Loss of breast epithelial marker hCLCA2 promotes epithelial to mesenchymal transition and indicates higher risk of metastasis

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFβ or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.

Reduced Il17a expression distinguishes a Ly6cloMHCIIhi macrophage population promoting wound healing

Macrophages are the main components of inflammation during skin wound healing. They are critical in wound closure and in excessive inflammation, resulting in defective healing observed in chronic wounds. Given the heterogeneity of macrophage phenotypes and functions, we here hypothesized that different subpopulations of macrophages would have different and sometimes opposing effects on wound healing. Using multimarker flow cytometry and RNA expression array analyses on macrophage subpopulations from wound granulation tissue, we identified a Ly6cloMHCIIhi “noninflammatory” subset that increased both in absolute number and proportion during normal wound healing and was missing in Ob/Ob and MYD88−/− models of delayed healing. We also identified IL17 as the main cytokine distinguishing this population from proinflammatory macrophages and demonstrated that inhibition of IL17 by blocking Ab or in IL17A−/− mice accelerated normal and delayed healing. These findings dissect the complexity of the role and activity of the macrophages during wound inflammation and may contribute to the development of therapeutic approaches to restore healing in chronic wounds.

miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data

miRDeep and its varieties are widely used to quantify known and novel micro RNA (miRNA) from small RNA sequencing (RNAseq). This article describes miRDeep*, our integrated miRNA identification tool, which is modeled off miRDeep, but the precision of detecting novel miRNAs is improved by introducing new strategies to identify precursor miRNAs. miRDeep* has a user-friendly graphic interface and accepts raw data in FastQ and Sequence Alignment Map (SAM) or the binary equivalent (BAM) format. Known and novel miRNA expression levels, as measured by the number of reads, are displayed in an interface, which shows each RNAseq read relative to the pre-miRNA hairpin. The secondary pre-miRNA structure and read locations for each predicted miRNA are shown and kept in a separate figure file. Moreover, the target genes of known and novel miRNAs are predicted using the TargetScan algorithm, and the targets are ranked according to the confidence score. miRDeep* is an integrated standalone application where sequence alignment, pre-miRNA secondary structure calculation and graphical display are purely Java coded. This application tool can be executed using a normal personal computer with 1.5 GB of memory. Further, we show that miRDeep* outperformed existing miRNA prediction tools using our LNCaP and other small RNAseq datasets. miRDeep* is freely available online at http://www.australianprostatecentre.org/research/software/mirdeep-star

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography

Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly

Cryo-electron tomography of Marburg Virus particles and their morphogenesis within infected cells

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.

A 250 μg/week dose of vitamin D was as effective as a 50 μg/d dose in healthy adults, but a regimen of four weekly followed by monthly doses of 1250 μg raised the risk of hypercalciuria

The risk of vitamin D insufficiency is increased in persons having limited sunlight exposure and dietary vitamin D. Supplementation compliance might be improved with larger doses taken less often, but this may increase the potential for side effects. The objective of the present study was to determine whether a weekly or weekly/monthly regimen of vitamin D supplementation is as effective as daily supplementation without increasing the risk of side effects. Participants were forty-eight healthy adults who were randomly assigned for 3 months to placebo or one of three supplementation regimens: 50 μg/d (2000 IU/d, analysed dose 70 μg/d), 250 μg/week (10 000 IU/week, analysed dose 331 μg/week) or 1250 μg/week (50 000 IU/week, analysed dose 1544 μg/week) for 4 weeks and then 1250 μg/month for 2 months. Daily and weekly doses were equally effective at increasing serum 25-hydroxyvitamin D, which was significantly greater than baseline in all the supplemented groups after 30 d of treatment. Subjects in the 1250 μg treatment group, who had a BMI >26 kg/m2, had a steady increase in urinary Ca in the first 3 weeks of supplementation, and, overall, the relative risk of hypercalciuria was higher in the 1250 μg group than in the placebo group (P= 0·01). Although vitamin D supplementation remains a controversial issue, these data document that supplementing with ≤ 250 μg/week ( ≤ 10 000 IU/week) can improve or maintain vitamin D status in healthy populations without the risk of hypercalciuria, but 24 h urinary Ca excretion should be evaluated in healthy persons receiving vitamin D3 supplementation in weekly single doses of 1250 μg (50 000 IU).

Do RNA viruses require genome cyclisation for replication?

Complementary sequences at the 5′ and 3′ ends of the dengue virus RNA genome are essential for viral replication, and are believed to cyclise the genome through long-range base pairing in cis. Although consistent with evidence in the literature, this view neglects possible biologically active multimeric forms that are equally consistent with the data. Here, we propose alternative multimeric structures, and suggest that multigenome noncovalent concatemers are more likely to exist under cellular conditions than single cyclised monomers. Concatemers provide a plausible mechanism for the dengue virus to overcome the single-stranded (+)-sense RNA virus dilemma, and can potentially assist genome transport from the virus-induced vesicles into the cytosol.

Transcriptome analysis of neutrophils after endurance exercise reveals novel signaling mechanisms in the immune response to physiological stress

Neutrophils serve as an intriguing model for the study of innate immune cellular activity induced by physiological stress. We measured changes in the transcriptome of circulating neutrophils following an experimental exercise trial (EXTRI) consisting of 1 h of intense cycling immediately followed by 1 h of intense running. Blood samples were taken at baseline, 3 h, 48 h, and 96 h post-EXTRI from eight healthy, endurance-trained, male subjects. RNA was extracted from isolated neutrophils. Differential gene expression was evaluated using Illumina microarrays and validated with quantitative PCR. Gene set enrichment analysis identified enriched molecular signatures chosen from the Molecular Signatures Database. Blood concentrations of muscle damage indexes, neutrophils, interleukin (IL)-6 and IL-10 were increased (P < 0.05) 3 h post-EXTRI. Upregulated groups of functionally related genes 3 h post-EXTRI included gene sets associated with the recognition of tissue damage, the IL-1 receptor, and Toll-like receptor (TLR) pathways (familywise error rate, P value < 0.05). The core enrichment for these pathways included TLRs, low-affinity immunoglobulin receptors, S100 calcium binding protein A12, and negative regulators of innate immunity, e.g., IL-1 receptor antagonist, and IL-1 receptor associated kinase-3. Plasma myoglobin changes correlated with neutrophil TLR4 gene expression (r = 0.74; P < 0.05). Neutrophils had returned to their nonactivated state 48 h post-EXTRI, indicating that their initial proinflammatory response was transient and rapidly counterregulated. This study provides novel insight into the signaling mechanisms underlying the neutrophil responses to endurance exercise, suggesting that their transcriptional activity was particularly induced by damage-associated molecule patterns, hypothetically originating from the leakage of muscle components into the circulation.

Population Dynamics of RNA viruses

Between 50 and 100 million people are infected with dengue viruses each year and more than 100,000 of these die. Dr Choudhury has demonstrated that populations of dengue viruses in individual patients are genetically and functionally very diverse and that this diversity changes significantly at the time of major outbreaks of disease. The results of his studies may inform strategies which will make dengue vaccines far more effective.

A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

Background Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation. Methods The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). Conclusions Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.

Circulating microRNAs involved in multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated, demyelinating and neurodegenerative disease of the central nervous system. After traumatic brain injury, it is the leading cause of neurology disability in young adults. Considerable advances have been made in identifying genes involved in MS but the genetic and phenotypic complexity associated with this disease significantly hinders any progress. A novel class of small RNA molecules, microRNAs (miRNAs) has acquired much attention because they regulate the expression of up to 30% of protein-coding genes and may play a pivotal role in the development of many, if not all, complex diseases. Seven published studies investigated miRNAs from peripheral blood mononuclear cells, CD4+, CD8+ T cell, B lymphocytes, peripheral blood leukocytes, whole blood and brain astrocytes with MS risk. The absence of MS studies investigating plasma miRNA prompted the current investigation of identifying a circulating miRNA signature in MS. We conducted a microarray analysis of over 900 known miRNA transcripts from plasma samples collected from four MS individuals and four sex-aged and ethnicity matched healthy controls. We identified six plasma miRNA (miR-614, miR-572, miR-648, miR-1826, miR-422a and miR-22) that were significantly up-regulated and one plasma miRNA (miR-1979) that was significantly down-regulated in MS individuals. Both miR-422a and miR-22 have previously been implicated in MS. The present study is the first to show a circulating miRNA signature involved in MS that could serve as a potential prognostic and diagnostic biomarker for MS.

Investigation of the 1758G>C and 2880A>G variants within the NCOA3 gene in a breast cancer affected Australian population

NCOA3 is a known low to moderate-risk breast cancer susceptibility gene, amplified in 5–10% and over expressed in about 60% of breast tumours. Additionally, this over expression is associated with Tamoxifen resistance and poor prognosis. Previously, two variants of NCOA3, 1758G > C and 2880A > G have been associated with breast cancer in two independent populations. Here we assessed the influence of the two NCOA3 variants on breast cancer risk by genotyping an Australian case–control study population. 172 cases and 178 controls were successfully genotyped for the 1758G > C variant and 186 cases and 182 controls were successfully genotyped for the 2880A > G variant using high-resolution melt analysis (HRM). The genotypes of the 1758G > C variant were validated by sequencing. χ2 tests were performed to determine if significant differences exist in the genotype and allele frequencies between the cases and controls. χ2 analysis returned no statistically significant difference (p > 0.05) for genotype frequencies between cases and controls for 1758G > C (χ2 = 0.97, p = 0.6158) or 2880A > G (χ2 = 2.09, p = 0.3516). Similarly, no statistical difference was observed for allele frequencies for 1758G > C (χ2 = 0.07, p = 0.7867) or 2880A > G (χ2 = 0.04, p = 0.8365). Haplotype analysis of the two SNPs also showed no difference between the cases and the controls (p = 0.9585). Our findings in an Australian Caucasian population composed of breast cancer sufferers and an age matched control population did not support the findings of previous studies demonstrating that these markers play a significant role in breast cancer susceptibility. Here, no significant difference was detected between breast cancer patients and healthy matched controls by either the genotype or allele frequencies for the investigated variants (all p ≥ 0.05). While an association of the two variants and breast cancer was not detected in our case–control study population, exploring these variants in a larger population of the same kind may obtain results in concordance with previous studies. Given the importance of NCOA3 and its involvement in biological processes involved in breast cancer and the possible implications variants of the gene could have on the response to Tamoxifen therapy, NCOA3 remains a candidate for further investigations.

Quantitative and qualitative changes in gene expression patterns characterize the activity of plaques in multiple sclerosis

Multiple sclerosis (MS) is a complex autoimmune disorder of the CNS with both genetic and environmental contributing factors. Clinical symptoms are broadly characterized by initial onset, and progressive debilitating neurological impairment. In this study, RNA from MS chronic active and MS acute lesions was extracted, and compared with patient matched normal white matter by fluorescent cDNA microarray hybridization analysis. This resulted in the identification of 139 genes that were differentially regulated in MS plaque tissue compared to normal tissue. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue<0.0001, ρ=0.73, by Spearman's ρ analysis); while 70 transcripts were uniquely differentially expressed (≥1.5-fold) in either acute or chronic active tissues. These results included known markers of MS such as the myelin basic protein (MBP) and glutathione S-transferase (GST) M1, nerve growth factors, such as nerve injury-induced protein 1 (NINJ1), X-ray and excision DNA repair factors (XRCC9 and ERCC5) and X-linked genes such as the ribosomal protein, RPS4X. Primers were then designed for seven array-selected genes, including transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), GSTP1, crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1) and tubulin β-5 (TBB5), and real time quantitative (Q)-PCR analysis was performed. The results of comparative Q-PCR analysis correlated significantly with those obtained by array analysis (r=0.75, Pvalue<0.01, by Pearson's bivariate correlation). Both chronic active and acute plaques shared the majority of factors identified suggesting that quantitative, rather than gross qualitative differences in gene expression pattern may define the progression from acute to chronic active plaques in MS.

Single-nucleotide polymorphism alleles in the insulin receptor gene are associated with typical migraine

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.

G-protein β3 subunit gene (GNB3) variant in causation of essential hypertension

Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein β3 subunit gene (GNB3) induces formation of a splice variant (Gβ3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (χ2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105±7, 109±16, and 128±28 mm Hg (mean±SD) for CC, CT, and 7T, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein β3 subunit gene in the causation of essential hypertension.