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1.  Cytogenetic relationships among Citrullus species in comparison with some genera of the tribe Benincaseae (Cucurbitaceae) as inferred from rDNA distribution patterns 
Comparative mapping of 5S and 45S rDNA by fluorescent in situ hybridization (FISH) technique is an excellent tool to determine cytogenetic relationships among closely related species.
In this study, the number and position of 5S and 45S rDNA loci in all Citrullus species and subspecies were determined. The cultivated watermelon (C. lanatus subsp. vulgaris), C. lanatus subsp. mucosospermus, C. colocynthis and C. naudinianus (or Acanthosicyos naudinianus) had two 45S rDNA loci and one 5S rDNA locus which was located syntenic to one of the 45S rDNA loci. C. ecirrhosus and C. lanatus subsp. lanatus had one 45S rDNA locus and two 5S rDNA loci, each located on a different chromosome. C. rehmii had one 5S and one 45S rDNA locus positioned on different chromosomes. The distribution of 5S and 45S rDNA in several species belonging to other genera in Benincaseae tribe was also investigated. The distribution pattern of rDNAs showed a great difference among these species.
The present study confirmed evolutionary closeness among cultivated watermelon (C. lanatus subsp. vulgaris), C. lanatus subsp. mucosospermus and C. colocynthis. Our result also supported that C. lanatus subsp. lanatus was not a wild form of the cultivated watermelon instead was a separate crop species. In addition, present cytogenetic analysis suggested that A. naudinianus was more closely related to Cucumis than to Citrullus or Acanthosicyos, but with a unique position and may be a link bridge between the Citrullus and the Cucumis.
PMCID: PMC4835933  PMID: 27090090
rDNA; Oligonucleotides probes; Citrullus species; Cytogenetic relationship; Fluorescence in situ hybridization
2.  Fitness-Balanced Escape Determines Resolution of Dynamic Founder Virus Escape Processes in HIV-1 Infection 
Journal of Virology  2015;89(20):10303-10318.
To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days postinfection. T-cell responses were screened by gamma interferon/interleukin-2 (IFN-γ/IL-2) FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individual's viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r = 0.43; P = 0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack.
IMPORTANCE Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used deep sequencing to comprehensively follow viral evolution over time in six linked HIV transmission pairs. We then mapped T-cell responses to explore if mutations arose due to adaption to the host and found that escape processes were often highly dynamic, with multiple mutations arising within targeted epitopes. When we explored the impact of these mutations on replicative capacity, we found that dynamic escape processes only resolve with the selection of mutations that conferred escape with no fitness cost to the virus. These results provide further understanding of the complicated viral-host interactions that occur during early HIV-1 infection and may help inform the design of future vaccine immunogens.
PMCID: PMC4580195  PMID: 26223634
3.  MLVA and MLST typing of Brucella from Qinghai, China 
The Qinghai-Tibet Plateau (QTP) of China is an extensive pastoral and semi-pastoral area, and because of poverty and bad hygiene conditions, Brucella is highly prevalent in this region. In order to adequately prevent this disease in the QTP region it is important to determine the identity of Brucella species that caused the infection.
A total of 65 Brucella isolates were obtained from human, livestock and wild animals in Qinghai, a Chinese province in east of the QTP. Two molecular typing methods, MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) were used to identify the species and genotypes of these isolates.
Both MLVA and MLST typing methods classified the 65 isolates into three species, B. melitensis, B. abortus and B. suis, which included 60, 4 and 1 isolates respectively. The MLVA method uniquely detected 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes for B. melitensis, B. abortus and B. suis, respectively. However, none of these genotypes exactly matched any of the genotypes in the Brucella2012 MLVA database. The MLST method identified five known ST types: ST7 and ST8 (B. melitensis), ST2 and ST5 (B. abortus), and ST14 (B. suis). We also detected a strain with a mutant type (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2). Extensive genotype-sharing events could be observed among isolates from different host species.
There were at least three Brucella (B. melitensis, B. abortus and B. suis) species in Qinghai, of which B. melitensis was the predominant species in the area examined. The Brucella population in Qinghai was very different from other regions of the world, possibly owing to the unique geographical characteristics such as extremely high altitude in QTP. There were extensive genotype-sharing events between isolates obtained from humans and other animals. Yaks, sheep and blue sheep were important zoonotic reservoirs of brucellosis causing species found in humans.
Electronic supplementary material
The online version of this article (doi:10.1186/s40249-016-0123-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4830052  PMID: 27072820
Brucella; Molecular identification; Genotype; Zoonotic host
4.  The effect of growing Rod treatment on coronal balance during serial lengthening surgeries in early onset scoliosis 
Gaining and maintaining spinal balance after surgery is of great importance for early onset scoliosis (EOS). However, tendency of balance on the coronal plane after growing rod surgery has not been studied before. This study evaluated the effect of growing rod treatment on coronal balance (CB) during serial lengthening surgeries in EOS.
All EOS patients treated with growing rod technique in our hospital from August 2002 to June 2014 were retrospectively reviewed. Radiographic data before the sixth lengthening surgery were measured on the posteroanterior X-ray images, including global CB (C7 plumbline-central sacral vertical line, C7PL-CSVL), regional CB (apical vertebrae-CSVL), Cobb angle of the main curve and pelvic inlet width (PIW). Global CB index and regional CB index were calculated as dividing global CB and regional CB by PIW, respectively. The changes of these parameters during repeated lengthening surgeries were analyzed.
Five hundred seventy Radiographs of 67 patients, including 134 images before and after growing rod insertion surgeries and 436 images pre- and post-lengthening surgeries were measured. Global CB and global CB index did not show significant differences between every two set points during lengthening procedures (P > 0.05). The percentage of patients with C7PL-CSVL distance more than 20 mm roughly ranged from 30 to 45 % during the lengthening process. With regards to regional CB and main curve Cobb angles, there were significant differences between every two adjacent set points during the first five lengthening surgeries (P < 0.05).
Global CB did not significantly change during serial lengthening surgeries and C7PL-CSVL distances of greater than 20 mm comprised of over one third of patients during growing rod treatment. However, worsening regional CB and Cobb angles of the main curve during lengthening intervals were corrected by lengthening manipulation and maintained at a stable level.
PMCID: PMC4830065  PMID: 27072316
Coronal balance; Growing rod; Early onset scoliosis; Lengthening surgery
5.  Effects of Xiaoyaosan on Stress-Induced Anxiety-Like Behavior in Rats: Involvement of CRF1 Receptor 
Background. Compared with antidepressant activity of Xiaoyaosan, the role of Xiaoyaosan in anxiety has been poorly studied. Objective. To observe the effects of Xiaoyaosan on anxiety-like behavior induced by chronic immobilization stress (CIS) and further explore whether these effects were related to CRF1R signaling. Methods. Adult male SD rats were randomly assigned to five groups (n = 12): the nonstressed control group, vehicle-treated (saline, p.o.) group, Xiaoyaosan-treated (3.854 g/kg, p.o.) group, vehicle-treated (surgery) group, and antalarmin-treated (surgery) group. Artificial cerebrospinal fluid (0.5 μL/side) or CRF1R antagonist antalarmin (125 ng/0.5 μL, 0.5 μL/side) was bilaterally administered into the basolateral amygdala in the surgery groups. Except for the nonstressed control group, the other four groups were exposed to CIS (14 days, 3 h/day) 30 minutes after treatment. On days 15 and 16, all animals were subjected to the elevated plus-maze (EPM) and novelty suppressed feeding (NSF) test. We then examined the expression of CRF1R, pCREB, and BDNF in the amygdala. Results. Chronic pretreatment with Xiaoyaosan or antalarmin significantly reversed elevated anxiety-like behavior and the upregulated level of CRF1R and BDNF in the amygdala of stressed rats. pCREB did not differ significantly among the groups. Conclusions. These results suggest that Xiaoyaosan exerts anxiolytic-like effects in behavioral tests and the effects may be related to CRF1R signaling in the amygdala.
PMCID: PMC4793091  PMID: 27042185
6.  Hematopoietic Stem Cell Capture and Directional Differentiation into Vascular Endothelial Cells for Metal Stent-Coated Chitosan/Hyaluronic Acid Loading CD133 Antibody 
Tissue Engineering. Part A  2014;21(5-6):1173-1183.
A series of metal stents coated with chitosan/hyaluronic acid (CS/HA) loading antibodies by electrostatic self-assembled method were prepared, and the types of cells captured by antibodies and their differentiation in vascular endothelial cells (ECs) evaluated by molecular biology and scanning electron microscope. The results showed that CD133 stent can selectively capture hematopoietic stem cells (HSC),which directionally differentiate into vascular ECs in peripheral blood by (CS/HA) induction, and simultaneously inhibit migration and proliferation of immune cells and vascular smooth muscle cells (MCs). CD34 stent can capture HSC, hematopoietic progenitor cells that differentiate into vascular ECs and immune cells, promoting smooth MCs growth, leading to thrombosis, inflammation, and rejection. CD133 stent can be implanted into miniature pig heart coronary and can repair vascular damage by capturing own HSC, thus contributing to the rapid natural vascular repair, avoiding inflammation and rejection, thrombosis and restenosis. These studies demonstrated that CD133 stent of HSC capture will be an ideal coated metal stent providing a new therapeutic approach for cardiovascular and cerebrovascular disease.
PMCID: PMC4356193  PMID: 25404533
7.  Connexin 43 Channels are Essential for Normal Bone Structure and Osteocyte Viability 
Connexin (Cx) 43 serves important roles in bone function and development. Targeted deletion of Cx43 in osteoblasts or osteocytes leads to increased osteocyte apoptosis, osteoclast recruitment, and reduced biomechanical properties. Cx43 forms both gap junction channels and hemichannels, which mediate the communication between adjacent cells or between cell and extracellular environments, respectively. Two transgenic mouse models driven by a DMP1 promoter with the overexpression of dominant negative Cx43 mutants were generated to dissect the functional contribution of Cx43 gap junction channels and hemichannels in osteocytes. The R76W mutant blocks gap junction channel, but not hemichannel function, and the Δ130-136 mutant inhibits activity of both types of channels. Δ130-136 mice showed a significant increase in bone mineral density compared to WT and R76W mice. MicroCT analyses revealed a significant increase in total tissue and bone area in midshaft cortical bone of Δ130-136 mice. The bone marrow cavity was expanded, whereas the cortical thickness was increased and associated with increased bone formation along the periosteal area. However, there is no significant alteration in the structure of trabecular bone. Histologic sections of the midshaft showed increased apoptotic osteocytes in Δ130-136, but not in WT and R76W, mice which correlated with altered biomechanical and estimated bone material properties. Osteoclasts were increased along the endocortical surface in both transgenic mice with a greater effect in Δ130-136 mice which likely contributed to the increased marrow cavity. Interestingly, the overall expression of serum bone formation and resorption markers were higher in R76W mice. These findings suggest that osteocytic Cx43 channels play distinctive roles in the bone; hemichannels play a dominant role in regulating osteocyte survival, endocortical bone resorption and periosteal apposition, and gap junction communication is involved in the process of bone remodeling.
PMCID: PMC4333056  PMID: 25270829
8.  Significance and prognostic value of increased serum direct bilirubin level for lymph node metastasis in Chinese rectal cancer patients 
World Journal of Gastroenterology  2016;22(8):2576-2584.
AIM: To determine the significance of increased serum direct bilirubin level for lymph node metastasis (LNM) in Chinese rectal cancer patients, after those with known hepatobiliary and pancreatic diseases were excluded.
METHODS: A cohort of 469 patients, who were treated at the China-Japan Friendship Hospital, Ministry of Health (Beijing, China), in the period from January 2003 to June 2011, and with a pathological diagnosis of rectal adenocarcinoma, were recruited. They included 231 patients with LNM (49.3%) and 238 patients without LNM. Follow-up for these patients was taken through to December 31, 2012.
RESULTS: The baseline serum direct bilirubin concentration was (median/inter-quartile range) 2.30/1.60-3.42 μmol/L. Univariate analysis showed that compared with patients without LNM, the patients with LNM had an increased level of direct bilirubin (2.50/1.70-3.42 vs 2.10/1.40-3.42, P = 0.025). Multivariate analysis showed that direct bilirubin was independently associated with LNM (OR = 1.602; 95%CI: 1.098-2.338, P = 0.015). Moreover, we found that: (1) serum direct bilirubin differs between male and female patients; a higher concentration was associated with poor tumor classification; (2) as the baseline serum direct bilirubin concentration increased, the percentage of patients with LNM increased; and (3) serum direct bilirubin was associated with the prognosis of rectal cancer patients and higher values indicated poor prognosis.
CONCLUSION: Higher serum direct bilirubin concentration was associated with the increased risk of LNM and poor prognosis in our rectal cancers.
PMCID: PMC4768203  PMID: 26937145
Rectal cancer; Lymph node metastasis; Direct bilirubin; Risk; Prognosis
9.  Higher Serum Uric Acid Is Associated with Higher Bone Mineral Density in Chinese Men with Type 2 Diabetes Mellitus 
Accumulating evidence suggests that oxidative stress is associated with osteoporosis. Serum uric acid (UA) is a strong endogenous antioxidant. Therefore, we investigated the relationship between the serum UA and BMD in Chinese men with T2DM. In this cross-sectional study of 621 men with T2DM, BMDs at lumbar spine (L2–4), femoral neck (FN), and total hip (TH) were measured by dual-energy X-ray absorptiometry (DXA). Serum levels of UA, calcium (Ca), 25-OH vitamin D3 (vitD3), parathyroid hormone (PTH), and creatinine (Cr) were also tested. Data analyses revealed that serum UA levels were positively associated with BMD at all sites (p < 0.05) in men with T2DM after adjusting for multiple confounders. The serum UA levels were positively correlated with body weight (r = 0.322), body mass index (BMI) (r = 0.331), Ca (r = 0.179), and Cr (r = 0.239) (p < 0.001) and were also positively associated with the concentrations of PTH (r = 0.10, p < 0.05). When compared with those in the lowest tertile of UA levels, men with T2DM in the highest tertile had a lower prevalence of osteoporosis or osteopenia (adjusted odds ratio 0.54, 95% confidence interval [CI] 0.31–0.95). These data suggest that higher serum levels of UA are associated with higher BMDs and lower risks of osteoporosis in Chinese men with T2DM.
PMCID: PMC4789039  PMID: 27022396
10.  Risk Factors for Steatorrhea in Chronic Pancreatitis: A Cohort of 2,153 Patients 
Scientific Reports  2016;6:21381.
This study aimed to investigate the occurrence of and determine the risk factors for steatorrhea in chronic pancreatitis (CP). It was based on analysis of both retrospectively and prospectively acquired database for CP patients admitted to our center from January 2000 to December 2013. Demographic data, course of disease, medical history, and follow-up evaluations of patients were documented in detail. Cumulative rate of steatorrhea was calculated by using the Kaplan–Meier method. For risk factor analysis, multivariate analysis by Cox proportional hazards regression model was performed. A total of 2,153 CP patients were included with a mean follow-up duration of 9.3 years. Approximately 14% (291/2,153) of CP patients presented with steatorrhea at diagnosis of CP. Cumulative rates of steatorrhea at 1, 5, 10, and 20 years after diagnosis of CP were 4.27% (95% CI: 3.42%–5.34%), 12.53% (95% CI: 10.74%–14.59%), 20.44% (95% CI: 17.37%–23.98%) and 30.82% (95% CI: 20.20%–45.21%), respectively. Male gender (HR = 1.771, p = 0.004), diabetes (HR = 1.923, p < 0.001), alcohol abuse (HR = 1.503, p = 0.025) and pancreaticoduodenectomy (HR = 2.901, p < 0.001) were independent risk factors for steatorrhea while CP in adolescents (HR = 0.433, p = 0.009) was a protective factor. In conclusion, male gender, adult, diabetes, alcohol abuse and pancreaticoduodenectomy lead to increased risk of steatorrhea in CP patients.
PMCID: PMC4753434  PMID: 26877248
11.  Intra-molecular Triplet Energy Transfer is a General Approach to Improve Organic Fluorophore Photostability 
Bright, long-lasting and non-phototoxic organic fluorophores are essential to the continued advancement of biological imaging. Traditional approaches towards achieving photostability, such as the removal of molecular oxygen and the use of small-molecule additives in solution, suffer from potentially toxic side effects, particularly in the context of living cells. The direct conjugation of small-molecule triplet state quenchers, such as cyclooctatetraene (COT), to organic fluorophores has the potential to bypass these issues by restoring reactive fluorophore triplet states to the ground state through intra-molecular triplet energy transfer. Such methods have enabled marked improvement in cyanine fluorophore photostability spanning the visible spectrum. However, the generality of this strategy to chemically and structurally diverse fluorophore species has yet to be examined. Here, we show that the proximal linkage of COT increases the photon yield of a diverse range of organic fluorophores widely used in biological imaging applications, demonstrating that the intra-molecular triplet energy transfer mechanism is a potentially general approach for improving organic fluorophore performance and photostability.
PMCID: PMC4749434  PMID: 26700693
12.  The Osteogenic Niche Promotes Early-Stage Bone Colonization of Disseminated Breast Cancer Cells 
Cancer cell  2015;27(2):193-210.
Breast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We further elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human datasets analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases.
In advanced stages, breast cancer bone metastases are driven by paracrine crosstalk among cancer cells, osteoblasts, and osteoclasts, which constitute a vicious osteolytic cycle. Current therapies targeting this process limit tumor progression, but do not improve patient survival. On the other hand, bone micrometastases may remain indolent for years before activating the vicious cycle, providing a therapeutic opportunity to prevent macrometastases. Here, we show that bone colonization is initiated in a microenvironment niche exhibiting active osteogenesis. Cancer and osteogenic cells form heterotypic adherens junctions, which enhance mTOR activity and drive early-stage bone colonization prior to osteolysis. These results reveal a strong connection between osteogenesis and micrometastasis and suggest potential therapeutic targets to prevent bone macrometastases.
PMCID: PMC4326554  PMID: 25600338
13.  Texture Feature Analysis for Computer-Aided Diagnosis on Pulmonary Nodules 
Journal of Digital Imaging  2014;28(1):99-115.
Differentiation of malignant and benign pulmonary nodules is of paramount clinical importance. Texture features of pulmonary nodules in CT images reflect a powerful character of the malignancy in addition to the geometry-related measures. This study first compared three well-known types of two-dimensional (2D) texture features (Haralick, Gabor, and local binary patterns or local binary pattern features) on CADx of lung nodules using the largest public database founded by Lung Image Database Consortium and Image Database Resource Initiative and then investigated extension from 2D to three-dimensional (3D) space. Quantitative comparison measures were made by the well-established support vector machine (SVM) classifier, the area under the receiver operating characteristic curves (AUC) and the p values from hypothesis t tests. While the three feature types showed about 90 % differentiation rate, the Haralick features achieved the highest AUC value of 92.70 % at an adequate image slice thickness, where a thinner or thicker thickness will deteriorate the performance due to excessive image noise or loss of axial details. Gain was observed when calculating 2D features on all image slices as compared to the single largest slice. The 3D extension revealed potential gain when an optimal number of directions can be found. All the observations from this systematic investigation study on the three feature types can lead to the conclusions that the Haralick feature type is a better choice, the use of the full 3D data is beneficial, and an adequate tradeoff between image thickness and noise is desired for an optimal CADx performance. These conclusions provide a guideline for further research on lung nodule differentiation using CT imaging.
PMCID: PMC4305062  PMID: 25117512
Lung CADx; Lung nodule analysis; Haralick features; Gabor features; LBP features
14.  Validation Study of Medicare Claims to Identify Older US Adults With CKD Using the Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study 
Healthcare claims data may provide a cost-efficient approach for studying chronic kidney disease (CKD).
Study Design
Prospective cohort study.
Setting & Participants
We compared characteristics and outcomes for individuals with CKD defined using laboratory measurements versus claims data from 6,982 Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study participants who had Medicare fee-for-service coverage.
Presence of CKD as defined by both the REGARDS Study (CKDREGARDS) and Medicare data (CKDMedicare), absence of CKD as defined by both, presence of CKDREGARDS but not CKDMedicare, and presence of CKDMedicare but not CKDREGARDS.
Mortality and incident end-stage renal disease (ESRD).
The research study definition of CKD (CKDREGARDS) included estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73m2 or albumin-creatinine ratio (ACR) > 30 mg/g at the REGARDS study visit. CKD in Medicare (CKDMedicare) was identified during the two years before each participant’s REGARDS visit using a claims-based algorithm.
Overall, 32% of participants had CKDREGARDS and 6% had CKDMedicare. The sensitivity, specificity, and positive and negative predictive values of CKDMedicare for identifying CKDREGARDS were 15.5% (95% CI, 14.0%–17.1%), 97.7% (95% CI, 97.2%–98.1%), 75.6% (95% CI, 71.4%–79.5%), and 71.5% (95% CI, 70.4%–72.6%), respectively. Mortality and ESRD incidence rates, expressed per 1,000 person-years, were higher for participants with versus without CKDMedicare (mortality: 72.5 [95% CI, 61.3–83.7] versus 33.3 [95% CI, 31.5–35.2]; ESRD: 16.4 [95% CI, 11.2–21.6] versus 1.3 [95% CI, 0.9–1.6]) and with versus without CKDREGARDS (mortality: 59.9 [95% CI, 55.4–64.4] versus 25.5 [95% CI, 23.6–27.4]; ESRD: 6.8 [95% CI, 5.4–8.3] versus 0.1 [95% CI, 0.0–0.3]). Among participants with CKDREGARDS, those with abdominal obesity, diabetes, anemia, a lower eGFR, more outpatient visits, a hospitalization and a nephrologist visit in the two years before their REGARDS visit were more likely to have CKDMedicare.
CKDREGARDS relied on eGFR and albuminuria assessed at a single visit.
CKD, whether defined in claims or through research study measurements, was associated with increased mortality and ESRD. However, individuals with CKD identified in claims may represent a select high-risk population.
PMCID: PMC4721899  PMID: 25242367
chronic kidney disease (CKD); health care claims data; sensitivity; specificity; predictive value; claims-based algorithm; albuminuria; estimated glomerular filtration rate (eGFR); end-stage renal disease (ESRD)
15.  Gap junction mediated miRNA intercellular transfer and gene regulation: A novel mechanism for intercellular genetic communication 
Scientific Reports  2016;6:19884.
Intercellular genetic communication is an essential requirement for coordination of cell proliferation and differentiation and has an important role in many cellular processes. Gap junction channels possess large pore allowing passage of ions and small molecules between cells. MicroRNAs (miRNAs) are small regulatory RNAs that can regulate gene expression broadly. Here, we report that miRNAs can pass through gap junction channels in a connexin-dependent manner. Connexin43 (Cx43) had higher permeability, whereas Cx30 showed little permeability to miRNAs. In the tested connexin cell lines, the permeability to miRNAs demonstrated: Cx43 > Cx26/30 > Cx26 > Cx31 > Cx30 = Cx-null. However, consistent with a uniform structure of miRNAs, there was no significant difference in permeability to different miRNAs. The passage is efficient; the miRNA level in the recipient cells could be up to 30% of the donor level. Moreover, the transferred miRNA is functional and could regulate gene expression in neighboring cells. Connexin mutation and gap junctional blockers could eliminate this miRNA intercellular transfer and gene regulation. These data reveal a novel mechanism for intercellular genetic communication. Given that connexin expression is cell-specific, this connexin-dependent, miRNA intercellular genetic communication may play an important role in synchronizing and coordinating proliferation and differentiation of specific cell types during multicellular organ development.
PMCID: PMC4728487  PMID: 26814383
16.  Connexin26 (GJB2) deficiency reduces active cochlear amplification leading to late-onset hearing loss 
Neuroscience  2014;0:719-729.
Connexin26 (Cx26, GJB2) mutations account for >50% of nonsyndromic hearing loss. The deafness is not always congenital. A large group of these patients (~30%) demonstrate a late-onset hearing loss, starting in childhood. They have normal hearing early in life and are therefore good candidates for applying protective and therapeutic interventions. However, the underlying deafness mechanism is unclear. In this study, we used a time-controlled, inducible gene knockout technique to knockout Cx26 expression in the cochlea after birth. We found that deletion of Cx26 after postnatal day 5 (P5) in mice could lead to late-onset hearing loss. Similar to clinical observations, the mice demonstrated progressive, mild to moderate hearing loss. The hearing loss initiated at high frequencies and then extended to the middle- and low-frequency range. The cochlea showed normal development and had no apparent hair cell loss. However, distortion product otoacoustic emission (DPOAE) was reduced. The reduction was also progressive and large at high-frequencies. Consistent with DPOAE reduction, we found that outer hair cell electromotility associated nonlinear capacitance was shifted to the right and the slope of voltage dependence was reduced. The endocochlear potential was reduced in Cx26 conditional knockout (cKO) mice but the reduction was not associated with progressive hearing loss. These data suggest that Cx26 deficiency may impair active cochlear amplification leading to late-onset hearing loss. Our study also helps develop newer protective and therapeutic interventions to this common nonsyndromic hearing loss.
PMCID: PMC4268423  PMID: 25451287
gap junction; connexin; Cx26; deafness; inner ear; active cochlear amplification
17.  Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway 
Toxicology and applied pharmacology  2014;282(2):227-236.
Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore, Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway.
PMCID: PMC4721529  PMID: 25450480
Ent-kaurene diterpenoid; Rabdosia rubescens; ROS; ATM; Cell cycle arrest
18.  Low Prognostic Nutritional Index Correlates with Worse Survival in Patients with Advanced NSCLC following EGFR-TKIs 
PLoS ONE  2016;11(1):e0147226.
This study was designed to demonstrate the prognostic value of prognostic nutritional index (PNI), a reflection systemic immunonutritional status, on the long-term survival of patients taking epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs).
In this retrospective study, eligible advanced NSCLC patients with sensitive EGFR mutations (exon 19 deletion or L858R in exon 21) were included to investigate the correlation between the PNI and overall survival (OS). The PNI was calculated as 10 x serum albumin value (g/dl) + 0.005 x peripheral lymphocyte count (per mm3). The prognostic significance of PNI and other clinicopathologic factors was identified by univariate and multivariate analysis.
Finally, 144 patients met the inclusion criteria. The optimal cut-off value of PNI for survival stratification was 48.78. Compared with high PNI group (n = 81), low PNI (n = 63) was significantly associated with elevated C-reactive protein (CRP) level and non-response to TKIs. Overall survival was superior in the high PNI group (HR, 0.44, p = 0.004), especially for patient with L858R (HR, 0.37, p = 0.009) rather than 19 deletion (HR, 0.69, p = 0.401). The independent prognostic value of PNI was validated by multivariate analysis.
This pilot investigation demonstrated that low prognostic nutritional index correlates with worse survival for patients with advanced NSCLC and taking EGFR-TKIs. The assessment of a convenient index, known as PNI, worth attention in routine clinical practice for patients following EGFR-TKIs treatment.
PMCID: PMC4718699  PMID: 26784943
19.  Crystal structure, conformational fixation, and entry-related interactions of mature ligand-free HIV-1 Env 
As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation, and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies, but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C-433C (DS) variant, specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like particle and soluble formats providing a new generation of vaccine antigens.
PMCID: PMC4706170  PMID: 26098315
20.  EFTUD2 Is a Novel Innate Immune Regulator Restricting Hepatitis C Virus Infection through the RIG-I/MDA5 Pathway 
Journal of Virology  2015;89(13):6608-6618.
The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway.
IMPORTANCE Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.
PMCID: PMC4468487  PMID: 25878102
21.  Clinical Severity in Lesch-Nyhan Disease: the Role of Residual Enzyme and Compensatory Pathways 
Mutations in the HPRT1 gene, which encodes the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt), cause Lesch-Nyhan disease (LND) and more mildly affected Lesch-Nyhan variants. Prior studies have suggested a strong correlation between residual hypoxanthine recycling activity and disease severity. However, the relevance of guanine recycling and compensatory changes in the de novo synthesis of purines have received little attention. In the current studies, fibroblast cultures were established for 21 healthy controls and 36 patients with a broad spectrum of disease severity related to HGprt deficiency. We assessed hypoxanthine recycling, guanine recycling, steady-state purine pools, and de novo purine synthesis. There was a strong correlation between disease severity and either hypoxanthine or guanine recycling. Intracellular purines were normal in the HGprt-deficient fibroblasts, but purine wasting was evident as increased purine metabolites excreted from the cells. The normal intracellular purines in the HGprt-deficient fibroblasts was likely due in part to a compensatory increase in purine synthesis, as demonstrated by a significant increase in purinosomes. However, the increase in purine synthesis did not appear to correlate with disease severity. These results refine our understanding of the potential sources of phenotypic heterogeneity in LND and its variants.
PMCID: PMC4277921  PMID: 25481104
Inherited metabolic disease; Genotype-phenotype correlation; Purine metabolism; Purinosome
22.  Energy Efficient Moving Target Tracking in Wireless Sensor Networks 
Moving target tracking in wireless sensor networks is of paramount importance. This paper considers the problem of state estimation for L-sensor linear dynamic systems. Firstly, the paper establishes the fuzzy model for measurement condition estimation. Then, Generalized Kalman Filter design is performed to incorporate the novel neighborhood function and the target motion information, improving with an increasing number of active sensors. The proposed measurement selection approach has some advantages in time cost. As such, if the desired accuracy has been achieved, the parameter initialization for optimization can be readily resolved, which maximizes the expected lifespan while preserving tracking accuracy. Through theoretical justifications and empirical studies, we demonstrate that the proposed scheme achieves substantially superior performances over conventional methods in terms of moving target tracking under the resource-constrained wireless sensor networks.
PMCID: PMC4732062  PMID: 26729129
wireless sensor networks; target tracking; generalized Kalman filter; neighborhood function; fuzzy
23.  Anaplastic Lymphoma Kinase Rearrangement in Digestive Tract Cancer: Implication for Targeted Therapy in Chinese Population 
PLoS ONE  2015;10(12):e0144731.
Anaplastic lymphoma kinase (ALK) rearrangements define a subgroup of lung cancer which is eligible to targeted kinase inhibition. The aim of this study is to observe the incidence rate of ALK fusion in a large cohort of Chinese digestive tract cancer patients.
Patients and Methods
Tissue microarray (TMA) was constructed from 808 digestive tract cancer cases, including 169 esophageal squamous cell carcinoma, 182 gastric cancer and 457 colorectal cancer (CRC) cases. We tested all cases for ALK expression via a fully automated immunohistochemistry (IHC) assay. The IHC-positive cases were subjected to fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (qRT-PCR), target gene enrichment and sequencing for confirmation of ALK gene rearrangement and discovery of novel fusion partner.
Among the tested cases, 2 (0.44%) CRC cases showed positive both by IHC and FISH. By qRT-PCR, EML4–ALK fusion was found in one IHC-positive CRC case. In another IHC-positive CRC case, target gene enrichment and sequencing revealed ALK was fused to a novel partner, spectrin beta non-erythrocytic 1 (SPTBN1). One gastric cancer case showed partially positive IHC result, but no fusion was found by FISH and gene sequencing.
The incidence rate of ALK gene fusion in Chinese CRC patients was 0.44%,but not detectable in gastric and esophageal cancers. The novel SPTBN1 -ALK fusion, together with other ALK fusion genes, may become a potential target for anti-ALK therapy.
PMCID: PMC4683076  PMID: 26678488
24.  Comparison of first-line chemotherapy based on irinotecan or other drugs to treat non-small cell lung cancer in stage IIIB/IV: a systematic review and meta-analysis 
BMC Cancer  2015;15:949.
To compare the efficacy and toxicity of irinotecan-based chemotherapy (IBC) and non-irinotecan-based chemotherapy (NIBC) as first-line treatment for stage IIIB/IV non-small cell lung cancer (NSCLC).
PubMed, EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL), abstracts from the annual meetings of ASCO and the ESMO up to 2014 were searched for randomized controlled trials (RCTs) that compared IBC with NIBC. Data on overall survival (OS) and progression-free survival (PFS) were meta-analyzed to provide hazard ratios (HRs), while data on overall response rate (ORR) and frequencies of toxicity were meta-analyzed to provide relative risk ratios (RR).
Seven RCTs (6 RCTs from Asian population and 1 from non-Asian population) involving 1473 patients with previously untreated stage IIIB/IV NSCLC were included in the meta-analysis. IBC and NIBC were associated with similar ORR (RR: 1.08, 95 %CI: 0.94 to 1.23, p = 0.30), OS (HR: 0.97, 95 %CI: 0.88 to 1.07, p = 0.56), and PFS (HR: 1.02, 95 %CI: 0.97 to 1.08, p = 0.38). However, the subgroups between Asian and non-Asian patients differed significantly in OS (HR: 0.94 vs 1.87, p = 0.007). There was no significant difference for hematological toxicity (RR: 0.79, 95 %CI: 0.60 to 1.04, p = 0.09) and significant worse for non-hematological toxicity (RR: 2.28, 95 %CI: 1.60 to3.24, p < 0.001), when IBC compared to NIBC.
As the available evidence suggests that IBC and NIBC are equivalent in terms of ORR, PFS, OS, at least in Asian patients, we recommend that IBC be considered as a first-line treatment in Asian patients with stage IIIB/IV NSCLC. However, the non-hematological toxicity of IBC must be considered.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1978-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4682247  PMID: 26673747
Irinotecan; Chemotherapy; Non-small cell lung cancer; Meta-analysis
25.  Therapeutic role of endoscopic ultrasound in pancreaticobiliary disease: A comprehensive review 
World Journal of Gastroenterology  2015;21(46):12996-13003.
With the development of technology and accessories, the role of endoscopic ultrasound (EUS) has evolved from diagnostics to therapeutics. In order to characterise the therapeutic role of EUS, we searched Web of Knowledge database and reviewed articles associated with therapeutic EUS. There are two modalities for the therapeutic purpose: drainage and fine-needle injection. EUS-guided drainage is a promising procedure for the treatment of peripancreatic fluid collection and biliary obstruction; EUS-guided fine-needle injections such as celiac plexus neurolysis, for the purpose of pain relief for pancreatic cancer and chronic pancreatitis, has emerged as a promising procedure. The aim of the study was to perform a comprehensive and conscientious review on the techniques, complications and clinical outcomes of those EUS-based procedures.
PMCID: PMC4674718  PMID: 26675538
Endoscopic ultrasound; Therapeutic; Biliary drainage; Peripancreatic fluid collection; Celiac plexus neurolysis

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