A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

Pepino mosaic virus (PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor (SlTXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. SlTXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamilyclosesttophosducin-likeprotein-3. InPepMV-infectedandhealthyNicotianabenthamiana plants,NbTXND9mRNAlevelswerecomparable,andexpressionlevelsremainedstableinbothlocal and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α-SlTXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27–35 kDa, in the 1000 and 30,000 g pellets, and the soluble fraction of extracts of healthy and PepMV-infected N. benthamiana leaves, but not in the cell wall. These bands likely consist of the homologous protein NbTXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labellingofultrathinsectionsofPepMV-infectedN.benthamianaleavesusingα-SlTXND9IgGrevealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress

Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro

[EN] Cucurbit chlorotic yellows virus (CCYV) is a new member of the genus Crinivirus (family Closteroviridae) with a bi-partite genome. CCYV RNA 1-encoded p22 has recently been reported to be a weak local suppressor of RNA silencing for which an interaction with cucumber SKP1LB1 through an F-box-like motif was demonstrated to be essential. Using a bacterially expressed maltose-binding protein (MBP) fusion of CCYV p22 in electrophoretic mobility shift assays (EMSA), we have examined in vitro its ability to bind different RNA templates. Our experiments showed that CCYV p22 is able to bind to ss and ds long RNAs, in addition to ss and ds small interfering (si) RNA molecules. CCYV p22 deletion mutants (MBP_CCYV DEL1-4) were produced that covered the entire protein, with MBP_CCYV DEL2 corresponding to the F-box motif and its flanking sequences. None of these deletions abolished the capacity of CCYV p22 to bind ss- and dsRNA molecules. However, deletions affecting the C-terminal half of the protein resulted in decreased binding efficiency for either ss- or dsRNA molecules indicating that essential elements for these interactions are located in this region. Taken together, our data add to current knowledge of the mode of action of suppressors of RNA silencing encoded by genes sited at the 3'-terminus of crinivirus genomic RNA 1, and shed light on the involvement of CCYV p22 in the suppression of RNA silencing and/or in another role in the virus life cycle via RNA binding.Salavert, F.; Navarro Bohigues, JA.; Owen, CA.; Khechmar, S.; Pallás Benet, V.; Livieratos, IC. (2020). Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro. Virus Research. 279:1-8. https://doi.org/10.1016/j.virusres.2020.197887S18279Abrahamian, P. E., Seblani, R., Sobh, H., & Abou-Jawdah, Y. (2013). Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR. Journal of Virological Methods, 193(2), 320-326. doi:10.1016/j.jviromet.2013.06.004Bananej, K., Menzel, W., Kianfar, N., Vahdat, A., & Winter, S. (2013). First Report of Cucurbit chlorotic yellows virus Infecting Cucumber, Melon, and Squash in Iran. Plant Disease, 97(7), 1005-1005. doi:10.1094/pdis-01-13-0125-pdnBrantley, J. D., & Hunt, A. G. (1993). The N-terminal protein of the polyprotein encoded by the potyvirus tobacco vein mottling virus is an RNA-binding protein. Journal of General Virology, 74(6), 1157-1162. doi:10.1099/0022-1317-74-6-1157BUCK, K. W., & KEMPSON-JONES, G. F. (1970). Three Types of Virus Particle in Penicillium stoloniferum. Nature, 225(5236), 945-946. doi:10.1038/225945a0Cañizares, M. C., Navas-Castillo, J., & Moriones, E. (2008). Multiple suppressors of RNA silencing encoded by both genomic RNAs of the crinivirus, Tomato chlorosis virus. Virology, 379(1), 168-174. doi:10.1016/j.virol.2008.06.020Chen, S., Sun, X., Shi, Y., Wei, Y., Han, X., Li, H., … Shi, Y. (2019). Cucurbit Chlorotic Yellows Virus p22 Protein Interacts with Cucumber SKP1LB1 and Its F-Box-Like Motif Is Crucial for Silencing Suppressor Activity. Viruses, 11(9), 818. doi:10.3390/v11090818Cuellar, W. J., Tairo, F., Kreuze, J. F., & Valkonen, J. P. T. (2008). Analysis of gene content in sweet potato chlorotic stunt virus RNA1 reveals the presence of the p22 RNA silencing suppressor in only a few isolates: implications for viral evolution and synergism. Journal of General Virology, 89(2), 573-582. doi:10.1099/vir.0.83471-0Cuellar, W. J., Kreuze, J. F., Rajamaki, M.-L., Cruzado, K. R., Untiveros, M., & Valkonen, J. P. T. (2009). Elimination of antiviral defense by viral RNase III. Proceedings of the National Academy of Sciences, 106(25), 10354-10358. doi:10.1073/pnas.0806042106GYOUTOKU, Y., OKAZAKI, S., FURUTA, A., ETOH, T., MIZOBE, M., KUNO, K., … OKUDA, M. (2009). Chlorotic yellows disease of melon caused by Cucurbit chlorotic yellows virus, a new crinivirus. Japanese Journal of Phytopathology, 75(2), 109-111. doi:10.3186/jjphytopath.75.109Herranz, M. C., & Pallás, V. (2004). RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus. Journal of General Virology, 85(3), 761-768. doi:10.1099/vir.0.19534-0Huang, L.-H., Tseng, H.-H., Li, J.-T., & Chen, T.-C. (2010). First Report of Cucurbit chlorotic yellows virus Infecting Cucurbits in Taiwan. Plant Disease, 94(9), 1168-1168. doi:10.1094/pdis-94-9-1168bKataya, A. R. A., Suliman, M. N. S., Kalantidis, K., & Livieratos, I. C. (2009). Cucurbit yellow stunting disorder virus p25 is a suppressor of post-transcriptional gene silencing. Virus Research, 145(1), 48-53. doi:10.1016/j.virusres.2009.06.010Klaassen, V. A., Mayhew, D., Fisher, D., & Falk, B. W. (1996). In VitroTranscripts from Cloned cDNAs of the Lettuce Infectious Yellows Closterovirus Bipartite Genomic RNAs Are Competent for Replication inNicotiana benthamianaProtoplasts. Virology, 222(1), 169-175. doi:10.1006/viro.1996.0407Kreuze, J. F., Savenkov, E. I., Cuellar, W., Li, X., & Valkonen, J. P. T. (2005). Viral Class 1 RNase III Involved in Suppression of RNA Silencing. Journal of Virology, 79(11), 7227-7238. doi:10.1128/jvi.79.11.7227-7238.2005Kubota, K., & Ng, J. C. K. (2016). Lettuce chlorosis virus P23 Suppresses RNA Silencing and Induces Local Necrosis with Increased Severity at Raised Temperatures. Phytopathology®, 106(6), 653-662. doi:10.1094/phyto-09-15-0219-rLandeo-Ríos, Y., Navas-Castillo, J., Moriones, E., & Cañizares, M. C. (2016). The p22 RNA silencing suppressor of the crinivirus Tomato chlorosis virus preferentially binds long dsRNAs preventing them from cleavage. Virology, 488, 129-136. doi:10.1016/j.virol.2015.11.008Marcos, J. F., Vilar, M., Pérez-Payá, E., & Pallás, V. (1999). In VivoDetection, RNA-Binding Properties and Characterization of the RNA-Binding Domain of the p7 Putative Movement Protein from Carnation Mottle Carmovirus (CarMV). Virology, 255(2), 354-365. doi:10.1006/viro.1998.9596Mashiko, T., Wang, W.-Q., Hartono, S., Suastica, G., Neriya, Y., Nishigawa, H., & Natsuaki, T. (2019). The p27 open reading frame of tomato infectious chlorosis virus encodes a suppressor of RNA silencing. Journal of General Plant Pathology, 85(4), 301-305. doi:10.1007/s10327-019-00850-0Navarro, J. A., Genovés, A., Climent, J., Saurí, A., Martínez-Gil, L., Mingarro, I., & Pallás, V. (2006). RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins. Virology, 356(1-2), 57-67. doi:10.1016/j.virol.2006.07.040Okuda, M., Okazaki, S., Yamasaki, S., Okuda, S., & Sugiyama, M. (2010). Host Range and Complete Genome Sequence of Cucurbit chlorotic yellows virus, a New Member of the Genus Crinivirus. Phytopathology®, 100(6), 560-566. doi:10.1094/phyto-100-6-0560Orfanidou, C., Maliogka, V. I., & Katis, N. I. (2014). First Report of Cucurbit chlorotic yellows virus in Cucumber, Melon, and Watermelon in Greece. Plant Disease, 98(10), 1446-1446. doi:10.1094/pdis-03-14-0311-pdnOrfanidou, C. G., Mathioudakis, M. M., Katsarou, K., Livieratos, I., Katis, N., & Maliogka, V. I. (2019). Cucurbit chlorotic yellows virus p22 is a suppressor of local RNA silencing. Archives of Virology, 164(11), 2747-2759. doi:10.1007/s00705-019-04391-xOrílio, A. F., Fortes, I. M., & Navas-Castillo, J. (2014). Infectious cDNA clones of the crinivirus Tomato chlorosis virus are competent for systemic plant infection and whitefly-transmission. Virology, 464-465, 365-374. doi:10.1016/j.virol.2014.07.032Pumplin, N., & Voinnet, O. (2013). RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nature Reviews Microbiology, 11(11), 745-760. doi:10.1038/nrmicro3120Richmond, K. E., Chenault, K., Sherwood, J. L., & German, T. L. (1998). Characterization of the Nucleic Acid Binding Properties of Tomato Spotted Wilt Virus Nucleocapsid Protein. Virology, 248(1), 6-11. doi:10.1006/viro.1998.9223Salem, N. M., Chen, A. Y. S., Tzanetakis, I. E., Mongkolsiriwattana, C., & Ng, J. C. K. (2009). Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2. Virology, 390(1), 45-55. doi:10.1016/j.virol.2009.04.025Serra-Soriano, M., Antonio Navarro, J., & Pallás, V. (2016). Dissecting the multifunctional role of the N-terminal domain of theMelon necrotic spot viruscoat protein in RNA packaging, viral movement and interference with antiviral plant defence. Molecular Plant Pathology, 18(6), 837-849. doi:10.1111/mpp.12448Simon, A. E., & Miller, W. A. (2013). 3′ Cap-Independent Translation Enhancers of Plant Viruses. Annual Review of Microbiology, 67(1), 21-42. doi:10.1146/annurev-micro-092412-155609Voinnet, O. (2005). Induction and suppression of RNA silencing: insights from viral infections. Nature Reviews Genetics, 6(3), 206-220. doi:10.1038/nrg1555Wang, J., Stewart, L. R., Kiss, Z., & Falk, B. W. (2010). Lettuce infectious yellows virus (LIYV) RNA 1-encoded P34 is an RNA-binding protein and exhibits perinuclear localization. Virology, 403(1), 67-77. doi:10.1016/j.virol.2010.04.006Weinheimer, I., Boonrod, K., Moser, M., Wassenegger, M., Krczal, G., Butcher, S. J., & Valkonen, J. P. T. (2014). Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus. Journal of General Virology, 95(2), 486-495. doi:10.1099/vir.0.058693-0Yeh, H.-H., Tian, T., Rubio, L., Crawford, B., & Falk, B. W. (2000). Asynchronous Accumulation of Lettuce Infectious Yellows Virus RNAs 1 and 2 and Identification of an RNA 1 trans Enhancer of RNA 2 Accumulation. Journal of Virology, 74(13), 5762-5768. doi:10.1128/jvi.74.13.5762-5768.200

Long-term efficacy and safety of first-line ibrutinib treatment for patients with CLL/SLL: 5 years of follow-up from the phase 3 RESONATE-2 study.

RESONATE-2 is a phase 3 study of first-line ibrutinib versus chlorambucil in chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Patients aged ≥65 years (n = 269) were randomized 1:1 to once-daily ibrutinib 420 mg continuously or chlorambucil 0.5-0.8 mg/kg for ≤12 cycles. With a median (range) follow-up of 60 months (0.1-66), progression-free survival (PFS) and overall survival (OS) benefits for ibrutinib versus chlorambucil were sustained (PFS estimates at 5 years: 70% vs 12%; HR [95% CI]: 0.146 [0.098-0.218]; OS estimates at 5 years: 83% vs 68%; HR [95% CI]: 0.450 [0.266-0.761]). Ibrutinib benefit was also consistent in patients with high prognostic risk (TP53 mutation, 11q deletion, and/or unmutated IGHV) (PFS: HR [95% CI]: 0.083 [0.047-0.145]; OS: HR [95% CI]: 0.366 [0.181-0.736]). Investigator-assessed overall response rate was 92% with ibrutinib (complete response, 30%; 11% at primary analysis). Common grade ≥3 adverse events (AEs) included neutropenia (13%), pneumonia (12%), hypertension (8%), anemia (7%), and hyponatremia (6%); occurrence of most events as well as discontinuations due to AEs decreased over time. Fifty-eight percent of patients continue to receive ibrutinib. Single-agent ibrutinib demonstrated sustained PFS and OS benefit versus chlorambucil and increased depth of response over time

8th International conference on management and rehabilitation of chronic respiratory failure: the long summaries – part 2

This paper summarizes the Part 2 of the proceedings of the 8th International Conference on Management and Rehabilitation of Chronic Respiratory Failure, held in Pescara, Italy, on 7 and 8 May, 2015. It summarizes the contributions from numerous experts in the field of chronic respiratory disease and chronic respiratory failure. The outline follows the temporal sequence of presentations. This paper (Part 2) includes sections regarding: Promoting Physical Activity across the Spectrum of COPD (Physical activity: definitions, measurements, and significance; Increasing Physical Activity through Pharmacotherapy in COPD); Pulmonary Rehabilitation in Critical Illness (Complex COPD with comorbidities and its impact during acute exacerbation; Collaborative Self-Management in COPD: A Double-Edged Sword?; and Pulmonary Rehabilitation in Critical Illness

What do older people do when sitting and why? Implications for decreasing sedentary behaviour

Background and Objectives: Sitting less can reduce older adults’ risk of ill health and disability. Effective sedentary behavior interventions require greater understanding of what older adults do when sitting (and not sitting), and why. This study compares the types, context, and role of sitting activities in the daily lives of older men and women who sit more or less than average. Research Design and Methods: Semistructured interviews with 44 older men and women of different ages, socioeconomic status, and objectively measured sedentary behavior were analyzed using social practice theory to explore the multifactorial, inter-relational influences on their sedentary behavior. Thematic frameworks facilitated between-group comparisons. Results: Older adults described many different leisure time, household, transport, and occupational sitting and non-sitting activities. Leisure-time sitting in the home (e.g., watching TV) was most common, but many non-sitting activities, including “pottering” doing household chores, also took place at home. Other people and access to leisure facilities were associated with lower sedentary behavior. The distinction between being busy/not busy was more important to most participants than sitting/not sitting, and informed their judgments about high-value “purposeful” (social, cognitively active, restorative) sitting and low-value “passive” sitting. Declining physical function contributed to temporal sitting patterns that did not vary much from day-to-day. Discussion and Implications: Sitting is associated with cognitive, social, and/or restorative benefits, embedded within older adults’ daily routines, and therefore difficult to change. Useful strategies include supporting older adults to engage with other people and local facilities outside the home, and break up periods of passive sitting at home

Evaluation Research and Institutional Pressures: Challenges in Public-Nonprofit Contracting

This article examines the connection between program evaluation research and decision-making by public managers. Drawing on neo-institutional theory, a framework is presented for diagnosing the pressures and conditions that lead alternatively toward or away the rational use of evaluation research. Three cases of public-nonprofit contracting for the delivery of major programs are presented to clarify the way coercive, mimetic, and normative pressures interfere with a sound connection being made between research and implementation. The article concludes by considering how public managers can respond to the isomorphic pressures in their environment that make it hard to act on data relating to program performance.This publication is Hauser Center Working Paper No. 23. The Hauser Center Working Paper Series was launched during the summer of 2000. The Series enables the Hauser Center to share with a broad audience important works-in-progress written by Hauser Center scholars and researchers

Long-term efficacy and safety of first-line ibrutinib treatment for patients with CLL/SLL: 5 years of follow-up from the phase 3 RESONATE-2 study

RESONATE-2 is a phase 3 study of first-line ibrutinib versus chlorambucil in chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Patients aged >= 65 years (n = 269) were randomized 1:1 to once-daily ibrutinib 420 mg continuously or chlorambucil 0.5-0.8 mg/kg for = 3 adverse events (AEs) included neutropenia (13%), pneumonia (12%), hypertension (8%), anemia (7%), and hyponatremia (6%); occurrence of most events as well as discontinuations due to AEs decreased over time. Fifty-eight percent of patients continue to receive ibrutinib. Single-agent ibrutinib demonstrated sustained PFS and OS benefit versus chlorambucil and increased depth of response over time

Sustained efficacy and detailed clinical follow-up of first-line ibrutinib treatment in older patients with chronic lymphocytic leukemia: extended phase 3 results from RESONATE-2.

Results of RESONATE-2 (PCYC-1115/1116) supported approval of ibrutinib for first-line treatment of chronic lymphocytic leukemia. Extended analysis of RESONATE-2 was conducted to determine long-term efficacy and safety of ibrutinib in older patients with chronic lymphocytic leukemia. A total of 269 patients aged ≥65 years with previously untreated chronic lymphocytic leukemia without del(17p) were randomized 1:1 to ibrutinib (n=136) or chlorambucil (n=133) on days 1 and 15 of a 28-day cycle for 12 cycles. Median ibrutinib treatment duration was 28.5 months. Ibrutinib significantly prolonged progression-free survival versus chlorambucil (median, not reached vs 15 months; hazard ratio, 0.12; 95% confidence interval, 0.07-0.20; P<0.0001). The 24-month progression-free survival was 89% with ibrutinib (97% and 89% in patients with del[11q] and unmutated immunoglobulin heavy chain variable region gene, respectively). Progression-free survival rates at 24 months were also similar regardless of age (<75 years [88%], ≥75 years [89%]). Overall response rate was 92% (125/136). Rate of complete response increased substantially from 7% at 12 months to 18% with extended follow up. Greater quality of life improvements occurred with ibrutinib versus chlorambucil in Functional Assessment of Chronic Illness Therapy-Fatigue (P=0.0013). The most frequent grade ≥3 adverse events were neutropenia (12%), anemia (7%), and hypertension (5%). Rate of discontinuations due to adverse events was 12%. Results demonstrated that first-line ibrutinib for elderly patients with chronic lymphocytic leukemia provides sustained response and progression-free survival benefits over chemotherapy, with depth of response improving over time without new toxicity concerns. This trial was registered at clinicaltrials.gov identifier 01722487 and 01724346