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1.  Clinical Characteristics and Outcomes of Peritoneal Dialysis-Related Peritonitis with Different Trends of Change in Effluent White Cell Count: A Longitudinal Study 
♦ Background: Effluent white cell count (WCC) is among the important prognostic factors for peritonitis outcome, but its trend has never been studied. We aimed to explore the clinical characteristics and outcomes of peritonitis episodes having different trends in effluent WCC change in the first 5 days.
♦ Methods: For each peritonitis episode, we examined the patient’s demographic and biochemical data, serial effluent WCC, and organisms cultured. Peritonitis-associated death and transfer to hemodialysis were defined as treatment failure.
♦ Results: Based on the trend of effluent WCC in the first 5 days, we divided 190 peritonitis episodes into group A (WCC persistently declined), group B (WCC declined after a transient increase), group C (WCC increased after a transient decline), and group D (WCC persistently increased). In group A, peritonitis was caused mostly by gram-positive organisms, and effluent WCC declined the most quickly, leading to a good prognosis. Although the elevation of effluent WCC was prolonged in group B, and the infections were, compared with those in group A, more often caused by gram-negative organisms, outcomes were not worse. In group C, the effluent WCC was more likely to be higher than 100/μL on day 5, and the infection was, compared with those in groups A and B, less likely to be caused by gram-positive organisms. Accordingly, membership in group C independently predicted the worst outcome of peritonitis even adjusted for age, sex, and causative organism.
♦ Conclusions: Different trends of change in effluent WCC during the early stage of peritonitis represent different clinical patterns and outcomes. Further investigation for optimizing outcomes is required.
PMCID: PMC3707723  PMID: 23733659
Peritonitis; peritoneal effluent; outcomes
2.  Enrichment of MTHFR 677 T in a Chinese long-lived cohort and its association with lipid modulation 
Variants in the Methylenetetrahydrofolate reductase (MTHFR) gene may result in a lowered catalytic activity and associate with subsequent elevated serum homocysteine (Hcy) concentration, abnormal DNA synthesis and methylation, cardiovascular risk, and unhealthy aging. Several investigations on the relationship of MTHFR C677T polymorphism with serum lipid profile and longevity have been conducted in some populations, but the findings remain mixed. Herein, we sought to look at the association between MTHFR C677T and lipid profile in a longevous cohort in Bama, a well-known home of longevity in China.
Genotyping of MTHFR C677T was undertaken in 516 long-lived inhabitants (aged 90 and older, long-lived group, LG) and 493 healthy controls (aged 60–75, non-long-lived group, non-LG) recruited from Bama area. Correlation between MTHFR genotypes and lipids was then evaluated.
T allele and TT genotype were significantly more prevalent in LG (P = 0.001 and 0.002, respectively), especially in females, than in non-LG. No difference in the tested lipid measures among MTHFR C677T genotypes was observed in LG, non-LG and total population (P > 0.05 for all). However, female but not male T carriers exhibited higher TC and LDL-C levels than did T noncarriers in the total population and in LG after stratification by sex (P < 0.05 for each). These differences did not however remain through further subdivision by hyperlipidemia and normolipidemia.
The higher prevalence of MTHFR 677 T genotypes and its modest unfavorable impact on lipids in Bama long-lived individuals may imply an existence of other protective genotypes which require further determination.
PMCID: PMC4092207  PMID: 24968810
3.  Engineered RNase P Ribozymes Effectively Inhibit Human Cytomegalovirus Gene Expression and Replication 
Viruses  2014;6(6):2376-2391.
RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.
PMCID: PMC4074932  PMID: 24932966
ribozyme; RNase P; gene targeting; cytomegalovirus
4.  Melatonin Inhibits Endoplasmic Reticulum Stress and Epithelial-Mesenchymal Transition during Bleomycin-Induced Pulmonary Fibrosis in Mice 
PLoS ONE  2014;9(5):e97266.
Several reports indicate that melatonin alleviates bleomycin (BLM)-induced pulmonary fibrosis in rodent animals. Nevertheless, the exact mechanism remains obscure. The present study investigated the effects of melatonin on endoplasmic reticulum (ER) stress and epithelial-mesenchymal transition (EMT) during BLM-induced lung fibrosis. For the induction of pulmonary fibrosis, mice were intratracheally injected with a single dose of BLM (5.0 mg/kg). Some mice were intraperitoneally injected with melatonin (5 mg/kg) daily for a period of 3 wk. Twenty-one days after BLM injection, lung fibrosis was evaluated. As expected, melatonin significantly alleviated BLM-induced pulmonary fibrosis, as evidenced by Sirius red staining. Moreover, melatonin significantly attenuated BLM-induced EMT to myofibroblasts, as determined by its repression of α-SMA expression. Further analysis showed that melatonin markedly attenuated BLM-induced GRP78 up-regulation and elevation of the cleaved ATF6 in the lungs. Moreover, melatonin obviously attenuated BLM-induced activation of pulmonary eIF2α, a downstream target of the PERK pathway. Finally, melatonin repressed BLM-induced pulmonary IRE1α phosphorylation. Correspondingly, melatonin inhibited BLM-induced activation of XBP-1 and JNK, two downstream targets of the IRE1 pathway. Taken together, these results suggest that melatonin alleviates ER stress and ER stress-mediated EMT in the process of BLM-induced pulmonary fibrosis.
PMCID: PMC4018327  PMID: 24818755
5.  Wavefront sensorless adaptive optics temporal focusing-based multiphoton microscopy 
Biomedical Optics Express  2014;5(6):1768-1777.
Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1μm fluorescent beads sealed in agarose gel at different depths is improved.
PMCID: PMC4052910  PMID: 24940539
(110.1080) Active or adaptive optics; (170.3880) Medical and biological imaging; (180.4315) Nonlinear microscopy
6.  Peritoneal Protein Leakage, Systemic Inflammation, and Peritonitis Risk in Patients on Peritoneal Dialysis 
♦ Background: Whether peritoneal protein leakage predicts risk for peritonitis in patients on peritoneal dialysis (PD) is unknown. In this observational cohort study, we aimed to determine that association and, further, to explore if it might be explained by systemic inflammation.
♦ Methods: We prospectively followed 305 incident PD patients to first-episode peritonitis, censoring, or the end of the study. Demographics, comorbidity score, biochemistry, and peritoneal protein clearance (PrC) were collected at baseline. The predictors of first-episode peritonitis were analyzed prospectively.
♦ Results: During follow-up, 14 868 patient months and 251 episodes of peritonitis were observed. The baseline PrC was 73.2 mL/day (range: 53.2 - 102 mL/day). Patients with a high PrC were prone to be older and malnourished. They also had a higher comorbidity score and higher C-reactive protein values. In 132 first episodes of peritonitis, baseline PrC was shown to be a significant independent predictor after adjustment for age, sex, body mass index, diabetes, residual renal function, hemoglobin, and peritoneal transport rate. Systemic inflammatory markers such as serum albumin, C-reactive protein, and interleukin-6 could not explain the association of PrC and high risk for peritonitis.
♦ Conclusions: Baseline peritoneal protein leakage was able to independently predict risk for peritonitis, which is not explained by systemic inflammation. The underlying mechanisms should be explored in future.
PMCID: PMC3649896  PMID: 23284072
Peritonitis; protein leakage; inflammation
7.  Helicobacter pylori isolates from ethnic minority patients in Guangxi: Resistance rates, mechanisms, and genotype 
AIM: To investigate the rate of Helicobacter pylori (H. pylori) resistance to clarithromycin among ethnic minority patients in Guangxi, explore the underlying mechanisms, and analyze factors influencing genotype distribution of H. pylori isolates.
METHODS: H. pylori strains were isolated, cultured and subjected to drug sensitivity testing. The 23S rRNA gene of H. pylori isolates was amplified by PCR and analyzed by PCR-RFLP and direct sequencing to detect point mutations. REP-PCR was used for genotyping of H. pylori isolates, and NTsys_2 software was used for clustering analysis based on REP-PCR DNA fingerprints. Factors potentially influencing genotype distribution of H. pylori isolates were analyzed.
RESULTS: The rate of clarithromycin resistance was 31.3%. A2143G and A2144G mutations were detected in the 23S rRNA gene of all clarithromycin-resistant H. pylori isolates. At a genetic distance of 78%, clarithromycin-resistant H. pylori isolates could be divided into six groups. Significant clustering was noted among H. pylori isolates from patients with peptic ulcer or gastritis.
CONCLUSION: The rate of clarithromycin resistance is relatively high in ethnic minority patients in Guangxi. Main mechanisms of clarithromycin resistance are A2143G and A2144G mutations in the 23S rRNA gene. Clarithromycin-resistant H. pylori isolates can be divided into six groups based on REP-PCR DNA fingerprints. Several factors such as disease type may influence the genotype distribution of H. pylori isolates.
PMCID: PMC4000514  PMID: 24782630
Helicobacter pylori; Antibiotic resistance; Mechanism; Clarithromycin; Genotype
8.  Dopamine D4 Receptor Excitation of Lateral Habenula Neurons via Multiple Cellular Mechanisms 
The Journal of Neuroscience  2013;33(43):16853-16864.
Glutamatergic lateral habenula (LHb) output communicates negative motivational valence to ventral tegmental area (VTA) dopamine (DA) neurons via activation of the rostromedial tegmental nucleus (RMTg). However, the LHb also receives a poorly understood DA input from the VTA, which we hypothesized constitutes an important feedback loop regulating DA responses to stimuli. Using whole-cell electrophysiology in rat brain slices, we find that DA initiates a depolarizing inward current (IDAi) and increases spontaneous firing in 32% of LHb neurons. IDAi was also observed upon application of amphetamine or the DA uptake blockers cocaine or GBR12935, indicating involvement of endogenous DA. IDAi was blocked by D4 receptor (D4R) antagonists (L745,870 or L741,742), and mimicked by a selective D4R agonist (A412997). IDAi was associated with increased whole-cell conductance and was blocked by Cs+ or a selective blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, ZD7288. IDAi was also associated with a depolarizing shift in half-activation voltage for the hyperpolarization-activated cation current (Ih) mediated by HCN channels. Recordings from LHb neurons containing fluorescent retrograde tracers revealed that IDAi was observed only in cells projecting to the RMTg and not the VTA. In parallel with direct depolarization, DA also strongly increased synaptic glutamate release and reduced synaptic GABA release onto LHb cells. These results demonstrate that DA can excite glutamatergic LHb output to RMTg via multiple cellular mechanisms. Since the RMTg strongly inhibits midbrain DA neurons, activation of LHb output to RMTg by DA represents a negative feedback loop that may dampen DA neuron output following activation.
PMCID: PMC3807019  PMID: 24155292
9.  Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation 
PLoS Genetics  2014;10(4):e1004261.
Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.
Author Summary
Cryptococcus neoformans var. grubii is a major human pathogen responsible for deadly meningoencephalitis in immunocompromised patients. Here, we report the sequencing and annotation of its genome. Evidence for extensive intron splicing, antisense transcription, non-coding RNAs, and alternative polyadenylation indicates the potential for highly intricate regulation of gene expression in this opportunistic pathogen. In addition, detailed molecular, genetic, and genomic studies were performed to characterize structural features of the genome, including centromeres and origins of replication. Finally, the phenotypic and genome re-sequencing analysis of a collection of isolates of the reference H99 strain resulting from laboratory passage revealed that microevolutionary processes during in vitro culturing of pathogenic fungi can impact virulence.
PMCID: PMC3990503  PMID: 24743168
10.  Synergistic effect of lidocaine with pingyangmycin for treatment of venous malformation using a mouse spleen model 
Aims: To explore whether lidocaine has the synergistic effect with pingyangmycin (PYM) in the venous malformations (VMs) treatment. Methods: The mouse spleen was chosen as a VM model and injected with different concentration of lidocaine or PYM or jointly treated with lidocaine and PYM. After 2, 5, 8 or 14 days, the mouse spleen tissues were acquired for hematoxylin-eosin (HE) staining, transmission electron microscopy (TEM) analysis, TUNEL assay and quantitative RT-PCR analysis to examine the toxicological effects of lidocaine and PYM on splenic vascular endothelial cells. Results: 0.4% of lidocaine mildly promoted the apoptosis of endothelial cells, while 2 mg/ml PYM significantly elevated the apoptotic ratios. However, the combination of 0.2% lidocaine and 0.5 mg/ml PYM notably elevated the apoptotic ratios of splenic cells and severely destroyed the configuration of spleen, compared to those of treatment with 0.5 mg/ml PYM alone. Conclusion: Lidocaine exerts synergistic effects with PYM in promoting the apoptosis of mouse splenic endothelial cells, indicating that lidocaine possibly promotes the therapeutic effects of PYM in VMs treatment via synergistically enhancing the apoptosis of endothelial cells of malformed venous lesions.
PMCID: PMC4069942  PMID: 24966943
Venous malformation; lidocaine; pingyangmycin; splenic endothelial cells; apoptosis
11.  Predicting Rotator Cuff Tears Using Data Mining and Bayesian Likelihood Ratios 
PLoS ONE  2014;9(4):e94917.
Rotator cuff tear is a common cause of shoulder diseases. Correct diagnosis of rotator cuff tears can save patients from further invasive, costly and painful tests. This study used predictive data mining and Bayesian theory to improve the accuracy of diagnosing rotator cuff tears by clinical examination alone.
In this retrospective study, 169 patients who had a preliminary diagnosis of rotator cuff tear on the basis of clinical evaluation followed by confirmatory MRI between 2007 and 2011 were identified. MRI was used as a reference standard to classify rotator cuff tears. The predictor variable was the clinical assessment results, which consisted of 16 attributes. This study employed 2 data mining methods (ANN and the decision tree) and a statistical method (logistic regression) to classify the rotator cuff diagnosis into “tear” and “no tear” groups. Likelihood ratio and Bayesian theory were applied to estimate the probability of rotator cuff tears based on the results of the prediction models.
Our proposed data mining procedures outperformed the classic statistical method. The correction rate, sensitivity, specificity and area under the ROC curve of predicting a rotator cuff tear were statistical better in the ANN and decision tree models compared to logistic regression. Based on likelihood ratios derived from our prediction models, Fagan's nomogram could be constructed to assess the probability of a patient who has a rotator cuff tear using a pretest probability and a prediction result (tear or no tear).
Our predictive data mining models, combined with likelihood ratios and Bayesian theory, appear to be good tools to classify rotator cuff tears as well as determine the probability of the presence of the disease to enhance diagnostic decision making for rotator cuff tears.
PMCID: PMC3986413  PMID: 24733553
12.  Glycemic Control and Radiographic Manifestations of Tuberculosis in Diabetic Patients 
PLoS ONE  2014;9(4):e93397.
Radiographic manifestations of pulmonary tuberculosis (TB) in patients with diabetes mellitus (DM) have previously been reported, with inconsistent results. We conducted a study to investigate whether glycemic control has an impact on radiographic manifestations of pulmonary TB.
Consecutive patients with culture-positive pulmonary TB who had DM in three tertiary care hospitals from 2005–2010 were selected for review and compared with a similar number without DM. Glycemic control was assessed by glycated haemoglobin A1C (HbA1C). A pre-treatment chest radiograph was read independently by two qualified pulmonologists blinded to patients’ diabetic status. Films with any discordant reading were read by a third reader.
1209 culture positive pulmonary TB patients (581 with DM and 628 without DM) were enrolled. Compared with those without DM, TB patients with DM were significantly more likely to have opacity over lower lung fields, extensive parenchymal lesions, any cavity, multiple cavities and large cavities (>3 cm). The relative risk of lower lung field opacities was 0.80 (95% CI 0.46–1.42) for those with DM with A1C<7%, 2.32 (95% CI 1.36 - 3.98) for A1C 7%–9%, and 1.62 (95% CI 1.12–2.36) for A1C>9%; and that of any cavity over no cavity was 0.87 (95% CI 0.46–1.62) for patients with DM with A1C<7%, 1.84 (95% CI 1.20–2.84) for A1C 7%–9%, and 3.71 (95% CI 2.64–5.22) for A1C>9%, relative to patients without DM.
Glycemic control significantly influenced radiographic manifestations of pulmonary TB in patients with DM.
PMCID: PMC3974751  PMID: 24699457
13.  In Vitro Evaluation of the Inhibitory Potential of Pharmaceutical Excipients on Human Carboxylesterase 1A and 2 
PLoS ONE  2014;9(4):e93819.
Two major forms of human carboxylesterase (CES), CES1A and CES2, dominate the pharmacokinetics of most prodrugs such as imidapril and irinotecan (CPT-11). Excipients, largely used as insert vehicles in formulation, have been recently reported to affect drug enzyme activity. The influence of excipients on the activity of CES remains undefined. In this study, the inhibitory effects of 25 excipients on the activities of CES1A1 and CES2 were evaluated. Imidapril and CPT-11 were used as substrates and cultured with liver microsomes in vitro. Imidapril hydrolase activities of recombinant CES1A1 and human liver microsomes (HLM) were strongly inhibited by sodium lauryl sulphate (SLS) and polyoxyl 40 hydrogenated castor oil (RH40) [Inhibition constant (Ki) = 0.04±0.01 μg/ml and 0.20±0.09 μg/ml for CES1A1, and 0.12±0.03 μg/ml and 0.76±0.33 μg/ml, respectively, for HLM]. The enzyme hydrolase activity of recombinant CES2 was substantially inhibited by Tween 20 and polyoxyl 35 castor oil (EL35) (Ki = 0.93±0.36 μg/ml and 4.4±1.24 μg/ml, respectively). Thus, these results demonstrate that surfactants such as SLS, RH40, Tween 20 and EL35 may attenuate the CES activity; such inhibition should be taken into consideration during drug administration.
PMCID: PMC3974814  PMID: 24699684
14.  Bile Salt Export Pump is Dysregulated with Altered Farnesoid X Receptor Isoform Expression in Patients with Hepatocellular Carcinoma 
Hepatology (Baltimore, Md.)  2013;57(4):1530-1541.
As a canalicular bile acid effluxer, bile salt export pump (BSEP) plays a vital role in maintaining bile acid homeostasis. BSEP deficiency leads to severe cholestasis and hepatocellular carcinoma (HCC) in young children. Regardless of the etiology, chronic inflammation is the common pathological process for HCC development. Clinical studies showed that bile acid homeostasis is disrupted in HCC patients with elevated serum bile acid level as a proposed marker for HCC. However, the underlying mechanisms remain largely unknown. In this study, we found that BSEP expression was severely diminished in HCC tissues and markedly reduced in adjacent non-tumor tissues. In contrast to mouse, human BSEP was regulated by farnesoid x receptor (FXR) in an isoform-dependent manner. FXRα2 exhibited a much more potent activity than FXRα1 in transactivating human BSEP in vitro and in vivo. The decreased BSEP expression in HCC was associated with altered relative expression of FXRα1 and FXRα2. The FXRα1/FXRα2 ratios were significantly increased with undetectable FXRα2 expression in one third of the HCC tumor samples. Similar correlation between BSEP and FXR isoform expression was confirmed in hepatoma Huh 7 and HepG2 cells. Further studies showed that intrahepatic proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were significantly elevated in HCC tissues. Treatment of Huh 7 cells with IL-6 and TNF-α resulted in a marked increase in the FXRα1/FXRα2 ratio concurrent with a significant decrease in BSEP expression. In conclusion, BSEP expression was severely diminished in HCC patients associated with alteration of FXR isoform expression induced by inflammation, and the restoration of BSEP expression through suppressing inflammation in the liver may re-establish the bile acid homeostasis.
PMCID: PMC3608797  PMID: 23213087
BSEP; FXR; HCC; Bile acids; Gene regulation
15.  Analysis of Spatial and Temporal Protein Expression in the Cerebral Cortex after Ischemia-Reperfusion Injury 
Background and Purpose
Hypoxia, or ischemia, is a common cause of neurological deficits in the elderly. This study elucidated the mechanisms underlying ischemia-induced brain injury that results in neurological sequelae.
Cerebral ischemia was induced in male Sprague-Dawley rats by transient ligation of the left carotid artery followed by 60 min of hypoxia. A two-dimensional differential proteome analysis was performed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to compare changes in protein expression on the lesioned side of the cortex relative to that on the contralateral side at 0, 6, and 24 h after ischemia.
The expressions of the following five proteins were up-regulated in the ipsilateral cortex at 24 h after ischemia-reperfusion injury compared to the contralateral (i.e., control) side: aconitase 2, neurotensin-related peptide, hypothetical protein XP-212759, 60-kDa heat-shock protein, and aldolase A. The expression of one protein, dynamin-1, was up-regulated only at the 6-h time point. The level of 78-kDa glucose-regulated protein precursor on the lesioned side of the cerebral cortex was found to be high initially, but then down-regulated by 24 h after the induction of ischemia-reperfusion injury. The expressions of several metabolic enzymes and translational factors were also perturbed soon after brain ischemia.
These findings provide insights into the mechanisms underlying the neurodegenerative events that occur following cerebral ischemia.
PMCID: PMC4017024  PMID: 24829593
reperfusion injury; proteomics; protein expression; cerebral ischemia; neurodegenerative mechanisms; gerontology
16.  Palmitoyl Acyltransferase, Zdhhc13, Facilitates Bone Mass Acquisition by Regulating Postnatal Epiphyseal Development and Endochondral Ossification: A Mouse Model 
PLoS ONE  2014;9(3):e92194.
ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.
PMCID: PMC3956893  PMID: 24637783
17.  The impact of antibodies on clinical outcomes in diseases treated with therapeutic protein: Lessons learned from infantile Pompe disease 
Enzyme replacement therapy with rhGAA (Myozyme®) has lead to improved survival, which is largely attributable to improvements in cardiomyopathy and skeletal muscle function. However, crossreactive immunologic material-negative patients have a poor clinical response to enzyme replacement therapy secondary to high sustained antibody titers. Furthermore, although the majority of crossreactive immunologic material-positive patients tolerize or experience a downtrend in anti-rhGAA antibody titers, antibody response is variable with some crossreactive immunologic material-positive infants also mounting high sustained antibody titers.
We retrospectively analyzed 34 infants with Pompe disease: 11 crossreactive immunologic material-negative patients, nine high-titer crossreactive immunologic material-positive patients, and 14 low-titer crossreactive immunologic material-positive patients. Clinical outcome measures were overall survival, ventilator-free survival, left ventricular mass index, Alberta Infant Motor Scale score, and urine Glc4 levels.
Clinical outcomes in the high-titer crossreactive immunologic material-positive group were poor across all areas evaluated relative to the low-titer crossreactive immunologic material-positive group. For the crossreactive immunologic material-negative and high-titer crossreactive immunologic material-positive groups, no statistically significant differences were observed for any outcome measures, and both patient groups did poorly.
Our data indicate that, irrespective of crossreactive immunologic material status, patients with infantile Pompe disease with high sustained antibody titer have an attenuated therapeutic response to enzyme replacement therapy. With the advent of immunomodulation therapies, identification of patients at risk for developing high sustained antibody titer is critical.
PMCID: PMC3954622  PMID: 21637107
Pompe disease; enzyme replacement therapy (ERT); crossreactive immunologic material (CRIM); CRIM negative (CN); high sustained antibody titers (HSAT); high-titer CRIM-positive (HTCP); low-titer CRIM-positive (LTCP); therapeutic proteins
18.  Patient and heath system delays in the diagnosis and treatment of new and retreatment pulmonary tuberculosis cases in Malawi 
BMC Infectious Diseases  2014;14:132.
Tuberculosis (TB) control remains a challenge in Malawi despite the National TB Control Program since 1984. This study aimed at measuring patient and health system delays and identifying factors associated with these delays.
A cross-sectional survey of 588 pulmonary TB patients was conducted in three TB centres in Blantyre, Lilongwe, and Mzuzu, between July and December 2011 using a semi-structured questionnaire. Patient delay was defined as the time interval between the onset of TB symptom(s) (a common symptom being coughing) to the first visit to any health provider. Health system delay was the interval from the first care-seeking visit at any health provider to the initiation of anti-tuberculosis treatment. Participants were invited to participate in the study during intensive phase of treatment. The characteristics associated with patient and health system delays were analyzed.
The median patient delay was 14 days for both new and retreatment TB cases (interquartile range [IQR] 14 – 28 and 7 – 21, respectively). The median health system delay was 59 days (IQR 26 – 108) for new and 40.5 days (IQR 21–90) for retreatment cases. Factors associated with longer patient delay in new cases included primary education (adjusted odds ratio [AOR] 2.2, 95% CI 1.3 – 3.9) and knowledge that more than three weeks of coughing is a sign of TB (AOR 1.9, 1.1 – 3.3). In retreatment cases, distance >10 Km (AOR 3.3, 1.1 – 9.6) and knowledge that more than three weeks of coughing is a sign of TB (AOR 3.7, 1.3 – 10.7; p < 0.05) were significant factors. Making the first visit to a health centre (OR 1.9, 0.9 – 3.8) or a drug store/ traditional healer (OR 5.1, 1.1 – 21.7) in new TB cases were associated with a longer health system delay (p < 0.05) while smear negative (OR 6.4, 1.5 – 28.3), and smear unknown or not done (OR 6.1, 1.3 – 26.9) among retreatment cases were associated with a longer health system delay (p < 0.05).
Effective management and new diagnostic techniques are needed especially among retreatment cases. It is also needed to address geographic barriers to accessing care and increasing TB awareness in the community.
PMCID: PMC3976046  PMID: 24606967
Patient delay; Health system delay; Tuberculosis; Case detection
19.  Integrative Annotation of Variants from 1092 Humans: Application to Cancer Genomics 
Science (New York, N.Y.)  2013;342(6154):1235587.
Interpreting variants, especially noncoding ones, in the increasing number of personal genomes is challenging. We used patterns of polymorphisms in functionally annotated regions in 1092 humans to identify deleterious variants; then we experimentally validated candidates. We analyzed both coding and noncoding regions, with the former corroborating the latter. We found regions particularly sensitive to mutations (“ultrasensitive”) and variants that are disruptive because of mechanistic effects on transcription-factor binding (that is, “motif-breakers”). We also found variants in regions with higher network centrality tend to be deleterious. Insertions and deletions followed a similar pattern to single-nucleotide variants, with some notable exceptions (e.g., certain deletions and enhancers). On the basis of these patterns, we developed a computational tool (FunSeq), whose application to ~90 cancer genomes reveals nearly a hundred candidate noncoding drivers.
PMCID: PMC3947637  PMID: 24092746
20.  Direct Assessment of Articular Cartilage and Underlying Subchondral Bone Reveals a Progressive Gene Expression Change in Human Osteoarthritic Knees 
To evaluate the interaction of articular cartilage (AC) and subchondral bone (SB) through analysis of osteoarthritis (OA)-related genes of site-matched tissue.
We developed a novel method for isolating site-matched overlying AC and underlying SB from three and four regions of interest respectively from the human knee tibial plateau (n=50). For each site, the severity of cartilage changes of OA were assessed histologically, and the severity of bone abnormalities were assessed by microcomputed tomography. An RNA isolation procedure was optimized that yielded high quality RNA from site-matched AC and SB tibial regions. Q-PCR analysis was performed to evaluate gene expression of 61 OA-associated genes for correlation with cartilage integrity and bone structure parameters.
A total of 27 (44%) genes were coordinately up or down regulated in both tissues. The expression levels of 19 genes were statistically significantly correlated with the severity of AC degeneration and changes of SB structure; these included: ADAMTS1, ASPN, BMP6, BMPER, CCL2, CCL8, COL5A1, COL6A3, COL7A1, COL16A1, FRZB, GDF10, MMP3, OGN, OMD, POSTN, PTGES, TNFSF11 and WNT1.
These results provide a strategy for identifying targets whose modification may have the potential to ameliorate pathological alterations and progression of disease in both AC and SB simultaneously. In addition, this is the first study, to our knowledge, to overcome the major difficulties related to isolation of high quality RNA from site-matched joint tissues. We expect this method to facilitate advances in our understanding of the coordinated molecular responses of the whole joint organ.
PMCID: PMC3593157  PMID: 23220557
Subchondral bone; cartilage; RNA isolation; Gene expression; Bone sectioning and grinding; Osteoarthritis
21.  Change of Body Weight and Macrophage Inhibitory Cytokine-1 during Chemotherapy in Advanced Gastric Cancer: What Is Their Clinical Significance? 
PLoS ONE  2014;9(2):e88553.
Weight loss in advanced gastric cancer (GC) has been widely acknowledged to be a predictor for poor survival. However, very few studies have investigated the weight loss that occurs during chemotherapy. Therefore, we focused on weight loss during chemotherapy in patients with advanced GC and investigated the concentrations of macrophage inhibitory cytokine-1 (MIC-1), which has been recognized as a probable etiological factor in anorexia and weight loss.
We analyzed 384 patients with inoperable locally advanced or metastatic GC receiving first-line chemotherapy. Patients were assigned to one of two groups on the basis of their weight change during chemotherapy: >3% weight loss and ≤3% weight loss. Serum MIC-1 and C-reactive protein (CRP) concentrations were also assessed in these patients.
The >3% weight loss group had shorter overall survival (OS; 12.0 months vs. 17.5 months, P = 0.000) than the ≤3% weight loss group, and the survival rates improved if the weight loss was reversed during chemotherapy. Although the MIC-1 concentrations were not correlated with weight loss before (P = 0.156) or during chemotherapy (P = 0.164), it correlated significantly with the CRP concentration (P = 0.001). Furthermore, elevated MIC-1 concentrations before chemotherapy (P = 0.017) and increased MIC-1 concentrations during chemotherapy (P = 0.001) were both found to be predictors of poor OS.
Changes in the body weight during chemotherapy could influence the prognosis in patients with advanced GC, and the MIC-1 might be a potential predictive and prognostic biomarker in those patients.
PMCID: PMC3938426  PMID: 24586342
22.  Systematic Review of Evidence-Based Guidelines on Medication Therapy for Upper Respiratory Tract Infection in Children with AGREE Instrument 
PLoS ONE  2014;9(2):e87711.
To summarize recommendations of existing guidelines on the treatment of upper respiratory tract infections (URTIs) in children, and to assess the methodological quality of these guidelines.
We searched seven databases and web sites of relevant academic agencies. Evidence-based guidelines on pediatric URTIs were included. AGREE II was used to assess the quality of these guidelines. Two researchers selected guidelines independently and extracted information on publication years, institutions, target populations, recommendations, quality of evidence, and strength of recommendations. We compared the similarities and differences of recommendations and their strength. We also analyzed the reasons for variation.
Thirteen guidelines meeting our inclusion criteria were included. Huge differences existed among these 13 guidelines concerning the categorization of evidence and recommendations. Nearly all of these guidelines lacked the sufficient involvement of stake holders. Further, the applicability of these guidelines still needs to be improved. In terms of recommendations, penicillin and amoxicillin were suggested for group A streptococcal pharyngitis. Amoxicillin and amoxicillin-clavulanate were recommended for acute bacterial rhinosinusitis (ABRS). An observation of 2–3 days prior to antibiotic therapy initiation for mild acute otitis media (AOM) was recommended with amoxicillin as the suggested first choice agent. Direct evidence to support strong recommendations on the therapy for influenza is still lacking. In addition, the antimicrobial durations for pharyngitis and ABRS were still controversial. No consensus was reached for the onset of antibiotics for ABRS in children.
Future guidelines should use a consistent grading system for the quality of evidence and strength of recommendations. More effort needs to be paid to seek the preference of stake holders and to improve the applicability of guidelines. Further, there are still areas in pediatric URTIs that need more research.
PMCID: PMC3930557  PMID: 24586287
23.  Common Handling Procedures Conducted in Preclinical Safety Studies Result in Minimal Hepatic Gene Expression Changes in Sprague-Dawley Rats 
PLoS ONE  2014;9(2):e88750.
Gene expression profiling is a tool to gain mechanistic understanding of adverse effects in response to compound exposure. However, little is known about how the common handling procedures of experimental animals during a preclinical study alter baseline gene expression. We report gene expression changes in the livers of female Sprague-Dawley rats following common handling procedures. Baseline gene expression changes identified in this study provide insight on how these changes may affect interpretation of gene expression profiles following compound exposure. Rats were divided into three groups. One group was not subjected to handling procedures and served as controls for both handled groups. Animals in the other two groups were weighed, subjected to restraint in Broome restrainers, and administered water via oral gavage daily for 1 or 4 days with tail vein blood collections at 1, 2, 4, and 8 hours postdose on days 1 and 4. Significantly altered genes were identified in livers of animals following 1 or 4 days of handling when compared to the unhandled animals. Gene changes in animals handled for 4 days were similar to those handled for 1 day, suggesting a lack of habituation. The altered genes were primarily immune function related genes. These findings, along with a correlating increase in corticosterone levels suggest that common handling procedures may cause a minor immune system perturbance.
PMCID: PMC3925150  PMID: 24551150
24.  Expansion of Anti-Mesothelin Specific CD4+ and CD8+ T Cell Responses in Patients with Pancreatic Carcinoma 
PLoS ONE  2014;9(2):e88133.
We aimed to assess the status of naturally occurring CD4+ and CD8+ T cell responses to a tumour associated antigen, Mesothelin, in patients with pancreatic carcinoma and study the effects of elevated IL-10 on Mesothelin-specific T cell responses. For that sake, short term T cell lines were generated from PBMCs of 16 healthy controls, 15 patients with benign pancreatic diseases and 25 patients with pancreatic carcinoma and Mesothelin-specific CD4+ and CD8+ T cell responses were analysed using intracellular cytokine assays for IFN-γ. Plasma levels of IL-10 and Mesothelin were measured using cytometric bead array and ELISA assay, respectively. The blocking assays were performed to assess the effects of IL-10 on Mesothelin-specific T cell responses. Here, we demonstrate that the plasma levels of Mesothelin and IL-10 are significantly increased in patients with pancreatic carcinoma. Additionally, we found that (a) Mesothelin-specific T cell responses are significantly expanded in cancer patients (p = 0.0053), (b) the multifunctional CD4+ T cell response is directed toward a broad repertoire of epitopes within the Mesothelin protein. (c) Mesothelin-specific CD4+ T cell response is directly inhibited by elevated IL-10 in cancer patients. These data provides evidence for the use of Mesothelin as an immunogen for tumour-specific T cell response.
PMCID: PMC3919833  PMID: 24520352
25.  The Cryptococcus neoformans Transcriptome at the Site of Human Meningitis 
mBio  2014;5(1):e01087-13.
Cryptococcus neoformans is the leading cause of fungal meningitis worldwide. Previous studies have characterized the cryptococcal transcriptome under various stress conditions, but a comprehensive profile of the C. neoformans transcriptome in the human host has not been attempted. Here, we extracted RNA from yeast cells taken directly from the cerebrospinal fluid (CSF) of two AIDS patients with cryptococcal meningitis prior to antifungal therapy. The patients were infected with strains of C. neoformans var. grubii of molecular type VNI and VNII. Using RNA-seq, we compared the transcriptional profiles of these strains under three environmental conditions (in vivo CSF, ex vivo CSF, and yeast extract-peptone-dextrose [YPD]). Although we identified a number of differentially expressed genes, single nucleotide variants, and novel genes that were unique to each strain, the overall expression patterns of the two strains were similar under the same environmental conditions. Specifically, yeast cells obtained directly from each patient’s CSF were more metabolically active than cells that were incubated ex vivo in CSF. Compared with growth in YPD, some genes were identified as significantly upregulated in both in vivo and ex vivo CSF, and they were associated with genes previously recognized for contributing to pathogenicity. For example, genes with known stress response functions, such as RIM101, ENA1, and CFO1, were regulated similarly in the two clinical strains. Conversely, many genes that were differentially regulated between the two strains appeared to be transporters. These findings establish a platform for further studies of how this yeast survives and produces disease.
Cryptococcus neoformans, an environmental, opportunistic yeast, is annually responsible for an estimated million cases of meningitis and over 600,000 deaths, mostly among HIV-infected patients in sub-Saharan Africa and Asia. Using RNA-seq, we analyzed the gene expression of two strains of C. neoformans obtained from the cerebrospinal fluid (CSF) of infected patients, thus creating a comprehensive snapshot of the yeasts’ genetic responses within the human body. By comparing the gene expression of each clinical strain under three conditions (in vivo CSF, ex vivo CSF, and laboratory culture), we identified genes and pathways that were uniquely regulated by exposure to CSF and likely crucial for the survival of C. neoformans in the central nervous system. Further analyses revealed genetic diversity between the strains, providing evidence for cryptococcal evolution and strain specificity. This ability to characterize transcription in vivo enables the elucidation of specific genetic responses that promote disease production and progression.
PMCID: PMC3950508  PMID: 24496797

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