Genomic Copy Research Articles

Aging2Cancer: an integrated resource for linking aging to tumor multi-omics data

BackgroundAging and tumorigenesis share intricate regulatory processes, that alter the genome, epigenome, transcriptome and immune landscape of tissues. Discovering the link between aging and cancer in terms of multiomics characteristics remains a challenge for biomedical researchers.MethodsWe collected high-throughput datasets for 57 human tumors and 20 normal tissues, including 23,125 samples with age information. On the basis of these sufficient omics data, we introduced six useful modules including genomic (somatic mutation and copy number variation), gene expression, DNA methylation, hallmarks (aging and cancer), immune landscape (immune infiltration, immune pathways, immune signatures, and antitumor immune activities) and survival analysis. Correlation and differential analyses were performed for the multiomic signatures associated with aging at the gene level.ResultsWe developed Aging2Cancer (http://210.37.77.200:8080/Aging2Cancer/index.jsp), which is a comprehensive database and analysis platform for revealing the associations between aging and cancer. Users can search for and visualize the results of genes of interest to explore the relationships between aging and cancer at the gene level for different omics levels.ConclusionsWe believe that Aging2Cancer is a valuable resource for identifying novel biomarkers and will serve as a bridge for linking aging to cancer.

ERBB2/HOXB13 co-amplification with interstitial loss of BRCA1 defines a unique subset of breast cancers

BackgroundThe HOXB13/IL17RB gene expression biomarker has been shown to predict response to adjuvant and extended endocrine therapy in patients with early-stage ER+ HER2- breast tumors. HOXB13 gene expression is the primary determinant driving the prognostic and endocrine treatment-predictive performance of the biomarker. Currently, there is limited data on HOXB13 expression in HER2+ and ER- breast cancers. Herein, we studied the expression of HOXB13 in large cohorts of HER2+ and ER- breast cancers.MethodsWe investigated gene expression, genomic copy number, mutational signatures, and clinical outcome data in the TGGA and METABRIC breast cancer cohorts. Genomic-based gene amplification data was validated with tri-colored fluorescence in situ hybridization.ResultsIn the TCGA breast cancer cohort, HOXB13 gene expression was significantly higher in HER2+ versus HER2- breast cancers, and its expression was also significantly higher in the ER- versus ER+ breast cancers. HOXB13 is frequently co-gained or co-amplified with ERBB2. Joint copy gains of HOXB13 and ERBB2 occurred with low-level co-gains or high-level co-amplifications (co-amp), the latter of which is associated with an interstitial loss that includes the tumor suppressor BRCA1. ERBB2/HOXB13 co-amp tumors with interstitial BRCA1 loss exhibit a mutational signature associated with APOBEC deaminase activity and copy number signatures associated with chromothripsis and genomic instability. Among ERBB2-amplified tumors of different tissue origins, ERBB2/HOXB13 co-amp with a BRCA1 loss appeared to be enriched in breast cancer compared to other tumor types. Lastly, patients with ERBB2/HOXB13 co-amplified and BRCA1 lost tumors displayed a significantly shorter progression-free survival (PFS) than those with ERBB2-only amplifications. The difference in PFS was restricted to the ER- subset patients and this difference in PFS was not solely driven by HOXB13 gene expression.ConclusionsHOXB13 is frequently co-gained with ERBB2 at both low-copy number level or as complex high-level amplification with relative BRCA1 loss. ERBB2/HOXB13 amplified, BRCA1-lost tumors are strongly enriched in breast cancer, and patients with such breast tumors experience a shortened PFS.

Genomic characterization and histologic analysis of uterine leiomyosarcoma arising from leiomyoma with bizarre nuclei.

Leiomyoma with bizarre nuclei (LM-BN) is a rare variant of leiomyoma with a benign clinical course. In contrast, leiomyosarcoma (LMS) is a high-grade, malignant neoplasm characterized by high recurrence rates and poor survival. While LM-BN and LMS show distinct morphologies, they share similar immunoprofiles and molecular alterations, with both considered 'genomically unstable'. Rare cases of LM-BN associated with LMS have been reported; however, the histogenesis and molecular relationship between these two tumors remains unclear. In this study, we assessed 11 cases of LMS arising in conjunction with LM-BN confirmed by histology and immunohistochemistry (IHC), further analyzed by clinical, histologic, and molecular characteristics of these distinct components. LM-BN and LMS had similar p16 and p53 IHC patterns, but LMS had a higher Ki-67 index and lower estrogen and progesterone eceptor expression. Digital image analysis based on cytologic features revealed spatial relationships between LMS and LM-BN. Genomic copy number alterations (CNAs) demonstrated the same clonal origin of LMS arising from existing LM-BN through conserved CNAs. LMS harbored highly complex CNAs and more frequent losses of the TP53, RB1, and PTEN genomic regions than LM-BN (p = 0.0031), with CDKN2A/B deletion identified in LMS only. Mutational profiling revealed many shared oncogenic alterations in both LM-BN and LMS; however, additional mutations were present within LMS, indicative of tumor progression through progressive genomic instability. Analysis of spatial transcriptomes defined uniquely expressed gene signatures that matched the geographic distribution of LM-BN, LMS, and other cell types. Our findings indicate for the first time that a subset of LMS arises from an existing LM-BN, and highly complex genomic alterations could be potential high risks associated with disease progression in LM-BN. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Rabies virus phosphoprotein exhibits thermoresponsive phase separation with a lower critical solution temperature.

Rabies virus (RABV) generates membrane-less liquid organelles (Negri bodies) in the cytoplasm of its host cell, where genome transcription and replication and nucleocapsid assembly take place, but the mechanisms of their assembly and maturation remain to be explained. An essential component of the viral RNA synthesizing machine, the phosphoprotein (P), acts as a scaffold protein for the assembly of these condensates. This intrinsically disordered protein forms star-shaped dimers with N-terminal negatively charged flexible arms and C-terminal globular domains exhibiting a large dipole moment. Our study shows that in vitro self-association of RABV P drives a complex thermoresponsive phase separation with a lower critical solution temperature. Protein dimers assemble already below the saturation concentration, and condensation is driven by attractive conformation-specific interactions leading to reentrant liquid phase separation over a narrow range of salt concentration. We propose a minimal molecular model in which P can adopt three limit conformational states and the disordered N-terminal arms control the interactions between giant dipoles that is consistent with our observations.

Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription

Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg2+ (site B) inducing formation of a negatively charged pocket where a second floating Mg2+ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg2+ and the primer 3′-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication.

Cryo-EM structure of Nipah virus L-P polymerase complex

Nipah virus (NiV) is a non-segmented, negative-strand (NNS) RNA virus, belonging to Paramyxoviridae. The RNA polymerase complex, composed of large (L) protein and tetrameric phosphoprotein (P), is responsible for genome transcription and replication by catalyzing NTP polymerization, mRNA capping and cap methylation. Here, we determine the cryo-electron microscopy (cryo-EM) structure of fully bioactive NiV L-P polymerase complex at a resolution of 3.19 Å. The L-P complex displays a conserved architecture like other NNS RNA virus polymerases and L interacts with the oligomerization domain and the extreme C-terminus region of P tetramer. Moreover, we elucidate that NiV is naturally resistant to the allosteric L-targeting inhibitor GHP-88309 due to the conformational change in the drug binding site. We also find that the non-nucleotide drug suramin can inhibit the NiV L-P polymerase activity at both the enzymatic and cellular levels. Our findings have greatly enhanced the molecular understanding of NiV genome replication and transcription and provided the rationale for broad-spectrum polymerase-targeted drug design.

Genetically-stable engineered optogenetic gene switches modulate spatial cell morphogenesis in two- and three-dimensional tissue cultures

Recent advances in tissue engineering have been remarkable, yet the precise control of cellular behavior in 2D and 3D cultures remains challenging. One approach to address this limitation is to genomically engineer optogenetic control of cellular processes into tissues using gene switches that can operate with only a few genomic copies. Here, we implement blue and red light-responsive gene switches to engineer genomically stable two- and three-dimensional mammalian tissue models. Notably, we achieve precise control of cell death and morphogen-directed patterning in 2D and 3D tissues by optogenetically regulating cell necroptosis and synthetic WNT3A signaling at high spatiotemporal resolution. This is accomplished using custom-built patterned LED systems, including digital mirrors and photomasks, as well as laser techniques. These advancements demonstrate the capability of precise spatiotemporal modulation in tissue engineering and open up new avenues for developing programmable 3D tissue and organ models, with significant implications for biomedical research and therapeutic applications.

IAV Antagonizes Host Innate Immunity by Weakening the LncRNA-LRIR2-Mediated Antiviral Functions

A growing number of studies have shown that long non-coding RNAs (lncRNAs) are implicated in many biological processes, including the regulation of innate immunity and IAV replication. In addition, IAV has been found to be able to hijack lncRNAs and thus antagonize host innate immunity. Nonetheless, whether IAV can antagonize host innate immunity by weakening the antiviral functions mediated by lncRNAs is unknown. In this study, we found that LncRNA-ENST00000491430 regulates IAV replication and named it LRIR2. Interestingly, we found that the expression of LRIR2 was suppressed during IAV infection. Importantly, LRIR2 overexpression inhibited IAV replication, suggesting that LRIR2 plays an antiviral role during IAV infection. Mechanistically, we demonstrated that LRIR2 inhibits the transcription and replication of the IAV genome. In addition, the antiviral function of LRIR2 is mainly dependent on the stem-loop structures of 1–118 nt and 575–683 nt. Taken together, IAV could antagonize host innate immunity by weakening the LncRNA-LRIR2-mediated antiviral functions. Our study provides novel perspectives into viral strategies to antagonize host innate immunity. It lays a theoretical foundation for the design of novel anti-IAV drugs that target host lncRNAs or the antagonism effect.

ARRDC3, a novel α-arrestin, modulates WSSV replication and AHPND pathogenesis in Litopeneaus vannamei

Although shrimp are a valuable protein source, shrimp aquaculture has numerous challenges from various infectious diseases and understanding molecular mechanisms of disease pathogenesis is crucial for disease management. In this study, a gene-to-gene correlation network generated from a transcriptomic database of the stomach of shrimp infected with acute hepatopancreatic necrosis disease (AHPND) was used to identify a new α-arrestin, termed arrestin domain containing-3 gene (LvARRDC3), with crucial roles in development of both AHPND and white spot disease (WSD). Double stranded RNA-mediated silencing or plasmid-mediated overexpression of LvARRDC3 gene significantly decreased expression of WSSV genes (IE1, VP28, and ICP11) and viral genome copy numbers. Nevertheless, in AHPND, silencing the LvARRDC3 gene increased the AHPND-associated plasmid and Pir toxins copy numbers, whereas overexpression of LvARRDC3 had the opposite effect. An in vitro pathogen binding assay with recombinant LvARRDC3 protein produced robust binding to WSSV virions and AHPND-causing V. parahaemolyticus. Moreover, based on immunofluorescence, LvARRDC3 was localized in the cytoplasm of Spodoptera frugiperda (Sf9) insect cells. Therefore, we inferred that LvARRDC3 has a role in pathogen internalization, making it a valuable target for addressing AHPND and WSD and also a biomarker for marker-associated shrimp breeding.

The glycogene alterations and potential effects in esophageal squamous cell carcinoma

BackgroundAberrant glycosylation is one of the hallmarks of cancer. The profile of glycoprotein expression caused by abnormal glycosylation has been revealed, while abnormal glycogenes that may disturb the structure of glycans have not yet been identified in esophageal squamous cell carcinoma (ESCC).MethodsGenomic alterations driven by differentially expressed glycogenes in ESCC were compared with matched normal tissues by multi-omics analysis. Immunohistochemistry, MTT, colony formation, transwell assays, subcutaneous tumor formation experiments and tail vein injection were used to study the expression and the effect on the proliferation and metastasis of the differentially expressed glycogenes POFUT1 and RPN1 in ESCC. In the alkyne fucose labeling experiment, AAL lectin affinity chromatography and immunoprecipitation were used to explore the mechanism of POFUT1 in ESCC.ResultsThe expression of the POFUT1 and RPN1 glycogenes were upregulated, as determined by genomic copy number gain and proteomics analysis. The overexpression of POFUT1 or RPN1 was associated with poor prognosis in ESCC patients and affected the proliferation and metastasis of ESCC in vivo and in vitro. The overexpression of POFUT1 increased the overall fucosylation level and activated the Notch signaling pathway, which partially mediated POFUT1 induced pro-migration in ESCC. The regulation of malignant progression of ESCC by RPN1 may be related to the TNF signaling pathway, p53 signaling pathway, etc.ConclusionsOur study fills a gap in the study of abnormal glycogenes and highlights the potential role of the POFUT1/Notch axis in ESCC. Moreover, our study identifies POFUT1 and RPN1 as promising anticancer targets in ESCC.

Viral Load Measurements for Kaposi Sarcoma Herpesvirus (KSHV/HHV8): Review and an Updated Assay.

"When you can measure what you are speaking about, and express it in numbers, you know something about it." is a famous quote attributed to Lord Kelvin. This sentiment puts viral load measurements at the center of virology. Viral load, or more precisely, DNA copy number measurements, are also used to follow infections with human herpesviruses, such as Kaposi sarcoma herpesvirus (KSHV) and Epstein-Barr Virus (EBV). EBV and KSHV are associated with human cancers, and determining their DNA copy numbers in the context of cancer prediction and progression on therapy is of fundamental scientific and translationalinterest. Yet, there is no generally accepted assay for KSHV DNA quantitation, and KSHV viral load is not used in clinical decision-making. Here, we review the history of KSHV DNA detection assays, explore factors that affect sensitivity and specificity, and describe an automated, high-throughput, real-time quantitative polymerase chain reaction (PCR) assay for KSHV and EBV. In conjunction with a digital PCR assay using the same primer/probe combination, we describe how to determine the absolute KSHV genome copy numbers in plasma, peripheral blood mononuclear cells, saliva, and other easily accessible body fluids.

Additional expression of T-cell engager in clinically tested oncolytic adeno-immunotherapy redirects tumor-infiltrated, irrelevant T cells against cancer cells to enhance antitumor immunity

BackgroundOncolytic adenoviruses (OAds) are the most clinically tested viral vectors for solid tumors. However, most clinically tested “Armed” OAds show limited antitumor effects in patients with various solid tumors even...

Transcription- and Replication-Competent Virus-like Particle Systems for Marburg Virus.

Here, we describe the transcription- and replication-competent virus-like particle (trVLP) system for Marburg virus (MARV), which recapitulates transcription and replication of the viral genome in addition to viral particle assembly, egress, and entry. This protocol includes instructions for transfections for producer and acceptor cells and the use of trVLPs for infection.

The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response.

The roles of host cell miRNAs have not been well studied in the context of BCoV replication and immune regulation. This study aimed to identify miRNA candidates that regulate essential host genes involved in BCoV replication, tissue tropism, and immune regulation. To achieve these goals, we used two isolates of BCoV (enteric and respiratory) to infect bovine endothelial cells (BECs) and Madine Darby Bovine Kidney (MDBK) cells. We determined the miRNA expression profiles of these cells after BCoV infection. The expression of miRNA16a is differentially altered during BCoV infection. Our data show that miRNA16a is a significantly downregulated miRNA in both in vitro and ex vivo models. We confirmed the miRNA16aexpression profile by qRT-PCR. Overexpression of pre-miRNA16ain the BEC and the MDBK cell lines markedly inhibited BCoV infection, as determined by the viral genome copy numbers measured by qRT‒PCR, viral protein expression (S and N) measured by Western blot, and virus infectivity using a plaque assay. Our bioinformatic prediction showed that Furin is a potential target of miRNA16a. We compared the Furin protein expression level in pre-miRNA16a-transfected/BCoV-infected cells to that in pre-miRNA-scrambled-transfected cells. Our qRT-PCR and Western blot data revealed marked inhibition of Furin expression at the mRNA and protein levels, respectively. BCoV-S protein expression was markedly inhibited at both the mRNA and protein levels. To further confirm the impact of the downregulation of the Furin enzyme on the replication of BCoV, we transfected cells with specific Furin-siRNAs parallel to the scrambled siRNA. Marked inhibition of BCoV replication was observed in the Furin-siRNA-treated group. To further validate Furin as a novel target for miRNA16a, we cloned the 3'UTR of bovine Furin carrying the seed region of miRNA16a in the dual luciferase vector. Our data showed that luciferase activity in pre-miRNA16a-transfected cells decreased by more than 50% compared to cells transfected with the construct carrying the mutated Furin seed region. Our data confirmed that miRNA16ainhibits BCoV replication by targeting the host cell line Furin and the BCoV-S glycoprotein. It also enhances the host immune response, which contributes to the inhibition of viral replication. This is the first study to confirm that Furin is a valid target of miRNA16a. Our findings highlight the clinical applications of host miRNA16a as a potential miRNA-based vaccine/antiviral therapy.

Cocaine-Induced DNA-Dependent Protein Kinase Relieves RNAP II Pausing by Promoting TRIM28 Phosphorylation and RNAP II Hyperphosphorylation to Enhance HIV Transcription.

Drug abuse continues to pose a significant challenge in HIV control efforts. In our investigation, we discovered that cocaine not only upregulates the expression of the DNA-dependent protein kinase (DNA-PK) but also augments DNA-PK activation by enhancing its phosphorylation at S2056. Moreover, DNA-PK phosphorylation triggers the higher localization of the DNA-PK into the nucleus. The finding that cocaine increases the nuclear localization of the DNA-PK provides further support to our observation of enhanced DNA-PK recruitment at the HIV long terminal repeat (LTR) following cocaine exposure. By activating and facilitating the nuclear localization of the DNA-PK, cocaine effectively orchestrates multiple stages of HIV transcription, thereby promoting HIV replication. Additionally, our study demonstrates that the cocaine-induced DNA-PK promotes the hyper-phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) at Ser5 and Ser2 sites, enhancing both the initiation and elongation phases, respectively, of HIV transcription. The cocaine-mediated enhancement of transcriptional initiation is supported by its activation of cyclin-dependent kinase 7 (CDK7). Additionally, the induction of transcriptional elongation is marked by higher LTR recruitment and the increased phosphorylation of CDK9, which indicates the stimulation of positive transcriptional elongation factor b (P-TEFb). We demonstrate for the first time that cocaine, through DNA-PK activation, promotes the specific phosphorylation of TRIM28 at serine 824 (p-TRIM28, S824). This modification converts TRIM28 from a transcriptional inhibitor to a transactivator for HIV transcription. Additionally, we observed that the phosphorylation of TRIM28 (p-TRIM28, S824) promotes the transition from the pausing phase to the elongation phase of HIV transcription, thereby facilitating the production of full-length HIV genomic transcripts. This finding corroborates the previously observed enhanced RNAP II CTD phosphorylation at Ser2, a marker of transcriptional elongation, following cocaine exposure. Accordingly, upon cocaine treatment, we observed the elevated recruitment of p-TRIM28-(S824) at the HIV LTR. Overall, our results unravel the intricate molecular mechanisms underlying cocaine-induced HIV transcription and gene expression. These findings hold promise for the development of highly targeted therapeutics aimed at mitigating the detrimental effects of cocaine in individuals living with HIV.

EANO guideline on molecular testing of meningiomas for targeted therapy selection.

Meningiomas are the most common primary intracranial tumors of adults. For meningiomas that progress or recur despite surgical resection and radiotherapy, additional treatment options are limited due to lack of proven efficacy. Meningiomas show recurring molecular aberrations, which may serve as predictive markers for systemic pharmacotherapies with targeted drugs or immunotherapy, radiotherapy or radioligand therapy. Here, we review the evidence for a predictive role of a wide range of molecular alterations and markers including NF2, AKT1, SMO, SMARCE1, PIK3CA, CDKN2A/B, CDK4/6, TERT, TRAF7, BAP1, KLF4, ARID1/2, SUFU, PD-L1, SSTR2A, PR/ER, mTOR, VEGFR, PDGFR, as well as homologous recombination deficiency (HRD), genomic copy number variations, DNA methylation classes and combined gene expression profiles. In our assessment based on the established ESMO ESCAT (European Society for Medical Oncology Scale for Clinical Actionability of molecular Targets) evidence level criteria, no molecular target reached ESCAT I ("ready for clinical use") classification and only mTOR pathway activation and NF2 alterations reached ESCAT II ("investigational") classification, respectively. Our evaluations may guide targeted therapy selection in clinical practice and clinical trial efforts and highlight areas for which additional research is warranted.

High-grade B-cell lymphoma not otherwise specified, with diffuse large B-cell lymphoma gene expression signatures: Genomic analysis and potential therapeutics.

High-grade B-cell lymphoma not otherwise specified (HGBCL, NOS) has overlapping morphological and genetic features with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL), leading to uncertainty in its diagnosis and clinical management. Using functional genomic approaches, we previously characterized HGBCL and NOS, that demonstrate gene expression profiling (GEP), and genetic signatures similar to BL. Herein, we characterize distinct HGBCL, NOS, cohort (n = 55) in adults (n = 45) and in children (n = 10), and compared the GEP, genomic DNA copy number (CN), and mutational spectrum with de novo DLBCL (n = 85) and BL (n = 52). This subgroup, representing ~60% of HGBCL, NOS, lack gene-expression signature of BL and double hit/dark zone lymphoma, but express DLBCL like signatures and are characterized by either GCB- or ABC-like mRNA signatures and exhibit higher genomic complexity, similar to de novo DLBCL, and show alteration in genes regulating B-cell activation (CD79B, MYD88, PRDM1, TBLIXR1, CARD11), epigenome (KMT2D, TET2) and cell cycle transition (TP53, ASPM). However, recurrent mutations in genes often mutated in BL (DDX3X, GNA13, CCND3), but rare in DLBCL, are also present in HGBCL-NOS, highlighting genetic heterogeneity. Consistent with mutation spectrum, frequent genomic CN alterations in genes regulating B-cell activation (del-PRDM1, gain-BCL6, -REL, -STAT3) and cell cycle regulators (del-TP53, del-CDKN2A, del-RB1, gain-CCND3) were observed. Pediatric cases showed GCB-DLBCL-like mRNA signatures, but also featured hallmark mutations of pediatric BL. Frequent oncogenic PIM1 mutations were present in adult HGBCL, NOS. In vitro analyses with pharmacologic or genetic inhibition of PIM1 expression triggered B-cell activation and NF-κB-induced apoptosis, suggesting that PIM1 is a rational therapeutic target.

Comparing Two Seawater Temperatures For Human Norovirus Depuration From Oysters

Despite regulations set up to monitor the microbiological quality of shellfish in producing areas, shellfish-borne gastroenteritis outbreaks still occur. Indeed, oyster depuration practices that are efficient to eliminate bacteria, fail to eliminate human norovirus from oyster flesh. In order to evaluate the impact of seawater temperature on the elimination of norovirus particles from oysters, large batches of oysters were contaminated using raw sewage containing norovirus and subjected to depuration at 8 °C or 18 °C. Over the experiment, quantitative RT-qPCR showed a one-log decrease of norovirus (both genogroups combined) genome copies per gram of digestive tissue after 41 days for oysters depurated at 8 °C and 24 days at 18 °C. The decrease of norovirus (both genogroups combined) in two batches of field-contaminated oysters depurated for two weeks at 18 °C was in the same range (21 and 23 days, respectively). All experiments showed a difference in genomic decay between the two norovirus genogroups, with norovirus genogroup I being more persistent in March/April compared to April/May.

ISWI1 complex proteins facilitate developmental genome editing in Paramecium.

One of the most extensive forms of natural genome editing occurs in ciliates, a group of microbial eukaryotes. Ciliate germline and somatic genomes are contained in distinct nuclei within the same cell. During the massive reorganization process of somatic genome development, ciliates eliminate tens of thousands of DNA sequences from a germline genome copy. Recently, we showed that the chromatin remodeler ISWI1 is required for somatic genome development in the ciliate Paramecium tetraurelia Here, we describe two high similarity paralogous proteins, ICOPa and ICOPb, essential for their genome editing. ICOPa and ICOPb are highly divergent from known proteins; the only domain detected showed distant homology to the WSD (WHIM2+WHIM3) motif. We show that both ICOPa and ICOPb interact with the chromatin remodeler ISWI1. Upon ICOP knockdown, changes in alternative DNA excision boundaries and nucleosome densities are similar to those observed for ISWI1 knockdown. We thus propose that a complex comprising ISWI1 and either or both ICOPa and ICOPb are needed for Paramecium's precise genome editing.

Plant Viral Synergism: Co-Expression of P1 and NIa-Pro Cistrons of Wheat Streak Mosaic Virus and Triticum Mosaic Virus Is Required for Synergistic Interactions in Wheat.

Synergistic interactions among unrelated viruses in mixed infections can cause significant yield losses, and viral determinants of these interactions are poorly understood. Wheat (Triticum aestivum L.) co-infection with wheat curl mite-transmitted wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) results in disease synergism with a drastically increased symptom phenotype of stunted growth, leaf bleaching, and enhanced titers of both viruses. In this study, we examined the viral determinants responsible for WSMV-TriMV disease synergism through transient expression of select cistrons of WSMV in wheat through TriMV and vice-versa. We found that expression of WSMV P1, NIa, or NIaPro cistrons in wheat through TriMV or vice-versa elicited moderate to severe symptoms with a moderate or no increase in virus titer. However, co-expression of P1 and NIaPro cistrons of WSMV in wheat through TriMV or vice-versa exhibited a WSMV-TriMV disease synergism-like phenotype. Additionally, we found that the P3 cistron of both viruses is dispensable for synergism, whereas HCPro and NIaVPg cistrons of WSMV and TriMV are not the primary determinants but might have a minor role in efficient synergism. In co-infected wheat, accumulation of vsiRNAs was increased, similar to viral genomic RNA copies, despite the presence of dual viral RNA silencing suppressors (VRSS), which function through sequestration of vsiRNAs. Our findings revealed that WSMV-TriMV disease synergism is not caused by the suppression of host post-transcriptional gene silencing by dual VRSS in co-infected wheat and the P1 and NIaPro cistrons of both viruses collectively drive synergistic interactions between WSMV and TriMV in wheat.