FIB-SEM imaging of carbon nanotubes in mouse lung tissue

Ultrastructural characterisation is important for understanding carbon nanotube (CNT) toxicity and how the CNTs interact with cells and tissues. The standard method for this involves using transmission electron microscopy (TEM). However, in particular, the sample preparation, using a microtome to cut thin sample sections for TEM, can be challenging for investigation of regions with agglomerations of large and stiff CNTs because the CNTs cut with difficulty. As a consequence, the sectioning diamond knife may be damaged and the uncut CNTs are left protruding from the embedded block surface excluding them from TEM analysis. To provide an alternative to ultramicrotomy and subsequent TEM imaging, we studied focused ion beam scanning electron microscopy (FIB-SEM) of CNTs in the lungs of mice, and we evaluated the applicability of the method compared to TEM. FIB-SEM can provide serial section volume imaging not easily obtained with TEM, but it is time-consuming to locate CNTs in the tissue. We demonstrate that protruding CNTs after ultramicrotomy can be used to locate the region of interest, and we present FIB-SEM images of CNTs in lung tissue. FIB-SEM imaging was applied to lung tissue from mice which had been intratracheally instilled with two different multiwalled CNTs; one being short and thin, and the other longer and thicker. FIB-SEM was found to be most suitable for detection of the large CNTs (Ø ca. 70 nm), and to be well suited for studying CNT agglomerates in biological samples which is challenging using standard TEM techniques. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-013-7566-x) contains supplementary material, which is available to authorized users

ERCC2 2251A>C genetic polymorphism was highly correlated with early relapse in high-risk stage II and stage III colorectal cancer patients: A preliminary study

<p>Abstract</p> <p>Background</p> <p>Early relapse in colorectal cancer (CRC) patients is attributed mainly to the higher malignant entity (such as an unfavorable genotype, deeper tumor invasion, lymph node metastasis and advance cancer stage) and poor response to chemotherapy. Several investigations have demonstrated that genetic polymorphisms in drug-targeted genes, metabolizing enzymes, and DNA-repairing enzymes are all strongly correlated with inter-individual differences in the efficacy and toxicity of many treatment regimens. This preliminary study attempts to identify the correlation between genetic polymorphisms and clinicopathological features of CRC, and evaluates the relationship between genetic polymorphisms and chemotherapeutic susceptibility of Taiwanese CRC patients. To our knowledge, this study discusses, for the first time, early cancer relapse and its indication by multiple genes.</p> <p>Methods</p> <p>Six gene polymorphisms functional in drug-metabolism – <it>GSTP1 </it>Ile105Val, <it>ABCB1 </it>Ile1145Ile, <it>MTHFR </it>Ala222Val, <it>TYMS </it>double (2R) or triple (3R) tandem repeat – and DNA-repair genes – <it>ERCC2 </it>Lys751Gln and <it>XRCC1 A</it>rg399Gln – were assessed in 201 CRC patients using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) technique and DNA sequencing. Patients were diagnosed as either high-risk stage II (T2 and 3 N0 M0) or III (any T N1 and 2 M0) and were administered adjuvant chemotherapy regimens that included 5-fluorouracil (5FU) and leucovorin (LV). The correlations between genetic polymorphisms and patient clinicopathological features and relapses were investigated.</p> <p>Results</p> <p>In this study, the distributions of <it>GSTP1 </it>(<it>P </it>= 0.003), <it>ABCB1 </it>(<it>P </it>= 0.001), <it>TYMS </it>(<it>P </it>< 0.0001), <it>ERCC2 </it>(<it>P </it>< 0.0001) and <it>XRCC1 </it>(<it>P </it>= 0.006) genotypes in the Asian population, with the exception of <it>MTHFR </it>(<it>P </it>= 0.081), differed significantly from their distributions in a Caucasian population. However, the unfavorable genotype <it>ERCC2 </it>2251A>C (<it>P </it>= 0.006), tumor invasion depth (<it>P </it>= 0.025), lymph node metastasis (<it>P </it>= 0.011) and cancer stage (<it>P </it>= 0.008) were significantly correlated with early relapse. Patients carrying the <it>ERCC2 </it>2251AC or2251CC genotypes had a significantly increased risk of early relapse (OR = 3.294, 95% CI, 1.272–8.532).</p> <p>Conclusion</p> <p>We suggest that <it>ERCC2 </it>2251A>C alleles may be genetic predictors of early CRC relapse.</p

Expression of homothorax and extradenticle mRNA in the legs of the crustacean Parhyale hawaiensis: evidence for a reversal of gene expression regulation in the pancrustacean lineage

In Drosophila leg development, the extradenticle (exd) gene is expressed ubiquitously and its co-factor homothorax (hth) is restricted to the proximal leg portion. This condition is conserved in other insect species but is reversed in chelicerates and myriapods. As the region of co-expression does not differ in the two groups and transcripts from both are necessary for function, this difference in expression is likely to be functionally neutral. Here, we report the expression patterns of exd and hth in a crustacean, the amphipod shrimp Parhyale hawaiensis. The patterns in P. hawaiensis are similar to the insect patterns, supporting the close relationship between crustaceans and insects in the taxon Tetraconata. However, mRNA expression of exd in P. hawaiensis is weak in the distal leg parts, thus being intermediate between the complete lack of distal exd expression in chelicerates and myriapods and the strong distal exd expression in insects. Our data suggest that the reversal of the gene expression regulation of hth and exd occurred in the pancrustacean lineage

A review of African horse sickness and its implications for Ireland

African horse sickness is an economically highly important non-contagious but infectious Orbivirus disease that is transmitted by various species of Culicoides midges. The equids most severely affected by the virus are horses, ponies, and European donkeys; mules are somewhat less susceptible, and African donkeys and zebra are refractory to the devastating consequences of infection. In recent years, Bluetongue virus, an Orbivirus similar to African horse sickness, which also utilises Culicoides spp. as its vector, has drastically increased its range into previously unaffected regions in northern Europe, utilising indigenous vector species, and causing widespread economic damage to the agricultural sector. Considering these events, the current review outlines the history of African horse sickness, including information concerning virus structure, transmission, viraemia, overwintering ability, and the potential implications that an outbreak would have for Ireland. While the current risk for the introduction of African horse sickness to Ireland is considered at worst ‘very low’, it is important to note that prior to the 2006 outbreak of Bluetongue in northern Europe, both diseases were considered to be of equal risk to the United Kingdom (‘medium-risk’). It is therefore likely that any outbreak of this disease would have serious socio-economic consequences for Ireland due to the high density of vulnerable equids and the prevalence of Culicoides species, potentially capable of vectoring the virus

Computational Analysis of mRNA Expression Profiles Identifies MicroRNA-29a/c as Predictor of Colorectal Cancer Early Recurrence

Colorectal cancer (CRC) is one of the leading malignant cancers with a rapid increase in incidence and mortality. The recurrences of CRC after curative resection are sometimes unavoidable and often take place within the first year after surgery. MicroRNAs may serve as biomarkers to predict early recurrence of CRC, but identifying them from over 1,400 known human microRNAs is challenging and costly. An alternative approach is to analyze existing expression data of messenger RNAs (mRNAs) because generally speaking the expression levels of microRNAs and their target mRNAs are inversely correlated. In this study, we extracted six mRNA expression data of CRC in four studies (GSE12032, GSE17538, GSE4526 and GSE17181) from the gene expression omnibus (GEO). We inferred microRNA expression profiles and performed computational analysis to identify microRNAs associated with CRC recurrence using the IMRE method based on the MicroCosm database that includes 568,071 microRNA-target connections between 711 microRNAs and 20,884 gene targets. Two microRNAs, miR-29a and miR-29c, were disclosed and further meta-analysis of the six mRNA expression datasets showed that these two microRNAs were highly significant based on the Fisher p-value combination (p = 9.14×10−9 for miR-29a and p = 1.14×10−6 for miR-29c). Furthermore, these two microRNAs were experimentally tested in 78 human CRC samples to validate their effect on early recurrence. Our empirical results showed that the two microRNAs were significantly down-regulated (p = 0.007 for miR-29a and p = 0.007 for miR-29c) in the early-recurrence patients. This study shows the feasibility of using mRNA profiles to indicate microRNAs. We also shows miR-29a/c could be potential biomarkers for CRC early recurrence

GLUT1 gene is a potential hypoxic marker in colorectal cancer patients

<p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is an important factor related to tumor resistance to radiotherapy and chemotherapy. This study investigated molecules synthesized in colorectal cancer cells during hypoxia to explore the possibility of developing molecular probes capable of detecting cell death and/or the efficiency of radiotherapy and chemotherapy.</p> <p>Methods</p> <p>At first, we incubated two human colorectal adenocarcinoma cell lines SW480 (UICC stage II) and SW620 (UICC stage III) cells in hypoxic (≤2% O₂, 93% N₂, and 5% CO₂) and normoxic conditions (20% O₂, 75% N₂, and 5% CO₂) for 24 h and 48 h. The relative expression ratio of GLUT1 mRNA in hypoxic conditions was analyzed by RT-PCR. Ten cancerous tissues collected from human colorectal cancer patients were examined. HIF-1α and HIF-2α levels were measured to indicate the degree of hypoxia, and gene expression under hypoxic conditions was determined. As a comparison, HIF-1α, HIF-2α, and GLUT1 levels were measured in the peripheral blood of 100 CRC patients.</p> <p>Results</p> <p>Hypoxia-induced lactate was found to be elevated 3.24- to 3.36-fold in SW480 cells, and 3.06- to 3.17-fold in SW620 cells. The increased relative expression ratio of GLUT1 mRNA, under hypoxic conditions was higher in SW620 cells (1.39- to 1.72-fold elevation) than in SW480 cells (1.24- to 1.66-fold elevation). HIF-1α and HIF-2α levels were elevated and GLUT1 genes were significantly overexpressed in CRC tissue specimens. The elevated ratio of GLUT1 was higher in stage III and IV CRC tissue specimens than in the stage I and II (2.97–4.73 versus 1.44–2.11). GLUT1 mRNA was also increased in the peripheral blood of stage II and III CRC patients as compared to stage I patients, suggesting that GLUT1 may serve as a hypoxic indicator in CRC patients.</p> <p>Conclusion</p> <p>In conclusion, this study demonstrated that GLUT1 has the potential to be employed as a molecular marker to indicate the degree of hypoxia experienced by tumors circulating in the blood of cancer patients.</p

Evolutionary relationships and divergence times among the native rats of Australia

Background The genus Rattus is highly speciose and has a complex taxonomy that is not fully resolved. As shown previously there are two major groups within the genus, an Asian and an Australo-Papuan group. This study focuses on the Australo-Papuan group and particularly on the Australian rats. There are uncertainties regarding the number of species within the group and the relationships among them. We analysed 16 mitochondrial genomes, including seven novel genomes from six species, to help elucidate the evolutionary history of the Australian rats. We also demonstrate, from a larger dataset, the usefulness of short regions of the mitochondrial genome in identifying these rats at the species level. Results Analyses of 16 mitochondrial genomes representing species sampled from Australo-Papuan and Asian clades of Rattus indicate divergence of these two groups ~2.7 million years ago (Mya). Subsequent diversification of at least 4 lineages within the Australo-Papuan clade was rapid and occurred over the period from ~ 0.9-1.7 Mya, a finding that explains the difficulty in resolving some relationships within this clade. Phylogenetic analyses of our 126 taxon, but shorter sequence (1952 nucleotides long), Rattus database generally give well supported species clades. Conclusions Our whole mitochondrial genome analyses are concordant with a taxonomic division that places the native Australian rats into the Rattus fuscipes species group. We suggest the following order of divergence of the Australian species. R. fuscipes is the oldest lineage among the Australian rats and is not part of a New Guinean radiation. R. lutreolus is also within this Australian clade and shallower than R. tunneyi while the R. sordidus group is the shallowest lineage in the clade. The divergences within the R. sordidus and R. leucopus lineages occurring about half a million years ago support the hypotheses of more recent interchanges of rats between Australia and New Guinea. While problematic for inference of deeper divergences, we report that the analysis of shorter mitochondrial sequences is very useful for species identification in rats

Differentiation of Schizophrenia Patients from Healthy Subjects by Mismatch Negativity and Neuropsychological Tests

BACKGROUND: Schizophrenia is a heterogeneous disorder with diverse presentations. The current and the proposed DSM-V diagnostic system remains phenomenologically based, despite the fact that several neurobiological and neuropsychological markers have been identified. A multivariate approach has better diagnostic utility than a single marker method. In this study, the mismatch negativity (MMN) deficit of schizophrenia was first replicated in a Han Chinese population, and then the MMN was combined with several neuropsychological measurements to differentiate schizophrenia patients from healthy subjects. METHODOLOGY/PRINCIPAL FINDINGS: 120 schizophrenia patients and 76 healthy controls were recruited. Each subject received examinations for duration MMN, Continuous Performance Test, Wisconsin Card Sorting Test, and Wechsler Adult Intelligence Scale Third Edition (WAIS-III). The MMN was compared between cases and controls, and important covariates were investigated. Schizophrenia patients had significantly reduced MMN amplitudes, and MMN decreased with increasing age in both patient and control groups. None of the neuropsychological indices correlated with MMN. Predictive multivariate logistic regression models using the MMN and neuropsychological measurements as predictors were developed. Four predictors, including MMN at electrode FCz and three scores from the WAIS-III (Arithmetic, Block Design, and Performance IQ) were retained in the final predictive model. The model performed well in differentiating patients from healthy subjects (percentage of concordant pairs: 90.5%). CONCLUSIONS/SIGNIFICANCE: MMN deficits were found in Han Chinese schizophrenia patients. The multivariate approach combining biomarkers from different modalities such as electrophysiology and neuropsychology had a better diagnostic utility

Identification of IGF1, SLC4A4, WWOX, and SFMBT1 as Hypertension Susceptibility Genes in Han Chinese with a Genome-Wide Gene-Based Association Study

Hypertension is a complex disorder with high prevalence rates all over the world. We conducted the first genome-wide gene-based association scan for hypertension in a Han Chinese population. By analyzing genome-wide single-nucleotide-polymorphism data of 400 matched pairs of young-onset hypertensive patients and normotensive controls genotyped with the Illumina HumanHap550-Duo BeadChip, 100 susceptibility genes for hypertension were identified and also validated with permutation tests. Seventeen of the 100 genes exhibited differential allelic and expression distributions between patient and control groups. These genes provided a good molecular signature for classifying hypertensive patients and normotensive controls. Among the 17 genes, IGF1, SLC4A4, WWOX, and SFMBT1 were not only identified by our gene-based association scan and gene expression analysis but were also replicated by a gene-based association analysis of the Hong Kong Hypertension Study. Moreover, cis-acting expression quantitative trait loci associated with the differentially expressed genes were found and linked to hypertension. IGF1, which encodes insulin-like growth factor 1, is associated with cardiovascular disorders, metabolic syndrome, decreased body weight/size, and changes of insulin levels in mice. SLC4A4, which encodes the electrogenic sodium bicarbonate cotransporter 1, is associated with decreased body weight/size and abnormal ion homeostasis in mice. WWOX, which encodes the WW domain-containing protein, is related to hypoglycemia and hyperphosphatemia. SFMBT1, which encodes the scm-like with four MBT domains protein 1, is a novel hypertension gene. GRB14, TMEM56 and KIAA1797 exhibited highly significant differential allelic and expressed distributions between hypertensive patients and normotensive controls. GRB14 was also found relevant to blood pressure in a previous genetic association study in East Asian populations. TMEM56 and KIAA1797 may be specific to Taiwanese populations, because they were not validated by the two replication studies. Identification of these genes enriches the collection of hypertension susceptibility genes, thereby shedding light on the etiology of hypertension in Han Chinese populations