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1.  An atlas of DNA methylomes in porcine adipose and muscle tissues 
Nature communications  2012;3:850.
It is evident that epigenetic factors, especially DNA methylation, play essential roles in obesity development. Using pig as a model, here we investigated the systematic association between DNA methylation and obesity. We sampled eight variant adipose and two distinct skeletal muscle tissues from three pig breeds living within comparable environments but displaying distinct fat level. We generated 1,381 gigabases (Gb) of sequence data from 180 methylated DNA immunoprecipitation (MeDIP) libraries, and provided a genome-wide DNA methylation map as well as a gene expression map for adipose and muscle studies. The analysis showed global similarity and difference among breeds, sexes and anatomic locations, and identified the differentially methylated regions (DMRs). The DMRs in promoters are highly associated with obesity development via expression repression of both known obesity-related genes and novel genes. This comprehensive map provides a solid basis for exploring epigenetic mechanisms of adipose deposition and muscle growth.
PMCID: PMC3508711  PMID: 22617290
2.  Genome-Wide Mapping of DNA Methylation in Chicken 
PLoS ONE  2011;6(5):e19428.
Cytosine DNA methylation is an important epigenetic modification termed as the fifth base that functions in diverse processes. Till now, the genome-wide DNA methylation maps of many organisms has been reported, such as human, Arabidopsis, rice and silkworm, but the methylation pattern of bird remains rarely studied. Here we show the genome-wide DNA methylation map of bird, using the chicken as a model organism and an immunocapturing approach followed by high-throughput sequencing. In both of the red jungle fowl and the avian broiler, DNA methylation was described separately for the liver and muscle tissue. Generally, chicken displays analogous methylation pattern with that of animals and plants. DNA methylation is enriched in the gene body regions and the repetitive sequences, and depleted in the transcription start site (TSS) and the transcription termination site (TTS). Most of the CpG islands in the chicken genome are kept in unmethylated state. Promoter methylation is negatively correlated with the gene expression level, indicating its suppressive role in regulating gene transcription. This work contributes to our understanding of epigenetics in birds.
PMCID: PMC3088676  PMID: 21573164
3.  Impact of enteral nutrition on energy metabolism in patients with Crohn’s disease 
AIM: To investigate the impact of enteral nutrition (EN) on the body composition and metabolism in patients with Crohn’s disease (CD).
METHODS: Sixty-one patients diagnosed with CD were enrolled in this study. They were given only EN (enteral nutritional suspension, TPF, non-elemental diet) support for 4 wk, without any treatment with corticosteroids, immunosuppressive drugs, infliximab or by surgical operation. Body composition statistics such as weight, body mass index, skeletal muscle mass (SMM), fat mass, protein mass and inflammation indexes such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and CD activity index (CDAI) were recorded before and after EN support.
RESULTS: The 61 patients were divided into three groups according to CDAI before and after EN support: A (active phase into remission via EN, n = 21), B (remained in active phase before and after EN, n = 19) and C (in remission before and after EN, n = 21). Patients in group A had a significant increase in SMM (22.11 ± 4.77 kg vs 23.23 ± 4.49 kg, P = 0.044), protein mass (8.01 ± 1.57 kg vs 8.44 ± 1.45 kg, P = 0.019) and decrease in resting energy expenditure (REE) per kilogram (27.42 ± 5.01 kcal/kg per day vs 22.62 ± 5.45 kcal/kg per day, P < 0.05). There was no significant difference between predicted and measured REE in active CD patients according to the Harris-Benedict equation. There was no linear correlation between the measured REE and CRP, ESR or CDAI in active CD patients.
CONCLUSION: EN could decrease the hypermetabolism in active CD patients by reducing the inflammatory response.
PMCID: PMC4306176  PMID: 25632205
Crohn’s disease; Enteral nutrition; Body composition; Metabolism
4.  Novel variation and de novo mutation rates in population-wide de novo assembled Danish trios 
Nature Communications  2015;6:5969.
Building a population-specific catalogue of single nucleotide variants (SNVs), indels and structural variants (SVs) with frequencies, termed a national pan-genome, is critical for further advancing clinical and public health genetics in large cohorts. Here we report a Danish pan-genome obtained from sequencing 10 trios to high depth (50 × ). We report 536k novel SNVs and 283k novel short indels from mapping approaches and develop a population-wide de novo assembly approach to identify 132k novel indels larger than 10 nucleotides with low false discovery rates. We identify a higher proportion of indels and SVs than previous efforts showing the merits of high coverage and de novo assembly approaches. In addition, we use trio information to identify de novo mutations and use a probabilistic method to provide direct estimates of 1.27e−8 and 1.5e−9 per nucleotide per generation for SNVs and indels, respectively.
The generation of a national pan-genome, a population-specific catalogue of genetic variation, may advance the impact of clinical genetics studies. Here the Besenbacher et al. carry out deep sequencing and de novo assembly of 10 parent–child trios to generate a Danish pan-genome that provides insight into structural variation, de novo mutation rates and variant calling.
PMCID: PMC4309431  PMID: 25597990
5.  Nonuniform gene expression pattern detected along the longitudinal axis in the matured rice leaf 
Scientific Reports  2015;5:8015.
Rice (Oryza sativa) is a staple crop that supports half the world's population and an important monocot model system. Monocot leaf matures in a basipetal manner, and has a well-defined developmental gradient along the longitudinal axis. However, little is known about its transcriptional dynamics after leaf maturation. In this study, we have reconstructed a high spatial resolution transcriptome for the matured rice leaf by sectioning the leaf into seven 3-cm fragments. We have performed strand-specific Illumina sequencing to generate gene expression profiles for each fragment. We found that the matured leaf contains a longitudinal gene expression gradient, with 6.97% (2,603) of the expressed genes showing differentially expression along the seven sections. The leaf transcriptome showed a gradual transition from accumulating transcripts related to primary cell wall and basic cellular metabolism at the base to those involved in photosynthesis and energy production in the middle, and catabolic metabolism process toward the tip.
PMCID: PMC4306128  PMID: 25619793
6.  Comparison of complications in one-stage bilateral total knee arthroplasty with and without drainage 
The aim of this meta-analysis was to compare the complication rates of one-stage bilateral total knee arthroplasty (TKA) with and without drainage in order to identify whether there was no clinical significance and the value of drainage.
Randomized controlled trials (RCTs) based on bilateral TKA with and without drainage were identified via a search of PubMed, EMBASE, Cochrane Central Register of Controlled Trials, Wanfang databases, and Google Scholar, which were published up to May 2014. Methodological quality was assessed by the Physiotherapy Evidence Database scale. After data extraction, we compared the outcomes using fixed-effects or random-effects models depending on the heterogeneity.
Three RCTs involving 125 one-stage bilateral TKA patients with an average follow-up of 14 months met the predetermined inclusion criteria. There were 56 total complications in TKA without drainage and 17 with drainage. Except for less erythema and ecchymosis around the wound in the drainage group, there were no statistical differences in wound healing, wound infection, swelling, and deep vein thrombosis in one-stage bilateral TKA with and without drainage.
The current evidences confirm that both drainage and non-drainage have similar clinical value in one-stage bilateral TKA. However, the conclusion should be used with caution due to the limitations of the current study.
PMCID: PMC4299130  PMID: 25585542
Drainage; One-stage; Bilateral; Total knee arthroplasty; Randomized controlled trials
7.  Annual rates of change in pre- vs. post- bronchodilator FEV1 and FVC over 4 years in moderate to very severe COPD 
Respiratory medicine  2013;107(12):1904-1911.
While the slope of decline in FEV1 has traditionally been calculated from the post- rather than the pre-bronchodilator measurement in COPD interventional trials, it is not clear whether and to what extent these two slopes differ in symptomatic patients with COPD. Therefore, we used data from the 4-year UPLIFT trial of tiotropium 18 mcg QD vs. placebo to compare annual rates of change in pre- vs. post-bronchodilator FEV1 in 5041 patients with moderate to very severe COPD (mean FEV1 48% pred) in whom the post-bronchodilator FEV1 was measured after 4 inhalations of two different classes of short-acting inhaled bronchodilators at baseline and 1 month and every 6 months post-randomization over 4 years. Linear mixed effects models were used to estimate annual rates of decline in FEV1 and FVC pre- and post- bronchodilator in each treatment group separately, after adjusting for height, gender, smoking status, baseline % predicted FEV1 or FVC, and baseline acute % improvement in lung function. The slopes of the post-bronchodilator FEV1 and FVC were significantly steeper than the pre-bronchodilator slopes regardless of treatment arm (p < 0.001), while the estimated variances of the slopes were similar. Post-bronchodilator increases in FEV1 and FVC diminished progressively and significantly (p < 0.0001) over the 4-year trial, suggesting a possible explanation for the significant differences between the pre- and post-bronchodilator slopes. While the reasons for these differences are not completely clear, they are important to consider when assessing treatment effects on rates of decline in FEV1 and FVC.
PMCID: PMC4284059  PMID: 23972968
slope of FEV1 decline; post-bronchodilator; COPD; UPLIFT
8.  Developing the Psychological Strain Scales (PSS): Reliability, Validity, and Preliminary Hypothesis Tests1 
Social indicators research  2012;115(1):337-361.
Since its inception, the Strain Theory of Suicide has been tested and supported in a number of empirical studies. This social psychological theory can be employed as a complementary conceptualization to account for suicidal behaviors as well as mental disorders. However, the lack of consistent measurements of the strains limits the application of the theory in scientific research. Our research team has developed such scales for future testing of the Strain Theory of Suicide in a more systematic approach. For the initial items to measure the four strains (value, aspiration, deprivation, and coping), we solicited approximately 40 items for each strain with high face validity by about 30 fellow researchers. A preliminary examination of about 160 items for consistency and validity, with a sample of about 300 college students, yielded 20 consistent items for each of the four strain scales. Then, a second study was conducted at a different university with approximately 500 students to further streamline each of the four strain scales and test the validity of each with corresponding established scales and variables. As a result, 15 items were selected for each of the four Psychological Strain Scales (PSS). In correlation and multiple regression analyses, we found support for the hypotheses regarding the positive associations between psychological strains measured by the PSS and psychopathology including suicidal ideation. Follow up research with the new scales needs to be carried out in order to test the effects of psychological strains on suicide and mental disorders for various populations.
PMCID: PMC3891678  PMID: 24443628
Psychological Strain; Reliability; Validity; Mental Disorder; Suicide
9.  Digital PCR quantification of miRNAs in sputum for diagnosis of lung cancer 
MicroRNAs (miRNAs) play important roles in the initiation and progression of lung cancer. Measuring miRNA expression levels in sputum could provide a potential approach for the diagnosis of lung cancer. The emerging digital PCR is a straightforward technique for precise, direct, and absolute quantification of nucleic acids. The objective of the study was to investigate whether digital PCR could be used to quantify miRNAs in sputum for lung cancer diagnosis.
We first determined and compared dynamic ranges of digital PCR and conventional quantitative reverse transcriptase PCR (qRT-PCR) for miRNA quantification using RNA isolated from sputum of five healthy individuals. We then used digital PCR to quantify copy number of two lung cancer-associated miRNAs (miR-31 and miR-210) in 35 lung cancer patients and 40 cancer-free controls.
Copy number of the miRNAs measured by digital PCR displayed a linear response to input cDNA amount in a twofold dilution series over seven orders of magnitude. miRNA quantification determined by digital PCR assay was in good agreement with that obtained from qRT-PCR analysis in sputum. Furthermore, combined quantification of miR-31 and miR-210 copy number by using digital PCR in sputum of the cases and controls provided 65.71 % sensitivity and 85.00 % specificity for lung cancer diagnosis.
As digital PCR becomes more established, it would be a robust tool for quantitative assessment of miRNA copy number in sputum for lung cancer diagnosis.
PMCID: PMC3898839  PMID: 24281335
Digital PCR; miRNAs; Sputum; Diagnosis; Lung cancer
10.  Pubertal status moderates the association between mother and child laboratory pain tolerance 
There is limited information regarding the relationship between parent and child responses to laboratory pain induction in the absence of experimental manipulation.
To assess the association between responses to cold and pressure pain tasks in 133 nonclinical mothers and children (mean age 13.0 years; 70 girls), and the moderating effects of child sex and pubertal status on these mother-child relationships.
Mothers and children independently completed the cold and pressure pain tasks. Multiple linear regression analyses examined the association between mothers’ and children’s laboratory pain responses. The moderating effects of child sex and pubertal status were tested in the linear models by examining the interaction among mother laboratory pain responses, and child sex and pubertal status.
Mothers’ cold pain anticipatory anxiety and pressure pain intensity were associated with children’s pressure pain anticipatory anxiety. Mothers’ pressure pain tolerance was associated with children’s pain tolerance for both the cold and pressure pain tasks. Mothers’ cold pain tolerance was associated with children’s pressure pain tolerance. Pubertal status moderated two of the three significant mother-child pain tolerance relationships, such that the associations held for early pubertal but not for late pubertal children. Sex did not moderate mother-child pain associations.
The results indicate that mother-child pain relationships are centred primarily on pain avoidance behaviour, particularly among prepubertal children. These findings may inform interventions focused on pain behaviours, with a particular emphasis on mothers of prepubertal children, to reduce acute pain responses in their children.
PMCID: PMC3938339  PMID: 24367794
Adolescents; Children; Cold pressor task; Experimental pain; Parents
11.  M-CSF cooperating with NFκB induces macrophage transformation from M1 to M2 by upregulating c-Jun 
Cancer Biology & Therapy  2013;15(1):99-107.
Increasing evidence suggests tumor-associated macrophages (TAMs) are polarized M2 subtype of macrophage that exerts pro-tumor effects and promote the malignancy of some cancers, but the concrete mechanism is not well defined. Our previous research exhibited that proto-oncogene AP-1 regulated IL-6 expression in macrophages and promoted the formation of M2 macrophages. In this study, we investigate whether extra-cellular stimulus M-CSF help this process or nuclear factor NFκB has a synergistic role in the activation state of polarized M2 subtype of macrophage. RAW 264.7 macrophage and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. Being co-cultured with 4T1 or its supernatant, the expression of c-Jun, the member of AP-1 family, has a dramatically increase both on the level of gene and on the protein in RAW 264.7 macrophages, but the expression of c-Fos does not increase neither on the level of gene nor on the protein. After co-cultured with 4T1, RAW 264.7 has a higher consumption of M-CSF than RAW 264.7 macrophages alone. With the stimulation of M-CSF, the mRNA of c-Jun increased significantly, but decreased remarkably after adding the anti-M-CSF. And at the same time, p50, the member of NFκB family, has a similar tendency to c-Jun. WB results suggest that with the stimulation of M-CSF, p-Jun in nuclear increases heavily but decreases after the neutralizing antibody added. Coimmunoprecipitation and immunoblotting techniques confirmed that c-Jun and p50 NFκB coprecipitated, and c-Jun protein expression is properly enhanced with rM-CSF effect. In conclusion, M-CSF induces macrophage transformation by upregulating c-Jun with a certain synergy of NFκB. Our study may present a novel therapeutic strategy against cancer.
PMCID: PMC3938528  PMID: 24100343
M-CSF; NFκB; c-Jun; co-culture; transformation
12.  Berberine Prevents Intestinal Mucosal Barrier Damage During Early Phase of Sepsis in Rat through the Toll-Like Receptors Signaling Pathway 
Our previous study has shown berberine prevents damage to the intestinal mucosal barrier during early phase of sepsis in rat through mechanisms independent of the NOD-like receptors signaling pathway. In this study, we explored the regulatory effects of berberine on Toll-like receptors during the intestinal mucosal damaging process in rats. Male Sprague-Dawlay (SD) rats were treated with berberine for 5 d before undergoing cecal ligation and puncture (CLP) to induce polymicrobial sepsis. The expression of Toll-like receptor 2 (TLR 2), TLR 4, TLR 9, the activity of nuclear factor-kappa B (NF-κB), the levels of selected cytokines and chemokines, percentage of cell death in intestinal epithelial cells, and mucosal permeability were investigated at 0, 2, 6, 12 and 24 h after CLP. Results showed that the tumor necrosis factor-α (TNF-α ) and interleukin-6 (IL-6) level were significantly lower in berberine-treated rats compared to the control animals. Conversely, the expression level of tight junction proteins, percentage of cell death in intestinal epithelial cells and the mucosal permeability were significantly higher in berberine-treated rats. The mRNA expression of TLR 2, TLR 4, and TLR 9 were significantly affected by berberine treatment. Our results indicate that pretreatment with berberine attenuates tissue injury and protects the intestinal mucosal barrier in early phase of sepsis and this may possibly have been mediated through the TLRs pathway.
PMCID: PMC4297756  PMID: 25605990
Berberine; Cecal ligation and puncture; Intestinal mucosal barrier; Intra-abdominal infections; Toll-like receptors
13.  Bovine FcRn-Mediated Human Immunoglobulin G Transfer across the Milk-Blood Barrier in Transgenic Mice 
PLoS ONE  2014;9(12):e115972.
Maternal-fetal IgGs transport occurs either prenatally or postnatally, which confers the newborns with passive immunity before their own immune system has matured. However, little is known about the mechanisms of postnatal IgGs passage in the mammary gland. To investigate how FcRn mediates the IgGs transport in the mammary gland, we first generated bFcRn and anti-HAV mAb transgenic mice, and then obtained HF transgenic mice expressing both transgenes by mating the above two strains. Transgene expression of bFcRn in the four lines was determined by qRT-PCR and western blot. We then localized the expression of bFcRn to the acinar epithelial cells in the mammary gland, and anti-HAV mAb was mainly detected in the acini with weak staining in the acinar epithelial cells. Human IgGs could be detected in both milk and serum of HF transgenic mice by western blot and ELISA. A significantly lower milk to serum ratio of human IgGs in HF mice compared with that of anti-HAV mAb mice, indicating that bFcRn could transport human IgGs across the milk-blood barrier from milk to serum during lactation in HF mice. While, there were no transport of murine IgGs, IgAs, or IgMs. These results provide understandings about the mechanisms of maternal-fetal immunity transfer in the mammary gland.
PMCID: PMC4278800  PMID: 25546424
14.  Protective effects of terminal ileostomy against bacterial translocation in a rat model of intestinal ischemia/reperfusion injury 
World Journal of Gastroenterology : WJG  2014;20(47):17905-17913.
AIM: To investigate the effects of terminal ileostomy on bacterial translocation (BT) and systemic inflammation after intestinal ischemia/reperfusion (I/R) injury in rats.
METHODS: Thirty-two rats were assigned to either the sham-operated group, I/R group, I/R + resection and anastomosis group, or the I/R + ileostomy group. The superior mesenteric artery was occluded for 60 min. After 4 h, tissue samples were collected for analysis. BT was assessed by bacteriologic cultures, intestinal permeability and serum levels of endotoxin; systemic inflammation was assessed by serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10, as well as by the activity of myeloperoxidase (MPO) and by intestinal histopathology.
RESULTS: Intestinal I/R injury not only caused morphologic damage to ileal mucosa, but also induced BT, increased MPO activity and promoted the release of TNF-α, IL-6, and IL-10 in serum. BT and ileal mucosa injuries were significantly improved and levels of TNF-α and IL-6 in serum were decreased in the I/R + ileostomy group compared with the I/R + resection and anastomosis group.
CONCLUSION: Terminal ileostomy can prevent the detrimental effects of intestinal I/R injury on BT, intestinal tissue, and inflammation.
PMCID: PMC4273140  PMID: 25548488
Bacterial reflux; Bacterial translocation; Intestinal ischemia/reperfusion; Terminal ileostomy
15.  Dissemination of Cephalosporin Resistance Genes between Escherichia coli Strains from Farm Animals and Humans by Specific Plasmid Lineages 
PLoS Genetics  2014;10(12):e1004776.
Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.
Author Summary
The rapid global rise of infections caused by Escherichia coli that are resistant to clinically relevant antimicrobials, including third-generation cephalosporins, is cause for concern. The intestinal tract of livestock, in particular poultry, is an important reservoir for drug resistant E. coli, but it is unknown to what extent these bacteria can spread to humans. Food is thought to be an important source because drug-resistant E. coli have been detected in animals raised for meat consumption and in meat products. Previous studies that used traditional, low-resolution, genetic typing methods found that drug resistant E. coli present in humans and poultry were indistinguishable from each other, suggesting dissemination of these bacteria through the food-chain to humans. However, by applying high-resolution, whole-genome sequencing methods, we did not find evidence for such transmission of bacteria through the food-chain. Instead, by employing a novel approach for the reconstruction of mobile genetic elements from whole-genome sequence data, we discovered that genetically unrelated E. coli isolates from both humans and animal sources carried nearly identical plasmids that encode third-generation cephalosporin resistance determinants. Our data suggest that cephalosporin resistance is mainly disseminated via the transfer of mobile genetic elements between animals and humans.
PMCID: PMC4270446  PMID: 25522320
16.  A comparison of upper airway parameters in postpartum patients: vaginal delivery vs. caesarean section 
Mallampati class has been shown to increase during labour. The influence of delivery mode on this change is not known yet. The aim of our study is to investigate the changes of upper airway parameters in parturients after caesarean section and vaginal delivery. Ninety parturients undergoing elective caesarean section and ninety parturients with vaginal delivery were enrolled. The parameters of Modified Mallampati test (MMT), inter-incisor distance, thyromental distance, sternomental distance, neck circumference were measured before labour (T0) and 1 h (T1), 6 h (T6) and 24 h (T24) after delivery. Overall, 39 parturients (45.3%) with increases in MMT class in the vaginal delivery group within 24 h After childbirth, were more than that in the caesarean section group [24 parturients (26.7%), P=0.01]. In the vaginal delivery group, the incidence of the increases in MMT class at T1 and T6 were similar, which were higher than that at T24 (P=0.015). In the caesarean section group, the incidence of MMT class increases at T6 was significantly higher than that at T1 (P=0.015) and T24 (P=0.015). Our findings indicate that increase in Mallamapti class may be more significant and may take place earlier in parturients undergoing vaginal delivery than in those undergoing elective caesarean section.
PMCID: PMC4307507  PMID: 25664060
Airway; anesthesia; labour; obstetric; Modified Mallampati test
17.  Fever of unknown origin: report of 107 cases in a university hospital 
Fever of unknown origin (FUO) is a challenging problem in clinical practice. Evaluation of patient’s characteristics may illustrate the etiologies of FUO. In present study, 107 patients with FUO hospitalized in our inpatient department between 2010 and 2011 were investigated. The median age of the patients was 48 years (15-94). The median fever duration was 8.5 weeks (3-104). The median hospital stay was 8.5 days (1-51). Etiologies of FUO were identified as follows: infectious diseases 32 (29.9%), malignancies 19 (17.8%), inflammatory rheumatic diseases 18 (16.8%), and miscellaneous diseases 15 (14.0%). In 23 (21.5%) patients, the diagnosis remained unclear. Infection group had relative shorter average fever duration and hospital stay than other groups. Shortened mean fever duration was observed in geriatric age group. In conclusion, as the most common cause of FUO in the present study, infectious cases had relative shorter average fever duration and hospital stay, and geriatric patients had shortened average fever duration as well.
PMCID: PMC4307568  PMID: 25664121
Fever of unknown origin; etiology; infection
18.  ESET histone methyltransferase regulates osteoblastic differentiation of mesenchymal stem cells during postnatal bone development 
FEBS letters  2013;587(24):3961-3967.
To investigate the effects of histone methyltransferase ESET (also known as SETDB1) on bone metabolism, we analyzed osteoblasts and osteoclasts in ESET knockout animals, and performed osteogenesis assays using ESET-null mesenchymal stem cells. We found that ESET deletion severely impairs osteoblast differentiation but has no effect on osteoclastogenesis, that co-transfection of ESET represses Runx2-mediated luciferase reporter while siRNA knockdown of ESET activates the luciferase reporter in mesenchymal cells, and that ESET is required for postnatal expression of Indian hedgehog protein in the growth plate. As the bone phenotype in ESET-null mice is 100% penetrant, these results support ESET as a critical regulator of osteoblast differentiation during bone development.
PMCID: PMC3947621  PMID: 24188826
21.  Domain Dissection of AvrRxo1 for Suppressor, Avirulence and Cytotoxicity Functions 
PLoS ONE  2014;9(12):e113875.
AvrRxo1, a type III effector from Xanthomonas oryzae pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) in rice, can be recognised by non-host resistance protein Rxo1. It triggers a hypersensitive response (HR) in maize. Little is known regarding the virulence function of AvrRxo1. In this study, we determined that AvrRxo1 is able to suppress the HR caused by the non-host resistance recognition of Xanthomonas oryzae pv. oryzae (Xoo) by Nicotiana benthamiana. It is toxic, inducing cell death from transient expression in N. benthamiana, as well as in yeast. Among the four AvrRxo1 alleles from different Xoc strains, we concluded that the toxicity is abolished by a single amino acid substitution at residue 344 in two AvrRxo1 alleles. A series of truncations from the carboxyl terminus (C-terminus) indicate that the complete C-terminus of AvrRxo1 plays an essential role as a suppressor or cytotoxic protein. The C-terminus was also required for the avirulence function, but the last two residues were not necessary. The first 52 amino acids of N-terminus are unessential for toxicity. Point mutagenesis experiments indicate that the ATP/GTP binding site motif A is required for all three functions of AvrRxo1, and NLS is required for both the avirulence and the suppression of non-host resistance. The putative thiol protease site is only required for the cytotoxicity function. These results determine that AvrRxo1 plays a role in the complex interaction with host proteins after delivery into plant cells.
PMCID: PMC4250038  PMID: 25437277
22.  CXCR3 Ligands Are Associated with the Continuum of Diffuse Alveolar Damage to Chronic Lung Allograft Dysfunction 
Rationale: After lung transplantation, insults to the allograft generally result in one of four histopathologic patterns of injury: (1) acute rejection, (2) lymphocytic bronchiolitis, (3) organizing pneumonia, and (4) diffuse alveolar damage (DAD). We hypothesized that DAD, the most severe form of acute lung injury, would lead to the highest risk of chronic lung allograft dysfunction (CLAD) and that a type I immune response would mediate this process.
Objectives: Determine whether DAD is associated with CLAD and explore the potential role of CXCR3/ligand biology.
Methods: Transbronchial biopsies from all lung transplant recipients were reviewed. The association between the four injury patterns and subsequent outcomes were evaluated using proportional hazards models with time-dependent covariates. Bronchoalveolar lavage (BAL) concentrations of the CXCR3 ligands (CXCL9/MIG, CXCL10/IP10, and CXCL11/ITAC) were compared between allograft injury patterns and “healthy” biopsies using linear mixed-effects models. The effect of these chemokine alterations on CLAD risk was assessed using Cox models with serial BAL measurements as time-dependent covariates.
Measurements and Main Results: There were 1,585 biopsies from 441 recipients with 62 episodes of DAD. An episode of DAD was associated with increased risk of CLAD (hazard ratio, 3.0; 95% confidence interval, 1.9–4.7) and death (hazard ratio, 2.3; 95% confidence interval, 1.7–3.0). There were marked elevations in BAL CXCR3 ligand concentrations during DAD. Furthermore, prolonged elevation of these chemokines in serial BAL fluid measurements predicted the development of CLAD.
Conclusions: DAD is associated with marked increases in the risk of CLAD and death after lung transplantation. This association may be mediated in part by an aberrant type I immune response involving CXCR3/ligands.
PMCID: PMC3863740  PMID: 24063316
lung transplantation; chronic lung allograft dysfunction; bronchiolitis obliterans syndrome; diffuse alveolar damage; CXC chemokines
23.  LncRNA profile study reveals a three-lncRNA signature associated with the survival of patients with oesophageal squamous cell carcinoma  
Gut  2014;63(11):1700-1710.
Oesophageal cancer is one of the most deadly forms of cancer worldwide. Long non-coding RNAs (lncRNAs) are often found to have important regulatory roles.
To assess the lncRNA expression profile of oesophageal squamous cell carcinoma (OSCC) and identify prognosis-related lncRNAs.
LncRNA expression profiles were studied by microarray in paired tumour and normal tissues from 119 patients with OSCC and validated by qRT-PCR. The 119 patients were divided randomly into training (n=60) and test (n=59) groups. A prognostic signature was developed from the training group using a random Forest supervised classification algorithm and a nearest shrunken centroid algorithm, then validated in a test group and further, in an independent cohort (n=60). The independence of the signature in survival prediction was evaluated by multivariable Cox regression analysis.
LncRNAs showed significantly altered expression in OSCC tissues. From the training group, we identified a three-lncRNA signature (including the lncRNAs ENST00000435885.1, XLOC_013014 and ENST00000547963.1) which classified the patients into two groups with significantly different overall survival (median survival 19.2 months vs >60 months, p<0.0001). The signature was applied to the test group (median survival 21.5 months vs >60 months, p=0.0030) and independent cohort (median survival 25.8 months vs >48 months, p=0.0187) and showed similar prognostic values in both. Multivariable Cox regression analysis showed that the signature was an independent prognostic factor for patients with OSCC. Stratified analysis suggested that the signature was prognostic within clinical stages.
Our results suggest that the three-lncRNA signature is a new biomarker for the prognosis of patients with OSCC, enabling more accurate prediction of survival.
PMCID: PMC4215280  PMID: 24522499
Oesophageal Cancer
24.  Substrate-Triggered Addition of Dioxygen to the Diferrous Cofactor of Aldehyde-Deformylating Oxygenase to form a Diferric-Peroxide Intermediate† 
Journal of the American Chemical Society  2013;135(42):10.1021/ja405047b.
Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or mono-unsaturated Cn fatty aldehydes to formate and the corresponding Cn-1 alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 co-substrate to the oxidation state of water and incorporates one O-atom from O2 into the formate co-product. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2III/III complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2III/III center with resolved sub-sites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t1/2 ~ 400 s at 5 °C) to produce a very modest yield of formate (< 0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazine (MeOPMS) to yield product, albeit at only ~ 50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or hetero-dinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2III/III-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1–C2 radical fragmentation and formation of the alk(a/e)ne and formate products.
PMCID: PMC3869994  PMID: 23987523
25.  Single-Cell Analysis of Proxy Reporter Allele-Marked Epithelial Cells Establishes Intestinal Stem Cell Hierarchy 
Stem Cell Reports  2014;3(5):876-891.
The recent development of targeted murine reporter alleles as proxies for intestinal stem cell activity has led to significant advances in our understanding of somatic stem cell hierarchies and dynamics. Analysis of these reporters has led to a model in which an indispensable reserve stem cell at the top of the hierarchy (marked by Bmi1 and Hopx reporters) gives rise to active intestinal stem cells (marked by an Lgr5 reporter). Despite these advances, controversy exists regarding the specificity and fidelity with which these alleles distinguish intestinal stem cell populations. Here, we undertake a comprehensive comparison of widely used proxy reporters including both CreERT2 and EGFP cassettes targeted to the Lgr5, Bmi1, and Hopx loci. Single-cell transcriptional profiling of these populations and their progeny reveals that reserve and active intestinal stem cells are molecularly and functionally distinct, supporting a two-stem-cell model for intestinal self-renewal.
Graphical Abstract
•Proxy intestinal stem cell reporter alleles often mark heterogeneous populations•Discrepancies exist between proxy reporter activity and endogenous transcripts•Reserve and active intestinal stem cells are molecularly distinct•Reserve intestinal stem cells give rise to active stem cells during homeostasis
In this study, Lengner and colleagues utilize single-cell profiling to provide a comparative analysis of epithelial cells marked by a number of putative intestinal stem cell proxy reporter alleles. They confirm the existence of two molecularly distinct stem cell populations governing homeostasis, with a long-term reserve stem cell giving rise to a short-term active stem cell.
PMCID: PMC4235148  PMID: 25418730

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