical course in real‐world settings. These findings expand our knowledge of the SARS‐CoV‐2 infection and may be helpful for management of the epidemic outbreak of COVID‐19 worldwide. Further studies concerning effective antiviral agents and vaccines are urgently needed.

Metformin is always used as the baseline antidiabetic therapy for patients with type 2 diabetes mellitus (T2DM) and hyperuricemia. Metformin is excreted into urine through active secretion mediated by rOCTs and rMATE1.The aim of this study was to identify the effects of high uric acid on the disposition and its mechanism. For the in vivo study, a hyperuricemic animal model was induced by intraperitoneal injection of potassium oxonate (250 mg/kg) in rats. Metformin (100 mg/kg) was administered orally to investigate the pharmacokinetics in control and hyperuricemic rats, respectively. For the in vitro study, HEK293 and HepaRG cells were used to investigate the effect of uric acid (15 mg/dl) on the expression of OCT1, OCT2 and MATE1 and the disposition of metformin, respectively. The in vivo study showed that the AUC0 → 600 of metformin was significantly decreased by 33.3%, whereas the cumulative urinary excretion of metformin was increased by 25.4% in hyperuricemic rats compared with that in control rats. The renal rOCT1, rOCT2 and rMATE1 and hepatic rMATE1 levels were increased in hyperuricemic rats compared with those in control rats, respectively. The in vitro study showed that uric acid could upregulate the expression of OCT2 and MATE1 in HEK293 cells and MATE1 in HepaRG cells and increase the intracellular metformin concentration in these two cell lines. These results demonstrated that a high uric acid level promoted urinary metformin excretion and decreased the plasma metformin concentration; the in vivo and in vitro studies provided a possible explanation being that high uric acid could upregulate the expression of renal metformin transporters OCTs and MATE1.

The kidney plays a major part in the elimination of many drugs and their metabolites, and drug-induced kidney injury commonly alters either glomerular filtration or tubular transport, or both. However, the renal excretion pathway of drugs has not been fully elucidated at different stages of renal injury. This study aimed to evaluate the alteration of renal excretion pathways in gentamicin (GEN)-induced renal injury in rats. Results showed that serum cystatin C, creatinine and urea nitrogen levels were greatly increased by the exposure of GEN (100mg kg(-1)), and creatinine concentration was increased by 39.7% by GEN (50mg kg(-1)). GEN dose-dependently upregulated the protein expression of rOCT1, downregulated rOCT2 and rOAT1, but not affected rOAT2. Efflux transporters, rMRP2, rMRP4 and rBCRP expressions were significantly increased by GEN(100), and the rMATE1 level was markedly increased by GEN(50) but decreased by GEN(100). GEN(50) did not alter the urinary excretion of inulin, but increased metformin and furosemide excretion. However, GEN(100) resulted in a significant decrease of the urinary excretion of inulin, metformin and p-aminohippurate. In addition, urinary metformin excretions in vivo were significantly decreased by GEN(100), but slightly increased by GEN(50). These results suggested that GEN(50) resulted in the induction of rOCTs-rMATE1 and rOAT3-rMRPs pathway, but not changed the glomerular filtration rate, and GEN(100)-induced acute kidney injury caused the downregulated function of glomerular filtration -rOCTs-rMATE1 and -rOAT1-rMRPs pathway. The exposure of GEN (100 mg kg(-1)) caused in an acute kidney injury. The negligible tubular injury and increased creatinine level were observed at 50 mg kg(-1) dose. GEN (50) did not alter the urinary excretion of inulin, but increased metformin and furosemide excretion. However, GEN (100) resulted in a significant decrease of the urinary excretion of inulin, metformin and p-aminohippurate. These results demonstrated that the function of rOCTs-rMATE1 and rOAT3-rMRPs pathways were significantly upregulated by GEN(50), and glomerular filtration-rOCTs-rMATE1 and -rOAT1-rMRPs pathways were markedly downregulated by GEN(100).

Drug interactions are one of the commonest causes of side effects, particularly in long-term therapy. The aim of the current study was to investigate the possible effects of metoprolol on the pharmacokinetics of metformin in rats and to clarify the mechanism of drug interaction. In this study, rats were treated with metformin alone or in combination with metoprolol. Plasma, urine and tissue concentrations of metformin were determined by HPLC. Western blotting and real-time qPCR were used to evaluate the expression of rOCTs and rMATE1. The results showed that, after single or 7-day repeated administration, the plasma concentrations of metformin in the co-administration group were significantly decreased compared with that in the metformin group. However, the parameter V/F of metformin in the co-administration group was markedly increased compared with that in the metformin group. The hepatic, renal and muscular Kp of metformin were markedly elevated after co-administration with metoprolol. Consistently, metformin uptake in rat kidney slices was significantly induced by metoprolol. In addition, multiple administrations of metoprolol significantly reduced the expression of rMATE1 in rat kidney as well as the urinary excretion of metformin. Importantly, after long-term administration, lactic acid and uric acid levels in the co-administration group were increased by 25% and 26%, respectively, compared with that in the metformin group. These results indicate that metoprolol can decrease the plasma concentration of metformin via the induction of hepatic, renal and muscular uptake, and long-term co-administration of metformin and metoprolol can cause elevated lactic acid and uric acid levels. Copyright © 2016 John Wiley & Sons, Ltd.

We reconstructed a phylogenetic tree of Chinese vascular plants (Tracheophyta) using sequences of the chloroplast genes atpB, matK, ndhF, and rbcL and mitochondrial matR. We produced a matrix comprising 6098 species and including 13 695 DNA sequences, of which 1803 were newly generated. Our taxonomic sampling spanned 3114 genera representing 323 families of Chinese vascular plants, covering more than 93% of all genera known from China. The comprehensive large phylogeny supports most relationships among and within families recognized by recent molecular phylogenetic studies for lycophytes, ferns (monilophytes), gymnosperms, and angiosperms. For angiosperms, most families in Angiosperm Phylogeny Group IV are supported as monophyletic, except for a paraphyletic Dipterocarpaceae and Santalaceae. The infrafamilial relationships of several large families and monophyly of some large genera are well supported by our dense taxonomic sampling. Our results showed that two species of Eberhardtia are sister to a clade formed by all other taxa of Sapotaceae, except Sarcosperma. We have made our phylogeny of Chinese vascular plants publically available for the creation of subtrees via SoTree (http://www.darwintree.cn/flora/index.shtml), an automated phylogeny assembly tool for ecologists.

Isoniazid (INH) and rifampicin (RFP), two front-line drugs used in tuberculosis therapy, may lead to seriously hepatotoxicity. The current study was carried out to investigate the hepatoprotective effects of hesperidin against INH- and RFP-induced oxidative damage. The liver injury animal model of rats was induced by INH (75 mg/kg) and RFP (150 mg/kg) coadministration for 7 days, and hesperidin, at the dose of 50, 100, and 200 mg/kg, was orally administered to rats 2 h before INH and RFP administration. The biochemical and pathologic examinations were performed after rats were sacrificed. Moreover, the serum and liver glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde (MDA), GSH peroxidase, and GSSG reductase were determined by test kits, and the expression of multidrug resistance proteins 2 (Mrp2) was determined by Western blotting and immunohistochemistry. The results showed that hesperidin significantly alleviated liver injury as indicated by the decreased levels of ALT, AST, bilirubin, total bile acid, and glutathione peroxidase and the increased levels of the GSH/GSSG ratio and the expression of Mrp2. Moreover, hesperidin could effectively reduce the pathological tissue damage. These results indicate that hesperidin can attenuate INH-and RFP-induced oxidative damage, and the underlying mechanism may have correlation with its effect on the upregulation of Mrp2. (C) 2016 Wiley Periodicals, Inc.

We estimated the molecular phylogenetic relationships of the Chinese members of the family Orchidaceae. Within the Tree of Life for the Genera of Chinese Vascular Plants using atpB, rbcL, matK, ndhF, and matR, the currently delimited subfamilies, tribes, and subtribes are highly supported as monophyletic except for the perplexing Epidendroideae. Five genes (rbcL, matK, psaB, ycf1, and Xdh), which are more universally used in Orchidaceae, were further analyzed to reconstruct the phylogeny of Epidendroideae. The reconstructed trees were in strong agreement and showed significant support for the tribal and subtribal clades. Based on the highly supported circumscription and arrangement of the suprageneric levels in the Tree of Life and reconstructed trees, we have proposed a new phylogenetic classification of Chinese Orchidaceae that includes five subfamilies, 17 tribes, and 21 subtribes.

A simple, specific and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of metoprolol (MET), -hydroxymetoprolol (HMT) and O-desmethylmetoprolol (DMT) in rat plasma. The plasma samples were prepared by protein precipitation, then the separation of the analytes was performed on an Agilent HC-C-18 column (4.6x250mm, 5 mu m) at a flow rate of 1.0mL/min, and post-column splitting (1:4) was used to give optimal interface flow rates (0.2mL/min) for MS detection; the total run time was 8.5min. Mass spectrometric detection was achieved using a triple-quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 3.42-7000ng/mL for MET, 2.05-4200ng/mL for HMT and 1.95-4000ng/mL for DMT. The analytical method was successfully applied to herb-drug interaction study of MET and breviscapine after administration of breviscapine (12.5mg/kg) and MET (40mg/kg). The results suggested that breviscapine have negligible effect on pharmacokinetics of MET in rats; the information may be beneficial for the application of breviscapine in combination with MET in clinical therapy. Copyright (c) 2015 John Wiley & Sons, Ltd.

Periictal autonomic dysregulation is best studied using a "polygraphic" approach: electroencephalography ([EEG]), 3-channel electrocardiography [ECG], pulse oximetry, respiration, and continuous noninvasive blood pressure [BP]), which may help elucidate agonal pathophysiologic mechanisms leading to sudden unexpected death in epilepsy (SUDEP). A number of autonomic phenomena have been described in generalized tonic-clonic seizures (GTCS), the most common seizure type associated with SUDEP, including decreased heart rate variability, cardiac arrhythmias, and changes in skin conductance. Postictal generalized EEG suppression (PGES) has been identified as a potential risk marker of SUDEP, and PGES has been found to correlate with post-GTCS autonomic dysregulation in some patients. Herein, we describe a patient with a GTCS in whom polygraphic measurements were obtained, including continuous noninvasive blood pressure recordings. Significant postictal hypotension lasting >60 s was found, which closely correlated with PGES duration. Similar EEG changes are well described in hypotensive patients with vasovagal syncope and a similar vasodepressor phenomenon, and consequent cerebral hypoperfusion may account for the PGES observed in some patients after a GTCS. This further raises the possibility that profound, prolonged, and irrecoverable hypotension may comprise one potential SUDEP mechanism.

The epilepsy community increasingly recognizes the need for a modern classification system that can also be easily integrated with effective informatics tools. The 2010 reports by the United States President's Council of Advisors on Science and Technology (PCAST) identified informatics as a critical resource to improve quality of patient care, drive clinical research, and reduce the cost of health services. An effective informatics infrastructure for epilepsy, which is underpinned by a formal knowledge model or ontology, can leverage an ever increasing amount of multimodal data to improve (1) clinical decision support, (2) access to information for patients and their families, (3) easier data sharing, and (4) accelerate secondary use of clinical data. Modeling the recommendations of the International League Against Epilepsy (ILAE) classification system in the form of an epilepsy domain ontology is essential for consistent use of terminology in a variety of applications, including electronic health records systems and clinical applications. In this review, we discuss the data management issues in epilepsy and explore the benefits of an ontology-driven informatics infrastructure and its role in adoption of a “data-driven” paradigm in epilepsy research.

The 2010 International League Against Epilepsy (ILAE) classification and terminology commission report proposed a much needed departure from previous classifications to incorporate advances in molecular biology, neuroimaging, and genetics. It proposed an interim classification and defined two key requirements that need to be satisfied. The first is the ability to classify epilepsy in dimensions according to a variety of purposes including clinical research, patient care, and drug discovery. The second is the ability of the classification system to evolve with new discoveries. Multi-dimensionality and flexibility are crucial to the success of any future classification. In addition, a successful classification system must play a central role in the rapidly growing field of epilepsy informatics. An epilepsy ontology, based on classification, will allow information systems to facilitate data-intensive studies and provide a proven route to meeting the two foregoing key requirements. Epilepsy ontology will be a structured terminology system that accommodates proposed and evolving ILAE classifications, the NIH/NINDS Common Data Elements, the ICD systems and explicitly specifies all known relationships between epilepsy concepts in a proper framework. This will aid evidence based epilepsy diagnosis, investigation, treatment and research for a diverse community of clinicians and researchers. Benefits range from systematization of electronic patient records to multi-modal data repositories for research and training manuals for those involved in epilepsy care. Given the complexity, heterogeneity and pace of research advances in the epilepsy domain, such an ontology must be collaboratively developed by key stakeholders in the epilepsy community and experts in knowledge engineering and computer science.

A novel pepsin inhibitor was isolated from Coriolus versicolor. The purification was carried out by a 2-step ultrafiltration followed by DEAE-52 and Mono Q ion-exchange chromatography. SDS-PAGE and gel filtration chromatography analysis showed that the isolated inhibitor was a 22.3 kDa protein with a single subunit. Heat stability of this inhibitor was estimated and only 7% of its inhibitory activity lost after treatment at 98 °C. The inhibitor was more specific against pepsin than several other proteases. The dissociation constant (Ki) and concentration required for 50% pepsin inhibition (IC50) were 5.84 × 10−5 M and 26.26 μg/mL, respectively. Apparent decrease of α-helix and increase of random coil were observed in the circular dichroism spectra of pepsin when an equimolar amount of the inhibitor was added. The inhibition mechanism of this inhibitor differs from the reported aspartic protease inhibitors, according to the secondary structure and the kinetic studies of this inhibitor. Practical Application: Specific inhibitors of aspartic protease have a wide range of applications in therapeutics (Coates and others 2002). Meanwhile, aspartic protease inhibitors can also be applied in agriculture and food industry, for example, to increase the stability of wheat gluten, beer foam.

In this paper, notions of fuzzy closure system and fuzzy closure L—system on L—ordered sets are introduced from the fuzzy point of view. We first explore the fundamental properties of fuzzy closure systems. Then the correspondence between fuzzy closure systems (fuzzy closure L—systems) and fuzzy closure operators is established. Finally, we study the connections between fuzzy closure systems and fuzzy Galois connections. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Background and Objectives Hyaluronic acid binding protein 1 (HABP1), a family of proteins interacting with hyaluronan (HA), had been associated with cell adhesion and tumor invasion. The aim of this study was to investigate the correlation between clinicopathologic factors and patient survival time with the expression of HABP1 in breast cancer patients. Methods Expression of HABP1 mRNA and protein were detected with real-time quantitative PCR and immunohistochemical staining in 63 breast cancer and non-cancerous matched tissues. Results The mRNA expression level of HABP1 was unrelated to the patient's age, tumor size, histological grade, TNM stage. However, it proved to be positively related to axillary nodes metastasis (P = 0.008). Furthermore, it was shown that the survival rate of patients with low HABP1 expression was significantly higher than that of patients with high HABP1 expression (P = 0.025). Multivariate analysis revealed that HABP1 mRNA expression level was a significant factor for predicting prognosis (P = 0.022). The immunohistochemistry results showed that the expression level of HABP1 in breast cancer cells was higher than that in normal breast cells. Conclusion HABP1 might be an independent predictive factor for breast cancer prognosis and up-regulation of HABP1 might play an important role in the metastasis of breast cancer. J. Surg. Oncol. 2009;100:382–386. © 2009 Wiley-Liss, Inc.