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1.  Nonlinear optical microscopy for immunoimaging: a custom optimized system of high-speed, large-area, multicolor imaging 
The nonlinear optical microscopy has become the current state-of-the-art for intravital imaging. Due to its advantages of high resolution, superior tissue penetration, lower photodamage and photobleaching, as well as intrinsic z-sectioning ability, this technology has been widely applied in immunoimaging for a decade. However, in terms of monitoring immune events in native physiological environment, the conventional nonlinear optical microscope system has to be optimized for live animal imaging. Generally speaking, three crucial capabilities are desired, including high-speed, large-area and multicolor imaging. Among numerous high-speed scanning mechanisms used in nonlinear optical imaging, polygon scanning is not only linearly but also dispersion-freely with high stability and tunable rotation speed, which can overcome disadvantages of multifocal scanning, resonant scanner and acousto-optical deflector (AOD). However, low frame rate, lacking large-area or multicolor imaging ability make current polygonbased nonlinear optical microscopes unable to meet the requirements of immune event monitoring.
We built up a polygon-based nonlinear optical microscope system which was custom optimized for immunoimaging with high-speed, large-are and multicolor imaging abilities.
Firstly, we validated the imaging performance of the system by standard methods. Then, to demonstrate the ability to monitor immune events, migration of immunocytes observed by the system based on typical immunological models such as lymph node, footpad and dorsal skinfold chamber are shown. Finally, we take an outlook for the possible advance of related technologies such as sample stabilization and optical clearing for more stable and deeper intravital immunoimaging.
This study will be helpful for optimizing nonlinear optical microscope to obtain more comprehensive and accurate information of immune events.
PMCID: PMC4312289
Nonlinear optical microscopy; immunoimaging; high-speed; large-area; multicolor
2.  A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging 
Nature Communications  2014;5:5573.
Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)3R2. We develop photo-modulatable organic fluorescent probes consisting of a (rR)3R2 peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.
Single-molecule localization microscopy depends on the use of photo-modulatable fluorescent probes; however, many cannot be used in live-cell studies due to poor cell permeability. Pan et al. present a strategy for constructing cell-permeable probes and use it to image actin filament dynamics and lysosomes.
PMCID: PMC4263135  PMID: 25410769
3.  Coexistence of Histologically Confirmed Hashimoto's Thyroiditis with Different Stages of Papillary Thyroid Carcinoma in a Consecutive Chinese Cohort 
Purpose. To determine the relationship between Hashimoto's thyroiditis (HT) and all stages of papillary thyroid carcinoma (PTC) with or without local lymph node metastasis (LNM). Methods. We conducted a retrospective study of thyroidectomies from 2008–2013 in First Affiliated Hospital of Nanjing Medical University. We categorized patients according to the presence of histopathologically proven HT. The prevalence of mPTC (maximum diameter ≤ 10 mm) and crPTC (clinical relevant PTC) and local LNM rates were compared. Results. We evaluated 6,432 consecutive thyroidectomies. In total, 1,328 specimens were confirmed as HT. The prevalence of PTC in this HT cohort was 43.8%, significantly higher than non-HT group. After adjustment of gender and age, the prevalence of PTC was still higher in HT group. HT was a risk factor for PTC in multivariate analysis with odds ratio 2.725 (95% CI, 2.390–3.109) (P < 0.001). However, no correlation was found between HT and LNM of PTC. Conclusion. HT was associated with an increased prevalence of all stages of PTC, independent of tumor size, gender, and age. In contrast, locally advanced disease defined by LNM was unrelated to HT. These data suggest an association of HT with low risk PTC and a potential protective immunologic effect from further disease progression.
PMCID: PMC4255062  PMID: 25505911
4.  Cancer stem cells, epithelial-mesenchymal transition, and drug resistance in high-grade ovarian serous carcinoma 
Human pathology  2013;44(11):2373-2384.
Although epithelial ovarian cancer cells are eliminated by debulking surgery and chemotherapy during initial treatment, it is believed that only a subset of cancer cells, that is, cancer stem cells, may be an important source of tumor recurrence and drug resistance. This review highlights our current understanding of high-grade serous carcinoma, ovarian cancer stem cells, common methods for enrichment of ovarian cancer stem cells, mechanisms involved in drug resistance, and potential strategies for overcoming drug resistance, with associated potential controversies and pitfalls. We also review the potential relationship between epithelial-to-mesenchymal transition and cancer stem cells and how we can induce cancer cells to differentiate into benign stromal fibroblasts in response to certain chemotherapy drugs.
PMCID: PMC3797876  PMID: 23850493
High-grade ovarian serous cancer; Cancer stem cell; Epithelial-mesenchymal transition
5.  Algisyl-LVR™ with coronary artery bypass grafting reduces left ventricular wall stress and improves function in the failing human heart☆,☆☆ 
International journal of cardiology  2013;168(3):2022-2028.
Left ventricular (LV) wall stress reduction is a cornerstone in treating heart failure. Large animal models and computer simulations indicate that adding non-contractile material to the damaged LV wall can potentially reduce myofiber stress. We sought to quantify the effects of a novel implantable hydrogel (Algisyl-LVR™) treatment in combination with coronary artery bypass grafting (i.e. Algisyl-LVR™+CABG) on both LV function and wall stress in heart failure patients.
Methods and results
Magnetic resonance images obtained before treatment (n=3), and at 3 months (n=3) and 6 months (n=2) afterwards were used to reconstruct the LV geometry. Cardiac function was quantified using end-diastolic volume (EDV), end-systolic volume (ESV), regional wall thickness, sphericity index and regional myofiber stress computed using validated mathematical modeling. The LV became more ellipsoidal after treatment, and both EDV and ESV decreased substantially 3 months after treatment in all patients; EDV decreased from 264±91 ml to 146±86 ml and ESV decreased from 184±85 ml to 86±76 ml. Ejection fraction increased from 32±8% to 47±18% during that period. Volumetric-averaged wall thickness increased in all patients, from 1.06±0.21 cm (baseline) to 1.3±0.26 cm (3 months). These changes were accompanied by about a 35% decrease in myofiber stress at end-of-diastole and at end-of-systole. Post-treatment myofiber stress became more uniform in the LV.
These results support the novel concept that Algisyl-LVR™+CABG treatment leads to decreased myofiber stress, restored LV geometry and improved function.
PMCID: PMC3748222  PMID: 23394895
Congestive heart failure; Dilated cardiomyopathy; Coronary artery bypass grafting; Left ventricular wall stress; Mathematical modeling; Magnetic resonance imaging
6.  The Wuhan-Zhuhai (WHZH) cohort study of environmental air particulate matter and the pathogenesis of cardiopulmonary diseases: study design, methods and baseline characteristics of the cohort 
BMC Public Health  2014;14(1):994.
Particulate air pollution has been recognized to be associated with a wide range of adverse health effects, including increased mortality, morbidity, exacerbation of respiratory conditions. However, earlier physiological or pathological changes or long-term bodies’ reaction to air pollutants have not been studied in depth in China. The Wuhan-Zhuhai (WHZH) cohort study is designed to investigate the association between air pollutants exposure and physiological or pathological reactions on respiratory and cardiovascular system.
The cohort is a community-based prospective study that includes 4812 individuals aged 18–80 years. The collections of data were conducted from April to May 2011 in Wuhan city and in May 2012 in Zhuhai city. At baseline, data on demographic and socioeconomic information, occupational history, family disease history, lifestyle, cooking mode, daily travel mode, physical activity and living condition have been collected by questionnaires. Participants underwent an extensive physical examination, including anthropometry, spirometry, electrocardiography, and measurements of blood pressure, heart rate, exhaled nitric oxide and carbon monoxide. Potential conditions in the lung, heart, liver, spleen, and skin were synchronously performed. In addition, samples of morning urine, fasting blood serum and plasma were collected during physical health examination. DNA were extracted and were stored at -80°C. Environment concentrations of particulate matter and chemicals were determined for 15 days in each of four seasons. Participants are followed for physiological or pathological changes or incidence of cardiopulmonary diseases every 3 years.
The results obtained in WHZH cohort study may increase a better understanding of the relationship between particulate air pollution and its components and possible health damages. And the potential mechanisms underlying the development of cardiopulmonary diseases has implications for the development of prevention and treatment strategies.
PMCID: PMC4247123  PMID: 25252923
Cohort study; Air pollutants; Particulate matter; Pulmonary function; Respiratory diseases; Cardiovascular diseases
7.  A Coupled Biventricular Finite Element and Lumped Parameter Circulatory System Model of Heart Failure 
Numerical modeling of the cardiovascular system is becoming an important tool for assessing the influence of heart disease and treatment therapies. In the current study we present an approach for modeling the interaction between the heart and circulatory system. This was accomplished by creating animal specific biventricular finite element models, which characterize the mechanical response of the heart, and coupling them to a lumped parameter model that represents the systemic and pulmonic circulatory system. In order to minimize computation time, the coupling was enforced in a weak (one-way) manner, where the ventricular pressure-volume relationships were generated by the finite element models and then passed into the circulatory system model to ensure volume conservation and physiological pressure changes. The models were first validated by tuning the parameters, such that the output of the models matched experimentally measured pressures and volumes. Then the models were used to examine cardiac function and the myofiber stress in a healthy canine heart and a canine heart with dilated cardiomyopathy. The results show good agreement with experimental measurements. The stress in the case of cardiomyopathy was found to increase significantly, while the pump function was decreased, compared to the healthy case. The total run time of the simulations is less than many fully-coupled models presented in the literature. This will allow for a much quicker evaluation of possible treatment strategies for combating the effects of heart failure, especially in optimization schemes that require numerous finite element simulations.
PMCID: PMC3367128  PMID: 22248290
Finite Element; Lumped Parameter; Cardiac Mechanics
8.  Patient-specific finite element modeling of the Cardiokinetix Parachute® device: Effects on left ventricular wall stress and function 
The Parachute® (Cardiokinetix, Inc., Menlo Park, California) is a catheter-based device intended to reverse left ventricular (LV) remodeling after antero-apical myocardial infarction. When deployed, the device partitions the LV into upper and lower chambers. To simulate its mechanical effects, we created a finite element LV model based on computed tomography (CT) images from a patient before and 6 months after Parachute® implantation. Acute mechanical effects were determined by in-silico device implantation (VIRTUAL-Parachute). Chronic effects of the device were determined by adjusting the diastolic and systolic material parameters to better match the 6-month post-implantation CT data and LV pressure data at end-diastole (ED) (POST-OP). Regional myofiber stress and pump function were calculated in each case. The principal finding is that VIRTUAL-Parachute was associated with a 61.2% reduction in the lower chamber myofiber stress at ED. The POST-OP model was associated with a decrease in LV diastolic stiffness, and a larger reduction in myofiber stress at the upper (27.1%) and lower chamber (78.4%) at ED. Myofiber stress at end-systole and stroke volume were little changed in the POST-OP case. These results suggest that the primary mechanism of Parachute® is a reduction in ED myofiber stress, which may reverse eccentric post-infarct LV hypertrophy.
PMCID: PMC4118670  PMID: 24793158
Myocardial Infarction; Remodeling; Finite element method; Surgical ventricular restoration
9.  Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways 
Science (New York, N.Y.)  2013;342(6154):118-123.
Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers “virulent” sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism.
PMCID: PMC4096153  PMID: 24092744
10.  C-kit and PDGFRA gene mutations in triple negative breast cancer 
In this study, we evaluated C-kit immunohistochemical expression and C-kit and platelet derived growth factor receptor A (PDGFRA) gene mutations in triple negative breast cancer. 171 cases were analyzed by immunohistochemical staining for the expression of C-kit and 45 cases, including 10 C-kit negative cases and 35 C-kit positive cases, were performed for C-kit gene mutations in exons 9, 11, 13 and 17 and PDGFRA gene mutations in exons 12 and 18. C-kit expression was detected in 42.1% of triple negative breast cancers. Only 1 activating mutation was detected in exon 11 of C-kit gene in 1 case. No activating mutations were found in the other 44 cases. C-kit expression is a frequent finding in triple negative breast cancers; 1 activating mutation which was also found in gastrointestinal stromal tumors was detected; a few cases might benefit from imatinib.
PMCID: PMC4129045  PMID: 25120810
C-KIT; PDGFRA; mutation; triple negative breast cancer; immunohistochemical expression
11.  The Proteasome Inhibitor, MG132, Attenuates Diabetic Nephropathy by Inhibiting SnoN Degradation In Vivo and In Vitro 
BioMed Research International  2014;2014:684765.
Transforming growth factor-β (TGF-β) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-β signaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF-β/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-β were observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF-β were significantly increased (P < 0.05), whereas SnoN was significantly decreased in the DC group (P < 0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P < 0.05), but Arkadia expression gradually increased due to high glucose stimulation (P < 0.05), which could be almost completely reversed by MG132 (P < 0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-β activation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN.
PMCID: PMC4070544  PMID: 25003128
12.  Biventricular Finite Element Modeling of the Acorn CorCap Cardiac Support Device on a Failing Heart 
The Annals of thoracic surgery  2013;95(6):2022-2027.
The Acorn CorCap Cardiac Support Device (CSD; Acorn Cardiovascular Inc, St. Paul, MN) is a woven polyester jacket that is placed around the heart and designed to reverse the progressive remodeling associated with dilated cardiomyopathy. However, the effects of the Acorn CSD on myofiber stress and ventricular function remain unknown. We tested the hypothesis that the Acorn CSD reduces end-diastolic (ED) myofiber stress.
A previously described weakly coupled biventricular finite element (FE) model and circulatory model based on magnetic resonance images of a dog with dilated cardiomyopathy was used. Virtual applications of the CSD alone (Acorn), CSD with rotated fabric fiber orientation (rotated), CSD with 5% prestretch (tight), and CSD wrapped only around the left ventricle (LV; LV-only) were performed, and the effect on myofiber stress at ED and pump function was calculated.
The Acorn CSD has a large effect on ED myofiber stress in the LV free wall, with reductions of 55%, 79%, 92%, and 40% in the Acorn, rotated, tight, and LV-only cases, respectively. However, there is a tradeoff in which the Acorn CSD reduces stroke volume at LV end-diastolic pressure of 8 mm Hg by 23%, 25%, 30%, and 7%, respectively, in the Acorn, rotated, tight, and LV-only cases.
The Acorn CSD significantly reduces ED myofiber stress. However, CSD wrapped only around the LV was the only case with minimal negative effect on pump function. Findings suggest that LV-only CSD and Acorn fabric orientation should be optimized to allow maximal myofiber stress reduction with minimal reduction in pump function.
PMCID: PMC3702266  PMID: 23643546
13.  The Molecular Architecture of the Bacteriophage T4 Neck 
Journal of molecular biology  2013;425(10):1731-1744.
A hexamer of the bacteriophage T4 tail terminator protein, gp15, attaches to the top of the phage tail stabilizing the contractile sheath and forming the interface for binding of the independently assembled head. Here we report the crystal structure of the gp15 hexamer, describe its interactions in T4 virions that have either an extended tail or a contracted tail, and discuss its structural relationship to other phage proteins. The neck of T4 virions is decorated by the “collar” and “whiskers”, made of fibritin molecules. Fibritin acts as a chaperone helping to attach the long tail fibers to the virus during the assembly process. The collar and whiskers are environment-sensing devices, regulating the retraction of the long tail fibers under unfavorable conditions, thus preventing infection. Cryo-electron microscopy analysis suggests that twelve fibritin molecules attach to the phage neck with six molecules forming the collar and six molecules forming the whiskers.
PMCID: PMC3746776  PMID: 23434847
bacteriophage T4; fibritin; gpwac; T4 collar and whiskers; tail terminator protein, gp15
14.  LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells 
Autophagy  2013;9(5):756-769.
LC3 is a marker protein that is involved in the formation of autophagosomes and autolysosomes, which are usually characterized and monitored by fluorescence microscopy using fluorescent protein-tagged LC3 probes (FP-LC3). FP-LC3 and even endogenous LC3 can also be incorporated into intracellular protein aggregates in an autophagy-independent manner. However, the dynamic process of LC3 associated with autophagosomes and autolysosomes or protein aggregates in living cells remains unclear. Here, we explored the dynamic properties of the two types of FP-LC3-containing puncta using fluorescence microscopy techniques, including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). The FRAP data revealed that the fluorescent signals of FP-LC3 attached to phagophores or in mature autolysosomes showed either minimal or no recovery after photobleaching, indicating that the dissociation of LC3 from the autophagosome membranes may be very slow. In contrast, FP-LC3 in the protein aggregates exhibited nearly complete recovery (more than 80%) and rapid kinetics of association and dissociation (half-time < 1 sec), indicating a rapid exchange occurs between the aggregates and cytoplasmic pool, which is mainly due to the transient interaction of LC3 and SQSTM1/p62. Based on the distinct dynamic properties of FP-LC3 in the two types of punctate structures, we provide a convenient and useful FRAP approach to distinguish autophagosomes from LC3-involved protein aggregates in living cells. Using this approach, we find the FP-LC3 puncta that adjacently localized to the phagophore marker ATG16L1 were protein aggregate-associated LC3 puncta, which exhibited different kinetics compared with that of autophagic structures.
PMCID: PMC3669184  PMID: 23482084
autophagosome; protein aggregate; inclusion body; LC3; FRAP; FRET
15.  Incidence of Microscopically Positive Proximal Margins in Adenocarcinoma of the Gastroesophageal Junction 
PLoS ONE  2014;9(2):e88010.
To investigate the incidence and risk factors of microscopically positive proximal margins in Chinese patients with adenocarcinoma of the gastroesophageal junction.
The medical records of 483 patients, who underwent surgical treatment with curative intent for adenocarcinoma of the gastroesophageal junction in a single high-volume tertiary medical center, were reviewed. Demographic, radiographic, endoscopic, pathologic, and treatment-related variables were evaluated. All proximal margins were re-evaluated by two experienced pathologists, and a positive proximal margin was defined as the microscopic presence of invasive tumor cells seen at the esophageal transaction margin submitted en face on final paraffin sections.
The incidence of positive proximal margins was 23.81% in this series. Siewert type, depth of tumor invasion, lymph node involvement, presence of vascular or lymphatic invasion, and presence of perineural invasion were significantly associated with positive proximal margins. On multivariate analysis, the presence of vascular or lymphatic invasion and advanced-stage disease were independent risk factors for positive proximal margins in patients with adenocarcinoma of the gastroesophageal junction.
Residual cancer at proximal resection margins remains a major issue for the surgical treatment of adenocarcinoma of the gastroesophageal junction in China.
PMCID: PMC3914864  PMID: 24505353
16.  ATM impacts IGF-1 signaling in skeletal muscle 
Experimental physiology  2012;98(2):526-535.
Reports that ataxia telangiectasia mutated (ATM) is required for full activation of Akt raise the hypothesis that ATM plays a role in IGF-1 signaling through the Akt/mTOR pathway. Differentiated C2C12 cells harboring either ATM-targeting shRNA or non-targeting shRNA and myotubes from a C2C12 lineage previously exposed to empty vector lentivirus were incubated in the presence or absence of 10 nM IGF-1 followed by western blot analysis. Parallel experiments were performed in isolated soleus muscles from mice expressing only one functional ATM allele (ATM+/−) compared to muscles from wild-type (ATM+/+) mice. IGF-1 increased phosphorylation of Akt S473, Akt T308, and p70 S6 kinase (S6K) in myotubes expressing non-targeting shRNA and in empty vector controls, but the IGF-1 effects were significantly reduced in myotubes with shRNA-mediated ATM knockdown. Likewise, IGF-1-stimulated phosphorylation of Akt S473, Akt T308, mTOR, and S6K was lower in isolated soleus muscles from ATM+/− mice compared to muscles from ATM+/+ mice. The ATM inhibitor KU55933 prevented stimulation of S6K phosphorylation in C2C12 myotubes exposed to IGF-1, suggesting that decreased IGF-1 action is not limited to chronic conditions of decreased ATM function. Stimulation of IRS-1 tyrosine 612 phosphorylation by IGF-1 was unaffected by ATM deficiency, though IGF-1 phosphatidylinositol 3-kinase activity tended to be lower in muscle from ATM haploinsufficient mice compared to wild type muscle. The data suggest that ATM is a modulator of IGF-1 signaling downstream of IRS-1 in skeletal muscle.
PMCID: PMC3553258  PMID: 22941977
Akt; IGF-1; mTOR
17.  Low False-Positives in an mLumin-Based Bimolecular Fluorescence Complementation System with a Bicistronic Expression Vector 
Sensors (Basel, Switzerland)  2014;14(2):3284-3292.
The simplicity and sensitivity of the bimolecular fluorescence complementation (BiFC) assay make it a powerful tool to investigate protein-protein interactions (PPIs) in living cells. However, non-specific association of the fluorescent protein fragments in a BiFC system can complicate evaluation of PPIs. Here, we introduced a bicistronic expression vector, pBudCE4.1, into an mLumin-based BiFC system, denoted as the BEVL-BiFC system. The BEVL-BiFC system achieved a 25-fold contrast in BiFC efficiency between positive (Fos/Jun) and negative (ΔFos/Jun) PPIs. The high BiFC efficiency was due to a low false-positive rate, where less than 2% of cells displayed BiFC in the negative control. K-Ras and its interactive proteins, Ras binding domain (RBD) of Raf-1 and Grb2 were used to confirm the accuracy of the BEVL-BiFC system. The results also provide direct evidence in individual cells that post-translational modification of K-Ras and its localization at the plasma membrane (PM) were not essential for the interaction of K-Ras and Raf-1, whereas the interaction of Grb2 and K-Ras did depend on the PM localization of K-Ras. Taken together, the BEVL-BiFC system was developed to reduce the false-positive phenomenon in BiFC assays, resulting in more robust and accurate measurement of PPIs in living cells.
PMCID: PMC3958255  PMID: 24556667
protein-protein interaction; bimolecular fluorescence complementation (BiFC); expression vector; false positive; fluorescent protein
18.  Influence of genetic abnormalities on semen quality and male fertility: A four-year prospective study 
Background: Wide range of disorders ranging from genetic disorders to coital difficulties can influence male fertility. In this regard, genetic factors are highlighted as the most frequent, contributed to 10-15%, of male infertility causes.
Objective: To investigate the influence of genetic abnormalities on semen quality and reproductive hormone levels of infertile men from Northeast China.
Materials and Methods: 2034 infertile men including 691 patients with abnormal sperm parameters were investigated retrospectively. Semen analysis was performed according to the World Health Organization guidelines. Y chromosome micro deletions were detected by polymerase chain reaction assays. Chromosome analysis was performed using G-banding.
Results: The incidence of abnormal chromosomal karyotype in the patients with abnormal sperm parameters was 12.01% (83/691). The most frequent cause was Klinefelter's syndrome 37.35% (31/83). As the same as chromosomal abnormalities group, the volumes of testes (p=0.000 and 0.000, respectively) and the levels of testosterone (T) (p=0.000), and testosterone/ luteinizing hormone (T/LH) (p=0.000) of patients with Y chromosome micro deletions were significantly lower than those of fertile group. In addition, the levels of follicle-stimulating hormone (FSH) (p=0.000), and luteinizing hormone (LH) (p=0.000) were significantly higher in patients with Y chromosome micro deletions than those in the fertile group. Translocation abnormalities displayed slight effect on sperm motility.
Conclusion: Y chromosome micro deletions and sex chromosome disorders particularly Klinefelter’s (47, XXY), have severe adverse influence on normal hormone levels, testicular volume and sperm count, whereas translocation abnormalities may inversely correlate with sperm motility.
PMCID: PMC4009560  PMID: 24799866
Chromosomal abnormalities; Male infertility; Semen quality; Y -chromosome microdeletions
19.  Urinary cytology with acridine orange fluorescence is highly valuable for predicting high-grade upper urinary tract urothelial carcinoma 
Objectives: To evaluate the clinical value of acridine orange fluorescent staining in urinary cytology for the diagnosis of upper urinary tract urothelial carcinoma. Methods and materials: A retrospective analysis was conducted with 510 cases of upper urinary tract urothelial carcinoma (UTUC) in terms of the results of acridine orange fluorescence (AO-F) staining of the exfoliated cells in urine. The percentage of positive AO-F result and the positive predictive value of AO-F for high-grade and muscle invasive urothelial carcinoma were calculated and analyzed in terms of clinical characteristics. Results: The overall percentage of positive AO-F result was 49% in the 510 patients, 54.1% for males and 40.6% for females. AO-F was positive in 51.9% of the patients with hematuria and 36.2% of the patients without hematuria. AO-F was positive in 56.4% of the patients with renal pelvis carcinoma and 42.8% of the patients with ureteral cancer; in 44.6% of the patients with non-muscle invasive carcinoma and 53.5% of the patients with muscle-invasive carcinoma. AO-F was positive in 26.8% of the cases with low-grade carcinoma and 55.3% of the patients with high-grade carcinoma. The positive predictive value of AO-F was 88% for high-grade cancer, and only 53.6% for muscle invasive carcinoma. Conclusions: Acridine orange fluorescence microscopy cannot increase the sensitivity of urine exfoliative cytology in the diagnosis of UTUC. It may be used as a predictor of high-grade UTUC. Acridine orange fluorescence microscopy in urinary cytodiagnosis does not show high value in predicting muscle invasive UTUC.
PMCID: PMC3925926  PMID: 24551302
Urothelial carcinoma; upper urinary tract; acridine orange fluorescence; cytodiagnosis
20.  Multi-Scale Optical Imaging of the Delayed Type Hypersensitivity Reaction Attenuated by Rapamycin 
Theranostics  2014;4(2):201-214.
Neutrophils and monocytes/macrophages (MMs) play important roles in the development of cell-mediated delayed type hypersensitivity (DTH). However, the dynamics of neutrophils and MMs during the DTH reaction and how the immunosuppressant rapamycin modulates their behavior in vivo are rarely reported. Here, we take advantage of multi-scale optical imaging techniques and a footpad DTH reaction model to non-invasively investigate the dynamic behavior and properties of immune cells from the whole field of the footpad to the cellular level. During the classic elicitation phase of the DTH reaction, both neutrophils and MMs obviously accumulated at inflammatory foci at 24 h post-challenge. Rapamycin treatment resulted in advanced neutrophil recruitment and vascular hyperpermeability at an early stage (4 h), the reduced accumulation of neutrophils (> 50% inhibition ratio) at 48 h, and the delayed involvement of MMs in inflammatory foci. The motility parameters of immune cells in the rapamycin-treated reaction at 4 h post-challenge displayed similar mean velocities, arrest durations, mean displacements, and confinements as the classic DTH reaction at 24 h. These results indicate that rapamycin treatment shortened the initial preparation stage of the DTH reaction and attenuated its intensity, which may be due to the involvement of T helper type 2 cells or regulatory T cells.
PMCID: PMC3900803  PMID: 24465276
Delayed type hypersensitivity; fluorescent imaging; motility; rapamycin; neutrophils; monocyte/macrophage.
21.  Punctal plugs versus artificial tears for treating dry eye: a comparative observation of their effects on contrast sensitivity 
This study aimed to compare the effects of treatment with punctal plugs versus artificial tears on visual function and tear film stability for dry eye. A total of 56 consecutive eyes of 28 dry eye patients observed at our clinic from May to October in 2009 were divided into two groups. One group (32 eyes of 16 patients) was treated with artificial tears, and punctal plugs were used in the other group (24 eyes of 12 patients). A questionnaire was used in these patients before treatment and was repeated 2 weeks after treatment. Fluorescent staining for tear film break-up time (BUT), the Schirmer test I (STI), and contrast sensitivity was performed at the same time. The questionnaire indicated that all patients complained about the uncomfortable symptoms associated with dry eye. These symptoms were relieved after the application of artificial tears or punctal plugs, and there was no significant difference between these two groups. We found that the corneal fluorescent staining disappeared after treatment. The BUT was improved significantly after treatment in both groups, but the improvement was greater in patients who received punctal plugs than those that received artificial tears. There was no remarkable change in the STI in the artificial tears group, but a significant change was observed in the punctal plugs group. The contrast sensitivities were greatly improved in simulated daylight, night, and glare disability conditions after treatment with artificial tears and punctal plugs. However, the changes in contrast sensitivity did not significantly differ between groups. Both artificial tears and punctal plugs relieved dry eye symptoms, repaired corneal lesions, enhanced tear film stability, and improved contrast sensitivity. Punctal plugs could improve tear film stability and elongate the BUT better than artificial tears.
PMCID: PMC3523103  PMID: 24288582
Dry eye; Punctal plugs; Artificial tears; Contrast sensitivity
22.  A retrospective study of prostate cancer cases mimicking urothelial cell carcinoma of the bladder 
Prostate cancer (PCa) originating from the prostate base may intrude into the urinary bladder and may be misdiagnosed as bladder cancer. In this retrospective study, we reviewed the clinic data on PCa cases which were initially misdiagnosed as bladder cancer in order to identify diagnostic methods that would allow a better differential diagnosis for PCa.
Out of a total of 455 patients treated for PCa at our hospital between April 2003 and June 2011, 14 patients (3.1%) had been initially misdiagnosed as urinary bladder urothelial cell carcinoma. The clinical data on these 14 cases was retrieved and analyzed.
Of the 14 patients, 11 patients were eventually diagnosed with PCa after MRI examination, and seven out of these had PCa with bladder neck invasion. Prostate needle biopsy or transurethral resection of prostate (TURP) revealed that all 14 patients had adenocarcinoma of prostate with Gleason scores ranging from 7 to 9. Nine patients received TURP for hematuria or lower urinary tract blockage. The mean follow-up was 37 months, during which six patients survived.
As clinical presentation and in emergency settings, prostate cancer originating from the prostate base can be confused with bladder cancer originating from the neck or the triangle region of the urinary bladder. Serum prostate specific antigen (PSA) levels and digital rectal examination, in combination with transrectal ultrasound (TRUS), MRI, and prostate needle biopsy are valuable tools for definitive differential diagnosis of the basal prostate cancer.
PMCID: PMC3851773  PMID: 24090000
Prostate cancer; Urinary bladder urothelial cell carcinoma; MRI; Differential diagnosis; Misdiagnosis
23.  Transcript Assembly and Quantification by RNA-Seq Reveals Differentially Expressed Genes between Soft-Endocarp and Hard-Endocarp Hawthorns 
PLoS ONE  2013;8(9):e72910.
Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn.
PMCID: PMC3764154  PMID: 24039819
24.  The Prevalence and Influence Factors of Inter-Ankle Systolic Blood Pressure Difference in Community Population 
PLoS ONE  2013;8(8):e70777.
The aim of this study was to investigate the prevalence of interankle systolic blood pressure difference (sIAND) and its influencing factors in community population.
This study included 2849 (65.1±9.4 y) subjects. Blood pressure (BPs) of four limbs was simultaneously measured with 4 electronic sphygmomanometers after 10 min rest in supine position. The difference of systolic BP (SBP) between two ankles was calculated as DETASBP. The criterion for abnormal sIAND was ≥10 mmHg of absolute DeltaSBP, in which the criterion for 1o sIAND was 10–19 mmHg and for 2o sIAND was ≥20 mmHg. Age, gender, smoking, hypertension, family histories of hypertension and diabetes were recorded. Fasting blood glucose and lipids, circumference of hip and waist, and body mass index (BMI) were measured.
The SBP was higher in the right ankle than in the left ankle (158.9±21.8 vs 157.3±21.6 mmHg, P<0.05) and mean DeltaSBP was 6.08±6.26 mmHg. Similar difference was found in both genders. The prevalence of abnormal was 18.5%, in which, the prevalence 1o sIAND was 15.3% and that of 2o sIAND was 3.1%. Multivariate regression analysis showed that age, waist circumference and blood glucose level were the positive factors for DeltaSBP. The normal upper limit for DeltaSBP was 16.7 mmHg in this population, the prevalence of sIAND by≥16 mmHg was 5.8%.
Aging, hypertension, obesity and abnormal glucose metabolism are positive factors for inter-ankle SBP difference.
PMCID: PMC3754990  PMID: 24015177
25.  Nesfatin-1 Action in the Brain Increases Insulin Sensitivity Through Akt/AMPK/TORC2 Pathway in Diet-Induced Insulin Resistance 
Diabetes  2012;61(8):1959-1968.
Nesfatin-1, derived from nucleobindin 2, was recently identified as an anorexigenic signal peptide. However, its neural role in glucose homeostasis and insulin sensitivity is unknown. To evaluate the metabolic impact and underlying mechanisms of central nesfatin-1 signaling, we infused nesfatin-1 in the third cerebral ventricle of high-fat diet (HFD)–fed rats. The effects of central nesfatin-1 on glucose metabolism and changes in transcription factors and signaling pathways were assessed during euglycemic-hyperinsulinemic clamping. The infusion of nesfatin-1 into the third cerebral ventricle markedly inhibited hepatic glucose production (HGP), promoted muscle glucose uptake, and was accompanied by decreases in hepatic mRNA and protein expression and enzymatic activity of PEPCK in both standard diet- and HFD-fed rats. In addition, central nesfatin-1 increased insulin receptor (InsR)/insulin receptor substrate-1 (IRS-1)/AMP-dependent protein kinase (AMPK)/Akt kinase (Akt)/target of rapamycin complex (TORC) 2 phosphorylation and resulted in an increase in Fos immunoreactivity in the hypothalamic nuclei that mediate glucose homeostasis. Taken together, these results reveal what we believe to be a novel site of action of nesfatin-1 on HGP and the PEPCK/InsR/IRS-1/AMPK/Akt/TORC2 pathway and suggest that hypothalamic nesfatin-1 action through a neural-mediated pathway can contribute to increased peripheral and hepatic insulin sensitivity by decreasing gluconeogenesis and promoting peripheral glucose uptake in vivo.
PMCID: PMC3402309  PMID: 22688332

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