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1.  Intranasal Delivery of Apelin-13 Is Neuroprotective and Promotes Angiogenesis After Ischemic Stroke in Mice 
ASN NEURO  2015;7(5):1759091415605114.
Apelin is a peptide originally isolated from bovine stomach tissue extracts and identified as an endogenous ligand of the APJ receptor; recent work showed that apelin ameliorates the ischemic injury in the heart and the brain. Being an analogue to the angiotensin II receptor, the apelin/APJ signaling may mediate angiogenesis process. We explored the noninvasive intranasal brain delivery method and investigated therapeutic effects of apelin-13 in a focal ischemic stroke model of mice. Intranasal administration of apelin-13 (4 mg/kg) was given 30 min after the onset of stroke and repeated once daily. Three days after stroke, mice received apelin-13 had significantly reduced infarct volume and less neuronal death in the penumbra. Western blot analyses showed upregulated levels of apelin, apelin receptor APLNR, and Bcl-2 and decreased caspase-3 activation in the apelin-13-treated brain. The proinflammatory cytokines tumor necrosis factor-alpha, interleukin-1β, and chemokine monocyte chemoattractant protein-1 mRNA increased in the ischemic brain, which were significantly attenuated by apelin-13. Apelin-13 remarkably reduced microglia recruitment and activation in the penumbra according to morphological features of Iba-1-positive cells 3 days after ischemia. Apelin-13 significantly increased the expression of angiogenic factor vascular endothelial growth factor and matrix metalloproteinase-9 14 days after stroke. Angiogenesis illustrated by collagen IV + /5-bromo-2′-deoxyuridin + colabeled cells was significantly increased by the apelin-13 treatment 21 days after stroke. Finally, apelin-13 promoted the local cerebral blood flow restoration and long-term functional recovery. This study demonstrates a noninvasive intranasal delivery of apelin-13 after stroke, suggesting that the reduced inflammatory activities, decreased cell death, and increased angiogenesis contribute to the therapeutic benefits of apelin-13.
PMCID: PMC4580122  PMID: 26391329
apelin-13; neuroprotection; angiogenesis; inflammation; functional recovery; ischemic stroke
2.  Synthesis and biological evaluation of panitumumab-IRDye800 conjugate as a fluorescence imaging probe for EGFR-expressing cancers 
MedChemComm  2014;5(9):1337-1346.
To investigate panitumumab-IRDye800 as an intraoperative optical imaging agent for epidermal growth factor receptor (EGFR)-expressing cancers, we developed clinical-quality panitumumab-IRDye800 and evaluated its specificity and sensitivity to visualize tumors by fluorescence imaging in a variety of mouse xenograft models with different levels of EGFR-expression. Panitumumab was chemically conjugated to NIR-dye (Li-COR 800CW) at well-defined and limited substitution ratio (1:1–2) for the characterization of fluorescence signals. Yield and purity of the conjugate was 80±5% and 95±2% respectively (n= 6). Quality control (QC) tests showed that product was suitable for clinical development. Female athymic nude xenograft tumor bearing mice (n=5 per tumor model) with very low (BT-474), moderate (MDA-MB-231), and high (MDA-MB-468) EGFR-expression levels were administered panitumumab-IRDye800 formulations (100 μg of mAb in 100 μL of 0.9% saline) via tail-vein injection. Animal imaging and biodistribution experiments were conducted on the FMT 2500 (Perkin Elmer) fluorescence scanner at 24, 48, 72, 96, and 144 hours post injection. Immuno-fluorescence images of panitumumab-IRDye conjugate recorded in mouse xenograft models showed a good correlation (R2 = 0.91) between EGFR-expression level and tumor uptake. Uptake of panitumumab labeled with IR-Dye or [89Zr] in different tumor xenografts with high, medium, and low EGFR expression, as measured by fluorescence or radioactive counts are highly correlated (r2= 0.99). This preclinical in-vivo study proved that panitumumab-IRDye800 is specific and optical imaging in conjunction with this probe is sensitive enough to detect EGFR-expressing tumors.
PMCID: PMC4241857  PMID: 25431648
3.  Therapeutic Effects of Pharmacologically Induced Hypothermia against Traumatic Brain Injury in Mice 
Journal of Neurotrauma  2014;31(16):1417-1430.
Preclinical and clinical studies have shown therapeutic potential of mild-to-moderate hypothermia for treatments of stroke and traumatic brain injury (TBI). Physical cooling in humans, however, is usually slow, cumbersome, and necessitates sedation that prevents early application in clinical settings and causes several side effects. Our recent study showed that pharmacologically induced hypothermia (PIH) using a novel neurotensin receptor 1 (NTR1) agonist, HPI-201 (also known as ABS-201), is efficient and effective in inducing therapeutic hypothermia and protecting the brain from ischemic and hemorrhagic stroke in mice. The present investigation tested another second-generation NTR1 agonist, HPI-363, for its hypothermic and protective effect against TBI. Adult male mice were subjected to controlled cortical impact (CCI) (velocity=3 m/sec, depth=1.0 mm, contact time=150 msec) to the exposed cortex. Intraperitoneal administration of HPI-363 (0.3 mg/kg) reduced body temperature by 3–5°C within 30–60 min without triggering a shivering defensive reaction. An additional two injections sustained the hypothermic effect in conscious mice for up to 6 h. This PIH treatment was initiated 15, 60, or 120 min after the onset of TBI, and significantly reduced the contusion volume measured 3 days after TBI. HPI-363 attenuated caspase-3 activation, Bax expression, and TUNEL-positive cells in the pericontusion region. In blood–brain barrier assessments, HPI-363 ameliorated extravasation of Evans blue dye and immunoglobulin G, attenuated the MMP-9 expression, and decreased the number of microglia cells in the post-TBI brain. HPI-363 decreased the mRNA expression of tumor necrosis factor-α and interleukin-1β (IL-1β), but increased IL-6 and IL-10 levels. Compared with TBI control mice, HPI-363 treatments improved sensorimotor functional recovery after TBI. These findings suggest that the second generation NTR-1 agonists, such as HPI-363, are efficient hypothermic-inducing compounds that have a strong potential in the management of TBI.
PMCID: PMC4132583  PMID: 24731132
blood–brain barrier; hypothermia; inflammation; neurotensin analogue; sensorimotor function; traumatic brain injury
5.  Levels of Urbanization and Parental Education in Relation to the Mortality Risk of Young Children 
Background: The establishment of the National Health Insurance program in Taiwan in 1995 effectively removed the financial barrier to access health care services of Taiwanese people. This population-based cohort study aimed to determine the independent and joint effects of parental education and area urbanization on the mortality risk among children under the universal health insurance coverage in Taiwan since 1995. Methods: We linked 1,501,620 births from 1996 to 2000 to the Taiwan Death Registry to estimate the neonatal, infant, and under-five mortality rates, according to the levels of parental education and urbanization of residential areas. We used a logistic regression model that considers data clustering to estimate the independent and joint effects. Results: Lower levels of parental education and area urbanization exerted an independent effect of mortality on young children, with a stronger magnitude noted for areas with lower levels of urbanization. Children whose parents had lower levels of education and who were born in areas with lower levels of urbanization experienced the highest risk for neonatal (odds ratio (OR) = 1.60, 95% CI = 1.46–1.76), infant (OR = 1.58, 95% CI = 1.48–1.70), and under-five (OR = 1.71, 95% CI = 1.61–1.82) mortality. Conclusions: Even with universal health insurance coverage, lower levels of area urbanization and parental education still exerted independent and joint effects on mortality in young children. This finding implies the inadequate accessibility to health care resources for children from socially disadvantaged families and less urbanized areas.
PMCID: PMC4515684  PMID: 26184248
neonatal mortality; infant mortality; under-five mortality; socioeconomic status; cohort studies
6.  Derivation of Cinnamon Blocks Leukocyte Attachment by Interacting with Sialosides 
PLoS ONE  2015;10(6):e0130389.
Molecules derived from cinnamon have demonstrated diverse pharmacological activities against infectious pathogens, diabetes and inflammatory diseases. This study aims to evaluate the effect of the cinnamon-derived molecule IND02 on the adhesion of leukocytes to host cells. The anti-inflammatory ability of IND02, a pentameric procyanidin type A polyphenol polymer isolated from cinnamon alcohol extract, was examined. Pretreatment with IND02 significantly reduced the attachment of THP-1 cells or neutrophils to TNF-α-activated HUVECs or E-selectin/ICAM-1, respectively. IND02 also reduced the binding of E-, L- and P-selectins with sialosides. Furthermore, IND02 could agglutinate human red blood cells (RBC), and the agglutination could be disrupted by sialylated glycoprotein. Our findings demonstrate that IND02, a cinnamon-derived compound, can interact with sialosides and block the binding of selectins and leukocytes with sialic acids.
PMCID: PMC4468131  PMID: 26076445
7.  Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, and In Vitro Disease Modeling 
Stem Cells International  2015;2015:105172.
Human pluripotent stem cells (hPSCs) derived from either blastocyst stage embryos (hESCs) or reprogrammed somatic cells (iPSCs) can provide an abundant source of human neuronal lineages that were previously sourced from human cadavers, abortuses, and discarded surgical waste. In addition to the well-known potential therapeutic application of these cells in regenerative medicine, these are also various promising nontherapeutic applications in toxicological and pharmacological screening of neuroactive compounds, as well as for in vitro modeling of neurodegenerative and neurodevelopmental disorders. Compared to alternative research models based on laboratory animals and immortalized cancer-derived human neural cell lines, neuronal cells differentiated from hPSCs possess the advantages of species specificity together with genetic and physiological normality, which could more closely recapitulate in vivo conditions within the human central nervous system. This review critically examines the various potential nontherapeutic applications of hPSC-derived neuronal lineages and gives a brief overview of differentiation protocols utilized to generate these cells from hESCs and iPSCs.
PMCID: PMC4454762  PMID: 26089911
8.  Mortality of Combined Serum Phosphorus and Parathyroid Hormone Concentrations and their Changes over Time in Hemodialysis Patients 
Bone  2014;61:201-207.
Mineral and bone disorder (MBD) is common and associated with mortality in patients with chronic kidney disease (CKD). Given that disarrays in serum phosphorus (P) and parathyroid hormone (PTH) levels and their changes over time are closely interrelated, modeling mortality-predictability of their combinations may help improve CKD patient management.
A historical cohort study was undertaken to evaluate the joint effect of serum P and PTH levels on mortality in 107,299 chronic hemodialysis (HD) patients. Changes in serum P and PTH levels over 6 months, in particular discordant changes, were also modeled with mortality.
HD patients were 64±15 (mean±SD) years old and included 45% women, 33% African–American, and 59% diabetic. Compared with serum P level ≥7.0 mg/dL and PTH level ≥600 pg/mL, adjusted hazard ratio (HR) tended to be lowest in patients with serum P level of 3.5–<5.5 mg/dL combined with PTH level of 150–<300 pg/mL (HR 0.64, 95% confidence interval 0.61–0.67). A change over time in serum P level towards the 3.5–<5.5 mg/dL range from higher or lower ranges was associated with a decreased mortality, whereas only change in PTH level from <150 pg/mL to 150–<300 pg/mL range was associated with a lower risk of mortality. Upon discordant changes of PTH and P, i.e., decrease in one of the two measures while the other increased, no change in mortality risk was observed.
In CKD–MBD management, patent survival is the greatest with controlling both serum P and PTH levels in balance. Tailoring an individualized treatment strategy in CKD-MBD may benefit patients. Further studies are needed.
PMCID: PMC4024455  PMID: 24486956
bone; hemodialysis; hyperparathyroidism; mineral; phosphorus
9.  Lewisy Promotes Migration of Oral Cancer Cells by Glycosylation of Epidermal Growth Factor Receptor 
PLoS ONE  2015;10(3):e0120162.
Aberrant glycosylation changes normal cellular functions and represents a specific hallmark of cancer. Lewisy (Ley) carbohydrate upregulation has been reported in a variety of cancers, including oral squamous cell carcinoma (OSCC). A high level of Ley expression is related to poor prognosis of patients with oral cancer. However, it is unclear how Ley mediates oral cancer progression. In this study, the role of Ley in OSCC was explored. Our data showed that Ley was upregulated in HSC-3 and OC-2 OSCC cell lines. Particularly, glycosylation of epidermal growth factor receptor (EGFR) with Ley was found in OC-2 cells, and this modification was absent upon inhibition of Ley synthesis. The absence of Ley glycosylation of EGFR weakened phosphorylation of AKT and ERK in response to epidermal growth factor (EGF). Additionally, EGF-triggered cell migration was reduced, but cell proliferation was not affected. Ley modification stabilized EGFR upon ligand activation. Conversely, absence of Ley glycosylation accelerated EGFR degradation. In summary, these results indicate that increased expression of Ley in OSCC cells is able to promote cell migration by modifying EGFR which in turn stabilizes EGFR expression and downstream signaling. Targeting Ley on EGFR could have a potential therapeutic effect on oral cancer.
PMCID: PMC4370659  PMID: 25799278
10.  Association between Thiopurine S-methyltransferase Polymorphisms and Thiopurine-Induced Adverse Drug Reactions in Patients with Inflammatory Bowel Disease: A Meta-Analysis 
PLoS ONE  2015;10(3):e0121745.
Thiopurine drugs are well established treatments in the management of inflammatory bowel disease (IBD), but their use is limited by significant adverse drug reactions (ADRs). Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in thiopurine metabolism. Several clinical guidelines recommend determining TPMT genotype or phenotype before initiating thiopurine therapy. Although several studies have investigated the association between TPMT polymorphisms and thiopurine-induced ADRs, the results are inconsistent. The purpose of this study is to evaluate whether there is an association between TPMT polymorphisms and thiopurine-induced ADRs using meta-analysis.
We explored PubMed, Web of Science and Embase for articles on TPMT polymorphisms and thiopurine-induced ADRs. Studies that compared TPMT polymorphisms with-ADRs and without-ADRs in IBD patients were included. Relevant outcome data from all the included articles were extracted and the pooled odds ratio (OR) with corresponding 95% confidence intervals were calculated using Revman 5.3 software.
Fourteen published studies, with a total of 2,206 IBD patients, which investigated associations between TPMT polymorphisms and thiopurine-induced ADRs were included this meta-analysis. Our meta-analysis demonstrated that TPMT polymorphisms were significantly associated with thiopurine-induced overall ADRs and bone marrow toxicity; pooled ORs were 3.36 (95%CI: 1.82–6.19) and 6.67 (95%CI: 3.88–11.47), respectively. TPMT polymorphisms were not associated with the development of other ADRs including hepatotoxicity, pancreatitis, gastric intolerance, flu-like symptoms and skin reactions; the corresponding pooled ORs were 1.27 (95%CI: 0.60–2.71), 0.97 (95%CI: 0.38–2.48), 1.82 (95%CI: 0.93–3.53), 1.28 (95%CI: 0.47–3.46) and 2.32 (95%CI: 0.86–6.25), respectively.
Our meta-analysis demonstrated an association of TPMT polymorphisms with overall thiopurine-induced ADRs and bone marrow toxicity, but not with hepatotoxicity, pancreatitis, flu-like symptoms, gastric intolerance and skin reactions. These findings suggest that pretesting the TPMT genotype could be helpful in clinical practice before initiating thiopurine therapy. However, white blood cell count analysis should be the mainstay for follow-up.
PMCID: PMC4370632  PMID: 25799415
11.  Identification and Characterization of Two Novel Spliced Genes Located in the orf47-orf46-orf45 Gene Locus of Kaposi's Sarcoma-Associated Herpesvirus 
Journal of Virology  2014;88(17):10092-10109.
The orf47-orf46-orf45 gene cluster of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracil DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47-orf46-orf45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, consist of only a partial region of gL (ORF47), a unique 7-amino-acid motif, and the complete tegument protein ORF45. Like the ORF45 protein, ORF47/45-A and ORF47/45-B expressed in cells sufficiently activate the phosphorylation of p90 ribosomal S6 kinase (RSK) and extracellular signal-regulated protein kinase (ERK). However, unlike ORF45, both ORF47/45-A and ORF47/45-B contain a signal peptide sequence and are localized at the endoplasmic reticulum (ER). Additionally, we found that ORF47/45-A and ORF47/45-B have an extra function that mediates the upregulation of GRP78, a master regulator of ER homeostasis. The important event regarding GRP78 upregulation can be observed in all tested KSHV-positive cell lines after viral reactivation, and knockdown of GRP78 in cells significantly impairs viral lytic cycle progression, especially at late lytic stages. Compared with some other viral glycoproteins synthesized through the ER, our results strongly implicate that the ORF47/45 proteins may serve as key effectors for controlling GRP78 expression and ER homeostasis in cells. Taken together, our findings provide evidence showing the reciprocal association between the modulation of ER homeostasis and the progression of the KSHV lytic cycle.
IMPORTANCE Emerging evidence has shown that several viruses appear to use different strategies to control ER homeostasis for supporting their productive infections. The two parts of this study identify two aspects of the association between the regulation of ER homeostasis and the progression of the KSHV lytic cycle. The first part characterizes the function of two early lytic cycle proteins, ORF47/45-A and ORF47/45-B, on the activation of a major ER chaperone protein, GRP78. In addition to the ability to promote GRP78 upregulation, the ORF47/45 proteins also activate the phosphorylation of RSK and ERK. The second part reveals that upregulation of GRP78 is essential for the progression of the KSHV lytic cycle, especially at late stages. We therefore propose that activation of GRP78 expression by viral proteins at the early lytic stage may aid with the protection of host cells from severe ER stress and may directly involve the assembly or release of virions.
PMCID: PMC4136336  PMID: 24965448
12.  Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATB0,+, LAT1 and LAT2 
Cancer Science  2015;106(3):279-286.
The efficacy of boron neutron capture therapy relies on the selective delivery of boron carriers to malignant cells. p-Boronophenylalanine (BPA), a boron delivery agent, has been proposed to be localized to cells through transporter-mediated mechanisms. In this study, we screened aromatic amino acid transporters to identify BPA transporters. Human aromatic amino acid transporters were functionally expressed in Xenopus oocytes and examined for BPA uptake and kinetic parameters. The roles of the transporters in BPA uptake were characterized in cancer cell lines. For the quantitative assessment of BPA uptake, HPLC was used throughout the study. Among aromatic amino acid transporters, ATB0,+, LAT1 and LAT2 were found to transport BPA with Km values of 137.4 ± 11.7, 20.3 ± 0.8 and 88.3 ± 5.6 μM, respectively. Uptake experiments in cancer cell lines revealed that the LAT1 protein amount was the major determinant of BPA uptake at 100 μM, whereas the contribution of ATB0,+ became significant at 1000 μM, accounting for 20–25% of the total BPA uptake in MCF-7 breast cancer cells. ATB0,+, LAT1 and LAT2 transport BPA at affinities comparable with their endogenous substrates, suggesting that they could mediate effective BPA uptake in vivo. The high and low affinities of LAT1 and ATB0,+, respectively, differentiate their roles in BPA uptake. ATB0,+, as well as LAT1, could contribute significantly to the tumor accumulation of BPA at clinical dose.
PMCID: PMC4376436  PMID: 25580517
Amino acid transporter; BNCT; BPA; cancer cell lines; drug delivery
13.  Ubiquitin-Binding Site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23 
Nature communications  2014;5:4638.
Polyglutamine repeat expansion in ataxin-3 causes neurodegeneration in the most common dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3). Since reducing levels of disease proteins improves pathology in animals, we investigated how ataxin-3 is degraded. Here we show that, unlike most proteins, ataxin-3 turnover does not require its ubiquitination, but is regulated by Ubiquitin-Binding Site 2 (UbS2) on its N terminus. Mutating UbS2 decreases ataxin-3 protein levels in cultured mammalian cells and in Drosophila melanogaster by increasing its proteasomal turnover. Ataxin-3 interacts with the proteasome-associated proteins Rad23A/B through UbS2. Knockdown of Rad23 in cultured cells and in Drosophila results in lower levels of ataxin-3 protein. Importantly, reducing Rad23 suppresses ataxin-3-dependent degeneration in flies. We present a mechanism for ubiquitination-independent degradation that is impeded by protein interactions with proteasome-associated factors. We conclude that UbS2 is a potential target through which to enhance ataxin-3 degradation for SCA3 therapy.
PMCID: PMC4237202  PMID: 25144244
Deubiquitinase; Drosophila; Neurodegeneration; Neuroprotection; Polyglutamine; Proteasome; hHR23; Spinocerebellar Ataxia; Ubiquitin
14.  Follow-up plasma apolipoprotein E levels in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL) cohort 
Alzheimer’s disease (AD) is a growing socioeconomic problem worldwide. Early diagnosis and prevention of this devastating disease have become a research priority. Consequently, the identification of clinically significant and sensitive blood biomarkers for its early detection is very important. Apolipoprotein E (APOE) is a well-known and established genetic risk factor for late-onset AD; however, the impact of the protein level on AD risk is unclear. We assessed the utility of plasma ApoE protein as a potential biomarker of AD in the large, well-characterised Australian Imaging, Biomarkers and Lifestyle Study of Ageing (AIBL) cohort.
Total plasma ApoE levels were measured at 18-month follow-up using a commercial bead-based enzyme-linked immunosorbent assay: the Luminex xMAP human apolipoprotein kit. ApoE levels were then analysed between clinical classifications (healthy controls, mild cognitive impairment (MCI) and AD) and correlated with the data available from the AIBL cohort, including but not limited to APOE genotype and cerebral amyloid burden.
A significant decrease in ApoE levels was found in the AD group compared with the healthy controls. These results validate previously published ApoE protein levels at baseline obtained using different methodology. ApoE protein levels were also significantly affected, depending on APOE genotypes, with ε2/ε2 having the highest protein levels and ε4/ε4 having the lowest. Plasma ApoE levels were significantly negatively correlated with cerebral amyloid burden as measured by neuroimaging.
ApoE is decreased in individuals with AD compared with healthy controls at 18-month follow-up, and this trend is consistent with our results published at baseline. The influence of APOE genotype and sex on the protein levels are also explored. It is clear that ApoE is a strong player in the aetiology of this disease at both the protein and genetic levels.
PMCID: PMC4391582  PMID: 25859282
15.  Impact of Age, Race and Ethnicity on Dialysis Patient Survival and Kidney Transplantation Disparities 
American journal of nephrology  2014;39(3):183-194.
Prior studies show that African-American and Hispanic dialysis patients have lower mortality risk than whites. Recent age-stratified analyses suggest this survival advantage may be limited to younger age groups, but did not concurrently compare Hispanic, African-American, and white patients, nor account for differences in nutritional and inflammatory status as potential confounders. Minorities experience inequities in kidney transplantation access, but it is unknown whether these racial/ethnic disparities differ across age groups.
The associations between race/ethnicity with all-cause mortality and kidney transplantation were separately examined among 130,909 adult dialysis patients from a large national dialysis organization (entry period 2001-2006, follow-up through 2009) within 7 age categories using Cox proportional hazard models adjusted for case-mix and malnutrition and inflammatory surrogates.
African-Americans had similar mortality vs. whites in younger age groups (18-40 years), but decreased mortality in older age groups (>40 years). In contrast, Hispanics had lower mortality vs. whites across all ages. In sensitivity analyses using competing risk regression to account for differential kidney transplantation rates across racial/ethnic groups, the African-American survival advantage was limited to >60 year old age categories. African-Americans and Hispanics were less likely to undergo kidney transplantation from all donor types vs. whites across all ages, and these disparities were even more pronounced for living donor kidney transplantations (LDKT).
Hispanic dialysis patients have greater survival vs. whites across all ages; in African-Americans, this survival advantage is limited to patients >40 years old. Minorities are less likely to undergo kidney transplantation, particularly LDKT, across all ages.
PMCID: PMC4024458  PMID: 24556752
Race; Ethnicity; Disparities; Survival; Transplantation
16.  Deep RNA sequencing reveals a high frequency of alternative splicing events in the fungus Trichoderma longibrachiatum 
BMC Genomics  2015;16(1):54.
Alternative splicing is crucial for proteome diversity and functional complexity in higher organisms. However, the alternative splicing landscape in fungi is still elusive.
The transcriptome of the filamentous fungus Trichoderma longibrachiatum was deep sequenced using Illumina Solexa technology. A total of 14305 splice junctions were discovered. Analyses of alternative splicing events revealed that the number of all alternative splicing events (10034), intron retentions (IR, 9369), alternative 5’ splice sites (A5SS, 167), and alternative 3’ splice sites (A3SS, 302) is 7.3, 7.4, 5.1, and 5.9-fold higher, respectively, than those observed in the fungus Aspergillus oryzae using Illumina Solexa technology. This unexpectedly high ratio of alternative splicing suggests that alternative splicing is important to the transcriptome diversity of T. longibrachiatum. Alternatively spliced introns had longer lengths, higher GC contents, and lower splice site scores than constitutive introns. Further analysis demonstrated that the isoform relative frequencies were correlated with the splice site scores of the isoforms. Moreover, comparative transcriptomics determined that most enzymes related to glycolysis and the citrate cycle and glyoxylate cycle as well as a few carbohydrate-active enzymes are transcriptionally regulated.
This study, consisting of a comprehensive analysis of the alternative splicing landscape in the filamentous fungus T. longibrachiatum, revealed an unexpectedly high ratio of alternative splicing events and provided new insights into transcriptome diversity in fungi.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1251-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4324775  PMID: 25652134
Alternative splicing; Fungi; RNA-Seq; Intron retention; Transcriptome; Trichoderma longibrachiatum
17.  Comparison of two different standards of care in detecting malignant thyroid nodules using thyroid fine-needle aspiration 
Molecular and Clinical Oncology  2015;3(3):682-686.
The aim of the present study was to verify the optimal method to obtain enough fine-needle aspiration (FNA) materials for detecting thyroid malignancy. A prospective study was performed by comparing two different regional standards of care. In one group a traditional FNA method mainly used in Asian countries, including China, was performed in which a single pass of a 22-G needle was applied with or without aspiration. In the other group, the method mainly used in Western countries was performed in which three passes of a 25-G needle with non-aspiration were undertaken for thyroid nodules. The study included 718 thyroid nodules from 695 patients. These nodules were allocated for three different methods of performing thyroid FNA. There were 332 thyroid nodules subjected to the traditional Asian FNA method using a 22-G needle with aspiration for 142 nodules and non-aspiration for 190 nodules. FNA using the Western method was performed with three passes of non-aspiration using 25 G for 386 nodules. All the FNAs were performed with the guidance of ultrasound. The components of the nodules were documented. All the samples were reported using the Bethesda System for Reporting Thyroid Cytopathology. Among the 22 G group, the non-diagnostic rate in the aspiration group was as high as 76.76%, which was significantly higher than 44.21% in the non-aspiration group (P<0.01). For the non-aspiration group, the non-diagnostic rate in the 25 G group was 34.97%, which was significantly lower than 44.21% in the 22 G group. In general, the non-diagnostic rate for the 25-G needle was 34.97%, which was significantly lower than 58.13% in all the 22 G groups. For the solid and mixed nodules, the non-diagnostic rate was lower in the 25-G needle group compared to the 22 G groups with statistical significance. The non-aspiration method using a 25-G needle with multiple passes can result in a higher diagnostic rate for thyroid FNA.
PMCID: PMC4471588  PMID: 26137287
thyroid nodule; fine-needle aspiration; techniques; Bethesda System; standards of care
18.  Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis 
Scientific Reports  2014;4:7521.
Increasing evidence points to a negative correlation between KRAS mutations and patients' responses to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo KRAS mutation detection to be eligible for treatment. High resolution melting analysis (HRM) is gaining increasing attention in KRAS mutation detection. However, its accuracy has not been systematically evaluated. We conducted a meta-analysis of published articles, involving 13 articles with 1,520 samples, to assess its diagnostic accuracy compared with DNA sequencing. The quality of included articles was assessed using the revised Quality Assessment for Studies of Diagnostic Accuracy (QUADAS-2) tools. Random effects models were applied to analyze the performance of pooled characteristics. The overall sensitivity and specificity of HRM were 0.99 (95% confidence interval [CI]: 0.98–1.00) and 0.96 (95%CI: 0.94–0.97), respectively. The area under the summary receiver operating characteristic curve was 0.996. High sensitivity and specificity, less labor, rapid turn-around and the closed-tube format of HRM make it an attractive choice for rapid detection of KRAS mutations in clinical practice. The burden of DNA sequencing can be reduced dramatically by the implementation of HRM, but positive results still need to be sequenced for diagnostic confirmation.
PMCID: PMC4268648  PMID: 25515911
19.  High Amylose Resistant Starch Diet Ameliorates Oxidative Stress, Inflammation, and Progression of Chronic Kidney Disease 
PLoS ONE  2014;9(12):e114881.
Inflammation is a major mediator of CKD progression and is partly driven by altered gut microbiome and intestinal barrier disruption, events which are caused by: urea influx in the intestine resulting in dominance of urease-possessing bacteria; disruption of epithelial barrier by urea-derived ammonia leading to endotoxemia and bacterial translocation; and restriction of potassium-rich fruits and vegetables which are common sources of fermentable fiber. Restriction of these foods leads to depletion of bacteria that convert indigestible carbohydrates to short chain fatty acids which are important nutrients for colonocytes and regulatory T lymphocytes. We hypothesized that a high resistant starch diet attenuates CKD progression. Male Sprague Dawley rats were fed a chow containing 0.7% adenine for 2 weeks to induce CKD. Rats were then fed diets supplemented with amylopectin (low-fiber control) or high fermentable fiber (amylose maize resistant starch, HAM-RS2) for 3 weeks. CKD rats consuming low fiber diet exhibited reduced creatinine clearance, interstitial fibrosis, inflammation, tubular damage, activation of NFkB, upregulation of pro-inflammatory, pro-oxidant, and pro-fibrotic molecules; impaired Nrf2 activity, down-regulation of antioxidant enzymes, and disruption of colonic epithelial tight junction. The high resistant starch diet significantly attenuated these abnormalities. Thus high resistant starch diet retards CKD progression and attenuates oxidative stress and inflammation in rats. Future studies are needed to explore the impact of HAM-RS2 in CKD patients.
PMCID: PMC4260945  PMID: 25490712
20.  Acute and Delayed Protective Effects of Pharmacologically Induced Hypothermia in an Intracerebral Hemorrhage Stroke Model of Mice 
Neuroscience  2013;252:489-500.
Hemorrhagic stroke, including intracerebral hemorrhage (ICH), is a devastating subtype of stroke; yet, effective clinical treatment is very limited. Accumulating evidence has shown that mild to moderate hypothermia is a promising intervention for ischemic stroke and ICH. Current physical cooling methods, however, are less efficient and often impractical for acute ICH patients. The present investigation tested pharmacologically induced hypothermia (PIH) using the second generation neurotensin receptor (NTR) agonist HPI-201 (formerly known as ABS-201) in an adult mouse model with ICH. Acute or delayed administrations of HPI-201 (2 mg/kg bolus injection followed by 2 injections of 1 mg/kg, i.p.) were initiated at 1 or 24 hrs after ICH. HPI-201 induced mild hypothermia within 30 min and maintained body and brain temperatures at 32.7±0.4°C for at least 6 hrs without causing observable shivering. With the 1 hr delayed treatment, HPI-201-induced PIH significantly reduced ICH-induced cell death and brain edema compared to saline-treated ICH animals. When HPI-201-induced hypothermia was initiated 24 hrs after the onset of ICH, it still significantly attenuated brain edema, cell death and blood brain barrier breakdown. HPI-201 significantly decreased the expression of MMP-9, reduced caspase-3 activation, and increased Bcl-2 expression in the ICH brain. Moreover, ICH mice received 1-hr delayed HPI-201 treatment performed significantly better in the neurological behavior test 48 hrs after ICH. All together, these data suggest that systemic injection of HPI-201 is an effective hypothermic strategy that protects the brain from ICH injury with a wide therapeutic window. The protective effect of this PIH therapy is partially mediated through the alleviation of apoptosis and neurovascular damage. We suggest that pharmacological hypothermia using the newly developed neurotensin analogs is a promising therapeutic treatment for ICH.
PMCID: PMC3961766  PMID: 23912033
Intracerebral hemorrhage; pharmacological hypothermia; PIH; neurotensin receptor; ABS-201; HPI-201
21.  Association of Serum Phosphorus Concentration with Mortality in Elderly and Non-Elderly Hemodialysis Patients 
Hypo- and hyperphosphatemia have each been associated with increased mortality in maintenance hemodialysis (MHD) patients. There has not been previous evaluation of a differential relationship between serum phosphorus level and death risk across varying age groups in MHD patients.
Design and Settings
In a 6-year cohort of 107,817 MHD patients treated in a large dialysis organization, we examined the association between serum phosphorus levels with all-cause and cardiovascular mortality within 5 age categories (15-<45, 45-<65, 65-<70, 70-<75 and ≥75 years old) using Cox proportional hazards model adjusted for case-mix covariates and malnutrition inflammation complex syndrome (MICS) surrogates.
Main outcome measure
all-cause and cardiovascular mortality.
The overall mean age of the cohort was 60±16 years, among whom there were 45% women, 35% Blacks and 58% diabetics. The time averaged serum phosphorus level (mean ± SD) within each age category was 6.26±1.4, 5.65±1.2, 5.26±1.1, 5.11±1.0 and 4.88±1.0 mg/dl, respectively (P for trend <0.001). Hyperphosphatemia (>5.5 mg/dl) was consistently associated with increased all-cause and cardiovascular mortality risks across all age categories including after adjustment for case-mix and MICS-related covariates. In fully adjusted models, a low serum phosphorus level (<3.5 mg/dl) was associated with increased all-cause mortality only in elderly MHD patients ≥65 years old (hazard ratio [HR] (95% confidence interval[CI]): 1.21(1.07-1.37), 1.13(1.02-1.25), and 1.28(1.2-1.37) for patients 65-<70, 70-<75, and ≥75 years old, respectively], but not in younger patients (<65 years old). A similar differential for cardiovascular mortality of serum phosphorus levels between old and young age groups was observed.
The association between hyperphosphatemia and mortality is similar across all age groups of MHD patients, whereas hypophosphatemia is associated with increased mortality only in elderly MHD patients. Preventing very low serum phosphorus levels in elderly dialysis patients may be associated with better outcomes, which needs to be examined in future studies.
PMCID: PMC3735629  PMID: 23631888
hemodialysis; mortality; phosphorus; elderly
22.  Nuclear Import of Transcription Factor BR-C Is Mediated by Its Interaction with RACK1 
PLoS ONE  2014;9(10):e109111.
The transcription factor Broad Complex (BR-C) is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB) domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1), a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.
PMCID: PMC4184850  PMID: 25280016
23.  Impact of age on survival predictability of bone turnover markers in hemodialysis patients 
Nephrology Dialysis Transplantation  2013;28(10):2535-2545.
Abnormalities in serum alkaline phosphatase (ALP) and intact parathyroid hormone (PTH) concentrations, as biochemical markers of bone turnover in dialysis patients, correlate with increased mortality in maintenance hemodialysis (MHD) patients. Changes in bone turnover rate vary with age. The mortality predictability of serum ALP and PTH levels in MHD patients may be different across ages.
We examined differences across four age groups (18 to <45, 45 to <65, 65 to <75 and ≥75 years) in the mortality predictability of serum ALP and PTH in 102 149 MHD patients using Cox models.
Higher serum ALP levels were associated with higher mortality across all ages; however, the ALP–mortality association was much stronger in young patients (<45 years) compared with older patients. The association between higher serum PTH levels and mortality was stronger in older patients compared with the younger groups. Serum PTH levels were incrementally associated with mortality only in middle-aged and elderly patients (≥45 years). Compared with patients with serum PTH 150 to <300pg/mL, the death risks were higher in patients with serum PTH 300 to <600pg/mL [HRs (95% CI): 1.05 (1.01–1.10), 1.15 (1.10–1.21) and 1.25 (1.19–1.31) for patients 45 to <65, 65 to <75 and ≥75 years, respectively], and ≥600pg/mL [HRs(95% CI): 1.07 (1.01–1.14), 1.31(1.21–1.42) and 1.45(1.33–1.59) for age categories 45 to <65, 65 to <75 and ≥75 years, respectively]. However, no significant association between higher serum PTH levels and mortality was observed in patients <45 years.
There are important differences in mortality-predictability of serum ALP and PTH in older MHD patients compared with their younger counterparts. The effect of age needs to be considered when interpreting the prognostic implications of serum ALP and PTH levels.
PMCID: PMC3784839  PMID: 23904397
age; alkaline phosphatase; bone turnover markers; hemodialysis; parathyroid hormone
24.  Preconditioning of bone marrow mesenchymal stem cells by prolyl hydroxylase inhibition enhances cell survival and angiogenesis in vitro and after transplantation into the ischemic heart of rats 
Poor cell survival and limited functional benefits have restricted the efficacy of bone marrow mesenchymal stem cells (BMSCs) in the treatment of myocardial infarction. We showed recently that hypoxia preconditioning of BMSCs and neural progenitor cells before transplantation can enhance the survival and therapeutic properties of these cells in the ischemic brain and heart. The present investigation explores a novel strategy of preconditioning BMSCs using the Hypoxia-inducible factor 1α (HIF-α) prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) to enhance their survival and therapeutic efficacy after transplantation into infarcted myocardium.
BMSCs from green fluorescent protein transgenic rats were cultured with or without 1 mM DMOG for 24 hours in complete culture medium before transplantation. Survival and angiogenic factors were evaluated in vitro by trypan blue staining, Western blotting, and tube formation test. In an ischemic heart model of rats, BMSCs with and without DMOG preconditioning were intramyocardially transplanted into the peri-infarct region 30 minutes after permanent myocardial ischemia. Cell death was measured 24 hours after engraftment. Heart function, angiogenesis and infarct size were measured 4 weeks later.
In DMOG preconditioned BMSCs (DMOG-BMSCs), the expression of survival and angiogenic factors including HIF-1α, vascular endothelial growth factor, glucose transporter 1 and phospho-Akt were significantly increased. In comparison with control cells, DMOG-BMSCs showed higher viability and enhanced angiogenesis in both in vitro and in vivo assays. Transplantation of DMOG-BMSCs reduced heart infarct size and promoted functional benefits of the cell therapy.
We suggest that DMOG preconditioning enhances the survival capability of BMSCs and paracrine effects with increased differentiation potential. Prolyl hydroxylase inhibition is an effective and feasible strategy to enhance therapeutic efficacy and efficiency of BMSC transplantation therapy after heart ischemia.
PMCID: PMC4535299  PMID: 25257482
25.  Biosensors for hepatitis B virus detection 
World Journal of Gastroenterology : WJG  2014;20(35):12485-12492.
A biosensor is an analytical device used for the detection of analytes, which combines a biological component with a physicochemical detector. Recently, an increasing number of biosensors have been used in clinical research, for example, the blood glucose biosensor. This review focuses on the current state of biosensor research with respect to efficient, specific and rapid detection of hepatitis B virus (HBV). The biosensors developed based on different techniques, including optical methods (e.g., surface plasmon resonance), acoustic wave technologies (e.g., quartz crystal microbalance), electrochemistry (amperometry, voltammetry and impedance) and novel nanotechnology, are also discussed.
PMCID: PMC4168081  PMID: 25253948
Biosensor; Hepatitis B virus; Diagnosis; Detection; Quartz crystal microbalance

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