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1.  Neuropeptide Y Stimulates Proliferation and Migration of Vascular Smooth Muscle Cells from Pregnancy Hypertensive Rats via Y1 and Y5 Receptors 
PLoS ONE  2015;10(7):e0131124.
The increased proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in pathophysiological remodeling of arteries during hypertension in pregnancy. However, the mechanisms involved in this process remain unclear. We hypothesized that Neuropeptide Y (NPY), which is a potent mitogenic peptide, participates in modulating proliferation and migration of VSMCs during hypertension in pregnancy. Using pregnant hypertensive rats, induced by intraperitoneal injection of L-nitro-arginine methylester (L-NAME), the plasma concentration of NPY was detected. Open angle, which reflects the non-uniform remodeling with high sensitivity, was used to detect the pathophysiological vascular remodeling in vivo. The results revealed that NPY concentration and artery open angle were both significantly increased in rats with hypertension in pregnant. The underlying mechanism of elevated NPY on vascular remodeling were further analyzed by using cultured VSMCs in vitro. In cultured VSMCs, NPY most effectively stimulated the migration and proliferation of VSMCs at 10-6 mol/L, similar to the plasma concentration in L-NAME hypertension in pregnant rats. NPY up-regulated the expressions of both Y1 and Y5 receptors, increased the phosphorylations of STAT3 on Tyr705 and Ser727 residues, and induced the expression of c-Fos. The NPY-induced VSMCs proliferation was reduced by Y5 receptor antagonist, and fully blocked by combinations with other antagonist, such as Y2+Y5, Y1+Y5, and Y1+Y2+Y5. In contrast, the NPY-induced VSMC migration was blocked by either Y receptor antagonist or any combination of Y receptor antagonists. These results suggest that the elevated plasma concentration of NPY during hypertension in pregnancy may induce VSMC proliferation mainly via Y5 receptor, which subsequently modulate STAT3 and c-Fos signaling pathways to result in the vascular remodeling. These results also suggest that NPY mainly acts on VSMCs in vitro via Y1, Y5 receptors and in vascular tissues in vivo via Y5 receptor.
PMCID: PMC4488588  PMID: 26131716
2.  Differences in Arbuscular Mycorrhizal Fungal Community Composition in Soils of Three Land Use Types in Subtropical Hilly Area of Southern China 
PLoS ONE  2015;10(6):e0130983.
Land use type is key factor in restoring the degraded soils due to its impact on soil chemical properties and microbial community. In this study, the influences of land use type on arbuscular mycorrhizal fungal (AMF) community and soil chemical properties were assessed in a long-run experimental station in subtropical hilly area of southern China. Soil samples were collected from forest land, orchard and vegetable field. Soil chemical properties were analyzed, and PCR-DGGE was performed to explore the AMF community structure. Cloning and sequencing of DGGE bands were conducted to monitor AMF community composition. Results indicate that the contents of total P, available P and available K were the highest while the contents of soil organic matter, total N, total K and available N were the lowest in vegetable field soils, with forest land soils vice versa. According to DGGE profiling, AMF community in forest soils was more closely related to that in orchard soils than that in vegetable field soils. Sequencing indicated that 45 out of 53 excised bands were AMF and 64.4% of AMF belonged to Glomeraceae, including some “generalists” present in all soils and some “specialists” present only in soils of particular land use. Category principle component analysis demonstrated that total N, soil organic matter and available P were the most important factors affecting AMF community, and some AMF phylotypes were closely associated with particular soil chemical properties. Our data suggest that AMF communities are different with different land use types.
PMCID: PMC4479462  PMID: 26107285
3.  Flos Puerariae Extract Ameliorates Cognitive Impairment in Streptozotocin-Induced Diabetic Mice 
Objective. The effects of Flos Puerariae extract (FPE) on cognitive impairment associated with diabetes were assessed in C57BL/6J mice. Methods. Experimental diabetic mice model was induced by one injection of 50 mg/kg streptozotocin (STZ) for 5 days consecutively. FPE was orally administrated at the dosages of 50, 100, or 200 mg/kg/day, respectively. The learning and memory ability was assessed by Morris water maze test. Body weight, blood glucose, free fatty acid (FFA) and total cholesterol (TCH) in serum, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and acetylcholinesterase (AChE) activities in cerebral cortex and hippocampus were also measured. Results. Oral administration of FPE significantly improved cognitive deficits in STZ-induced diabetic mice. FPE treatment also maintained body weight and ameliorated hyperglycemia and dyslipidemia in diabetic mice. Additionally, decreased MDA level, enhanced CAT, and GSH-Px activities in cerebral cortex or hippocampus, as well as alleviated AChE activity in cerebral cortex, were found in diabetic mice supplemented with FPE. Conclusion. This study suggests that FPE ameliorates memory deficits in experimental diabetic mice, at least partly through the normalization of metabolic abnormalities, ameliorated oxidative stress, and AChE activity in brain.
PMCID: PMC4427852  PMID: 26060502
4.  A Wind Energy Powered Wireless Temperature Sensor Node 
Sensors (Basel, Switzerland)  2015;15(3):5020-5031.
A wireless temperature sensor node composed of a piezoelectric wind energy harvester, a temperature sensor, a microcontroller, a power management circuit and a wireless transmitting module was developed. The wind-induced vibration energy harvester with a cuboid chamber of 62 mm × 19.6 mm × 10 mm converts ambient wind energy into electrical energy to power the sensor node. A TMP102 temperature sensor and the MSP430 microcontroller are used to measure the temperature. The power management module consists of LTC3588-1 and LT3009 units. The measured temperature is transmitted by the nRF24l01 transceiver. Experimental results show that the critical wind speed of the harvester was about 5.4 m/s and the output power of the harvester was about 1.59 mW for the electrical load of 20 kΩ at wind speed of 11.2 m/s, which was sufficient to power the wireless sensor node to measure and transmit the temperature every 13 s. When the wind speed increased from 6 m/s to 11.5 m/s, the self-powered wireless sensor node worked normally.
PMCID: PMC4435174  PMID: 25734649
wireless sensor node; temperature; energy harvesting; wind-induced vibration; piezoelectricity
5.  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
6.  Structure and Specificity of the Bacterial Cysteine Methyltransferase Effector NleE Suggests a Novel Substrate in Human DNA Repair Pathway 
PLoS Pathogens  2014;10(11):e1004522.
Enteropathogenic E. coli (EPEC) and related enterobacteria rely on a type III secretion system (T3SS) effector NleE to block host NF-κB signaling. NleE is a first in class, novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates a zinc-coordinating cysteine in the Npl4-like Zinc Finger (NZF) domains in TAB2/3 adaptors in the NF-κB pathway, but its mechanism of action and other human substrates are unknown. Here we solve crystal structure of NleE-SAM complex, which reveals a methyltransferase fold different from those of known ones. The SAM, cradled snugly at the bottom of a deep and narrow cavity, adopts a unique conformation ready for nucleophilic attack by the methyl acceptor. The substrate NZF domain can be well docked into the cavity, and molecular dynamic simulation indicates that Cys673 in TAB2-NZF is spatially and energetically favorable for attacking the SAM. We further identify a new NleE substrate, ZRANB3, that functions in PCNA binding and remodeling of stalled replication forks at the DNA damage sites. Specific inactivation of the NZF domain in ZRANB3 by NleE offers a unique opportunity to suggest that ZRANB3-NZF domain functions in DNA repair processes other than ZRANB3 recruitment to DNA damage sites. Our analyses suggest a novel and unexpected link between EPEC infection, virulence proteins and genome integrity.
Author Summary
Pathogens often manipulate host functions by posttranslational modifications such as ubiquitination and methylation. The NF-κB pathway is most critical for immune defense against infection, thereby frequently targeted by bacterial virulence factors. NleE, a virulence effector from EPEC, is a SAM-dependent methyltransferase that modifies a zinc-finger cysteine in TAB2/3 in the NF-κB pathway. NleE is not homologous to any known methyltransferases. We present the crystal structure of SAM-bound NleE that shows a novel methyltransferase fold with a unique SAM-binding mode. Computational docking and molecular dynamics simulation illustrate a structural and chemical mechanism underlying NleE recognition of the NZF and catalyzing site-specific cysteine methylation. Subsequent substrate specificity analyses identify an N-terminal region in TAB3 required for efficient NleE recognition as well as another NZF protein ZRANB3 being a new substrate of NleE. NleE-catalyzed cysteine methylation also disrupts the ubiquitin chain-binding of ZRANB3-NZF domain, providing new insights into ZRANB3-NZF functioning in DNA damage repair. These results reinforce the idea of harnessing bacterial effectors as a tool for dissecting eukaryotic functions.
PMCID: PMC4239114  PMID: 25412445
7.  A structural mechanism for bacterial autotransporter glycosylation by a dodecameric heptosyltransferase family 
eLife  null;3:e03714.
A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. Here we demonstrate that TibC from ETEC harbors a heptosyltransferase activity on TibA and AIDA-I, defining a large family of bacterial autotransporter heptosyltransferases (BAHTs). The crystal structure of TibC reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal β-barrel, a catalytic domain, a β-hairpin thumb, and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through β-hairpin thumb-mediated hand-in-hand contact. The structure of ADP-D-glycero-β-D-manno-heptose (ADP-D,D-heptose)-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant. Electron-cryomicroscopy analyses uncover a TibC–TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate. The complex structure also highlights a high efficient hyperglycosylation of six autotransporter substrates simultaneously by the dodecamer enzyme complex.
eLife digest
Bacteria release proteins known as virulence factors to help them infect host cells. Many bacteria are surrounded by two membranes, so virulence factors must be able to pass through both of these membranes.
Autotransporters are a group of virulence factors that pass through the inner membrane and anchor themselves in the outer membrane; this allows part of the autotransporter to project from the surface of the bacterial cell and stick to the surface of the cell that the bacterium is about to infect. Many autotransporters are coated with sugar molecules and this increases their ability to adhere to cells. Enzymes called glycosyltransferases ensure that this sugar coating process takes place.
Autotransporters contain two sections: the passenger domain and the beta domain. The passenger domain is important for virulence, while the beta domain forms a pore in the outer membrane that the passenger domain passes through to reach the outer surface of the bacterium. TibA and AIDA-1 are autotransporters associated with two types of bacteria that infect the intestines and cause diarrhea. Similar autotransporters are found in a wide range of bacteria, but the precise details of how these autotransporters are coated with sugar molecules are not fully understood.
Yao et al. now show that a glycosyltransferase called TibC, which is found in many bacteria, adds large numbers of sugar molecules to the passenger domains of both TibA and AIDA-1. To learn more about this process Yao et al. used X-ray diffraction to work out the structure of TibC. Strikingly, this revealed that TibC proteins come together to form a large circular structure that contains two rings, each made of six TibC proteins. The integrity of this structure is maintained by the presence of iron atoms, which also gives TibC a characteristic brown colour.
Yao et al. also studied what happens when TibC binds to TibA; a technique called electron cryo-microscopy revealed that six TibA molecules are distributed along the inner surface of the circular TibC structure, with each TibA protein binding to two TibC proteins. This arrangement allows for the efficient transfer of sugar molecules from the glycosyltransferase to the autotransporter.
PMCID: PMC4358343  PMID: 25310236
bacterial autotransporters; glycosyltransferase; bacterial pathogenesis; cryo-EM; enzyme complex; enzyme catalysis; E. coli
8.  Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene 
PLoS ONE  2014;9(10):e108877.
The genotypic diversity of insoluble macromolecules degraded myxobacteria, provided an opportunity to discover new bacterial resources and find new ecological functions. In this study, we developed a semi-nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to determine the presence and genotypic diversity of myxobacteria in soil. After two rounds of PCR with myxobacteria-specific primers, an 194 bp fragment of mglA, a key gene involved in gliding motility, suitable for DGGE was obtained. A large number of bands were observed in DGGE patterns, indicating diverse myxobacteria inhabiting in soils. Furthermore, sequencing and BLAST revealed that most of the bands belonged to the myxobacteria-group, and only three of the twenty-eight bands belonged to other group, i.e., Deinococcus maricopensis. The results verified that myxobacterial strains with discrepant sequence compositions of gene mglA could be discriminated by DGGE with myxobacteria-specific primers. Collectively, the developed semi-nested-PCR-DGGE strategy is a useful tool for studying the diversity of myxobacteria.
PMCID: PMC4184826  PMID: 25280065
9.  Metadherin contribute to BCR signaling in chronic lymphocytic leukemia 
We have reported Metadherin (MTDH) was proven to be overexpression and involved in malignance of chronic lymphocytic leukemia (CLL) via Wnt signaling pathway. In this study, we further investigate the role of MTDH in regulation of BCR signaling pathway in CLL. Six CLL samples whose cells were proliferation after BCR activation were chosen from patients with unmutated IgVH. CCK-8 method used to evaluate the proliferation rate. MTDH expression was measured by quantitative PCR and Western blot. After BCR activation, there exist upregulation of MTDH expression in mRNA and protein level in all six CLL patients (P<0.05). In cell line MEC-1, we observed the same pro-proliferation effect accompanying with elevated MTDH expression. The proliferation effects of BCR activation to MEC-1 can be inhibited by MTDH interference. The results of this study indicate that MTDH involved in the pro-proliferation effect of BCR activation in CLL. And the results imply that MTDH can be a potential therapy target of CLL.
PMCID: PMC4014239  PMID: 24817955
Chronic lymphocytic leukemia; B cell receptor; Metadherin
10.  Detection and Analysis of Human Papillomavirus (HPV) DNA in Breast Cancer Patients by an Effective Method of HPV Capture 
PLoS ONE  2014;9(3):e90343.
Despite an increase in the number of molecular epidemiological studies conducted in recent years to evaluate the association between human papillomavirus (HPV) and the risk of breast carcinoma, these studies remain inconclusive. Here we aim to detect HPV DNA in various tissues from patients with breast carcinoma using the method of HPV capture combined with massive paralleled sequencing (MPS). To validate the confidence of our methods, 15 cervical cancer samples were tested by PCR and the new method. Results showed that there was 100% consistence between the two methods.DNA from peripheral blood, tumor tissue, adjacent lymph nodes and adjacent normal tissue were collected from seven malignant breast cancer patients, and HPV type 16(HPV16) was detected in 1/7, 1/7, 1/7and 1/7 of patients respectively. Peripheral blood, tumor tissue and adjacent normal tissue were also collected from two patients with benign breast tumor, and 1/2, 2/2 and 2/2 was detected to have HPV16 DNA respectively. MPS metrics including mapping ratio, coverage, depth and SNVs were provided to characterize HPV in samples. The average coverage was 69% and 61.2% for malignant and benign samples respectively. 126 SNVs were identified in all 9 samples. The maximum number of SNVs was located in the gene of E2 and E4 among all samples. Our study not only provided an efficient method to capture HPV DNA, but detected the SNVS, coverage, SNV type and depth. The finding has provided further clue of association between HPV16 and breast cancer.
PMCID: PMC3948675  PMID: 24614680
13.  A Subanesthetic Dose of Isoflurane during Postconditioning Ameliorates Zymosan-Induced Neutrophil Inflammation Lung Injury and Mortality in Mice 
Mediators of Inflammation  2013;2013:479628.
Anesthetic isoflurane (ISO) has immunomodulatory effects. In the present study, we investigated whether a subanesthetic dose of ISO (0.7%) protected against zymosan (ZY) induced inflammatory responses in the murine lung and isolated neutrophils. At 1 and 6 hrs after ZY administration intraperitoneally, ISO was inhaled for 1 hr, and 24 hrs later, lung inflammation and injury were assessed. We found that ISO improved the survival rate of mice and mitigated lung injury as characterized by the histopathology, wet-to-dry weight ratio, protein leakage, and lung function index. ISO significantly attenuated ZY-induced lung neutrophil recruitment and inflammation. This was suggested by the downregulation of (a) endothelial adhesion molecule expression and myeloperoxidase (MPO) activity in lung tissue and polymorphonuclear neutrophils (b) chemokines, and (c) proinflammatory cytokines in BALF. Furthermore, ZY-induced nuclear translocation and DNA-binding activity of NF-κB p65 were also reduced by ISO. ISO treatment inhibited iNOS expression and activity, as well as subsequent nitric oxide generation. Consistent with these in vivo observations, in vitro studies confirmed that ISO blocked NF-κB and iNOS activation in primary mouse neutrophils challenged by ZY. These results provide evidence that 0.7% ISO ameliorates inflammatory responses in ZY-treated mouse lung and primary neutrophils.
PMCID: PMC3863458  PMID: 24369446
14.  Biomechanical effects of semi-constrained integrated artificial discs on zygapophysial joints of implanted lumbar segments 
This study aimed to optimize the design and application of semi-constrained integrated artificial discs (SIADs) using a finite element (FE) analysis following implantation, wherein the zygapophysial joints of the segment were biomechanically reconstructed. An FE model of the L4–L5 segment was constructed. Variations in the stresses on the discs and zygapophysial joints were observed during 5° anteflexion, 5° extension and 5° rotation under the 400-N applied axial load. Stresses and load translation analyses of the discs and zygapophysial joints were conducted during anteflexion, extension and rotation under the 400-N applied axial load. Following implantation of the lumbar segments, the stresses on the SIAD zygapophysial joints were not significantly different from those of physiological discs during anteflexion, and these were both marginally greater compared with those of non-constrained artificial discs (NADs). During extension, the increase in the stress on the SIAD zygapophysial joints was less than that on NAD zygapophysial joints. Stresses on the NAD zygapophysial joints were higher than those on SIAD and physiological discs during rotation. The stress on the SIAD zygapophysial joints was not significantly different from that on physiological discs during rotation. For SIADs and NADs, the stresses on the zygapophysial joints and the displacements of the discs were greater compared with those of the physiological discs during extension. The SIADs affected the variations in the stresses on the implanted segment more than the NADs, and the SIADs protected the zygapophysial joints of the implanted segment to a higher degree than the NADs.
PMCID: PMC3829729  PMID: 24255672
lumbar vertebrae; zygapophysial joints; artificial disc; semi-constrained disc; biomechanics
15.  Palliative surgery for primary sarcoma in the abdominal aorta: A case report and review of the literature 
Oncology Letters  2013;6(6):1738-1740.
Primary sarcoma of the aorta is extremely rare and accounts for <1% of all sarcomas. The present study describes the case of a 45-year-old male who presented with lower limb and abdominal pain. Abdominal computed tomography (CT) and magnetic resonance (MR) arteriography revealed a tumor that extended from the infrarenal aorta to the aortic bifurcation. The external and internal iliac arteries were occluded by the tumor incursion. Palliative surgery was performed for the sarcoma since the patient refused a radical resection. To improve the blood supply to the lower limbs, an axillary bifemoral bypass was established. Following the surgery, the pain was significantly reduced. However, the patient succumbed due to extensive metastasis 6 months after this surgery. Aortic sarcoma is an extremely rare disease with a poor prognosis. A diagnosis at a relatively early stage is necessary for a longer survival time. Radical surgery is the most significant treatment. Patients at advanced stages should consider palliative surgery in order to improve their quality of life.
PMCID: PMC3834868  PMID: 24273607
abdominal aortic aneurysm; palliative surgery
17.  Isolation and Identification of Myxobacteria from Saline-Alkaline Soils in Xinjiang, China 
PLoS ONE  2013;8(8):e70466.
Fifty-eight terrestrial and salt-tolerant myxobacteria were isolated from the saline-alkaline soils collected from Xinjiang, China. Based on the morphologies and the 16S rRNA gene sequences, these isolates were assigned into 6 genera, Myxococcus, Cystobacter, Corallococcus, Sorangium, Nannocystis and Polyangium. All the strains grew better with 1% NaCl than without NaCl. Some Myxococcus strains were able to grow at 2% NaCl concentration, suggesting that these strains may be particular type of terrestrial myxobacteria.
PMCID: PMC3735578  PMID: 23936436
18.  Type-Specific Detection of 30 Oncogenic Human Papillomaviruses by Genotyping both E6 and L1 Genes 
Journal of Clinical Microbiology  2013;51(2):402-408.
Human papillomavirus (HPV) is the principal cause of invasive cervical cancer and benign genital lesions. There are currently 30 HPV types linked to cervical cancer. HPV infection also leads to other types of cancer. We developed a 61-plex analysis of these 30 HPV types by examining two genes, E6 and L1, using MassARRAY matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (PCR-MS). Two hundred samples from homosexual males (HM) were screened by PCR-MS and MY09/MY11 primer set-mediated PCR (MY-PCR) followed by sequencing. One hundred thirty-five formalin-fixed, paraffin-embedded (FFPE) cervical cancer samples were also analyzed by PCR-MS, and results were compared to those of the commercially available GenoArray (GA) assay. One or more HPV types were identified in 64.5% (129/200) of the samples from HM. Comprising all 30 HPV types, PCR-MS detected 51.9% (67/129) of samples with multiple HPV types, whereas MY-PCR detected only one single HPV type in these samples. All PCR-MS results were confirmed by MY-PCR. In the cervical cancer samples, PCR-MS and GA detected 97% (131/135) and 90.4% (122/135) of HPV-positive samples, respectively. PCR-MS and GA results were fully concordant for 122 positive and 4 negative samples. The sequencing results for the 9 samples that tested negative by GA were completely concordant with the positive PCR-MS results. Multiple HPV types were identified in 25.2% (34/135) and 55.6% (75/135) of the cervical cancer samples by GA and PCR-MS, respectively, and results were confirmed by sequencing. The new assay allows the genotyping of >1,000 samples per day. It provides a good alternative to current methods, especially for large-scale investigations of multiple HPV infections and degraded FFPE samples.
PMCID: PMC3553860  PMID: 23152557
19.  Hepatitis B Virus Surface Antigen (HBsAg)-Positive and HBsAg-Negative Hepatitis B Virus Infection among Mother-Teenager Pairs 13 Years after Neonatal Hepatitis B Virus Vaccination 
It is unclear whether a mother who is negative for hepatitis B virus surface antigen (HBsAg) but positive for hepatitis B virus (HBV) is at potential risk for mother-to-child transmission of HBV. This study, using a paired mother-teenager population, aimed to assess whether maternal HBsAg-negative HBV infection (hnHBI) is a significant source of child HBV infection (HBI). A follow-up study with blood collection has been conducted on the 93 mother-teenager pairs from the initial 135 pregnant woman-newborn pairs 13 years after neonatal HBV vaccination. Serological and viral markers of HBV have been tested, and phylogenetic analysis of HBV isolates has been done. The HBI prevalence was 1.9% (1 hnHBI/53) for teenage children of non-HBI mothers, compared with 16.7% (1 hnHBI/6) for those of hnHBI mothers and 2.9% (1 HBsAg-positive HBV infection [hpHBI]/34) for those of hpHBI mothers. Similar viral sequences have been found in one pair of whom both the mother and teenager have had hnHBI. In comparison with the hpHBI cases, those with hnHBI had a lower level of HBV load and a higher proportion of genotype-C strains, which were accompanied by differentiated mutations (Q129R, K141E, and Y161N) of the “a” determinant of the HBV surface gene. Our findings suggest that mother-to-teenager transmission of hnHBI can occur among those in the neonatal HBV vaccination program.
PMCID: PMC3571284  PMID: 23254298
20.  Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP 
Cell Research  2013;23(6):775-787.
Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.
PMCID: PMC3674391  PMID: 23588383
Legionella; Rab GTPases; GTPase-activating protein (GAP); type IV secretion system; TBC GAP; membrane trafficking
21.  Neoadjuvant rh-endostatin, docetaxel and epirubicin for breast cancer: efficacy and safety in a prospective, randomized, phase II study 
BMC Cancer  2013;13:248.
Recombinant human endostatin (rh-endostatin) is a novel antiangiogenesis drug developed in China. Previous experiments have shown that rh-endostatin can inhibit the proliferation and migration of endothelial cells and some types of tumor cells. In this study, we evaluated the efficacy and safety profiles of combination therapy of rh-endostatin and neoadjuvant chemotherapy for breast cancer patients in a prospective, randomized, controlled, phase II trial.
Sixty-eight patients with core-biopsy confirmed breast cancer were allocated randomly to two groups to receive 3 cycles of intravenous administration of either neoadjuvant DE (docetaxel: 75 mg/m2, d1, epirubicin: 75 mg/m2, d1, every 3 weeks), or neoadjuvant DE combined with rh-endostatin (7.5 mg/m2, d1-d14, every 3 weeks). The primary end point was clinical response based upon Response Evaluation Criteria in Solid Tumors, and the secondary end point was safety and quality of life.
All patients were assessable for toxicity and 64 (94.2%) were assessable for efficacy evaluation. The objective response rate was 67.7% for chemotherapy (n = 31) and 90.9% for rh-endostatin plus chemotherapy (n = 33) (P = 0.021). A retrospective subset analysis revealed that rh-endostatin was more effective in premenopausal patients and patients with ECOG score of zero (P = 0.002 and P = 0.049, respectively). Five patients in the rh-endostatin plus chemotherapy arm achieved pathologic complete response compared with 2 in the chemotherapy arm (P = 0.428). No significant difference was identified in quality of life score and side effects (P > 0.05).
The combination of rh-endostatin with chemotherapy produced a higher tumor response rate without increasing toxicity in breast cancer patients.
Trial registration Identifier, NCT00604435
PMCID: PMC3664086  PMID: 23693018
Breast cancer; Recombinant human endostatin; Neoadjuvant chemotherapy; Clinical trial
22.  Distribution and Properties of the Genes Encoding the Biosynthesis of the Bacterial Cofactor, Pyrroloquinoline Quinone† 
Biochemistry  2012;51(11):2265-2275.
Pyrroloquinoline quinone (PQQ) is a small, redox-active molecule that serves as a cofactor for several bacterial dehydrogenases, introducing pathways for carbon utilization that confer a growth advantage. Early studies had implicated a ribosomally translated peptide as the substrate for PQQ production. This study presents a sequence and structure based analysis of the components of the pqq operon. We find the necessary components for PQQ production are present in 126 prokaryotes, most of which are Gram- negative and a number of which are pathogens. A total of five gene products, PqqA, PqqB, PqqC, PqqD and PqqE, are concluded to be obligatory for PQQ production. Three of the gene products in the pqq operon, PqqB, PqqC and PqqE, are members of large protein superfamilies. By combining evolutionary conservation patterns with information from three-dimensional structures, we are able to differentiate the gene products involved in PQQ biosynthesis from those with divergent functions. The observed persistence of a conserved gene order within analyzed operons strongly suggests a role for protein/protein interactions in the course of cofactor biosynthesis. These studies propose previously unidentified roles for several of the gene products as well as possible new targets for antibiotic design and application.
PMCID: PMC3334298  PMID: 22324760
pyrroloquinoline; quinone; pathogenicity; phylogenomic analysis; metallo-beta-lactamase; radical SAM domain; cofactorless oxidase
23.  Induction of Thoracic Aortic Remodeling by Endothelial-Specific Deletion of MicroRNA-21 in Mice 
PLoS ONE  2013;8(3):e59002.
MicroRNAs (miRs) are known to have an important role in modulating vascular biology. MiR21 was found to be involved in the pathogenesis of proliferative vascular disease. The role of miR21 in endothelial cells (ECs) has well studied in vitro, but the study in vivo remains to be elucidated. In this study, miR21 endothelial-specific knockout mice were generated by Cre/LoxP system. Compared with wild-type mice, the miR21 deletion in ECs resulted in structural and functional remodeling of aorta significantly, such as diastolic pressure dropping, maximal tension depression, endothelium-dependent relaxation impairment, an increase of opening angles and wall-thickness/inner diameter ratio, and compliance decrease, in the miR21 endothelial-specific knockout mice. Furthermore, the miR21 deletion in ECs induced down-regulation of collagen I, collagen III and elastin mRNA and proteins, as well as up-regulation of Smad7 and down-regulation of Smad2/5 in the aorta of miR21 endothelial-specific knockout mice. CTGF and downstream MMP/TIMP changes were also identified to mediate vascular remodeling. The results showed that miR21 is identified as a critical molecule to modulate vascular remodeling, which will help to understand the role of miR21 in vascular biology and the pathogenesis of vascular diseases.
PMCID: PMC3601125  PMID: 23527070
24.  MicroRNA-195 plays a tumor-suppressor role in human glioblastoma cells by targeting signaling pathways involved in cellular proliferation and invasion 
Neuro-Oncology  2012;14(3):278-287.
Accumulating evidence has implicated the deregluation of miRNAs in tumorigenesis. Previous studies have reported that microRNA-195 (miR-195) is markedly down-regulated in human glioblastoma cells, compared with normal brain tissue, but the biological role of miR-195 in glioblastoma development is currently unknown. In this study, we define a tumor-suppressor role for miR-195 in human glioblastoma cells. Over-expression of miR-195 in glioblastoma cell lines robustly arrested cell cycle progression and significantly repressed cellular invasion. We identified E2F3 and CCND3 as functional downstream targets of miR-195 in glioblastoma cells. Through knockdown studies, we demonstrated that E2F3 was the dominant effector of miR-195-mediated cell cycle arrest and that CCND3 was a key mediator of miR-195–induced inhibition of glioblastoma cell invasion. Furthermore, we showed that p27Kip1 was an important regulator downstream of CCND3 and that the accumulation of p27Kip1 in the cytoplasm might be responsible for the miR-195–mediated cell invasion inhibition in glioblastoma cells. This work provides evidence for the initial mechanism by which miR-195 negatively regulates both the proliferation and invasion of glioblastoma cells, suggesting that the down-regulation of miR-195 might contribute to the malignant transformation of glioblastoma cells and could be a molecular signature associated with glioblastoma progression.
PMCID: PMC3280801  PMID: 22217655
glioblastoma; invasion; microRNA-195; proliferation; tumor-suppressor
25.  Involvement of Rab28 in NF-κB Nuclear Transport in Endothelial Cells 
PLoS ONE  2013;8(2):e56076.
Our previous proteomic analysis revealed the expression of Rab28 in arteries of rats. However, the function of Rab28 in mammalian cells, and its role in vessels are still unknown. Coarctation of abdominal aorta above left kidney artery in rat was used as hypertensive animal model. FX-4000 cyclic strain loading system was used to mimic the mechanical condition on vascular cells during hypertension in vitro. Immunofluorescence and co-immunoprecipitation (Co-IP) were used to identify distribution and interaction of Rab28 and nuclear factor kappa B (NF-κB). Rab28 expression was significantly increased in carotid arteries of hypertensive rats. High cyclic strain induced Rab28 expression of endothelial cells (ECs) through a paracrine control of vascular smooth muscles cells (VSMCs), which at least partly via angiotensin II (Ang II). Rab28 knockdown decreased proliferation of ECs, while increased apoptosis and migration. Immunofluorescence revealed that Ang II stimulated the co-translocation of Rab28 and NF-κB from cytoplasm into nucleus. Knockdown of Rab28 attenuated NF-κB activation. Co-IP of NF-κB p65 and Rab28 indicated their interaction. Our results revealed that Rab28, as a novel regulator of NF-κB nuclear transport, might participate in the disturbance of EC homeostasis.
PMCID: PMC3573041  PMID: 23457503

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