N-Myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor gene, which plays an important role in controlling tumor growth. The aim of this study was to investigate the expression of NDRG2 gene in bladder cancer (BC) tissues and several bladder cancer cell lines, and to seek its clinical and pathological significance. Ninety-seven bladder carcinoma and 15 normal bladder tissue sections were analyzed retrospectively with immunohistochemistry. The human bladder cancer cell line T24 was infected with LEN-NDRG2 or LEN-LacZ. The effects of NDRG2 overexpression on T24 cells and T24 nude mouse xenografts were measured via cell growth curves, tumor growth curves, flow cytometric analysis, western blot and Transwell assay. NDRG2 was highly expressed in normal bladder tissue, but absent or rarely expressed in cacinomatous tissues (χ2=8.761, p < 0.01). The NDRG2 level was negatively correlated with tumor grade and pathologic stage(r=-0.248, p < 0.05), as well as increased c-myc level (r=-0.454, p< 0.001). The expression of NDRG2 was low in the three BC cell lines. T24 cells infected with LEN-NDRG2 showed inhibition of proliferation both in vitro and in vivo, and NDRG2 overexpression can inhibit tumor growth and invasion in vitro.
Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.
Previous studies indicated that a novel influenza A virus (H17N10) was circulating in fruit bats from Guatemala (Central America). Herein, we investigated whether similar viruses are present in bat species from South America. Analysis of rectal swabs from bats sampled in the Amazon rainforest region of Peru identified another new influenza A virus from bats that is phylogenetically distinct from the one identified in Guatemala. The genes that encode the surface proteins of the new virus from the flat-faced fruit bat were designated as new subtype H18N11. Serologic testing of blood samples from several species of Peruvian bats indicated a high prevalence of antibodies to the surface proteins. Phylogenetic analyses demonstrate that bat populations from Central and South America maintain as much influenza virus genetic diversity in some gene segments as all other mammalian and avian species combined. The crystal structures of the hemagglutinin and neuraminidase proteins indicate that sialic acid is not a receptor for virus attachment nor a substrate for release, suggesting a novel mechanism of influenza A virus attachment and activation of membrane fusion for entry into host cells. In summary, our findings indicate that bats constitute a potentially important reservoir for influenza viruses.
The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.
Zooplankton are relatively small in size in the subtropical regions. This characteristic has been attributed to intense predation pressure, high nutrient loading and cyanobacterial biomass. To provide further information on the effect of predation and cyanobacteria on zooplankton size structure, we analyzed data from 96 shallow aquaculture lakes along the Yangtze River. Contrary to former studies, both principal components analysis and multiple regression analysis showed that the mean zooplankton size was positively related to fish yield. The studied lakes were grouped into three types, namely, natural fishing lakes with low nutrient loading (Type1), planktivorous fish-dominated lakes (Type 2), and eutrophic lakes with high cyanobacterial biomass (Type 3). A marked difference in zooplankton size structure was found among these groups. The greatest mean zooplankton size was observed in Type 2 lakes, but zooplankton density was the lowest. Zooplankton abundance was highest in Type 3 lakes and increased with increasing cyanobacterial biomass. Zooplankton mean size was negatively correlated with cyanobacterial biomass. No obvious trends were found in Type 1 lakes. These results were reflected by the normalized biomass size spectrum, which showed a unimodal shape with a peak at medium sizes in Type 2 lakes and a peak at small sizes in Type 3 lakes. These results indicated a relative increase in medium-sized and small-sized species in Types 2 and 3 lakes, respectively. Our results suggested that fish predation might have a negative effect on zooplankton abundance but a positive effect on zooplankton size structure. High cyanobacterial biomass most likely caused a decline in the zooplankton size and encouraged the proliferation of small zooplankton. We suggest that both planktivorous fish and cyanobacteria have substantial effects on the shaping of zooplankton community, particularly in the lakes in the eastern plain along the Yangtze River where aquaculture is widespread and nutrient loading is high.
Neurorestorative therapy targets multiple types of parenchymal cells in the intact tissue of the injured brain tissue to increase neurogenesis, angiogenesis, oligodendrogenesis, and axonal remodeling during recovery from neurological injury. In our laboratory, we tested thymosin β4 (Tβ4) as a neurorestorative agent to treat models of neurological injury. This review discusses our results demonstrating that Tβ4 improves neurological functional outcome in a rat model of embolic stroke, a mouse model of multiple sclerosis, and a rat model of traumatic brain injury. Tβ4 is a pleiotropic peptide exhibiting many actions in several different types of tissues. One mechanism associated with improvement of neurological improvement from Tβ4 treatment is oligodendrogenesis involving the differentiation of oligodendrocyte progenitor cells to mature myelin-secreting oligodendrocytes. Moreover, our preclinical data provide a basis for movement of Tβ4 into clinical trials for treatment of these devastating neurological diseases and injuries.
thymosin β4; stroke; multiple sclerosis; traumatic brain injury; rat
Pseudomonas sp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designated nox) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans. The nox gene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteria Escherichia coli DH5α (DH-nox) and Pseudomonas putida KT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that the nox gene is expressed in both DH5α and KT2440, suggesting that additional factors required for nicotine degradation are present in a Pseudomonas strain(s), but not in E. coli. The mutant of strain HZN6 with nox disrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that the nox gene is responsible for the first step of nicotine degradation. The (RS)-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.
Germination of Bacillus subtilis spores can be triggered by the binding of specific nutrients, called germinants, to germinant receptors (GRs) in the spore's inner membrane. This interaction eventually initiates, with variable time delays, the release of dipicolinic acid and cations from the spore core—a key step in spore germination. The kinetics of this process are highly heterogeneous for individual spores. In this work, we sought to investigate how the germination heterogeneity was controlled. In particular, we tested whether the rates of germination were determined by GR levels, which vary from spore to spore due to stochastic gene expression. Both the expression levels of GRs and the germination rate were measured in single spores, and the experimental results were compared to theoretical predictions. Our results indicated that the variation in the expression levels of GRs was not the primary factor that controls spore germination heterogeneity. Two alternative hypotheses are discussed in light of this experimental discovery.
Manganese (Mn), an established neurotoxicant, is a common component of welding fume. The neurological phenotype associated with welding exposures has not been well described. Prior epidemiologic evidence linking occupational welding to parkinsonism is mixed, and remains controversial.
This was a cross-sectional and nested case–control study to investigate the prevalence and phenotype of parkinsonism among 811 shipyard and fabrication welders recruited from trade unions. Two reference groups included 59 non-welder trade workers and 118 newly diagnosed, untreated idiopathic PD patients. Study subjects were examined by a movement disorders specialist using the Unified Parkinson Disease Rating Scale motor subsection 3 (UPDRS3). Parkinsonism cases were defined as welders with UPDRS3 score ≥15. Normal was defined as UPDRS3 < 6. Exposure was classified as intensity adjusted, cumulative years of welding. Adjusted prevalence ratios for parkinsonism were calculated in relation to quartiles of welding years.
The overall prevalence estimate of parkinsonism was 15.6% in welding exposed workers compared to 0% in the reference group. Among welders, we observed a U-shaped dose–response relation between weighted welding exposure-years and parkinsonism. UPDRS3 scores for most domains were similar between welders and newly diagnosed idiopathic Parkinson disease (PD) patients, except for greater frequency of rest tremor and asymmetry in PD patients.
This work-site based study among welders demonstrates a high prevalence of parkinsonism compared to nonwelding-exposed workers and a clinical phenotype that overlaps substantially with PD.
Parkinsonism; Parkinson disease; Welding; Manganese; Occupational exposures
To establish an ultra-performance liquid chromatography (UPLC) fingerprinting method for quality control of Phragmitis rhizoma from Baiyangdian.
Materials and Methods:
Ultrasonic extraction with 70% methanol was performed on 10 samples of P. rhizoma collected from 10 different villages in Baiyangdian. The sample solutions were analyzed by Waters UPLC equipped with the ACQUITY UPLC BEH C18 column and photodiode array (PDA) detector, and gradient eluted with acetonitrile/water as the mobile phase. The flow rate was set to 0.1 mL/min; the column temperature was set to 25°C; and the detection wavelength was set to 285 nm.
The chromatograms of the 10 samples showed 27 common peaks, of which one was identified as the ferulic acid standard. The similarity indexes were all above 0.82. Hierarchical cluster analysis showed that the constituents and their quantities differed according to the diameter of the original plant, which is related to its age.
The UPLC fingerprinting method had the advantages of being fast, accurate, and highly efficient; this indicated that it can be used for quality control of P. rhizoma produced in Baiyangdian. Also, the relation between the quality and diameter/age of the plant needs to be further investigated.
Baiyangdian; fingerprint; Phragmitis rhizoma; ultra-performance liquid chromatography
Renal accumulation of reactive carbonyl compounds (RCCs) has been linked to the progression of diabetic nephropathy. We previously demonstrated that carbonyl stress induces the formation of amino-carbonyl cross-links and sharply increases the content of β-sheet-rich structures, which is the seed of insoluble aggregates formation, and tea catechin (-)-epigallocatechin 3-gallate (EGCG) can reverse this process in vitro and in vivo. In this study, methylated derivative (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3”Me) was hypothesized to neutralize carbonyl stress mediating the formation of insoluble ubiquitinated protein (IUP) aggregates, and reduce the early development of diabetic nephropathy.
Methods and results
Diabetes was induced in mice by intraperitoneally injecting alloxan monohydrate (200 mg/kg/d) twice and administering EGCG3”Me by gavage for 15 d. Reagent case and western blot results showed that, in diabetic kidneys, the carbonyl proteins in the serum increased; and in insoluble protein fraction, 4-hydroxynonenal-modified proteins, IUP aggregates and p62 accumulated; FT-IR study demonstrated that the lipid content, anti-parallel β-sheet structure and aggregates increased. EGCG3”Me treatment could effectively reverse this process, even better than the negative control treatment.
EGCG3”Me exhibiting anti-β-sheet-rich IUP aggregate properties, maybe represents a new strategy to impede the progression of diabetic nephropathy and other diabetic complications.
Saffold cardiovirus (SAFV) is a new human cardiovirus with 11 identified genotypes. Little is known about the natural history and pathogenicity of SAFVs.
We sequenced the genome of five SAFV-1 strains which were identified from fecal samples taken from children with viral diarrhea in Beijing, China between March 2006 and November 2007, and analyzed the phylogenetic and phylodynamic properties of SAFVs using the genome sequences of every known SAFV genotypes. We identified multiple recombination events in our SAFV-1 strains, specifically recombination between SAFV-2, -3, -4, -9, -10 and the prototype SAFV-1 strain in the VP4 region and recombination between SAFV-4, -6, -8, -10, -11 and prototype SAFV-1 in the VP1/2A region. Notably, recombination in the structural gene VP4 is a rare event in Cardiovirus. The ratio of nonsynonymous substitutions to synonymous substitutions indicates a purifying selection of the SAFV genome. Phylogenetic and molecular clock analysis indicates the existence of at least two subclades of SAFV-1 with different origins. Subclade 1 includes two strains isolated from Pakistan, whereas subclade 2 includes the prototype strain and strains isolated in China, Pakistan, and Afghanistan. The most recent common ancestor of all SAFV genotypes dates to the 1710s, and SAFV-1, -2, and -3 to the 1940s, 1950s, and 1960s, respectively. No obvious relationship between variation and pathogenicity exists in the critical domains of the CD and EF loops of viral capsid proteins or the multi-functional proteins L based on animo acid sequence identity comparison between SAFV genotypes.
Our findings suggest that intertypic recombination plays an important role in the diversity of SAFVs, highlighting the diversity of the five strains with the previously described SAFV-1 strains.
New megafossil and microfossil data indicate four episodes in the diversification of Silurian–Early Carboniferous land plants of South China, a relatively continuous regional record. Plant diversity increased throughout, but the rising curve was punctuated by three major falls. There were peaks of origination in the Ludlow–Pragian, Givetian, late Famennian and Visean and peaks of extinction in the Pragian–Emsian, Givetian and early Tournaisian. Speciation and extinction rates were highest in the Lochkovian–Pragian and became progressively lower in subsequent stages. High correlation coefficients indicate that these events are associated with the availability of land habitat contingent on eustatic variations and increasing numbers of cosmopolitan genera. Meanwhile, proportions of endemic genera declined gradually. Due to less endemism and more migrations, both speciation and species extinction rates reduced. The changes of diversity and the timing of the three extinctions of land plants in South China are similar to those known already from Laurussia. However, the largest events in the Lochkovian–Pragian and subsequent smaller ones have not been seen in the global pattern of plant evolution. These land plant events do not correspond well temporally with those affecting land vertebrates or marine invertebrates. In South China, the diversity curve of land plants is generally opposite to that of marine faunas, showing a strong effect of eustatic variations. The increasing diversity of both land vertebrates and plants was punctuated above the Devonian–Carboniferous boundary, known as Romer's Gap, implying common underlying constraints on macroevolution of land animals and plants.
Chronic obstructive pulmonary disease (COPD) is a common disease that leads to huge economic and social burden. Efficient and effective management of stable COPD is essential to improve quality of life and reduce medical expenditure. The Internet of Things (IoT), a recent breakthrough in communication technology, seems promising in improving health care delivery, but its potential strengths in COPD management remain poorly understood. We have developed a mobile phone-based IoT (mIoT) platform and initiated a randomized, multicenter, controlled trial entitled the ‘MIOTIC study’ to investigate the influence of mIoT among stable COPD patients. In the MIOTIC study, at least 600 patients with stable GOLD group C or D COPD and with a history of at least two moderate-to-severe exacerbations within the previous year will be randomly allocated to the control group, which receives routine follow-up, or the intervention group, which receives mIoT management. Endpoints of the study include (1) frequency and severity of acute exacerbation; (2) symptomatic evaluation; (3) pre- and post-bronchodilator forced expiratory volume in 1 second (FEV1) and FEV1/forced vital capacity (FVC) measurement; (4) exercise capacity; and (5) direct medical cost per year. Results from this study should provide direct evidence for the suitability of mIoT in stable COPD patient management.
Internet of Things; mobile phone; chronic obstructive pulmonary disease; efficacy
Borderline personality disorder (BPD) is characterized by an inability to regulate emotional responses. The amygdala is important in learning about the valence (goodness and badness) of stimuli and has been reported to function abnormally in BPD.
Event-related functional MRI (fMRI) was employed in three groups: unmedicated BPD (n=33) and schizotypal personality disorder (SPD;n=28) participants and healthy controls (n=32) during a task involving an intermixed series of unpleasant, neutral, and pleasant pictures each presented twice within their respective trial block/run. The amygdala was hand-traced on each participant’s structural-MRI scan which was co-registered to their BOLD-scan. Amygdala responses were examined with a mixed-model MANOVA with repeated measures.
Compared with both control groups, BPD patients showed greater amygdala activation, particularly to the repeated emotional but not neutral pictures and a prolonged return to baseline for the overall BOLD response averaged across all pictures. Despite amygdala overactivation, BPD patients showed a blunted response on the self-report ratings of emotional but not neutral pictures. Fewer dissociative symptoms in both patient groups were associated with greater amygdala activation to repeated unpleasant pictures.
The increased amygdala response to the repeated emotional pictures observed in BPD was not observed in SPD patients suggesting diagnostic specificity. This BPD-related abnormality is consistent with the well-documented clinical feature of high sensitivity to emotional stimuli with unusually strong and long-lasting reactions. The finding of a mismatch between physiological and self-report measures of emotion reactivity in BPD patients suggests they may benefit from treatments which help them recognize emotions.
borderline personality disorder; schizotypal personality disorder; amygdala; emotion; fMRI; arousal; valence
Sex-determining region Y-box 2 (SOX2) is proposed to be a key transcription factor in embryonic stem cells. The known roles of SOX2 in development and cell differentiation suggest that it is relevant to the aberrant growth of tumor cells. Thus, SOX2 may play an important role in tumor progression. However, its clinical significance in human ovarian carcinoma has been uncertain until recently. The aim of the present study was to clarify the clinical role of SOX2 expression in ovarian carcinoma. Immunohistochemical staining of 540 human ovarian carcinoma samples for SOX2 was performed using tissue microarray. The associations among SOX2 expression and clinical factors (diagnosis, tumor grade, International Federation of Gynecology and Obstetrics stage, and response to chemotherapy), overall survival, and disease-free survival were analyzed. We observed SOX2 expression in 15% of the ovarian carcinoma samples. Use of the Fisher exact test suggested that SOX2 expression was associated with high-grade carcinoma (P = .009), especially high-grade serous carcinoma (P = .048); International Federation of Gynecology and Obstetrics stage (II-IV, P = .005); and malignant mixed müllerian tumors (P = .048). SOX2 expression was also associated with decreased disease-free survival durations (P = .035; log-rank test). Our results showed that SOX2 expression may be a potential marker of related to tumor recurrence, as implicated to its role in cancer stem cells.
Sex-determining region Y-box 2 (SOX2); Immunohistochemistry; Ovarian carcinoma; Prognosis
Mycobacterium tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand (FL) that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid (pIRES-epitope-peptides-FL) encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine (prime) and intranasal administration of the epitope peptides (boost) was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Th1 cytokines (IFN-γ and IL-12), the number of IFN-γ+ T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant pIRES-epitope-peptides-FL plasmid is a useful DNA vaccine for preventing Mycobacterium tuberculosis infection.
early secretory antigenic target-6 (ESAT-6); fms-like tyrosine kinase 3 ligand (FL); Mycobacterium tuberculosis; recombinant plasmid; T cell epitopes
The present study investigates cytogenetic damage in lymphocytes, derived from three victims who were unfortunately exposed to cobalt-60 (60Co) radiation (the 1999 accident occurred in a village in China’s Henan province). Case A of the three victims was exposed to a higher dose of 60Co radiation than Cases B and C. The chromosomal aberrations, cytokinesis-block micronucleus (CBMN, the CBMN assay), and DNA double-strand breaks (DSBs, the comet assay) examined in this study are biomarkers for cytogenetic abnormalities. After the lymphocytes collected from the victims were cultured, the frequencies of dicentric chromosomes and rings (dic + r) and CBMN in the first mitotic division detected in the lymphocytes of Case A were found to be substantially higher than in Cases B and C. Similarly, the DNA-DSB level found in the peripheral blood collected from Case A was much higher than those of Cases B and C. These results suggest that an acutely enhanced induction of the 60Co-induced cytogenetic abnormality frequency in humans depends on the dose of 60Co radiation. This finding is supported by the data obtained using practical techniques to evaluate early lymphoid-tissue abnormalities induced after exposure to acute radiation.
radiation accident; chromosome aberration; micronucleus assay; comet assay; DNA-DSB
To compare visual performance of wavefront-guided laser in situ keratomileusis (LASIK) with iris-registration (Wg-LASIK group) and conventional LASIK (LASIK group) one year after surgery and analyze the correlation between wavefront aberrations and visual performance.
Eight hundred and fifty-two myopic eyes of 430 patients were enrolled in this prospective study and divided into two groups: Wg-LASIK group (436 eyes) and LASIK group (416 eyes). A Wavescan Wavefront aberrometer was used to analyze Zernike coefficients and the root-mean-square (RMS) of higher order aberrations, and Optec 6500 visual function instrument was used to measure contrast sensitivity (CS) before and 3, 6, 12 months after surgery.
The mean spherical equivalent (SE) in Wg-LASIK group was significantly better than those in LASIK group one year after surgery (P=0.024). Wg-LASIK eyes showed better CS values than LASIK eyes at all spatial frequencies with and without glare after surgery (P all<0.01). Moreover, the increase of higher RMS (RMSh), coma, RMS3, RMS4, RMS5 in Wg-LASIK group were significantly lower than those in LASIK group 1 year after surgery (P all<0.05). The increase of coma, spherical aberration (SA), RMS3 and RMS4 in Wg-LASIK and coma and RMS3 in LASIK group were negatively correlated with reduction of contrast sensitivity 1 year after surgery.
A significant better visual performance is got in Wg-LASIK group compared with LASIK group 1 year after surgery, and the Wg-LASIK is particularly suitable for eyes with high-magnitude RMSh.
aberration; contrast sensitivity; visual performance; laser in situ keratomileusis
The speed of a separation defines the best time resolution possible in online measurements using chromatography. The desired time resolution multiplied by the flow rate of the stream of analyte being sampled defines the maximum volume of sample per injection. The best concentration sensitivity in chromatography is obtained by injecting the largest volume of sample that is consistent with achieving a satisfactory separation, and thus measurement accuracy. Taking these facts together, it is easy to understand that separation speed and concentration sensitivity are linked in this type of measurement. To address the problem of how to achieve the best sensitivity and shortest measurement time simultaneously, we have combined recent approaches to the optimization of the separation itself with an analysis of method sensitivity. This analysis leads to the column diameter becoming an important parameter in the optimization process. We use these ideas in one particular problem presented by online microdialysis sampling/liquid chromatography/electrochemical detection for measuring concentrations of serotonin in the dialysate. In this case the problem becomes the optimization of conditions to yield maximum signal for a given sample volume under the highest speed conditions with a certain required number of theoretical plates. It turns out that the observed concentration sensitivity at an electrochemical detector can be regulated by temperature, particle size, injection volume/column diameter and void time. The theory was successfully used for optimization of neurotransmitter serotonin measurement by capillary HPLC when sampling from a microdialysis flow stream. The final conditions are: 150 μm i.d., 3.1 cm long columns with 1.7 μm particle diameter working at a flow rate of 12 μL/min, an injection volume of 500 nL, and a temperature of 343 K. The retention time for serotonin is 22.7 s, the analysis time is about 36 s (which allows for determination of 3-methoxytyramine), and the sampling time is about 0.8 minutes with a perfusion flow rate of 0.6 μL/min.
HPLC system optimization; high speed analysis; column diameter; detection sensitivity; microdialysis; serotonin
In addition to amyloid beta (Aβ) and tau, α-synuclein, best known for its role in Parkinson’s disease (PD), has been suggested to be involved in cognition and pathogenesis of Alzheimer’s disease (AD). We investigate the potential of α-synuclein in cerebrospinal fluid (CSF) as a biomarker of cognitive decline in AD, and its prodromal phase, mild cognitive impairment (MCI). Using an established, sensitive Luminex assay, we measured α-synuclein levels in the CSF of a cohort of close to 400 healthy control, MCI and AD subjects obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and factored in APOE genotype in data analysis. CSF α-synuclein levels were significantly higher in the MCI (P = 0.005) and AD (P < 0.001) groups, compared to controls. However, receiver operating characteristic (ROC) curve analysis suggests that CSF α-synuclein level on its own only offered modest sensitivity (65 %) and specificity (74 %) as a diagnostic marker of AD, with an area under the curve (AUC) value of 0.719 for AD vs controls. The effect of APOE genotype, if any, was quite subtle. However, there was a significant correlation between α-synuclein and cognition (P = 0.001), with increased α-synuclein levels associated with decreased MMSE scores. Our results support a role for α-synuclein even in MCI, the early phase of AD, in addition to being a potential contributor in MCI and AD diagnosis or monitoring of disease progression.
alpha-synuclein; Alzheimer’s disease; Mild cognitive impairment; biomarkers; cerebrospinal fluid
Protein tyrosine phosphatase 1B (PTP1B) is a key molecule in modulating low-degree inflammatory conditions such as diabetes. The role of PTP1B in other chronic inflammations, however, remains unknown. Here, we report that PTP1B deficiency ameliorates Dextran Sulfate Sodium (DSS)-induced murine experimental colitis via expanding CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs). Employing DSS-induced murine experimental colitis as inflammatory animal model, we found that, compared with wild-type littermates, PTP1B-null mice demonstrated greater resistance to DSS-induced colitis, as reflected by slower weight-loss, greater survival rates and decreased PMN and macrophage infiltration into the colon. The evidence collectively also demonstrated that the resistance of PTP1B-null mice to DSS-induced colitis is based on the expansion of MDSCs. First, PTP1B-null mice exhibited a greater frequency of MDSCs in the bone marrow (BM), peripheral blood and spleen when compared with wild-type littermates. Second, PTP1B levels in BM leukocytes were significantly decreased after cells were induced into MDSCs by IL-6 and GM-CSF, and the MDSC induction occurred more rapidly in PTP1B-null mice than in wild-type littermates, suggesting PTP1B as a negative regulator of MDSCs. Third, the adoptive transfer of MDSCs into mice with DSS-colitis significantly attenuated colitis, which accompanies with a decreased serum IL-17 level. Finally, PTP1B deficiency increased the frequency of MDSCs from BM cells likely through enhancing the activities of signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2). In conclusion, our study provides the first evidences that PTP1B deficiency ameliorates murine experimental colitis via expanding MDSCs.
The intestinal microbiota have critical roles in immune system and metabolic homeostasis, but they must be tolerated by the host to avoid inflammatory responses that can damage the epithelial barrier separating the host from the luminal contents1-6. Breakdown of this regulation and the resulting inappropriate immune response to commensals are thought to lead to the development of inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis7. We hypothesized that the intestinal immune system is instructed by the microbiota to limit responses to luminal antigens. We demonstrate that, at steady state, the microbiota inhibit the transport of both commensal and pathogenic bacteria from the lumen to a key immune inductive site, the mesenteric lymph node (MLN). However, in the absence of Myd88 or under conditions of antibiotic-induced dysbiosis, non-invasive bacteria trafficked to the MLN in a CCR7-dependent manner and induced both T cell responses and IgA production. Trafficking was carried out by CX3CR1hi mononuclear phagocytes, an intestinal cell population previously reported to be non-migratory8. These findings define a central role for commensals in regulating the migration to the MLN of CX3CR1hi mononuclear phagocytes endowed with the ability to capture luminal bacteria, thereby compartmentalizing the intestinal immune response to avoid inflammation.
Recent studies have demonstrated the power of deep re-sequencing of the whole genome or exome in understanding cancer genomes. However, targeted capture of selected genomic whole gene-body regions, rather than the whole exome, have several advantages: 1) the genes can be selected based on biology or a hypothesis; 2) mutations in promoter and intronic regions, which have important regulatory roles, can be investigated; and 3) less expensive than whole genome or whole exome sequencing. Therefore, we designed custom high-density oligonucleotide microarrays (NimbleGen Inc.) to capture approximately 1.7 Mb target regions comprising the genomic regions of 28 genes related to colorectal cancer including genes belonging to the WNT signaling pathway, as well as important transcription factors or colon-specific genes that are over expressed in colorectal cancer (CRC). The 1.7 Mb targeted regions were sequenced with a coverage ranged from 32× to 45× for the 28 genes. We identified a total of 2342 sequence variations in the CRC and corresponding adjacent normal tissues. Among them, 738 were novel sequence variations based on comparisons with the SNP database (dbSNP135). We validated 56 of 66 SNPs in a separate cohort of 30 CRC tissues using Sequenom MassARRAY iPLEX Platform, suggesting a validation rate of at least 85% (56/66). We found 15 missense mutations among the exonic variations, 21 synonymous SNPs that were predicted to change the exonic splicing motifs, 31 UTR SNPs that were predicted to occur at the transcription factor binding sites, 20 intronic SNPs located near the splicing sites, 43 SNPs in conserved transcription factor binding sites and 32 in CpG islands. Finally, we determined that rs3106189, localized to the 5′ UTR of antigen presenting tapasin binding protein (TAPBP), and rs1052918, localized to the 3′ UTR of transcription factor 3 (TCF3), were associated with overall survival of CRC patients.
Manganese (Mn) is a common occupational exposure worldwide. Recent studies indicate clinical and imaging evidence of neurotoxicity in chronically exposed workers. The pathologic significance of these findings is unclear. South Africa produces over 80% of the world’s Mn from mines from a desert region in the Northern Cape Province. An autopsy program at the National Institute for Occupational Health (NIOH) in South Africa has provided compensation to families for mining-related lung diseases for almost 100 years. Building on this, we implemented a brain autopsy program to investigate the feasibility of obtaining brains from South African Mn miners and non-exposed reference miners to investigate neuropathologic consequences of chronic Mn exposure. Employing an experienced occupational health nurse, we identified deceased miners within 100 square km of the Mn mines. The nurse was notified of any Mn (case) or other (reference) miner or ex-miner death by local medical practitioners, occupational health and mine physicians, and community members, and families were approached for consent to remove the brains in addition to the cardio-respiratory organs. Families of deceased miners who had an autopsy at the NIOH in Johannesburg were also approached. To confirm exposure in Mn miners, mean pallidal indices were compared between Mn miners and non-exposed reference miners. Sixty-eight potential brain donors were identified; we obtained consent from the families to remove 51 (75%). The mean autopsy interval was seven days. With optimized fixation methods, the tissue quality of the brains for gross and regular microscopic examination was excellent. Ex-vivo MRI demonstrated increased pallidal index in Mn miners compared to reference miners. We conclude that obtaining brain tissue from deceased miners in South Africa is highly successful with only a modest investment in local infrastructure. Tissue quality was excellent and should be ideal to investigate the neuropathologic consequences of chronic occupational Mn exposure.
Manganese; parkinsonism; neuropathology; MRI
Age-associated arterial remodeling involves arterial wall collagen deposition and elastin fragmentation, and an increase in arterial pressure. This arterial remodeling is linked to proinflammatory signaling, including transforming growth factor-beta1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), and proendothelin-1(Pro-ET-1), activated by extracellular matrix metalloproteinases (MMPs) and orchestrated, in part, by the transcriptional factor ets-1. We tested the hypothesis that inhibition of MMP activation can decelerate the age-associated arterial proinflammation and its attendant increase in arterial pressure. Indeed, chronic administration of a broad spectrum MMP inhibitor, PD166739, via a daily gavage, to 16-mo-old rats for 8 months markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by (1) inhibition of the age-associated increases in aortic gelatinase and interstitial collagenase activity in situ; (2) preservation of the elastic fiber network integrity; (3) a reduction of collagen deposition; (4) a reduction of MCP-1 and TGF-β1 activation; (5) a diminution in the activity of the pro-fibrogenic signaling molecule, SMAD-2/3 phosphorylation; (6) inhibition of pro-ET-1 activation; and (7) down-regulation of expression of ets-1. Acute exposure of cultured vascular smooth muscle cells (VSMC) in vitro to pro-ET-1 increased both the transcription and translation of ets-1, and these effects were markedly reduced by MMP inhibition. Furthermore, infection of VSMC with an adenovirus harboring a full-length ets-1 cDNA increased activities of both TGF-β1 and MCP-1. Collectively, our results indicate that MMP inhibition retards age-associated arterial proinflammatory signaling, and this is accompanied by perseveration of intact elastin fibers, a reduction in collagen, and blunting of an age-associated increase in blood pressure.
Aging; Arterial remodeling; Matrix metalloproteinase inhibitor; Endothelin-1; Transforming growth factor-beta 1; ets-1; Monocyte chemoattractant protein-1; Blood pressure