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1.  The Growing Season, but Not the Farming System, Is a Food Safety Risk Determinant for Leafy Greens in the Mid-Atlantic Region of the United States 
Small- and medium-size farms in the mid-Atlantic region of the United States use varied agricultural practices to produce leafy greens during spring and fall, but the impact of preharvest practices on food safety risk remains unclear. To assess farm-level risk factors, bacterial indicators, Salmonella enterica, and Shiga toxin-producing Escherichia coli (STEC) from 32 organic and conventional farms were analyzed. A total of 577 leafy greens, irrigation water, compost, field soil, and pond sediment samples were collected. Salmonella was recovered from 2.2% of leafy greens (n = 369) and 7.7% of sediment (n = 13) samples. There was an association between Salmonella recovery and growing season (fall versus spring) (P = 0.006) but not farming system (organic or conventional) (P = 0.920) or region (P = 0.991). No STEC was isolated. In all, 10% of samples were positive for E. coli: 6% of leafy greens, 18% of irrigation water, 10% of soil, 38% of sediment, and 27% of compost samples. Farming system was not a significant factor for levels of E. coli or aerobic mesophiles on leafy greens but was a significant factor for total coliforms (TC) (P < 0.001), with higher counts from organic farm samples. Growing season was a factor for aerobic mesophiles on leafy greens (P = 0.004), with higher levels in fall than in spring. Water source was a factor for all indicator bacteria (P < 0.001), and end-of-line groundwater had marginally higher TC counts than source samples (P = 0.059). Overall, the data suggest that seasonal events, weather conditions, and proximity of compost piles might be important factors contributing to microbial contamination on farms growing leafy greens.
PMCID: PMC4357962  PMID: 25616798
3.  AmyM, a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. Strain EGB 
A novel α-amylase, AmyM, was purified from the culture supernatant of Corallococcus sp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of Corallococcus coralloides DSM 2259, the AmyM gene was identified and cloned into Escherichia coli. amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases. amyM was expressed in E. coli BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in E. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The Km and Vmax of recombinant AmyM for soluble starch were 6.61 mg ml−1 and 44,301.5 μmol min−1 mg−1, respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.
PMCID: PMC4345388  PMID: 25576603
4.  Involvement of the Cytochrome P450 System EthBAD in the N-Deethoxymethylation of Acetochlor by Rhodococcus sp. Strain T3-1 
Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a widely applied herbicide with potential carcinogenic properties. N-Deethoxymethylation is the key step in acetochlor biodegradation. N-Deethoxymethylase is a multicomponent enzyme that catalyzes the conversion of acetochlor to 2′-methyl-6′-ethyl-2-chloroacetanilide (CMEPA). Fast detection of CMEPA by a two-enzyme (N-deethoxymethylase–amide hydrolase) system was established in this research. Based on the fast detection method, a three-component enzyme was purified from Rhodococcus sp. strain T3-1 using ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular masses of the components of the purified enzyme were estimated to be 45, 43, and 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the results of peptide mass fingerprint analysis, acetochlor N-deethoxymethylase was identified as a cytochrome P450 system, composed of a cytochrome P450 oxygenase (43-kDa component; EthB), a ferredoxin (45 kDa; EthA), and a reductase (11 kDa; EthD), that is involved in the degradation of methyl tert-butyl ether. The gene cluster ethABCD was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). Resting cells of a recombinant E. coli strain showed deethoxymethylation activity against acetochlor. Subcloning of ethABCD showed that ethABD expressed in E. coli BL21(DE3) has the activity of acetochlor N-deethoxymethylase and is capable of converting acetochlor to CMEPA.
PMCID: PMC4345396  PMID: 25595756
5.  Amygdala Connectivity Differs Among Chronic, Early Course, and Individuals at Risk for Developing Schizophrenia 
Schizophrenia Bulletin  2013;40(5):1105-1116.
Alterations in circuits involving the amygdala have been repeatedly implicated in schizophrenia neuropathology, given their role in stress, affective salience processing, and psychosis onset. Disturbances in amygdala whole-brain functional connectivity associated with schizophrenia have yet to be fully characterized despite their importance in psychosis. Moreover, it remains unknown if there are functional alterations in amygdala circuits across illness phases. To evaluate this possibility, we compared whole-brain amygdala connectivity in healthy comparison subjects (HCS), individuals at high risk (HR) for schizophrenia, individuals in the early course of schizophrenia (EC-SCZ), and patients with chronic schizophrenia (C-SCZ). We computed whole-brain resting-state connectivity using functional magnetic resonance imaging at 3T via anatomically defined individual-specific amygdala seeds. We identified significant alterations in amygdala connectivity with orbitofrontal cortex (OFC), driven by reductions in EC-SCZ and C-SCZ (effect sizes of 1.0 and 0.97, respectively), but not in HR for schizophrenia, relative to HCS. Reduced amygdala-OFC coupling was associated with schizophrenia symptom severity (r = .32, P < .015). Conversely, we identified a robust increase in amygdala connectivity with a brainstem region around noradrenergic arousal nuclei, particularly for HR individuals relative to HCS (effect size = 1.54), but not as prominently for other clinical groups. These results suggest that deficits in amygdala-OFC coupling could emerge during the initial episode of schizophrenia (EC-SCZ) and may present as an enduring feature of the illness (C-SCZ) in association with symptom severity but are not present in individuals with elevated risk for developing schizophrenia. Instead, in HR individuals, there appears to be increased connectivity in a circuit implicated in stress response.
PMCID: PMC4133672  PMID: 24366718
schizophrenia; prefrontal cortex; amygdala; connectivity; first episode; risk for schizophrenia
6.  FlyVar: a database for genetic variation in Drosophila melanogaster 
FlyVar is a publicly and freely available platform that addresses the increasing need of next generation sequencing data analysis in the Drosophila research community. It is composed of three parts. First, a database that contains 5.94 million DNA polymorphisms found in Drosophila melanogaster derived from whole genome shotgun sequencing of 612 genomes of D. melanogaster. In addition, a list of 1094 dispensable genes has been identified. Second, a graphical user interface (GUI) has been implemented to allow easy and flexible queries of the database. Third, a set of interactive online tools enables filtering and annotation of genomic sequences obtained from individual D. melanogaster strains to identify candidate mutations. FlyVar permits the analysis of next generation sequencing data without the need of extensive computational training or resources.
Database URL:
PMCID: PMC4541374  PMID: 26289428
7.  The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold 
eLife  null;4:e06925.
B lymphocytes use B cell receptors (BCRs) to sense the physical features of the antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here, we addressed this question using a double-stranded DNA-based tension gauge tether system serving as a predefined mechanical force gauge ranging from 12 to 56 pN. We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect. In contrast, the activation of isotype-switched IgG- or IgE-BCR only requires a low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold. These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs.
eLife digest
The immune system protects us from invading bacteria and other microbes. Immune cells called B cells help the immune system to identify the microbes so they can be destroyed. These cells make proteins called antibodies that bind to molecules from the microbes known as antigens. The B cells only start to produce antibodies when they bind to a specific antigen via a large group or ‘complex’ of proteins on the surface of the B cell called the ‘B cell receptor’. After the body has defeated the microbe, some of the B cells will become memory B cells, which are primed to rapidly respond to the antigen if the same microbe tries to invade again in future.
Previous work on the B cell receptor has largely focused on the chemical features of the antigens. However, recent research suggests that B cell receptors are also influenced by physical cues from the antigen. For example, the stiffness of the surface of the host cell or microbe that is displaying the antigen may exert a mechanical force on the B cell receptor as it binds to the antigen. However, it is not clear what role these mechanical forces play in triggering B cell activation and antibody production.
Before a B cell encounters an antigen, it expresses a type of B cell receptor called the IgM-BCR, but memory B cells express different types of B cell receptors. Wan et al. investigated how the different B cell receptors are activated using a technique involving a DNA-based tension gauge. The experiments show that the activation of IgM-BCRs depends on the amount of mechanical force applied. Low levels of mechanical force only weakly activated the receptors, while higher levels of force resulted in more robust activation.
In contrast, small amounts of mechanical force were sufficient to strongly activate the other two types of B cell receptors, IgG-BCR and IgE-BCR, on memory B cells. This may help memory B cells to be activated more quickly than other B cells that haven't encountered an antigen before. The next challenge is to understand why the B cell receptors on memory B cells are less dependent on mechanical forces than IgM-BCRs.
PMCID: PMC4555871  PMID: 26258882
BCR activation; IgM-BCR; IgG-BCR; mechanical force; TGT; human; mouse
8.  The Prostaglandin EP4 Receptor Mediates Angiotensin II-Induced (Pro)Renin Receptor Expression in the Rat Renal Medulla 
Hypertension  2014;64(2):369-377.
AngII stimulates (pro)renin receptor (PRR) expression in the renal collecting duct (CD), triggering the local renin response in the distal nephron. Our recent study provided evidence for involvement of COX-2-PGE2 pathway in AngII-dependent stimulation of PRR expression in the CD. Here we tested the role of EP subtypes acting downstream of COX-2 in this phenomenon. In primary rat inner medullary collecting duct (IMCD) cells, AngII treatment for 12 h induced a 1.8-fold increase in the full-length PRR protein expression. To assess the contribution of EP receptor, the cell were pre-treated with specific EP receptor antagonists: SC-51382 (for EP1), L-798106 (for EP3), and L-161982 (for EP4) and ONO-AE3-208 (ONO, a structurally distinct EP4 antagonist). The upregulation of PRR expression by Ang II was consistently abolished by L-161982 and ONO, and partially suppressed by SC-51382, but was unaffected by L-798106. The PRR expression was also significantly elevated by the EP4 agonist CAY10598 in the absence of AngII. Sprague-Daley rats were subsequently infused for 1 or 2 weeks with vehicle, AngII alone or in combination with ONO. AngII infusion induced parallel increases in renal medullary PRR protein, and renal medullary and urinary renin activity and total renin content, all of which were blunted by ONO. Both tail cuff plethysmography and telemetry demonstrated attenuation of AngII hypertension by ONO. Overall, these results have established a crucial role of the EP4 receptor in mediating the upregulation of renal medullary PRR expression and renin activity during AngII hypertension.
PMCID: PMC4445967  PMID: 24866147
(Pro)renin receptor; prostaglandin E2; EP4 receptors; renin activity; inner medullary collecting duct
9.  Effect of miR-142-3p on the M2 Macrophage and Therapeutic Efficacy Against Murine Glioblastoma 
The immune therapeutic potential of microRNAs (miRNAs) in the context of tumor-mediated immune suppression has not been previously described for monocyte-derived glioma-associated macrophages, which are the largest infiltrating immune cell population in glioblastomas and facilitate gliomagenesis.
An miRNA microarray was used to compare expression profiles between human glioblastoma-infiltrating macrophages and matched peripheral monocytes. The effects of miR-142-3p on phenotype and function of proinflammatory M1 and immunosuppressive M2 macrophages were determined. The therapeutic effect of miR-142-3p was ascertained in immune-competent C57BL/6J mice harboring intracerebral GL261 gliomas and in genetically engineered Ntv-a mice bearing high-grade gliomas. Student t test was used to evaluate the differences between ex vivo datasets. Survival was analyzed with the log-rank test and tumor sizes with linear mixed models and F test. All statistical tests were two-sided.
miR-142-3p was the most downregulated miRNA (approximately 4.95-fold) in glioblastoma-infiltrating macrophages. M2 macrophages had lower miR-142-3p expression relative to M1 macrophages (P = .03). Overexpression of miR-142-3p in M2 macrophages induced selective modulation of transforming growth factor beta receptor 1, which led to subsequent preferential apoptosis in the M2 subset (P = .01). In vivo miR-142-3p administration resulted in glioma growth inhibition (P = .03, n = 5) and extended median survival (miR-142-3p–treated C57BL/6J mice vs scramble control: 31 days vs 23.5 days, P = .03, n = 10; miR-142-3p treated Ntv-a mice vs scramble control: 32 days vs 24 days, P = .03, n = 9), with an associated decrease in infiltrating macrophages (R 2 = .303).
These data indicate a unique role of miR-142-3p in glioma immunity by modulating M2 macrophages through the transforming growth factor beta signaling pathway.
PMCID: PMC4271080  PMID: 24974128
10.  Cardiac telocytes are double positive for CD34/PDGFR-α 
Telocytes (TCs) are a distinct type of interstitial cells, which are featured with a small cellular body and long and thin elongations called telopodes (Tps). TCs have been widely identified in lots of tissues and organs including heart. Double staining for CD34/PDGFR-β (Platelet-derived growth factor receptor β) or CD34/Vimentin is considered to be critical for TC phenotyping. It has recently been proposed that CD34/PDGFR-α (Platelet-derived growth factor receptor α) is actually a specific marker for TCs including cardiac TCs although the direct evidence is still lacking. Here, we showed that cardiac TCs were double positive for CD34/PDGFR-α in primary culture. CD34/PDGFR-α positive cells (putative cardiac TCs) also existed in mice ventricle and human cardiac valves including mitral valve, tricuspid valve and aortic valve. Over 87% of cells in a TC-enriched culture of rat cardiac interstitial cells were positive for PDGFR-α, while CD34/PDGFR-α double positive cells accounted for 30.25% of the whole cell population. We show that cardiac TCs are double positive for CD34/PDGFR-α. Better understanding of the immunocytochemical phenotypes of cardiac TCs might help using cardiac TCs as a novel source in cardiac repair.
PMCID: PMC4549054  PMID: 26082061
telocytes; myocardium; CD34; PDGFR-α, β; interstitial cells; cardiac repair
11.  High-level expression of human arginase I in Pichia pastoris and its immobilization on chitosan to produce L-ornithine 
BMC Biotechnology  2015;15:66.
L-ornithine (L-Orn), is an intermediate metabolite in the urea cycle that plays a significant role in humans. L-Orn can be obtained from the catalysis of L-arginine (L-Arg) by arginase. The Pichia pastoris expression system offers the possibility of generating a large amount of recombinant protein. The immobilized enzyme technology can overcome the difficulties in recovery, recycling and long-term stability that result from the use of free enzyme.
The recombinant human arginase I (ARG I) was obtained using an optimized method with the Pichia pastoris GS115 as the host strain. Chitosan paticles were cross-linked with glutaraldehyde and rinsed exhaustively. Then the expressed ARG I was immobilized on the crosslinked chitosan particles, and the enzymatic properties of both the free and immobilized enzymes were evaluated. At last, the immobilized ARG I was employed to catalyze L-Arg to L-Orn.
The results indicated that these two states both exhibited optimal activity under the same condition of pH10 at 40 °C. However, the immobilized ARG I exhibited the remarkable thermal and long-term stability as well as broad adaptability to pH, suggesting its potential for wide application in future industry. After a careful analysis of its catalytic conditions, immobilized ARG I was employed to catalyze the conversion of L-Arg to L-Orn under optimal condition of 1 % glutaraldehyde, 1 mM Mn2+, 40 °C, pH10 and an L-arginine (L-Arg) concentration of 200 g/L, achieving a highly converted content of 149.g/L L-Orn.
In this work, ARG Ι was abundantly expressed, and an efficient, facile and repeatable method was developed to synthesize high-quality L-Orn. This method not only solved the problem of obtaining a large amount of arginase, but also provided a promising alternative for the future industrial production of L-Orn.
PMCID: PMC4521451  PMID: 26227111
L-ornithine; Recombinant human arginase I; Immobilization; Chitosan; L-arginine; Transformation
12.  Comparison of Therapeutic Effect of Recombinant Tissue Plasminogen Activator by Treatment Time after Onset of Acute Ischemic Stroke 
Scientific Reports  2015;5:11743.
We aimed to compare the therapeutic effect of recombinant tissue plasminogen activator (rt-PA) administered at different time windows within the first 6 hours after onset of acute ischemic stroke (AIS). A retrospective analysis was performed of data collected from 194 patients who received rt-PA thrombolysis within 4.5 hours after AIS onset and from 29 patients who received rt-PA thrombolysis between 4.5–6 hours after AIS onset. The National Institutes of Health Stroke Scale (NIHSS) scores were statistically decreased in both groups (P < 0.05) at 24 hours and 7 days after onset. There was no statistical difference in the modified Rankin score or mortality at day 90 after treatment between the two groups (P > 0.05). In conclusion, AIS patients who received rt-PA treatment between 4.5–6 hours after onset were similar in therapeutic efficacy to those who received rt-PA within 4.5 hours after onset. Our results suggest that intravenous thrombolytic therapy for AIS within 4.5–6 hours after onset is effective and safe.
PMCID: PMC4513278  PMID: 26206308
13.  All-carbon based graphene field effect transistor with graphitic electrodes fabricated by e-beam direct writing on PMMA 
Scientific Reports  2015;5:12198.
A so called all-carbon based graphene field effect transistor (GFET) in which the electrodes are composed of graphite-like nano-sheets instead of metals in the traditional devices is fabricated by one-step e-beam direct writing (EBDW). It is also found that the graphite-like nano-sheets in electrodes are perpendicular to the channel graphene, which is confirmed by the transmission electron microscopy (HRTEM). The one-step fabrication of the carbonaceous electrodes is more convenient and lower-cost comparing to the preparation of traditional metal electrodes and can be applied to many other nano-electronic devices.
PMCID: PMC4508849  PMID: 26195033
14.  Targeted multidrug-resistance reversal in tumor based on PEG-PLL-PLGA polymer nano drug delivery system 
The study investigated the reversal of multidrug resistance (MDR) and the biodistribution of nanoparticles (NPs) that target leukemia cells in a nude mice model via a surface-bound transferrin (Tf). The cytotoxic cargo of daunorubicin (DNR) and tetrandrine (Tet) was protected in the NPs by an outer coat composed of polyethylene glycol (PEG)-poly-l-lysine (PLL)-poly(lactic-co-glycolic acid) (PLGA) NPs. Injection of DNR-Tet-Tf-PEG-PLL-PLGA NPs into nude mice bearing MDR leukemia cell K562/A02 xenografts was shown to inhibit tumor growth, and contemporaneous immunohistochemical analysis of tumor tissue showed the targeted NPs induced apoptosis in tumor cells. Targeted tumor cells exhibited a marked increase in Tf receptor expression, with noticeable decreases in P-glycoprotein, MDR protein, and nuclear factor κB, as assessed by quantitative real-time polymerase chain reaction and Western blot analysis. Moreover, the concentration of DNR was shown to increase in plasma, tumor tissue, and major organs. Flow cytometry analysis with a near-infrared fluorescent (NIRF) dye, NIR797, was used to study the effectiveness of Tf as a targeting group for leukemia cells, a finding that was supported by NIRF imaging in tumor-bearing nude mice. In summary, our studies show that DNR-Tet-Tf-PEG-PLL-PLGA NPs provide a specific and effective means to target cytotoxic drugs to MDR tumor cells.
PMCID: PMC4509529  PMID: 26213467
PEG-PLL-PLGA nanoparticles; transferrin; tetrandrine; multidrug resistance
15.  Acute pulmonary embolism with electrocardiagraphic changes mimicking acute coronary syndrome: a case report and literature review 
Acute pulmonary embolism (PE) is potentially a life threatening emergency that needs prompt management to reduce preventable deaths. Symptoms like dyspnoea and chest discomfort often lack specificity and overlap with acute coronary syndrome (ACS). Importantly, electrocardiographic changes associated with PE are reported to be variable with some ECG patterns mimicking ACS, posing problems in the differential diagnosis. More recently, precordial T wave inversion has been described to be a clue to suggest PE. However, this ECG change is more likely to present in ACS. We herein reported a case of a 78-year-old man presenting with progressive shortness of breath on exertion secondary to submassive pulmonary embolism which was initially misdiagnosed as ACS due to diffuse T wave inversion in both precordial leads V1-6 and inferior Leads II, III and aVF. Here, we discussed the diagnosis of this case and reviewed the medical literature with an emphasis on the limitations of ECG for the differentiation between PE and ACS.
PMCID: PMC4565285  PMID: 26379902
Acute pulmonary embolism; acute coronary syndrome; T wave inversion
16.  Fasting blood glucose levels affect hospitalization time and relapse and mortality rates of cerebral infarction patients 
Objective: This study determined the relationship between fasting glucose levels of cerebral infarction patients and the hospitalization time and relapse and mortality rates. Methods: A retrospective study was conducted between February 1996 and December 2006 involving 974 inpatients with cerebral infarctions. Fasting blood glucose and lipid levels and blood pressure were measured the morning after hospitalization. The length of hospital stay, and data obtained from telephone follow-up interviews regarding relapse and complications were recorded. The data were analyzed using multiple linear regressions, logistic regression, the chi-square test, and the Kruskal-Wallis analysis of variance of ranks test. Results: Our data show that the duration of hospitalization and relapse and mortality rates of patients with cerebral infarctions correlate with the admission fasting blood glucose levels. Cerebral infarction patients with fasting blood glucose levels > 11.1 mmol/L and LDL levels > 3.5 mmol/L have higher mortality rates (50.00%). Patients with fasting blood glucose levels > 11.1 mmol/L combined with a diastolic pressure < 80 mmHg or > 100 mmHg also have high mortality rates (33.33% and 30.00%, respectively). Conclusions: Fasting glucose levels of inpatients with cerebral infarctions are closely related to the duration of hospitalization and relapse and mortality rates. Higher fasting blood glucose levels exacerbate damage to cerebral blood vessels caused by alterations in blood lipid levels and blood pressure. Therefore, blood glucose levels should be monitored during the early stage of cerebral infarction and intervention should be provided promptly to decrease the length of hospital stay and the risk of relapse and mortality.
PMCID: PMC4565354  PMID: 26379971
Cerebral infarction; blood glucose; relapse; mortality
17.  A late diagnosed case of Spontaneous esophageal perforation in an elderly patient 
Spontaneous esophageal perforation, also known as Boerhaave’s syndrome, is a rare but potentially life-threatening condition, especially in elderly patients with more complications, speedy development and higher mortality. Successful handling of the disease depends on a timely diagnosis and the appropriate choice of treatment. Unfortunately, late diagnosis is common because of the non-specific clinical presentation. We here present a 72-year-old patient of spontaneous esophageal perforation who complained of chest pain, but sharply deteriorated with septic shock. With a vomiting history and gastrointestinal-genic bacterium identified in the chest fluid, the patient was highly suspected for esophageal perforation, though the oral methylene blue test was negative for three times. The diagnosis was finally established by esophagoscopy on the 10th day. The perforation was successfully healed by active conservative management and the patient was discharged home on the 43rd day eating normal diet.
PMCID: PMC4565371  PMID: 26379988
Spontaneous esophageal perforation; Boerhaave’s syndrome; chest pain; septic shock; conservative management
18.  SALL4 is a new target in endometrial cancer 
Oncogene  2013;34(1):63-72.
Aggressive cancers and embryonic stem (ES) cells share a common gene expression signature. Identifying the key factors/pathway(s) within this ES signature responsible for the aggressiveness of cancers can lead to a potential cure. In this study, we find that SALL4, a gene involved in the maintenance of ES cell self-renewal, is aberrantly expressed in 47.7% of primary human endometrial cancer samples. It is not expressed in normal or hyperplastic endometrial. More importantly, SALL4 expression is positively correlated with worse patient survival and aggressive features such as metastasis in endometrial carcinoma. Further functional studies have shown that loss of SALL4 inhibits endometrial cancer cell growth in vitro and tumorigenicity in vivo, as a result of inhibition of cell proliferation and increased apoptosis. In addition, down-regulation of SALL4 significantly impedes the migration and invasion properties of endometrial cancer cells in vitro and their metastatic potential in vivo. Furthermore, manipulation of SALL4 expression can affect drug sensitivity of endometrial cancer cells to carboplatin. Moreover, we show that SALL4 specifically binds to the c-Myc promoter region in endometrial cancer cells. While down-regulation of SALL4 leads to a decreased expression of c-Myc at both protein and mRNA levels, ectopic SALL4 overexpression causes increased c-Myc protein and mRNA expression, indicating that c-Myc is one of the SALL4 downstream targets in endometrial tumorigenesis. In summary, we are the first to demonstrate that SALL4 plays functional role(s) in metastasis and drug resistance in aggressive endometrial cancer. As a consequence of its functional roles in cancer cell and absence in normal tissue, SALL4 is a potential novel therapeutic target for the high risk endometrial cancer patient population.
PMCID: PMC4059794  PMID: 24336327
Endometrial cancer; SALL4; metastasis; chemoresistance
19.  Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris 
PLoS ONE  2015;10(7):e0131757.
The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR.
PMCID: PMC4489709  PMID: 26134129
20.  Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion 
PLoS Pathogens  2015;11(7):e1004958.
The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50 / μg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210 – 220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.
Author Summary
The transmissible spongiform encephalopathies (TSEs) are a group of infectious neurodegenerative diseases affecting both humans and animals. The prion hypothesis postulates that prions are protein conformation based infectious agents responsible for TSE infectivity. Prions have been synthetically generated in vitro, but it remains unclear whether the properties of synthetically generated prion are the same as those of TSE agents and whether the disease caused by synthetically generated prion is identical to naturally occurring TSEs. In this study, we demonstrated that similar to the classical TSE agents, the synthetically generated prion has a titratable infectivity and is able to cause prion disease in wild-type mice via routes other than direct intra-cerebral inoculation. More importantly, we showed that the synthetically generated prion induced pathological changes, including the dissemination of disease-specific prion protein accumulation and the route and mechanism of neuroinvasion, were all typical of classical TSEs. These results demonstrate the similarity of synthetically generated prion to the infectious agent in TSEs, providing strong evidence supporting the prion hypothesis.
PMCID: PMC4489884  PMID: 26136122
21.  Genomic Diversity and Virulence Profiles of Historical Escherichia coli O157 Strains Isolated from Clinical and Environmental Sources 
Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha, terD, and hlyA) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers (iha, terD, hlyA, and espP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.
PMCID: PMC4277581  PMID: 25381234
22.  Telocytes in regenerative medicine 
Telocytes (TCs) are a distinct type of interstitial cells characterized by a small cell body and extremely long and thin telopodes (Tps). The presence of TCs has been documented in many tissues and organs (go to Functionally, TCs form a three-dimensional (3D) interstitial network by homocellular and heterocellular communication and are involved in the maintenance of tissue homeostasis. As important interstitial cells to guide or nurse putative stem and progenitor cells in stem cell niches in a spectrum of tissues and organs, TCs contribute to tissue repair and regeneration. This review focuses on the latest progresses regarding TCs in the repair and regeneration of different tissues and organs, including heart, lung, skeletal muscle, skin, meninges and choroid plexus, eye, liver, uterus and urinary system. By targeting TCs alone or in tandem with stem cells, we might promote regeneration and prevent the evolution to irreversible tissue damage. Exploring pharmacological or non-pharmacological methods to enhance the growth of TCs would be a novel therapeutic strategy besides exogenous transplantation for many diseased disorders.
PMCID: PMC4511344  PMID: 26059693
telocytes; regenerative medicine; regeneration; stem cells; progenitor cells; interstitial cells
23.  A Novel Audiovisual Brain-Computer Interface and Its Application in Awareness Detection 
Scientific Reports  2015;5:9962.
Currently, detecting awareness in patients with disorders of consciousness (DOC) is a challenging task, which is commonly addressed through behavioral observation scales such as the JFK Coma Recovery Scale-Revised. Brain-computer interfaces (BCIs) provide an alternative approach to detect awareness in patients with DOC. However, these patients have a much lower capability of using BCIs compared to healthy individuals. This study proposed a novel BCI using temporally, spatially, and semantically congruent audiovisual stimuli involving numbers (i.e., visual and spoken numbers). Subjects were instructed to selectively attend to the target stimuli cued by instruction. Ten healthy subjects first participated in the experiment to evaluate the system. The results indicated that the audiovisual BCI system outperformed auditory-only and visual-only systems. Through event-related potential analysis, we observed audiovisual integration effects for target stimuli, which enhanced the discriminability between brain responses for target and nontarget stimuli and thus improved the performance of the audiovisual BCI. This system was then applied to detect the awareness of seven DOC patients, five of whom exhibited command following as well as number recognition. Thus, this audiovisual BCI system may be used as a supportive bedside tool for awareness detection in patients with DOC.
PMCID: PMC4485169  PMID: 26123281
24.  Circulating High-Molecular-Weight (HMW) Adiponectin Level Is Related with Breast Cancer Risk Better than Total Adiponectin: A Case-Control Study 
PLoS ONE  2015;10(6):e0129246.
The level of total adiponectin, a mixture of different adiponectin forms, has been reported associated with breast cancer risk with inconsistent results. Whether the different forms play different roles in breast cancer risk prediction is unclear. To examine this, we measured total and high molecular weight (HMW) adiponectin in a case-control study (1167 sets). Higher circulating HMW adiponectin was negatively associated with breast cancer risk after adjusting for menopausal status and family history of breast cancer (P=0.024). We analyzed the relationship between adiponectin and breast cancer risk in 6 subgroups. Higher circulating HMW adiponectin was also negatively associated with breast cancer risk (P=0.020, 0.014, 0.035) in the subgroups of postmenopausal women, negative family history of breast cancer, BMI>=24.0. Total adiponectin was positively associated with breast cancer (P=0.028) in the subgroup of BMI<=24.0. Higher HMW/total adiponectin ratio was negatively associated with breast cancer (P=0.019) in the subgroup of postmenopausal women. Interestingly, in the subgroup of women with family history of breast cancer, higher circulating total and HMW adiponectin were positively associated with breast cancer risk (P=0.034, 0.0116). This study showed different forms of circulating adiponectin levels might play different roles in breast cancer risk. A higher circulating HMW adiponectin is associated with a decreased breast cancer risk, especially in postmenopausal, without family history of breast cancer or BMI>=24.0 subgroups, whereas higher circulating HMW adiponectin levels is a risk factor in women with a family history of breast cancer. Further investigation of different forms of adiponectin on breast cancer risk is needed.
PMCID: PMC4466435  PMID: 26070203
25.  Acidic Fibroblast Growth Factor Promotes Endothelial Progenitor Cells Function via Akt/FOXO3a Pathway 
PLoS ONE  2015;10(6):e0129665.
Acidic fibroblast growth factor (FGF1) has been suggested to enhance the functional activities of endothelial progenitor cells (EPCs). The Forkhead homeobox type O transcription factors (FOXOs), a key substrate of the survival kinase Akt, play important roles in regulation of various cellular processes. We previously have shown that FOXO3a is the main subtype of FOXOs expressed in EPCs. Here, we aim to determine whether FGF1 promotes EPC function through Akt/FOXO3a pathway. Human peripheral blood derived EPCs were transduced with adenoviral vectors either expressing a non-phosphorylable, constitutively active triple mutant of FOXO3a (Ad-TM-FOXO3a) or a GFP control (Ad-GFP). FGF1 treatment improved functional activities of Ad-GFP transduced EPCs, including cell viability, proliferation, antiapoptosis, migration and tube formation, whereas these beneficial effects disappeared by Akt inhibitor pretreatment. Moreover, EPC function was declined by Ad-TM-FOXO3a transduction and failed to be attenuated even with FGF1 treatment. FGF1 upregulated phosphorylation levels of Akt and FOXO3a in Ad-GFP transduced EPCs, which were repressed by Akt inhibitor pretreatment. However, FGF1 failed to recover Ad-TM-FOXO3a transduced EPCs from dysfunction. These data indicate that FGF1 promoting EPC function is at least in part mediated through Akt/FOXO3a pathway. Our study may provide novel ideas for enhancing EPC angiogenic ability and optimizing EPC transplantation therapy in the future.
PMCID: PMC4463857  PMID: 26061278

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