Early mRNA Research Articles

Myocyte TLR4 enhances enteric and systemic inflammation driving late murine endotoxic ileus.

Myocytes are nonhemopoietic in origin and functionally essential in generating gastrointestinal motility. In endotoxemia, a rapid-onset nonhemopoietic mechanism potently triggers early ileus in a Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88)-dependent manner. Moreover, synergistically with hemopoietic cells, nonhemopoietic cells escalate late ileus via an IL-6 receptor-dependent inflammation-driven pathway. We therefore specifically investigated the role of myocytes in TLR4-triggered inflammation and ileus. TLR4(+/+), TLR4(-/-), bmTLR4(+/+)/TLR4(-/-) chimera, SM22-Cre(-/-)TLR4(flox/flox), and selective myocyte TLR4-deficient (SM22-Cre(+/-)TLR4(flox/flox)) mice were injected intraperitoneally with purified lipopolysaccharide. SM22-driven Cre recombinase activity was selectively detected in cardiac, gastrointestinal, skeletal, and vascular myocytes, of small-sized vessels in a two-color fluorescent Cre reporter mouse. In contrast to nonhemopoietic TLR4 deficiency, deletion of myocyte TLR4 signaling prevented neither endotoxin-induced suppression of spontaneous jejunal contractility in vitro nor early ileus in vivo at 6 h. Circulating plasma colony-stimulating factor 3 was greatly elevated during endotoxemia, independent of myocyte TLR4 signaling or time. TLR4 activation of myocytes contributed significantly to an early enteric IL-6 mRNA induction and systemic IL-6 release, as well as to a late increase in circulating chemokine (C-X-C motif) ligand 1 (CXCL1) and IL-17. Consequently, inhibition of myocyte TLR4 signaling allowed functional recovery of motility by preventing inflammation-driven late ileus at 24 h. Direct TLR4 activation of myocytes is not responsible for nonhemopoietic-mediated early ileus. However, myocytes are proinflammatory cells that potently drive enteric and systemic inflammation, subsequently fueling late mediator-triggered ileus. Specifically, the myocyte TLR4-dependent inflammatory signature of elevated plasma IL-6, CXCL1, and IL-17 is strongly associated with late rodent ileus.

Toxicogenomics profiling of bone marrow from rats treated with topotecan in combination with oxaliplatin: a mechanistic strategy to inform combination toxicity.

Combinations of anticancer agents may have synergistic anti-tumor effects, but enhanced hematological toxicity often limit their clinical use. We examined whether “microarray profiles” could be used to compare early molecular responses following a single dose of agents administered individually with that of the agents administered in a combination. We compared the mRNA responses within bone marrow of Sprague-Dawley rats after a single 30 min treatment with topotecan at 4.7 mg/kg or oxaliplatin at 15 mg/kg alone to that of sequentially administered combination therapy or vehicle control for 1, 6, and 24 h. We also examined the histopathology of the bone marrow following all treatments. Drug-related histopathological lesions were limited to bone marrow hypocellularity for animals dosed with either agent alone or in combination. Lesions had an earlier onset and higher incidence for animals given topotecan alone or in combination with oxaliplatin. Severity increased from mild to moderate when topotecan was administered prior to oxaliplatin compared with administering oxaliplatin first. Notably, six patterns of co-expressed genes were detected at the 1 h time point that indicate regulatory expression of genes that are dependent on the order of the administration. These results suggest alterations in histone biology, chromatin remodeling, DNA repair, bone regeneration, and respiratory and oxidative phosphorylation are among the prominent pathways modulated in bone marrow from animals treated with an oxaliplatin/topotecan combination. These data also demonstrate the potential for early mRNA patterns derived from target organs of toxicity to inform toxicological risk and molecular mechanisms for agents given in combination.

Pressure-limited sustained inflation vs. gradual tidal inflations for resuscitation in preterm lambs.

Support of the mechanically complex preterm lung needs to facilitate aeration while avoiding ventilation heterogeneities: whether to achieve this gradually or quickly remains unclear. We compared the effect of gradual vs. constant tidal inflations and a pressure-limited sustained inflation (SI) at birth on gas exchange, lung mechanics, gravity-dependent lung volume distribution, and lung injury in 131-day gestation preterm lambs. Lambs were resuscitated with either 1) a 20-s, 40-cmH2O pressure-limited SI (PressSI), 2) a gradual increase in tidal volume (Vt) over 5-min from 3 ml/kg to 7 ml/kg (IncrVt), or 3) 7 ml/kg Vt from birth. All lambs were subsequently ventilated for 15 min with 7 ml/kg Vt with the same end-expiratory pressure. Lung mechanics, gas exchange and spatial distribution of end-expiratory volume (EEV), and tidal ventilation (electrical impedance tomography) were recorded regularly. At 15 min, early mRNA tissue markers of lung injury were assessed. The IncrVt group resulted in greater tissue hysteresivity at 5 min (P = 0.017; two-way ANOVA), higher alveolar-arterial oxygen difference from 10 min (P < 0.01), and least uniform gravity-dependent distribution of EEV. There were no other differences in lung mechanics between groups, and the PressSI and 7 ml/kg Vt groups behaved similarly throughout. EEV was more uniformly distributed, but Vt least so, in the PressSI group. There were no differences in mRNA markers of lung injury. A gradual increase in Vt from birth resulted in less recruitment of the gravity-dependent lung with worse oxygenation. There was no benefit of a SI at birth over mechanical ventilation with 7 ml/kg Vt.

The mechanisms of genome-wide target gene regulation by TCF7L2 in liver cells

In the liver Wnt-signaling contributes to the metabolic fate of hepatocytes, but the precise role of the TCF7L2 in this process is unknown. We employed a temporal RNA-Seq approach to examine gene expression 3–96 h following Tcf7l2 silencing in rat hepatoma cells, and combined this with ChIP-Seq to investigate mechanisms of target gene regulation by TCF7L2. Silencing Tcf7l2 led to a time-dependent appearance of 406 differentially expressed genes (DEGs), including key regulators of cellular growth and differentiation, and amino acid, lipid and glucose metabolism. Direct regulation of 149 DEGs was suggested by strong proximal TCF7L2 binding (peak proximity score > 10) and early mRNA expression changes (≤18 h). Indirect gene regulation by TCF7L2 likely occurred via alternate transcription factors, including Hnf4a, Foxo1, Cited2, Myc and Lef1, which were differentially expressed following Tcf7l2 knock-down. Tcf7l2-silencing enhanced the expression and chromatin occupancy of HNF4α, and co-siRNA experiments revealed that HNF4α was required for the regulation of a subset of metabolic genes by TCF7L2, particularly those involved in lipid and amino-acid metabolism. Our findings suggest TCF7L2 is an important regulator of the hepatic phenotype, and highlight novel mechanisms of gene regulation by TCF7L2 that involve interplay between multiple hepatic transcriptional pathways.

Abstract 4529: The PI3Kδ inhibitor, idelalisib, inhibits transcription and translation through PI3K/Akt pathway in mantle cell lymphoma

Abstract PI3Kδ is the major PI3K family class I isoform expressed in B-cells that is known to promote cancer cell growth and survival. PI3Kδ pathway is activated through cell surface receptors, e.g. B-cell receptor signaling. This suggests that PI3Kδ is a therapeutic target for B-cell hematological malignancies. Idelalisib is a potent and selective PI3Kδ inhibitor (IC50=19nM) that is currently under investigation in Phase II/III trials in B-cell neoplasms. In solid tumors, it has been established that PI3K/Akt axis regulates transcription and translation processes. Based on the overexpression and hyperactivity of PI3Kδ in B-cell lymphoma such as mantle cell lymphoma (MCL), and PI3Kδ-specific inhibition by idelalisib, we hypothesized that idelalisib treatment will 1) impact the transcription and translation processes that are PI3K/Akt-dependent, 2) disrupt RNA and protein synthetic capacity, and 3) decrease short-lived oncogenic mRNA and proteins levels that are important for MCL cell survival. Idelalisib (0.5 to 5μM) induced modest levels of apoptosis (&amp;lt;15%) in MCL cell lines and primary cells. A reverse-phase protein array (RPPA) study suggested that idelalisib treatment resulted in a decrease in phosphorylation of transcription regulators (GSK-3αβ and c-Myc), and protein synthesis regulators (S6, p70S6K, 4E-BP1, eIF4 and mTOR) in MCL cell lines and primary cells. Immunoblot analysis suggests that a 4hr treatment with idelalisib (0.5 or 1μM) resulted in a decrease of phosphorylated Akt (T308 and S473) levels in both JeKo-1 and Mino cells following IgM stimulation. In parallel, phospho-levels of GSK-3β (S9), 4E-BP1 (S65) and S6 (S235/236) were also decreased. In IgM-stimulated MCL cell lines, moderate to significant decrease in global RNA synthesis (10-15% decrease in JeKo-1, 50% decrease in Mino) and protein synthesis (15% decrease in JeKo-1, &amp;gt;20% decrease in Mino) were detected with 72hr incubation of idelalisib (0.5 to 1μM). Consistent with cell line data, in B-cell lymphoma primary cells, transcription levels were decreased by 20% or &amp;gt;50% following a 24hr treatment with 1 or 5µM idelalisib, respectively; and a 40% decrease of translation level was detected with 5µM idelalisib. In Jeko-1, early response genes MCL1 mRNA level was reduced following 24hr idelalisib treatment, whereas the other mRNAs (PIM2, MYC, CCND1) were decreased to a lesser extent. Mcl-1 and cyclin D1 protein levels were decreased in JeKo-1 and Mino cells following short incubation of idelalisib (0.5 or 1µM). In summary, idelalisib was effective in reducing phosphorylation of Akt and its downstream effectors in MCL cell lines (GSK-3β, 4E-BP1 and S6). Idelalisib inhibits global RNA and protein synthesis, and decreases the levels of short-lived survival proteins. Collectively, these data imply that similar to solid tumors, PI3K regulates key factors involved in gene transcription and protein translation in MCL. Citation Format: Qingshan Yang, Lisa S. Chen, Sattva S. Neelapu, Varsha Gandhi. The PI3Kδ inhibitor, idelalisib, inhibits transcription and translation through PI3K/Akt pathway in mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4529. doi:10.1158/1538-7445.AM2014-4529

Complexin-1 and Foxp1 Expression Changes Are Novel Brain Effects of Alpha-Synuclein Pathology.

As the second most frequent neurodegenerative disorder of the aging population, Parkinson’s disease (PD) is characterized by progressive deficits in spontaneous movement, atrophy of dopaminergic midbrain neurons and aggregation of the protein alpha-synuclein (SNCA). To elucidate molecular events before irreversible cell death, we studied synucleinopathy-induced expression changes in mouse brain and identified 49 midbrain/brainstem-specific transcriptional dysregulations. In particular complexin-1 (Cplx1), Rabl2a and 14-3-3epsilon (Ywhae) downregulation, as well as upregulation of the midbrain-specific factor forkhead box P1 (Foxp1) and of Rabgef1, were interesting as early mRNA level effects of alpha-synuclein triggered pathology. The protein levels of complexin-1 were elevated in midbrain/brainstem tissue of mice with A53T-SNCA overexpression and of mice with SNCA-knockout. The response of CPLX1 and Foxp1 levels to SNCA deficiency supports the notion that these factors are regulated by altered physiological function of alpha-synuclein. Thus, their analysis might be useful in PD stages before the advent of Lewy pathology. Because both alpha-synuclein and complexin-1 modulate vesicle release, our findings support presynaptic dysfunction as an early event in PD pathology.

Oral and vaginal epithelial cell lines bind and transfer cell-free infectious HIV-1 to permissive cells but are not productively infected.

The majority of HIV-1 infections worldwide are acquired via mucosal surfaces. However, unlike the vaginal mucosa, the issue of whether the oral mucosa can act as a portal of entry for HIV-1 infection remains controversial. To address potential differences with regard to the fate of HIV-1 after exposure to oral and vaginal epithelium, we utilized two epithelial cell lines representative of buccal (TR146) and pharyngeal (FaDu) sites of the oral cavity and compared them with a cell line derived from vaginal epithelium (A431) in order to determine (i) HIV-1 receptor gene and protein expression, (ii) whether HIV-1 genome integration into epithelial cells occurs, (iii) whether productive viral infection ensues, and (iv) whether infectious virus can be transferred to permissive cells. Using flow cytometry to measure captured virus by HIV-1 gp120 protein detection and western blot to detect HIV-1 p24 gag protein, we demonstrate that buccal, pharyngeal and vaginal epithelial cells capture CXCR4- and CCR5-utilising virus, probably via non-canonical receptors. Both oral and vaginal epithelial cells are able to transfer infectious virus to permissive cells either directly through cell-cell attachment or via transcytosis of HIV-1 across epithelial cells. However, HIV-1 integration, as measured by real-time PCR and presence of early gene mRNA transcripts and de novo protein production were not detected in either epithelial cell type. Importantly, both oral and vaginal epithelial cells were able to support integration and productive infection if HIV-1 entered via the endocytic pathway driven by VSV-G. Our data demonstrate that under normal conditions productive HIV-1 infection of epithelial cells leading to progeny virion production is unlikely, but that epithelial cells can act as mediators of systemic viral dissemination through attachment and transfer of HIV-1 to permissive cells.

Circadian Perinatal Photoperiod Has Enduring Effects on Retinal Dopamine and Visual Function

Visual system development depends on neural activity, driven by intrinsic and light-sensitive mechanisms. Here, we examined the effects on retinal function due to exposure to summer- and winter-like circadian light cycles during development and adulthood. Retinal light responses, visual behaviors, dopamine content, retinal morphology, and gene expression were assessed in mice reared in seasonal photoperiods consisting of light/dark cycles of 8:16, 16:8, and 12:12 h, respectively. Mice exposed to short, winter-like, light cycles showed enduring deficits in photopic retinal light responses and visual contrast sensitivity, but only transient changes were observed for scotopic measures. Dopamine levels were significantly lower in short photoperiod mice, and dopaminergic agonist treatment rescued the photopic light response deficits. Tyrosine hydroxylase and Early Growth Response factor-1 mRNA expression were reduced in short photoperiod retinas. Therefore, seasonal light cycles experienced during retinal development and maturation have lasting influence on retinal and visual function, likely through developmental programming of retinal dopamine.

Nerve growth factor expression and nerve regeneration in monkey corneas after LASIK.

To determine whether changes in cornea nerve growth factor (NGF) protein and gene expression correlate with corneal nerve regeneration after LASIK in rhesus monkeys. Ten rhesus monkeys underwent randomly assigned LASIK procedures to correct -8.0 diopters of myopia in both eyes and two additional monkeys (with no surgery) served as controls. The central corneas of the experimental animals were excised 3 and 7 days and 1, 3, and 6 months after the surgery. Nerve regeneration, NGF mRNA, and protein expression were analyzed by gold chloride staining, real-time polymerase chain reaction, and enzyme-linked immune sorbent assay analysis, respectively. Compared to controls, the LASIK-treated animals had a significantly diminished nerve plexus in the sub-basal region of the cornea at all times after the surgery (P < .001). NGF protein levels decreased significantly on days 3 and 7 after LASIK (P < .001), but returned to control levels 1 month later. NGF mRNA levels increased 5.4-fold on day 3 after the surgery (P < .001), then reduced to two-fold (P < .05) above control levels on day 7 and were back to normal at 3 months and beyond. After LASIK, the changes of early NGF protein and NGF mRNA levels correlated with the density of the corneal nerve plexuses. The results showed that the LASIK procedure in non-human primates was associated with changes in NGF protein and mRNA levels in the cornea. Such changes may be related to the initiation of nerve regeneration and the final recovery of nerve plexuses in the cornea.

Transcription in Pronuclei and One- to Four-Cell Embryos Drives Early Development in a Nematode

A long-standing view of development is that transcription is silenced in the oocyte until early divisions in the embryo. The point at which major transcription is reactivated varies between organisms, but is usually after the two-cell stage. However, this model may not be universal. We used RNA-seq and exploited the protracted development of the parasitic nematode Ascaris suum to provide a comprehensive time course of mRNA expression, degradation, and translation during early development. Surprisingly, we find that ∼4,000 genes are transcribed prior to pronuclear fusion and in the one- to four-cell embryos. Intriguingly, we do not detect maternal contribution of many orthologs of maternal C. elegans mRNAs, but instead find that these are newly transcribed in the A. suum zygote prior to pronuclear fusion. Ribosome profiling demonstrates that, in general, early embryonic mRNAs are not stored for subsequent translation, but are directly translated after their synthesis. The role of maternally contributed and zygotically transcribed genes differs between the nematodes A. suum and C. elegans despite the fact that the two nematodes appear to exhibit highly similar morphological patterns during early development. Our study indicates that major transcription can occur immediately after fertilization and prior to pronuclear fusion in metazoa, suggesting that newly transcribed genes appear to drive A. suum early development. Furthermore, the mechanisms used for controlling the timing of the expression of key conserved genes has been altered between the two nematodes, illustrating significant plasticity in the regulatory networks that play important roles in developmental outcomes in nematodes.

Effects of curcumin on changes of Egr-1mRNA, IL-1β and TNF-α in paraquat-induced acute lung injury in rats

Objective To observe the changes of early growth response factor-1 mRNA (Egr-1 mRNA),interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in rat with paraquat (PQ)-induced acute lung injury,and the potential protective effect of curcumin on this injury.Methods Adult male Sprague-Dauley (SD) rats were randomly (random number) divided into control,PQ-poisoning and curcumin-intervention groups.At 3 h,6 h,24 h,3 d,7 d after treatment,each group respectively further divided into 5 subgroups.Rats in PQ-poisoning group were intraperitoneally (IP) administered with PQ 15 mg/kg,and rats in cureumin-intervention group were administered with PQ 15 mg/kg IP at 15 min after curcumin 200 mg/kg IP.Rats in control group were given normal saline (NS) 1 mL IP instead.The expression of Egr-1 mRNA in lungs tissue were measured with Fluorescence real-time quantitative PCR device.The levels of IL-1β and TNF-α in bronchoalveolar lavage fluid (BALF) were measured with enzyme-hnked immunosorbent assay (ELISA).Pathological changes in lung were observed under light microscope.Experimental data were treated with single-factor analysis of variance and compared between groups with Bonferroni method.Results Compared with the control group,Egr-1 mRNA expression in lungs tissue and TNF-α level in BALF in PQ-poisoning group and curcumin-intervention group,IL-1β level in BALF in PQ-poisoning group increased significantly at 6 h,24 h,3 d,7 d after PQ exposure (P ＜ 0.05 or P ＜ 0.01),and IL-1β level in BALF increased significantly at 24 h after PQ exposure in curcuminintervention group (P ＜ 0.05).Compared with the PQ-poisoning group,Egr-1 mRNA expression in lungs tissue,TNF-α and IL-1 β level in BALF decreased significantly at 24 h,3 d,7 d after PQ exposure in curcumin-intervention group (P ＜ 0.05 or P ＜ 0.01).Pathological changes of lung tissue were more serious in PQ-poisoning group than those in curcumin-intervention group.Conclusions The up-regulated expression of Egr-1 mRNA increased releasing ofIL-1β and TNF-α,and initiating inflammation may be one of the mechanism of PQ-induced acute lung injury in rats.Curcumin may have beneficial protective effects on lung in this above described pathway of signaling. Key words: Paraquat ; Early growth response factor-1 mRNA ; Interleukin-1β ; Tumor necrosis factor-α; Lung injury; Curcumin; Poison; Rat

Effect of sustained inflation vs. stepwise PEEP strategy at birth on gas exchange and lung mechanics in preterm lambs

Sustained inflation (SI) at birth facilitates establishment of functional residual capacity (FRC) in the preterm lung, but the ideal lung recruitment strategy is unclear. We have compared the effect of SI and a stepwise positive end-expiratory pressure (PEEP; SEP) strategy in a preterm model. 127 d gestation lambs received either 20-s SI (n = 9) or 2 cmH2O stepwise PEEP increases to 20 cmH2O every 10 inflations, and then decreases to 6 cmH2O (n = 10). Ventilation continued for 70 min, with surfactant administered at 10 min. Alveolar-arterial oxygen gradient (AaDO2), compliance (C(dyn)), end-expiratory thoracic volume (EEVRIP; respiratory inductive plethysmography), and EEV and C(dyn) in the gravity-dependent and nondependent hemithoraces (electrical impedance tomography) were measured throughout. Early mRNA markers of lung injury were analyzed using quantitative real-time PCR. From 15 min of life, AaDO2 was lower in SEP group (P < 0.005; two-way ANOVA). SEP resulted in higher and more homogeneous C(dyn) (P < 0.0001). Mean (SD) EEVRIP at 5 min was 18 (9) ml/kg and 6 (5) ml/kg following SEP and SI, respectively (P = 0.021; Bonferroni posttest); this difference was due to a greater nondependent hemithorax EEV. There was no difference in markers of lung injury. An SEP at birth improved gas exchange, lung mechanics, and EEV, without increasing lung injury, compared to the SI strategy used.

A Cyclopropane-Derived Stable Analog Of Fd-895 Induces Apoptosis and Inhibition Of mRNA Splicing In Lymphoma and Chronic Lymphocytic Leukemia: A Novel Therapeutic Approach

Abnormalities in RNA splicing have been implicated in tumorigenesis and cancer progression. Recent studies have defined RNA splicing as in attractive target for development of effective anticancer therapy. However, significant hurdles need to be solved before spliceosome inhibitors are ready for clinical trial applications. The work presented here addresses a major obstacle, specifically the design of active and metabolically stable spliceosome / RNA splicing inhibitors.We previously reported that FD-895 and Pladienolide B (PLAD-B) induce apoptosis viathe inhibition of RNA splicing in chronic lymphocytic leukemia (Kashyap et al, Blood 2012; 120: 3890). However, FD-895 and PLAD-B hydrolyze relatively rapidly in aqueous solution. To address this issue, we applied semi-synthesis and total synthesis to reveal key structure activity relationships (SARs) regulating the RNA splicing inhibition of FD-895. This information was applied to synthesize a panel of cyclopropane analogs (CypB) that showed improved biochemical stability and potent pro-apoptotic activity in a panel of lymphoma cell lines and primary lymphoma - leukemia cells from patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL).A series of cyclopropane analogs and FD-895 were tested for hydrolysis and stability using a combination of NMR and mass spectral methods. Based on stability, we selected a first generation cyclopropane analog, CypB, to conduct our experiments. Primary leukemia - lymphoma cells as well as cell lines were incubated with increasing concentrations of FD-895, and CypB over a gradient of 10-1,000 nM. We observed rapid development of intron retention within 15 min after incubation at concentrations ranging from 10-100 nM using a series of spliceosome related genes (DNAJB1, RIOK3, and BRD2). Contrary to that, intron retention was not observed after treatment with chemotherapy controls (fludarabine and etoposide). By using qRT-PCR, we were able to quantify unspliced mRNA forms induced by FD-895, and CypB but not by chemotherapy agents.In addition, we observed a close correlation between the level of intron retention and development of apoptosis induced by FD-895 and CypB in all cell lines and primary leukemia - lymphoma samples (IC50 range: 3-1,000 nM). Apoptosis induced by these compounds was also observed in chemotherapy resistant patient samples independently of TP53 function, deletion or mutational status.To understand the molecular mechanism of FD-895 and CypB induced apoptosis, we analyzed the expression profile of alternative spliced forms of genes related to apoptosis (mcl-1 and Bcl-X) by RT-PCR. Interestingly, we observed that FD-895, and CypB induced the expression of small alternative spliced forms that are known to be pro-apoptotic. This was not observed after incubation with control chemotherapy agents.In conclusion, using a SAR strategy we were able to optimize novel FD-895 based cyclopropane analogs. These compounds induce early mRNA intron retention in different lymphoma cell lines and primary lymphoma - leukemia samples derived from patients. Furthermore, these analogs induced apoptosis in a TP53 independent manner and induce the expression of pro-apoptotic alternatively spliced RNA genes. Our studies showed that combining medicinal chemistry and cancer biology, we were able to discover labile functional epoxide groups present in FD-895 that can be removed enhancing its stability and preserving the pro-apoptotic activity of this compound. This synthesis and optimization strategy provides new avenues for analog design in an effort to develop spliceosome inhibitors that can be used for cancer treatment. Current efforts are underway to complete these translational studies. Disclosures:Kipps:Bristol-Myers Squibb: Research Funding.

Differential Role for Host Translation Factors in Host and Viral Protein Synthesis during Human Cytomegalovirus Infection

The host eIF4F translation initiation complex plays a critical role the translation of capped mRNAs. Although human cytomegalovirus (HCMV) infection increases the abundance and activity of the host eIF4F complex, the requirement for eIF4F components in HCMV replication and mRNA translation has not been directly tested. In this study, we found that decreasing the abundance or activity of eIF4F from the start of infection inhibits HCMV replication. However, as infection progresses, viral mRNA translation and replication becomes increasingly resistant to eIF4F inhibition. During the late stage of infection the association of representative immediate-early, early, and late mRNAs with polysomes was not affected by eIF4F disruption. In contrast, eIF4F inhibition decreased the translation of representative host eIF4F-dependent mRNAs during the late stage of infection. A global analysis of the translation efficiency of HCMV mRNAs during the late stage of infection found that eIF4F disruption had a minimal impact on the association of HCMV mRNAs with polysomes but significantly diminished the translation efficiency of eIF4F-dependent host transcripts. Together, our data show that the translation of host eIF4F-dependent mRNAs remains dependent on eIF4F activity during HCMV infection. However, during the late stage of infection the translation efficiency of viral mRNAs does not correlate with the abundance or activity of the host eIF4F complex.

Eight Nucleotide Substitutions Inhibit Splicing to HPV-16 3′-Splice Site SA3358 and Reduce the Efficiency by which HPV-16 Increases the Life Span of Primary Human Keratinocytes

The most commonly used 3′-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16.

Human embryonic poly(A)-binding protein (EPAB) alternative splicing is differentially regulated in human oocytes and embryos

Oocyte maturation is associated with suppression of transcriptional activity. Consequently, gene expression during oocyte maturation, fertilization and early embryo development, until zygotic genome activation (ZGA) is primarily regulated by translational activation of maternally derived mRNAs. Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse and human oocytes and early embryos prior to ZGA. EPAB plays a key role in polyadenylation-dependent translational activation of mRNAs by stabilizing polyadenylated mRNAs and by stimulating their translation. Epab-knockout female mice are sterile, fail to generate mature oocytes and display impaired cumulus expansion and ovulation. Consistent with its role during gametogenesis and early embryo development, Xenopus and mouse Epab mRNA is expressed exclusively in oocytes and early embryos, and is undetectable following ZGA or in somatic tissues. Herein, we demonstrate that although EPAB is expressed in human somatic tissues, its transcripts largely consist of an alternatively spliced form lacking the first 58 bp of exon 8, which leads to the formation of a premature stop codon 6 amino acids downstream on exon 8, and omission of the functionally critical poly(A)-binding domain. Moreover, 8-cell and blastocyst stage human embryos also express only the alternatively spliced form of EPAB. On the other hand, the full-length form of EPAB mRNA is exclusively expressed in oocytes. In conclusion, in contrast with the transcriptional regulation in Xenopus and mouse, oocyte- and early embryo-specific expression of EPAB in human is regulated by a post-transcriptional mechanism.

Detection of molecular alterations in methamphetamine‐activated Fos‐expressing neurons from a single rat dorsal striatum using fluorescence‐activated cell sorting (FACS)

Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a fluorescence-activated cell sorting (FACS)-based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS-based method to characterize molecular alterations in Fos-expressing dorsal striatal neurons from a single rat using a multiplex pre-amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 5-6% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p.) while less than 0.5% of neurons were activated by saline injections. We used FACS to separate NeuN-labeled neurons into Fos-positive and Fos-negative neurons and assessed mRNA expression using RT-qPCR from as little as five Fos-positive neurons. Methamphetamine induced 3-20-fold increases of immediate early genes arc, homer-2, c-fos, fosB, and its isoforms (ΔfosB and a novel isoform ΔfosB-2) in Fos-positive but not Fos-negative neurons. Immediate early gene mRNA induction was 10-fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models. Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We here report an improved method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. We used FACS along with targeted PCR pre-amplification to assess acute methamphetamine-induced gene expression from as few as 5 Fos-expressing neurons from a single rat dorsal striatum. Methamphetamine induced 3-20-fold increases of immediate early genes (IEGs) in Fos-positive but not Fos-negative neurons. Targeted PCR pre-amplification makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models.

Acute EBV infection masquerading as "In-situ Follicular Lymphoma": a pitfall in the differential diagnosis of this entity

We present the case of a 30 year-old man who was referred for evaluation of diffuse lymphadenopathy. Six weeks prior, he noticed darkening of his urine associated with pale stools, nausea and an eventual 30 lb weight loss within a month. The initial laboratory findings showed elevation of the liver enzymes. A CT scan showed mesenteric and periaortic lymphadenopathy with the largest lymph node measuring 2.8 cm. Other laboratory results were otherwise unremarkable (including a normal LDH) with the exception of positive serum antibodies against Epstein-Barr virus (EBV) associated antigens (IgM+ and IgG+). An excisional biopsy of 4 of the small neck lymph nodes showed a normal architecture with prominent follicles and an intact capsule. But, by immunohistochemistry two of the follicles showed aberrant coexpression of BCL-2, in addition to CD10 and BCL-6. In-situ hybridization for early Epstein-Barr virus mRNA (EBER) and immunohistochemistry for latent membrane protein-1 (LMP-1) stained both scattered positive cells, as well as BCL-2 positive B-cells. Although an original diagnosis of in-situ follicular lymphoma was favored at an outside facility, additional interphase fluorescence in situ hybridization (FISH) studies for t(14;18);(IGH-BCL2) rearrangement (performed on the BCL-2 + follicles microdissected from the tissue block; Abott probe dual colour fusion) and molecular studies (IGH gene rearrangement by PCR, also performed on the microdissected follicles) were negative. Serologic studies (positive EBV antibodies) and immunostains in conjunction with the molecular studies confirmed the reactive nature of the changes. Our case also shows direct immunopathogenic evidence of BCL-2 expression among the EBV-infected cells, which has to our knowledge not been previously documented in vivo. A diagnosis of EBV infection should, therefore, be considered when confronted with BCL-2 expression in germinal centers, particularly in younger individuals, as the diagnosis of FLIS may lead to extensive and invasive haematologic work-ups.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1323656318940068

MiRNA regulation of BK polyomavirus replication during early infection

Viral microRNAs (miRNAs) play an important role during infection by posttranscriptionally regulating both host and viral gene expression. However, the function of many viral miRNAs remains poorly understood. In this study, we investigated the role of the BK polyomavirus (BKPyV) miRNA in regulating virus replication. The function of the polyomavirus miRNA was investigated in archetype BKPyV, which is the transmissible form of the virus and thought to establish a persistent infection in the host urinary tract. In agreement with previous studies, we show that the BKPyV miRNA targets early mRNAs. Importantly, we show that the miRNA plays a significant role in limiting archetype BKPyV replication in a natural host cell model of infection. This regulation is accomplished through the balance of regulatory elements located within the noncoding control region that control early gene expression and miRNA expression before genome replication. We therefore provide evidence for a unique function of the polyomavirus miRNA that may have important implications for the mechanism of viral persistence.

Phosphorylation of mRNA Decapping Protein Dcp1a by the ERK Signaling Pathway during Early Differentiation of 3T3-L1 Preadipocytes

BackgroundTurnover of mRNA is a critical step in the regulation of gene expression, and an important step in mRNA decay is removal of the 5′ cap. We previously demonstrated that the expression of some immediate early gene mRNAs is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes.Methodology/Principal FindingsHere we show that the mouse decapping protein Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of preadipocytes. Mass spectrometry analysis and site-directed mutagenesis combined with a kinase assay identified ERK pathway–mediated dual phosphorylation at Ser 315 and Ser 319 of Dcp1a. To understand the functional effects of Dcp1a phosphorylation, we examined protein-protein interactions between Dcp1a and other decapping components with co-immunoprecipitation. Dcp1a interacted with Ddx6 and Edc3 through its proline-rich C-terminal extension, whereas the conserved EVH1 (enabled vasodilator-stimulated protein homology 1) domain in the N terminus of Dcp1a showed a stronger interaction with Dcp2. Once ERK signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1), whose mRNA stability was analyzed under the overexpression of Dcp1a constructs in the Dcp1a knockdown 3T3-L1 cells.Conclusions/SignificanceOur findings suggest that ERK-phosphorylated Dcp1a enhances its interaction with the decapping enzyme Dcp2 during early differentiation of 3T3-L1 cells.