EBV noncoding RNA binds nascent RNA to drive host PAX5 to viral DNA.

EBER2 is an abundant nuclear noncoding RNA expressed by the Epstein-Barr virus (EBV). Probing its possible chromatin localization by CHART revealed EBER2's presence at the terminal repeats (TRs) of the latent EBV genome, overlapping previously identified binding sites for the B cell transcription factor PAX5. EBER2 interacts with PAX5 and is required for the localization of PAX5 to the TRs. EBER2 knockdown phenocopies PAX5 depletion in upregulating the expression of LMP2A/B and LMP1, genes nearest the TRs. Knockdown of EBER2 also decreases EBV lytic replication, underscoring the essential role of the TRs in viral replication. Recruitment of the EBER2-PAX5 complex is mediated by base-pairing between EBER2 and nascent transcripts from the TR locus. The interaction is evolutionarily conserved in the related primate herpesvirus CeHV15 despite great sequence divergence. Using base-pairing with nascent RNA to guide an interacting transcription factor to its DNA target site is a previously undescribed function for a trans-acting noncoding RNA.

Mammalian 5'-capped microRNA precursors that generate a single microRNA.

MicroRNAs (miRNAs) are short RNA gene regulators typically produced from primary transcripts that are cleaved by the nuclear microprocessor complex, with the resulting precursor miRNA hairpins exported by exportin 5 and processed by cytoplasmic Dicer to yield two (5p and 3p) miRNAs. Here, we document microprocessor-independent 7-methylguanosine (m(7)G)-capped pre-miRNAs, whose 5' ends coincide with transcription start sites and 3' ends are most likely generated by transcription termination. By establishing a small RNA Cap-seq method that employs the cap-binding protein eIF4E, we identified a group of murine m(7)G-capped pre-miRNAs genome wide. The m(7)G-capped pre-miRNAs are exported via the PHAX-exportin 1 pathway. After Dicer cleavage, only the 3p-miRNA is efficiently loaded onto Argonaute to form a functional microRNP. This unusual miRNA biogenesis pathway, which differs in pre-miRNA synthesis, nuclear-cytoplasmic transport, and guide strand selection, enables the development of shRNA expression constructs that produce a single 3p-siRNA.

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity.

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

Pseudouridylation of Epstein-Barr virus noncoding RNA EBER2 facilitates lytic replication.

Epstein-Barr virus (EBV) expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2, which are preserved in all clinical isolates of EBV, thus underscoring their essential function in the viral life cycle. Recent epitranscriptomics studies have uncovered a vast array of distinct RNA modifications within cellular as well as viral noncoding RNAs that are instrumental in executing their function. Here we show that EBER2 is marked by pseudouridylation, and by using HydraPsiSeq the modification site was mapped to a single nucleotide within the 3' region of EBER2. The writer enzyme was identified to be the snoRNA-dependent pseudouridine synthase Dyskerin, which is the catalytic subunit of H/ACA small nucleolar ribonucleoprotein complexes, and is guided to EBER2 by SNORA22. Similar to other noncoding RNAs for which pseudouridylation has a positive effect on RNA stability, loss of EBER2 pseudouridylation results in a decrease in RNA levels. Furthermore, pseudouridylation of EBER2 is required for the prolific accumulation of progeny viral genomes, suggesting that this single modification in EBER2 is important for efficient viral lytic replication. Taken together, our findings add to the list of RNA modifications that are essential for noncoding RNAs to implement their physiological roles.

The Analysis of in vitro Fertilization Outcomes after Fertility-Preserving Therapy for Endometrial Hyperplasia or Carcinoma.

OBJECTIVES: This study aimed to evaluate the clinical efficacy of fertility-preserving therapy through in vitro fertilization (IVF) procedures in women who were pathologically diagnosed with endometrial hyperplasia or carcinoma. DESIGN: A retrospective cohort study on fertility-preserving therapy was conducted. Participants/Materials, Setting: A total of 82 women were enrolled who had simple endometrial hyperplasia (SH), complex hyperplasia (CH), complex atypical hyperplasia (CAH), and endometrioid endometrial carcinoma stage IA (EC IA) and underwent IVF at Gangnam CHA fertility center between January 2008 and December 2020. METHODS: The primary endpoints were oncologic outcomes and subsequent reproductive outcomes of patients who underwent fertility-preserving treatments analyzed by χ2 test or Fisher's exact test. RESULTS: Of the 82 patients, 33 had a cumulative clinical pregnancy (40.2%), and 25 had a cumulative live birth (30.5%) through IVF procedures following pathologic confirmation of complete remission or non-progressive status. The cumulative clinical pregnancy rates and live birth rates for SH were 50.0% and 30.0%, for CH were 37.8% and 28.9%, for CAH were 25.0% and 25.0%, and for EC were 38.5% and 38.5%, respectively. There were no significant differences in cumulative clinical pregnancy rates or live birth rates when comparing the four groups. There was a difference in endometrial thickness between medroxyprogesterone acetate (MPA) treatment group and intrauterine device (IUD) group (p = 0.036); however, there were no significant differences in clinical pregnancy rates among MPA, IUD, and MPA+IUD groups. LIMITATIONS: Because of the retrospective nature of the study, many factors relevant to the treatment decision were not strictly controlled. CONCLUSIONS: All endometrial hyperplasia and carcinoma groups had competent cumulative live birth rates by IVF procedures. There may be differences in endometrial thickness depending on the treatment methods, but this does not affect clinical pregnancy rates. Therefore, the fertility-preserving treatment for endometrial hyperplasia and carcinoma is a safe and feasible method that results in good IVF outcomes.

Local changes in viral RNA sequence drive global changes in influenza nucleoprotein binding.

The genome of influenza A viruses (IAV) consists of eight negative-sense RNA segments that are coated by viral nucleoprotein (NP). Until recently, it was assumed that NP binds viral genomic RNA (vRNA) uniformly along the entire segment. However, genome-wide studies have revised the original model in that NP instead binds preferentially to certain regions of vRNA, while others are depleted for NP binding. Even strains with high sequence similarity exhibit distinct NP-binding profiles. Thus, it remains unknown how NP-binding specificity to vRNA is established. Here we introduced nucleotide changes to vRNA to examine whether primary sequence can affect NP binding. Our findings demonstrate that NP binding is indeed susceptible to sequence alterations, as NP peaks can be lost or appear de novo at mutated sites. Unexpectedly, nucleotide changes not only affect NP binding locally at the site of mutation, but also impact NP binding at distal regions that have not been modified. Taken together, our results suggest that NP binding is not regulated by primary sequence alone, but that a network formed by multiple segments governs the deposition of NP on vRNA.

Viral noncoding RNAs: more surprises.

Eukaryotic cells produce several classes of long and small noncoding RNA (ncRNA). Many DNA and RNA viruses synthesize their own ncRNAs. Like their host counterparts, viral ncRNAs associate with proteins that are essential for their stability, function, or both. Diverse biological roles--including the regulation of viral replication, viral persistence, host immune evasion, and cellular transformation--have been ascribed to viral ncRNAs. In this review, we focus on the multitude of functions played by ncRNAs produced by animal viruses. We also discuss their biogenesis and mechanisms of action.

Prevalence and morphological features of molar-root incisor malformation in children attending a specialist paediatric dental unit.

BACKGROUND: Molar-root incisor malformation (MRIM) is a rare dental anomaly featuring constricted cervical margins and tapered, narrow root and pulp morphology, often associated with severe toothache and infection. AIM: The aim of this study was to determine the prevalence of MRIM in children seen in a specialist paediatric dental unit of a tertiary referral hospital and to describe the characteristics of affected individuals. DESIGN: This study was an audit of children attending from November 2020 to November 2021. Radiographs were used to identify individuals with MRIM, and clinical data were collated. In addition, histology and microcomputed tomography (microCT) imaging were performed on teeth extracted from an affected individual. RESULTS: The prevalence of MRIM was five cases of 1054 children examined (0.47% or 1:210). The permanent first molars were affected in all five children and the primary second molars in two children; all children had medical comorbidities and multiple exposures to general anesthesia before 4 years of age. In addition, histological and microCT analyses displayed numerous microchannels connecting the pulp chamber to the external surface of the tooth at the furcation. CONCLUSIONS: Molar-root incisor malformation is an uncommon dental anomaly affecting paediatric patients with multiple comorbidities and is characterized by porosities extending from the pulp chamber to the external tooth surface, predisposing the risk of bacterial ingress from the oral cavity into the pulp chamber. Early detection may prevent atypical odontogenic facial pain and infection.

5-methylcytosine modification of an Epstein-Barr virus noncoding RNA decreases its stability.

Many cellular noncoding RNAs contain chemically modified nucleotides that are essential for their function. The Epstein-Barr virus expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2. To examine whether these viral RNAs contain modified nucleotides, we purified native EBERs from EBV-infected cells and performed mass spectrometry analysis. While EBER2 contains no modified nucleotides at stoichiometric amounts, EBER1 was found to carry 5-methylcytosine (m5C) modification. Bisulfite sequencing indicated that a single cytosine of EBER1 is methylated in ∼95% of molecules, and the RNA methyltransferase NSUN2 was identified as the EBER1-specific writer. Intriguingly, ablation of NSUN2 and thus loss of m5C modification resulted in an increase in EBER1 levels. We further found that EBER1 is a substrate for the RNase Angiogenin and cleavage in vivo is dependent on the presence of m5C, providing an explanation as to why loss of m5C increases EBER1 levels. Taken together, our observations indicate that m5C, a modification previously shown for tRNAs to oppose Angiogenin-mediated degradation, can also adversely affect RNA stability.

Effect of pulsed electromagnetic fields stimulation on ischemic skin model.

This study aimed to evaluate the effect of Pulsed Electromagnetic Fields (PEMF) in improving blood flow reduction and tissue necrosis of ischemic animal induced by skin flap. In each experiment, twenty rats (280-320 g) were randomly divided into control group (n = 10) and PEMF (n = 10) group. All of the rats were performed skin flap in back. In the PEMF group, PEMF (1 Hz, 10 mT) was performed in each experiment. In Experiment-1 (n = 20), PEMF was performed for 90 minutes. In Experiment-2 (n = 20), additionally, a blocking film was inserted, and suture was performed to induce necrosis. PEMF was performed for 30 minutes each day for 7 days. As a result of Speckle-Flow Index (SFI) analysis, in the control group, blood flow continued to decrease immediately after the procedure. In the PEMF group, blood flow was remained constant after 30 minutes and increased after 60 minutes. The blood flow in a specific region substantially increased from the initial state. As a result of skin necrosis analysis, the progression rate in the PEMF group was slower than that of the control group. The rate of necrosis in the PEMF group decreased dramatically from the 6th day, and there was a statistically significant difference between the two groups at the 7th day (p < .05). In this study, it was confirmed that PEMF (1 Hz, 10 mT) has a blood flow improvement and skin tissue necrosis alleviation in the ischemic flap animal model.

The many ways Epstein-Barr virus takes advantage of the RNA tool kit.

Epstein-Barr virus (EBV) was the first human cancer-causing virus to be discovered over fifty years ago. Given its relatively large genome size for a virus and hence the capacity to store more than mere protein-coding information, EBV also harbours genetic material for producing an array of distinct noncoding (nc)RNAs. The double-stranded nature of its DNA genome allows the utilization of the whole gamut of ncRNA types, including both RNA polymerase II and III transcripts, in devising a sophisticated strategy to ensure its replication upon infection in host cells and evasion of host immune responses. Owing to the development of sensitive technologies in recent years, mostly entailing next-generation sequencing, the list of ncRNA types generated by EBV has expanded now to include two RNAs (EBER1 and EBER2) best categorized as long ncRNAs, dozens of microRNAs, one small nucleolar RNA, stable intronic sequence RNAs, and the most recently discovered circular RNAs. With the application of cutting-edge technology, the molecular mechanisms of some of these noncoding transcripts are beginning to emerge, while others remain yet to be elucidated. As viruses often take advantage of existing molecular pathways established by the host, it is likely that further novel concepts of the greatly unexplored noncoding world can be learned from studying the many EBV ncRNAs.

Circular DNA tumor viruses make circular RNAs.

Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) cause ∼2% of all human cancers. RNase R-resistant RNA sequencing revealed that both gammaherpesviruses encode multiple, uniquely stable, circular RNAs (circRNA). EBV abundantly expressed both exon-only and exon-intron circRNAs from the BamHI A rightward transcript (BART) locus (circBARTs) formed from a spliced BART transcript and excluding the EBV miRNA region. The circBARTs were expressed in all verified EBV latency types, including EBV-positive posttransplant lymphoproliferative disease, Burkitt lymphoma, nasopharyngeal carcinoma, and AIDS-associated lymphoma tissues and cell lines. Only cells infected with the B95-8 EBV strain, with a 12-kb BART locus deletion, were negative for EBV circBARTs. Less abundant levels of EBV circRNAs originating from LMP2- and BHLF1-encoding genes were also identified. The circRNA sequencing of KSHV-infected primary effusion lymphoma cells revealed a KSHV-encoded circRNA from the vIRF4 locus (circvIRF4) that was constitutively expressed. In addition, KSHV polyadenylated nuclear (PAN) RNA locus generated a swarm (>100) of multiply backspliced, low-abundance RNase R-resistant circRNAs originating in both sense and antisense directions consistent with a novel hyperbacksplicing mechanism. In EBV and KSHV coinfected cells, exon-only EBV circBARTs were located more in the cytoplasm, whereas the intron-retaining circBARTs were found in the nuclear fraction. KSHV circvIRF4 and circPANs were detected in both nuclear and cytoplasmic fractions. Among viral circRNAs tested, none were found in polysome fractions from KSHV-EBV coinfected BC1 cells, although low-abundance protein translation from viral circRNAs could not be excluded. The circRNAs are a new class of viral transcripts expressed in gammaherpesvirus-related tumors that might contribute to viral oncogenesis.

Risk of occult atypical hyperplasia or cancer in women with nonatypical endometrial hyperplasia.

AIM: The aim of this study was to identify subsets of patients diagnosed with nonatypical endometrial hyperplasia (NAEH) by endometrial biopsy who had high risk for occult atypical endometrial hyperplasia (AEH) or endometrial cancer (EC). METHODS: We retrospectively reviewed the medical records of 281 patients who underwent hysterectomy within 6 months after a diagnosis of NAEH. We collected data on age, body mass index, menopausal status, tamoxifen use, previous history of NAEH, details of endometrial biopsy (location, curettage vs. pipelle sampling), NAEH subtype (simple vs. complex), interval between endometrial biopsy and hysterectomy, indication of hysterectomy and the presence of occult AEH or EC in hysterectomy specimen. Associations between variables and occult AEH or EC were analyzed. Risk of occult AEH or EC in subsets were calculated and visualized using a heatmap. RESULTS: Among 281 patients, 34 (12.1%) and 9 (3.2%) had occult AEH and EC in hysterectomy specimens, respectively. Using univariate analysis, we found age, menopausal status and subtype were associated with occult AEH or EC. Using multivariate analysis, older age (odds ratio = 1.09, P < 0.01) and complex subtype (odds ratio = 3.34, P < 0.01) were independent risk factors. Patients at an age ≥ 51 years with complex NAEH had about 50% risk of occult AEH or EC. CONCLUSION: Women at an age ≥ 51 years with complex NAEH had high risk for occult AEH or EC and surgical treatment can be considered for these patients.

GZ-11608, a Vesicular Monoamine Transporter-2 Inhibitor, Decreases the Neurochemical and Behavioral Effects of Methamphetamine.

Despite escalating methamphetamine use and high relapse rates, pharmacotherapeutics for methamphetamine use disorders are not available. Our iterative drug discovery program had found that R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), a selective vesicular monoamine transporter-2 (VMAT2) inhibitor, specifically decreased methamphetamine's behavioral effects. However, GZ-793A inhibited human-ether-a-go-go-related gene (hERG) channels, suggesting cardiotoxicity and prohibiting clinical development. The current study determined if replacement of GZ-793A's piperidine ring with a phenylalkyl group to yield S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11608) diminished hERG interaction while retaining pharmacological efficacy. VMAT2 inhibition, target selectivity, and mechanism of GZ-11608-induced inhibition of methamphetamine-evoked vesicular dopamine release were determined. We used GZ-11608 doses that decreased methamphetamine-sensitized activity to evaluate the potential exacerbation of methamphetamine-induced dopaminergic neurotoxicity. GZ-11608-induced decreases in methamphetamine reinforcement and abuse liability were determined using self-administration, reinstatement, and substitution assays. Results show that GZ-11608 exhibited high affinity (Ki = 25 nM) and selectivity (92-1180-fold) for VMAT2 over nicotinic receptors, dopamine transporter, and hERG, suggesting low side-effects. GZ-11608 (EC50 = 620 nM) released vesicular dopamine 25-fold less potently than it inhibited VMAT2 dopamine uptake. GZ-11608 competitively inhibited methamphetamine-evoked vesicular dopamine release (Schild regression slope = 0.9 ± 0.13). GZ-11608 decreased methamphetamine sensitization without altering striatal dopamine content or exacerbating methamphetamine-induced dopamine depletion, revealing efficacy without neurotoxicity. GZ-11608 exhibited linear pharmacokinetics and rapid brain penetration. GZ-11608 decreased methamphetamine self-administration, and this effect was not surmounted by increasing methamphetamine unit doses. GZ-11608 reduced cue- and methamphetamine-induced reinstatement, suggesting potential to prevent relapse. GZ-11608 neither served as a reinforcer nor substituted for methamphetamine, suggesting low abuse liability. Thus, GZ-11608, a potent and selective VMAT2 inhibitor, shows promise as a therapeutic for methamphetamine use disorder. SIGNIFICANCE STATEMENT: GZ-11608 is a potent and selective vesicular monoamine transporter-2 inhibitor that decreases methamphetamine-induced dopamine release from isolated synaptic vesicles from brain dopaminergic neurons. Results from behavioral studies show that GZ-11608 specifically decreases methamphetamine-sensitized locomotor activity, methamphetamine self-administration, and reinstatement of methamphetamine-seeking behavior, without exhibiting abuse liability. Tolerance does not develop to the efficacy of GZ-11608 to decrease the behavioral effects of methamphetamine. In conclusion, GZ-11608 is an outstanding lead in our search for a therapeutic to treat methamphetamine use disorder.

Postoperative Cancer Surveillance Following Oncoplastic Surgery with Latissimus Dorsi Flap: a Matched Case-Control Study.

BACKGROUND: The latissimus dorsi (LD) myocutaneous flap is a widely used local option in oncoplastic surgery for avoiding breast deformities; however, concerns exist regarding its influence in monitoring recurrence. In this study, we evaluated the impact of this flap on postoperative cancer surveillance. METHODS: Each patient receiving oncoplastic surgery with LD flap after partial mastectomy were matched in age, cancer stage, and body mass index with patients receiving partial mastectomy alone. Twenty-nine patients with the oncoplastic LD flap received 99 mammograms and 139 ultrasonograms, while 29 patients with partial mastectomy alone underwent 92 mammograms and 129 ultrasonograms. Mammographic and ultrasonographic findings were classified by Breast Imaging Reporting and Data System (BI-RADS) category and reviewed. Any recommendations for additional evaluation and recurrence were documented. RESULTS: During an average follow-up period of 44 months, although the oncoplastic group demonstrated more newly developed benign calcifications (control 14% vs. oncoplastic 41%; p = 0.019) on mammography, the percentage of recall for additional imaging in category 0, and the short-interval follow-up in category 3, was not different between the control and oncoplastic group. Regarding ultrasonography, BI-RADS category was also not different between the two groups; however, the control group showed more fluid collections than the oncoplastic group (control 21% vs. oncoplastic 0%; p = 0.023). One case of local recurrence was observed in the control group. CONCLUSION: Although there was an increase in benign calcifications in the oncoplastic group, there were no additional abnormal findings requiring further intervention. We concluded that the LD flap for oncoplastic surgery does not interfere with cancer surveillance, and even decreases the rate of fluid collection.

Muscarinic agonist, (±)-quinuclidin-3-yl-(4-fluorophenethyl)(phenyl)carbamate: High affinity, but low subtype selectivity for human M1 - M5 muscarinic acetylcholine receptors.

Novel quinuclidinyl N-phenylcarbamate analogs were synthesized, and binding affinities at M1-M5 muscarinic acetylcholine receptor (mAChR) subtypes were determined using Chinese hamster ovary (CHO) cell membranes stably expressing one specific subtype of human mAChR. Although not subtype selective, the lead analog (±)-quinuclidin-3-yl-(4-fluorophenethyl)(phenyl)carbamate (3c) exhibited the highest affinity (Ki = 2.0, 13, 2.6, 2.2, 1.8 nM) at each of the M1-M5 mAChRs, respectively. Based on results from the [3H]dopamine release assay using rat striatal slices, 3c acted as an agonist at mAChRs. The effect of 3c was inhibited by the nonselective mAChR antagonist, scopolamine, and 3c augmented release evoked by oxotremorine. A potent analog from the same scaffold, (±)-quinuclidin-3-yl-(4-methoxyphenethyl)(phenyl)-carbamate (3b) exhibited the greatest selectivity (17-fold) at M3 over M2 mAChRs. These analogs could serve as leads for further discovery of novel subtype-selective muscarinic ligands with the goal of providing therapeutics for substance use disorders and chronic obstructive pulmonary disease.

Practice patterns of surgery for advanced ovarian cancer: analysis from international surveys.

BACKGROUND: We investigated the practice patterns of surgery for advanced ovarian cancer (AOC) through relevant international surveys. METHODS: After searching for 878 studies on surgery for AOC till 2017, we extracted 18 questions with similar query and answer formats from eight studies. Among them, 5 and 13 were classified as comprehensive and procedure information. RESULTS: In comprehensive information, there was a higher preference for optimal cytoreduction defined as no visible tumor (44%) compared with residual tumors <1 cm (38%) or <2 cm (2%) and omental disease involving the spleen or pancreas was more important as an intraoperative finding precluding optimal cytoreduction (35%) since 2010. The preference for neoadjuvant chemotherapy was the highest at its use for 1-10% (36%), which was preferred in Europe over USA. The positive expectation of preoperative determination of optimal cytoreduction was higher in Europe than in USA (44 vs. 27%; P < 0.05). In procedure information, conventional gynecological surgery was mainly performed by gynecological oncologists, whereas more than 50% of upper abdominal or urological surgeries were conducted by other surgeons. European clinicians showed a higher response rate of diaphragmatic stripping and resection than those from USA (88 vs. 60%; 69 vs. 24%; P < 0.05). CONCLUSION: No visible tumor as the criterion for optimal cytoreduction was preferred in AOC, and aggressive surgery beyond conventional gynecological surgery tended to be performed by other surgeons. Moreover, the preference of neoadjuvant chemotherapy and the positive expectation of preoperative determination of optimal cytoreduction were higher in Europe than in USA.

Genome-wide analysis of influenza viral RNA and nucleoprotein association.

Influenza A virus (IAV) genomes are composed of eight single-stranded RNA segments that are coated by viral nucleoprotein (NP) molecules. Classically, the interaction between NP and viral RNA (vRNA) is depicted as a uniform pattern of 'beads on a string'. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), we identified the vRNA binding profiles of NP for two H1N1 IAV strains in virions. Contrary to the prevailing model for vRNA packaging, NP does not bind vRNA uniformly in the A/WSN/1933 and A/California/07/2009 strains, but instead each vRNA segment exhibits a unique binding profile, containing sites that are enriched or poor in NP association. Intriguingly, both H1N1 strains have similar yet distinct NP binding profiles despite extensive sequence conservation. Peaks identified by HITS-CLIP were verified as true NP binding sites based on insensitivity to DNA antisense oligonucleotide-mediated RNase H digestion. Moreover, nucleotide content analysis of NP peaks revealed that these sites are relatively G-rich and U-poor compared to the genome-wide nucleotide content, indicating an as-yet unidentified sequence bias for NP association in vivo. Taken together, our genome-wide study of NP-vRNA interaction has implications for the understanding of influenza vRNA architecture and genome packaging.

EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression.

Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.

WNT5A induces castration-resistant prostate cancer via CCL2 and tumour-infiltrating macrophages.

BACKGROUND: Although the standard treatment for the patients with recurrent and metastatic prostate cancer (CaP) is androgen deprivation therapy, castration-resistant prostate cancer (CRPC) eventually emerges. Our previous report indicated that bone morphogenetic protein 6 (BMP6) induced CRPC via tumour-infiltrating macrophages. In a separate line of study, we have observed that the WNT5A/BMP6 loop in CaP bone metastasis mediates resistance to androgen deprivation in tissue culture. Simultaneously, we have reported that BMP6 induced castration resistance in CaP cells via tumour-infiltrating macrophages. Therefore, our present study aims to investigate the mechanism of WNT5A and its interaction with macrophages on CRPC. METHODS: Doxycycline inducible WNT5A overexpression prostate cancer cell line was used for detailed mechanical study. RESULTS: WNT5A was associated with increased expression of chemokine ligand 2 (CCL2) in the human CaP cell line, LNCaP. Mechanistically, this induction of CCL2 by WNT5A is likely to be mediated via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling pathway. Our in vivo experiments demonstrated that the overexpression of WNT5A in LNCaP cells promoted castration resistance. Conversely, this resistance was inhibited with the removal of macrophages via clodronate liposomes. When patient-derived CaP LuCaP xenografts were analysed, high levels of WNT5A were correlated with increased levels of CCL2 and BMP6. In addition, higher levels of CCL2 and BMP6 were more commonly observed in intra-femoral transplanted tumours as compared to subcutaneous-transplanted tumours in the patient-derived PCSD1 bone-niche model. CONCLUSIONS: These findings collectively suggest that WNT5A may be a key gene that induces CRPC in the bone niche by recruiting and regulating macrophages through CCL2 and BMP6, respectively.