In silico interrogation of the miRNAome of infected hematopoietic cells to predict processes important for human cytomegalovirus latent infection.

Human cytomegalovirus (HCMV) latency in CD34+ progenitor cells is the outcome of a complex and continued interaction of virus and host that is initiated during very early stages of infection and reflects pro- and anti-viral activity. We hypothesized that a key event during early infection could involve changes to host miRNAs, allowing for rapid modulation of the host proteome. Here, we identify 72 significantly upregulated miRNAs and three that were downregulated by 6hpi of infection of CD34+ cells which were then subject to multiple in silico analyses to identify potential genes and pathways important for viral infection. The analyses focused on the upregulated miRNAs and were used to predict potential gene hubs or common mRNA targets of multiple miRNAs. Constitutive deletion of one target, the transcriptional regulator JDP2, resulted in a defect in latent infection of myeloid cells; interestingly, transient knockdown in differentiated dendritic cells resulted in increased viral lytic IE gene expression, arguing for subtle differences in the role of JDP2 during latency establishment and reactivation of HCMV. Finally, in silico predictions identified clusters of genes with related functions (such as calcium signaling, ubiquitination, and chromatin modification), suggesting potential importance in latency and reactivation. Consistent with this hypothesis, we demonstrate that viral IE gene expression is sensitive to calcium channel inhibition in reactivating dendritic cells. In conclusion, we demonstrate HCMV alters the miRNAome rapidly upon infection and that in silico interrogation of these changes reveals new insight into mechanisms controlling viral gene expression during HCMV latency and, intriguingly, reactivation.

Evasion of a Human Cytomegalovirus Entry Inhibitor with Potent Cysteine Reactivity Is Concomitant with the Utilization of a Heparan Sulfate Proteoglycan-Independent Route of Entry.

The dependence of viruses on the host cell to complete their replicative cycle renders cellular functions potential targets for novel antivirals. We screened a panel of broadly acting cellular ion channel inhibitors for activity against human cytomegalovirus (HCMV) and identified the voltage-gated chloride ion channel inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) as a potent inhibitor of HCMV replication. Time-of-addition studies demonstrated that DIDS inhibited entry via direct interaction with the virion that impeded binding to the plasma membrane. Synthesis and analysis of pharmacological variants of DIDS suggested that intrinsic cysteine, and not lysine, reactivity was important for activity against HCMV. Although sequencing of DIDS-resistant HCMV revealed enrichment of a mutation within UL100 (encoding glycoprotein M) and a specific truncation of glycoprotein RL13, these did not explain the DIDS resistance phenotype. Specifically, only the introduction of the RL13 mutant partially phenocopied the DIDS resistance phenotype. Serendipitously, the entry of DIDS-resistant HCMV also became independent of heparan sulfate proteoglycans (HSPGs), suggesting that evasion of DIDS lowered dependence on an initial interaction with HSPGs. Intriguingly, the DIDS-resistant virus demonstrated increased sensitivity to antibody neutralization, which mapped, in part, to the presence of the gM mutation. Taken together the data characterize the antiviral activity of a novel HCMV inhibitor that drives HCMV infection to occur independently of HSPGs and the generation of increased sensitivity to humoral immunity. The data also demonstrate that compounds with cysteine reactivity have the potential to act as antiviral compounds against HCMV via direct engagement of virions.IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen of nonimmunocompetent individuals that remains in need of new therapeutic options. Here, we identify a potent antiviral compound (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid [DIDS]), its mechanism of action, and the chemical properties required for its activity. In doing so, the data argue that cysteine-reactive compounds could have the capacity to be developed for anti-HCMV activity. Importantly, the data show that entry of DIDS-resistant virus became independent of heparan sulfate proteoglycans (HSPGs) but, concomitantly, became more sensitive to neutralizing antibody responses. This serendipitous observation suggests that retention of an interaction with HSPGs during the entry process in vivo may be evolutionarily advantageous through better evasion of humoral responses directed against HCMV virions.

Lack of correlation between predicted and actual off-target effects of short-interfering RNAs targeting the human papillomavirus type 16 E7 oncogene.

BACKGROUND: When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells. METHODS: We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo. RESULTS: We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types. CONCLUSION: These data do not support the assumption that actual OTEs correlate well with predicted OTEs.

DICER1 hotspot mutations in non-epithelial gonadal tumours.

BACKGROUND: Non-epithelial gonadal tumours largely comprise sex cord-stromal tumours (SCSTs) and germ cell tumours (GCTs). Specific somatic mutations in DICER1, a microRNA maturation pathway gene, have been identified in these tumours. We conducted a study that aimed to confirm, refine and extend the previous observations. METHODS: We used Sanger sequencing to sequence the RNase IIIa and IIIb domains of DICER1 in 154 gonadal tumours from 135 females and 19 males, as well as 43 extra-gonadal GCTs from 26 females and 17 males. RESULTS: We identified heterozygous non-synonymous mutations in the RNase IIIb domain of DICER1 in 14/197 non-epithelial tumours (7.1%). Mutations were found in 9/28 SCSTs (32%), 5/118 gonadal GCTs (4.2%), 0/43 extra-gonadal GCTs and 0/8 miscellaneous tumours. The 14 mutations affected only five residues: E1705, D1709, E1788, D1810 and E1813. In all five patients where matched and constitutional DNA was available, the mutations were only somatic. There were no mutations found in the RNase IIIa domain. CONCLUSION: More than half (8/15) of Sertoli-Leydig cell tumours (SLCTs) harbour DICER1 mutations in the RNase IIIb domain, while mutations are rarely found in GCTs. Genetic alterations in SLCTs may aid in classification and provide new approaches to therapy.

Auxological changes in UK survivors of childhood acute lymphoblastic leukaemia treated without cranial irradiation.

BACKGROUND: As most children with acute lymphoblastic leukaemia (ALL) achieve long-term survival, minimising late effects of treatment is a priority. Acute lymphoblastic leukaemia survivors treated historically with protocols including cranial irradiation demonstrate increased weight gain. METHODS: We retrospectively studied all 134 patients treated on the MRC/UKALL97 protocol (without cranial irradiation as standard therapy) at a single centre, with 77 inclusions. Height-, weight- and body mass index (BMI) standard-deviation scores (SDS) were recorded at diagnosis and annually until 3 years out (YO) from end of treatment (EoT); changes across time were explored using a univariate model (significance P ≤ 0.001 to account for multiple comparisons). RESULTS: Whole-group height SDS was lower from 1 year into treatment until 2 YO, whereas weight- and BMI-SDS remained higher until 3 YO. In females, height-SDS was lower until EoT, but higher weight- and BMI-SDS persisted until 3 YO. In males, height-SDS was lower at EoT and at 2 YO; differences in BMI-SDS had resolved by 2 YO. By WHO criteria, more patients were overweight or obese at 3 YO than at diagnosis (P=0.01). CONCLUSION: Survivors of childhood ALL, particularly females, exhibit adverse changes in height-, weight- and BMI-SDS, which arise during treatment and persist into follow-up. Patients should be supported with appropriate dietary and lifestyle advice during ALL treatment and follow-up, which may minimise these changes and reduce associated long-term morbidity.

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

BACKGROUND: Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. METHODS: A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. RESULTS: Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. CONCLUSION: Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

α-Fetoprotein.

α-Fetoprotein (AFP) measurements have clinical implications in fetal medicine and, in infants and older children, in detection, differential diagnosis and monitoring of malignant disease. Maternal serum AFP levels constitute part of a multiple-marker test used in early second-trimester screening to predict risk of fetal chromosomal abnormalities. Those individuals with increased risk are offered further definitive diagnostic investigation. Second-trimester screening is now increasingly being superseded by first-trimester screening with other serum markers and ultrasound. As AFP is only produced physiologically during fetal development, elevated serum levels after the first two post-natal years usually indicate the presence of a malignant disease process. Before this time, levels may be purely physiological and therefore serial values should be plotted on a logarithmic chart to ensure that they are falling appropriately, with a typical half-life of ∼5-6 days. If not, further investigation should be undertaken. Serum AFP is raised in a significant proportion of germ cell tumours (GCTs), hepatoblastoma and hepatocellular carcinoma (HCC). In suspected cases of GCT, serum human choriogonadotropin (HCG) estimation should also be performed. For possible intracranial GCTs, both serum and cerebrospinal fluid levels of AFP and HCG should be measured, ideally before neurosurgical biopsy. In malignant conditions, serum AFP may be used for diagnosis, treatment monitoring, surveillance for disease recurrence and prognostication. Immunohistochemistry for AFP using antibody staining is routinely used to assist pathological diagnosis on tissue sections where the differential includes GCT, hepatoblastoma and/or HCC. Elevations of serum AFP also occur in non-malignant conditions such as chronic liver disease.

Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass.

Aneuploidy has been well-documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening in a 24-h protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, parental source of aneuploidy and distinguishes certain meiotic from mitotic errors. In this study, our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from 17 couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e. one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of 51 (96.1%) ICM samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that TE karyotype is an excellent predictor of ICM karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations.

Use of the ACTH challenge test to identify the predominant glucocorticoid in the southern sea otter (Enhydra lutris nereis).

After nearly being hunted to extinction during the fur trade of the late 20th Century, sea otter (Enhydra lutris) populations have recovered to varying degrees of their historical range. While overall population numbers and range have increased, there are regions in which expansion has occurred at a slower rate and/or animal numbers have decreased, which may be a result of chronic stress from a variety of sources. Some have employed glucocorticoid analysis in their attempts to validate these explanations. Our goal was to conduct a controlled study using sea otters managed under human care to validate the use of serum glucocorticoid analysis to monitor stress physiology in the sea otter. We used a standard ACTH challenge test to compare cortisol and corticosterone responses, thereby identifying the primary glucocorticoid in the sea otter. Fourteen sea otters of both sexes (five males, nine females), including juveniles, sub-adults and adults, participated in the study. The results of the testing supported cortisol as the primary glucocorticoid in the sea otter. Sex and age did not affect how the individual responded to the ACTH or saline injection. Interestingly, the saline injection not only confirmed the effects of the ACTH on glucocorticoid release from the adrenal glands but also provided information on how long it takes the sea otter's glucocorticoid levels to return to baseline after capture and sedation. The insight gained from this study will aid in future efforts to better understand the role of stress in free-ranging sea otter populations. Recognition of the primary glucocorticoid will facilitate evaluation of more stable biological material, such as fur or whiskers, which tend to be less affected by the diurnal cycling of glucocorticoids.

Two classes of PDGF receptor recognize different isoforms of PDGF.

Previous studies involving platelet-derived growth factor (PDGF) have been based on the premise that a single cell-surface receptor binds all three isoforms of PDGF (AA, BB, and AB). It is now shown that two populations of PDGF receptor exist and can be distinguished by their ligand binding specificity. The B receptor binds only the BB dimer, whereas the A/B receptor binds AA, BB, and AB dimers. Human dermal fibroblasts appear to express seven times as much B receptor as A/B receptor. The B receptor is responsible for most PDGF receptor phosphorylation.

Mediastinal masses masquerading as common respiratory conditions of childhood: a case series.

INTRODUCTION: Leukaemia and lymphoma may present with symptoms and signs mimicking common respiratory conditions of childhood such as asthma or croup. The UK National Institute for Clinical Excellence guidelines for referral for suspected cancer state that "the primary healthcare professional should be ready to review the initial diagnosis in patients in whom common symptoms do not resolve as expected" and "must be alert to the possibility of cancer when confronted by unusual symptom patterns" (National Institute for Health and Clinical Excellence, 2005). RESULTS AND DISCUSSION: A child with an undiagnosed mediastinal mass presenting with signs and symptoms suggestive of asthma or croup may be given oral systemic steroids. We report four such illustrative cases presenting to a single institution within the last 3 years. CONCLUSION: We highlight key points from the history and examination findings which should lead to review of the original diagnosis, the benefit of early chest X-ray in such cases and the dangers of steroid pretreatment.

Platelet isoforms of platelet-derived growth factor stimulate fibroblasts to contract collagen matrices.

Fibroplasia and angiogenesis are essential components of tissue repair when substantial tissue has been lost at a site of injury. Platelets and monocyte/macrophages accumulate at these sites and release a variety of growth factors that are thought to initiate and sustain the repair. Often the involved tissue contracts, a process that can markedly reduce the amount of fibroplasia and angiogenesis necessary for the reestablishment of organ integrity. Such tissue contraction occurs over hours or days, a much slower time course than the rapid, reversible contraction of muscle tissue. Fibroblasts, which are rich in f-actin bundles, appear to be responsible for wound contraction. However, the signals that stimulate contraction are not known. Using cultured fibroblasts, which are also rich in f-actin bundles, we demonstrate the platelet and monocyte isoforms of platelet-derived growth factor (PDGF; AB and BB) but not PDGF-AA, can stimulate fibroblasts to contract collagen matrix in a time course similar to that of wound contraction. In addition, PDGF appears to be the predominant fibroblast/collagen gel contraction activity released from platelets. Vasoactive agonists known to stimulate smooth and striated muscle contraction do not stimulate fibroblast-driven collagen gel contraction.

Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera.

The transcriptional promoter of the Harvey sarcoma virus long terminal repeat has been used to construct a biologically active dihydrofolate reductase chimera. The construction placed the long terminal repeat at the 5' end of a dihydrofolate reductase cDNA. This chimera mediated methotrexate resistance when introduced into wild-type NIH3T3 mouse cells by transfection. The chimeric sequences were expressed in the form of polyadenylated RNA and dihydrofolate reductase protein and were amplified when the methotrexate-resistant transfectants were selected to grow in increasing methotrexate concentrations. This chimera was dominant acting and able to confer a methotrexate-resistant phenotype on wild-type NIH3T3 cells. It has been used in cotransfection experiments with DNA from human tumor cells to obtain foci of methotrexate-resistant transformed NIH3T3 cells resulting from uptake of exogenous DNA. The transfected methotrexate-resistant cells carried double minute chromosomes that appeared to contain DNA acquired during transfection.

Effects of roundabout on growth cone dynamics, filopodial length, and growth cone morphology at the midline and throughout the neuropile.

roundabout (robo) encodes an axon guidance receptor that controls midline crossing in the Drosophila CNS. In robo mutants, axons that normally project ipsilaterally can cross and recross the midline. Growth cones expressing Robo are believed to be repelled from the midline by the interaction of Robo and its ligand Slit, an extracellular protein expressed by the midline glia. To help understand the cellular basis for the midline repulsion mediated by Robo, we used time-lapse observations to compare the growth cone behavior of the ipsilaterally projecting motorneuron RP2 in robo and wild-type embyros. In wild-type embryos, filopodia can project across the midline but are quickly retracted. In robo mutants, medial filopodia can remain extended for longer periods and can develop into contralateral branches. In many cases RP2 produces both ipsilateral and contralateral branches, both of which can extend into the periphery. The growth cone also exhibits longer filopodia and more extensive branching both at the midline and throughout the neuropile. Cell injections in fixed stage 13 embryos confirmed and quantified these results for both RP2 and the interneuron pCC. The results suggest that Robo both repels growth cones at the midline and inhibits branching throughout the neuropile by promoting filopodial retraction.

Metal-polynucleotide interactions. A comparison of carcinogenic and non-carcinogenic metals in vitro.

The effects of divalent cations on mixing curves of synthetic polyribonucleotides in solution are described. Manganese and cadmium chlorides at 10(-3) M induce changes suggestive of base mispairing. MnCl2 induces mispairing in complexes formed between both poly(I) and poly(C,U) and poly(I) and poly(C,A) while CdCl2 affects base pairing between poly(I) and poly(C,U) only. By contrast, the chlorides of magnesium and zinc show no mispairing effects with either polymer pair. Manganese and cadmium are both reported carcinogens in animals while magnesium and zinc are not. The possibility that direct metal-nucleic acid interaction may be involved in metal carcinogenesis is discussed.

Statistical processing of large image sequences.

The dynamic estimation of large-scale stochastic image sequences, as frequently encountered in remote sensing, is important in a variety of scientific applications. However, the size of such images makes conventional dynamic estimation methods, for example, the Kalman and related filters, impractical. In this paper, we present an approach that emulates the Kalman filter, but with considerably reduced computational and storage requirements. Our approach is illustrated in the context of a 512 x 512 image sequence of ocean surface temperature. The static estimation step, the primary contribution here, uses a mixture of stationary models to accurately mimic the effect of a nonstationary prior, simplifying both computational complexity and modeling. Our approach provides an efficient, stable, positive-definite model which is consistent with the given correlation structure. Thus, the methods of this paper may find application in modeling and single-frame estimation.

Biology of childhood germ cell tumours, focussing on the significance of microRNAs.

Genomic and protein-coding transcriptomic data have suggested that germ cell tumours (GCTs) of childhood are biologically distinct from those of adulthood. Global messenger RNA profiles segregate malignant GCTs primarily by histology, but then also by age, with numerous transcripts showing age-related differential expression. Such differences are likely to account for the heterogeneous clinico-pathological behaviour of paediatric and adult malignant GCTs. In contrast, as global microRNA signatures of human tumours reflect their developmental lineage, we hypothesized that microRNA profiles would identify common biological abnormalities in all malignant GCTs owing to their presumed shared origin from primordial germ cells. MicroRNAs are short, non-protein-coding RNAs that regulate gene expression via translational repression and/or mRNA degradation. We showed that all malignant GCTs over-express the miR-371-373 and miR-302/367 clusters, regardless of patient age, histological subtype or anatomical tumour site. Furthermore, bioinformatic approaches and subsequent Gene Ontology analysis revealed that these two over-expressed microRNAs clusters co-ordinately down-regulated genes involved in biologically significant pathways in malignant GCTs. The translational potential of this finding has been demonstrated with the detection of elevated serum levels of miR-371-373 and miR-302/367 microRNAs at the time of malignant GCT diagnosis, with levels falling after treatment. The tumour-suppressor let-7 microRNA family has also been shown to be universally down-regulated in malignant GCTs, because of abundant expression of the regulatory gene LIN28. Low let-7 levels resulted in up-regulation of oncogenes including MYCN, AURKB and LIN28 itself, the latter through a direct feedback mechanism. Targeting LIN28, or restoring let-7 levels, both led to effective inhibition of this pathway. In summary, paediatric malignant GCTs show biological differences from their adult counterparts at a genomic and protein-coding transcriptome level, whereas they both display very similar microRNA expression profiles. These similarities and differences may be exploited for diagnostic and/or therapeutic purposes.

Dysregulated expression of ebaf, a novel molecular defect in the endometria of patients with infertility.

We recently described the expression of ebaf, a novel member of the transforming growth factor-beta superfamily in human endometrium. ebaf messenger ribonucleic acid was expressed in late secretory and menstrual endometria. Here, we show that ebaf is secreted as 42-, 34-, 28-, and 14-kDa proteins into the conditioned medium of transfected cells, endometrial fluid, and serum. The amount of secreted proteins was markedly reduced during the implantation window in the endometria and sera of normal fertile subjects. The expression of ebaf was dysregulated in the endometria of a subset of women with infertility during the receptive phase of the menstrual cycle. Abundant secreted protein was present in the endometria of these women during the implantation window. During the critical period of endometrial receptivity, ebaf protein was more abundant in patients with endometriosis who did not conceive than in patients who became pregnant. These findings show that ebaf is a secreted product and is released into body fluids. Some types of infertility are associated with dysregulated expression of ebaf in human endometrium, suggesting that a molecular defect in uterine receptivity may be identified using such a marker protein.

A prospective, randomized study of endometrial telomerase during the menstrual cycle.

The purpose of this study was to characterize telomerase activity during the menstrual cycle, focusing on the luteal phase. A total of 84 endometrial biopsy samples were obtained from 72 participants. Daily urinary LH testing (OvuQuick, Quidel) was used to establish the day of the LH rise, and participants were randomized to return during the secretory phase. Twelve women returned on the identical day during the luteal phase of a subsequent cycle to allow intercycle comparisons of telomerase activity. Telomerase activity was evaluated using a modified TRAP-eze (Intergen) detection protocol. At the time of each endometrial biopsy, serum estrogen and progesterone were measured. Proliferative phase endometrium showed high telomerase activity. At the onset of the luteal phase telomerase activity was high, but it decreased during the early luteal phase, disappeared by the midluteal phase (6 d after LH surge detected), and then rose to moderate levels in the late luteal phase beginning on luteal d 10. Serum progesterone levels were inversely related to telomerase activity. In conclusion, endometrial telomerase activity is dynamic: high during the proliferative phase but inhibited during the midsecretory phase of the menstrual cycle. The timing of expression coincides with the rise and fall of progesterone levels and the time period of maximal uterine receptivity for embryo implantation. This supports a relationship between sex steroid levels and telomerase regulation.