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Viruses pose a great threat to animal and plant health worldwide, with many being dependent on insect vectors for transmission between hosts. While the virus-host arms race has been well established, how viruses and insect vectors adapt to each other remains poorly understood. Begomoviruses comprise the largest genus of plant-infecting DNA viruses and are exclusively transmitted by the whitefly Bemisia tabaci. Here, we show that the vector Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway plays an important role in mediating the adaptation between the begomovirus tomato yellow leaf curl virus (TYLCV) and whiteflies. We found that the JAK/STAT pathway in B. tabaci functions as an antiviral mechanism against TYLCV infection in whiteflies as evidenced by the increase in viral DNA and coat protein (CP) levels after inhibiting JAK/STAT signaling. Two STAT-activated effector genes, BtCD109-2 and BtCD109-3, mediate this anti-TYLCV activity. To counteract this vector immunity, TYLCV has evolved strategies that impair the whitefly JAK/STAT pathway. Infection of TYLCV is associated with a reduction of JAK/STAT pathway activity in whiteflies. Moreover, TYLCV CP binds to STAT and blocks its nuclear translocation, thus, abrogating the STAT-dependent transactivation of target genes. We further show that inhibition of the whitefly JAK/STAT pathway facilitates TYLCV transmission but reduces whitefly survival and fecundity, indicating that this JAK/STAT-dependent TYLCV-whitefly interaction plays an important role in keeping a balance between whitefly fitness and TYLCV transmission. This study reveals a mechanism of plant virus-insect vector coadaptation in relation to vector survival and virus transmission.
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Begomovirus , Hemípteros , Vírus de Plantas , Solanum lycopersicum , Animais , Antivirais , Begomovirus/genética , DNA Viral , Hemípteros/fisiologia , Janus Quinases/genética , Solanum lycopersicum/genética , Doenças das Plantas , Vírus de Plantas/genética , Fatores de Transcrição STAT/genética , Transdução de SinaisRESUMO
A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.
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PURPOSE: Long-term study to evaluate the clinical and surgical outcomes of scleral buckle (SB) surgery for primary rhegmatogenous retinal detachment (RRD) at a large tertiary eye center. METHODS: Noncomparative, retrospective case series of 589 eyes of 569 patients with primary RRD who underwent SB surgery between 2004 and 2022 with a median follow-up of 6 months. The main outcome measures were best-corrected visual acuity, surgical outcomes, complications, and classification of RRD. RESULTS: At baseline, 447/589 (76.1%) round hole RRD, and 133/589 (22.7%) retinal dialysis RRD. Overall primary SB success rate was 83.7% for all retinal detachment subtypes, with round hole retinal detachment 84.8% and dialysis RRD 81.2%. Overall, the baseline best-corrected visual acuity was 0.42 logarithm of the minimum angle of resolution (logMAR) and the final best-corrected visual acuity was 0.26 logMAR ( P < 0.0001). In macula-off RRD, the best-corrected visual acuity significantly improved from 0.79 to 0.48 logMAR ( P < 0.0001). In patients with macula-on RRD, it improved from 0.19 to 0.12 logMAR ( P = 0.014). Binary logistic regression showed registrar surgeon grade (odds ratio [OR] 0.09, 95% confidence interval [CI] 0.01-0.55), and partial or complete posterior vitreous detachment (OR 0.21, 95% CI 0.10-0.49) was associated with reduced odds of primary success. Higher surgical failure was associated with low pre-fellowship SB surgeon experience ( P = 0.024). CONCLUSION: Favorable visual and functional outcomes have been reported in a large series of SB for primary retinal detachment, mainly for patients with round hole RRD and retinal dialysis RRD.
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Descolamento Retiniano , Recurvamento da Esclera , Acuidade Visual , Humanos , Recurvamento da Esclera/métodos , Descolamento Retiniano/cirurgia , Descolamento Retiniano/fisiopatologia , Descolamento Retiniano/diagnóstico , Estudos Retrospectivos , Acuidade Visual/fisiologia , Feminino , Masculino , Pessoa de Meia-Idade , Seguimentos , Adulto , Idoso , Resultado do Tratamento , Adulto Jovem , Complicações Pós-OperatóriasRESUMO
Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells-part of a phenomenon known as the "Warburg effect"- and is mediated by monocarboxylate transporters (MCTs). However, the role of MCTs has yet to be studied in EBV-associated malignancies, which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1, respectively. Functionally, MCT1 was required for early B cell proliferation, and MCT4 up-regulation promoted acquired resistance to MCT1 antagonism in LCLs. However, dual MCT1/4 inhibition led to LCL growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significantly reduced oxygen consumption rates (OCRs) and NAD+/NADH ratios, contrary to previous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/4-inhibited cancer cells. Furthermore, U-13C6-glucose labeling of MCT1/4-inhibited LCLs revealed depleted glutathione pools that correlated with elevated reactive oxygen species. Finally, we found that dual MCT1/4 inhibition also sensitized LCLs to killing by the electron transport chain complex I inhibitors phenformin and metformin. These findings were extended to viral lymphomas associated with EBV and the related gammaherpesvirus KSHV, pointing at a therapeutic approach for targeting both viral lymphomas.
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Linfoma/metabolismo , Linfoma/virologia , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Linfócitos B/virologia , Linhagem Celular Tumoral , Proliferação de Células , Infecções por Vírus Epstein-Barr/virologia , Glucose/metabolismo , Glutationa/metabolismo , Herpesvirus Humano 4/fisiologia , Herpesvirus Humano 8/fisiologia , Humanos , Ácido Láctico/metabolismo , Linfoma/patologia , Metformina/farmacologia , NAD/metabolismo , Consumo de Oxigênio , Fenformin/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Bemisia tabaci New World (NW) (Gennadius) (Hemiptera: Aleyrodidae), a whitefly in the B. tabaci species complex, is polyphagous on many plant species. Yet, it has been displaced, albeit not entirely, by other whitefly species. Potential causes could include issues with adaptation, feeding, and the colonization of new-hosts; however, insights that would help clarify these possibilities are lacking. Here, we sought to address these gaps by performing electropenetrography (EPG) recordings of NW whiteflies, designated "Napus" and "Rapa," reared on 2 colony hosts, Brassica napus and B. rapa, respectively. Analysis of 17 probing and pathway (pw) phase-related EPG variables revealed that the whiteflies exhibited unique probing behaviors on their respective colony hosts, with some deterrence being encountered on B. rapa. Upon switching to B. rapa and B. napus, the probing patterns of Napus and Rapa whiteflies, respectively, adapted quickly to these new-hosts to resemble that of whiteflies feeding on their colony hosts. Results for 3 of the EPG variables suggested that B. rapa's deterrence against Napus whitefly was significant prior to the phloem phase. This also suggested that adaptation by Rapa whitefly improved its pw probing on B. rapa. Based on analysis of 24 phloem phase-related EPG variables, Napus and Rapa whiteflies performed equally well once they entered phloem phase and exhibited comparable phloem acceptability on both the colony- and new-hosts. These findings demonstrate that NW whiteflies reared on a colony host are highly adaptable to feeding on a new host despite encountering some deterrence during the nonphloem phases in B. rapa plant.
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Brassica napus , Comportamento Alimentar , Hemípteros , Animais , Hemípteros/fisiologia , Brassica napus/parasitologia , Adaptação Fisiológica , Brassica rapa , Feminino , HerbivoriaRESUMO
A 71-year-old woman previously on rituximab treatment for rheumatoid arthritis presented with 2 years of progressive neurologic symptoms. She was found to have persistent hypogammaglobulinemia and B cell depletion despite rituximab discontinuation a year prior. MRI revealed diffuse meningeal enhancement along the entire neuroaxis. LP showed a CSF lymphocytic pleocytosis, elevated protein, and presence of enterovirus by PCR. The patient was hospitalized several times for progressive clinical and radiologic decline, though she had transient improvements following treatment with immunoglobulin therapy. Her CSF remained positive for enterovirus PCR for at least 12 months. Though two brain biopsies were non-diagnostic, pan-Enterovirus was ultimately identified using a high-throughput next-generation sequencing technique. She was treated with compassionate-use pocapavir with clinical stabilization at 4-month follow-up; however, she expired 8 months later from a bacterial pneumonia.
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Infecções por Enterovirus , Enterovirus , Meningoencefalite , Idoso , Enterovirus/genética , Infecções por Enterovirus/tratamento farmacológico , Feminino , Humanos , Imunização Passiva , Éteres Fenílicos , Rituximab/uso terapêuticoRESUMO
In June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases surged in Liberia. SARS-CoV-2 sequences from patients hospitalized during March-July 2021 revealed the Delta variant was in Liberia in early March and was dominant in June, irrespective of geography. Mutations and deletions suggest multiple SARS-CoV-2 Delta variant introductions.
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COVID-19 , SARS-CoV-2 , Humanos , Libéria/epidemiologia , Análise de SequênciaRESUMO
Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60â% (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41â% (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.
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Proteínas do Capsídeo/metabolismo , Crinivirus/fisiologia , Hemípteros/virologia , Insetos Vetores/virologia , Nicotiana/virologia , Doenças das Plantas/virologia , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Crinivirus/genética , Sistema Digestório/virologia , Engenharia Genética , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Mutação , Plantas Geneticamente Modificadas/virologia , Vírion/fisiologiaRESUMO
Post-transplant lymphoproliferative disorders (PTLD) are a group of lesions that can complicate solid organ or hematopoietic stem cell transplantation and are often associated with Epstein-Barr virus (EBV). The treatment of PTLD is dependent on the type of lesion and includes a wide range of therapies, but chimeric antigen receptor (CAR) T-cell therapy has not previously been reported as a treatment option for PTLD. We present a patient who developed refractory PTLD in her right retroperitoneum, right inguinal and iliac chains, and right axillary region shortly after heart transplantation and was treated with CAR T-cell therapy. She could not tolerate complete discontinuation of immunosuppression due to the risk of rejection of a life-supporting graft. The patient's PTLD responded to CAR T-cell therapy, and her heart was monitored throughout the treatment course without any signs of rejection or ventricular dysfunction. CAR T-cell therapy may be a viable treatment option in patients who develop PTLD after a solid organ transplant.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Transplante de Coração , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/terapia , Complicações Pós-Operatórias/imunologia , Complicações Pós-Operatórias/terapia , Receptores de Antígenos Quiméricos/imunologia , Adolescente , Feminino , HumanosRESUMO
BACKGROUND: Novel cancer immunotherapy seeks to harness the body's own immune system and tip the balance in favour of antitumour activity. The intracellular enzyme indoleamine 2,3-dioxygenase (IDO) is a critical regulator of the tumour microenvironment (TME) via tryptophan metabolism. The potential immunotherapeutic role of IDO in head and neck squamous cell carcinoma (HNSCC) requires further exploration. We aim to assess the evidence on IDO in HNSCC. METHODS: A systematic review of literature and clinical trials databases. RESULTS: We included 40 studies: seven involved cell lines: eight assessed tumour immunohistochemistry: ten measured IDO gene transcription: 15 reported on clinical trials. Increased cell line IDO expression was postulated to adversely affect tumour metabolism and apoptosis. Immunohistochemical IDO expression correlated with worse survival. Gene transcription studies associated IDO with positive PD-L1 and human papillomavirus (HPV) status. Phase I/II clinical trials showed (a) overall response (34%-55%) and disease control rates (62%-70%) for IDO1 inhibitor in combination with a PD-1 inhibitor, (b) similar safety profiles when both are used in combination therapy compared to each as monotherapies and (c) IDO gene expression as a predictive biomarker for response to PD-L1 therapy. CONCLUSIONS: IDO expression is increased in the TME of HNSCC, which correlates with poor prognosis. However, the exact mechanism of IDO-driven immune modulation in the TME is an enigma. Future translational studies should map IDO activity during HNSCC treatment and elucidate its precise role in the TME, such research will underpin the development of clinical trials establishing the efficacy of IDO inhibitors in HNSCC.
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Imunoterapia/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/enzimologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Microambiente TumoralRESUMO
Pediatric recipients of HCT may have a high susceptibility for overweight and obesity, and obesity may negatively impact post-transplant mortality and survival. This is a single-center retrospective analysis of 297 pediatric patients who received HCT between 2005 and 2018. Patients were classified as UW, NW, OW, or OB based on age-adjusted BMI. A mixed-effects linear regression model controlling for patient, disease, and transplant-related characteristics was used to trend weight longitudinally. Comparisons were made between weight category and post-transplant outcomes. In the pretransplant period, 5.4%, 54.5%, 22.2%, and 17.8% of patients were UW, NW, OW, and OB, respectively. Five years post-transplantation, those numbers were 10.6%, 48.2%, 16.5%, and 24.7%. Overall, BMI increased 0.00094 ± 0.0001 kg/m2 each day post-transplant (P < .001), with older individuals demonstrating greater rates of increase. Further, there was a larger BMI increase in patients without TBI compared with those who received TBI (1.29 ± 0.49, P = .008). Rates of acute GVHD, chronic GVHD, and viral infections, in addition to time to platelet and neutrophil engraftment and 5-year survival estimates, were not significantly different based on pretransplant BMI. Overweight and obese individuals had poorer 5-year survival based on 100-day post-transplant BMI (P = .02). Overall, pediatric HCT recipients are at risk of developing obesity, which is associated with decreased survival. Adolescents and young adults demonstrate the highest risk of weight gain, representing a vulnerable population that requires close monitoring, additional interventions, and further research.
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Índice de Massa Corporal , Previsões , Doença Enxerto-Hospedeiro/complicações , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Obesidade/etiologia , Sobrepeso/etiologia , Transplantados , Adolescente , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Incidência , Lactente , Masculino , Obesidade/epidemiologia , Sobrepeso/epidemiologia , Estudos Retrospectivos , Medição de Risco/métodos , Fatores de Risco , Resultado do Tratamento , Estados Unidos/epidemiologia , Adulto JovemRESUMO
This review discusses the current state of the viral metabolism field and gaps in knowledge that will be important for future studies to investigate. We discuss metabolic rewiring caused by viruses, the influence of oncogenic viruses on host cell metabolism, and the use of viruses as guides to identify critical metabolic nodes for cancer anabolism. We also discuss the need for more mechanistic studies identifying viral proteins responsible for metabolic hijacking and for in vivo studies of viral-induced metabolic rewiring. Improved technologies for detailed metabolic measurements and genetic manipulation will lead to important discoveries over the next decade.
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Neoplasias/metabolismo , Vírus/metabolismo , Interações Hospedeiro-Patógeno , Redes e Vias Metabólicas , Neoplasias/virologiaRESUMO
PURPOSE: The aim of this study was to evaluate both surgical and patient-centered stomal complications after stoma formation, with emphasis on underreported symptoms and complaints. DESIGN: Prospective, single-group study. SUBJECTS AND SETTING: Patients undergoing emergency and elective ostomy surgery between January 1, 1999, and June 1, 2016, in 3 acute care hospitals were followed up by stoma care nurse specialists in NHS Lanarkshire, Scotland. METHODS: Data were collected on surgery type (emergency or elective), stoma type (ileostomy or colostomy), stoma-related complications including surgical complications (stenosis, retractions, hernia, and prolapse) and so-called "patient-centered" complications (skin changes, odor, leakage, soiling, and nighttime emptying) at 5 time points: 10 days, 3 months, 6 months, 1 year, and 2 years postoperatively. For this study, we report comparisons at 10 days and 2 years, using frequencies reported as percentages. RESULTS: Data from 3509 consecutive stoma surgeries were analyzed. Complication rates were similar in both emergency and elective cases. The nighttime symptoms of leakage and soiling were significantly greater in the ileostomy group and worsened over the 2-year period. The parastomal hernia rate was 34.5% at 2 years, a finding more common in the colostomy group (46.4% vs 20.1%, P < .001). However, the rate of clinically significant hernia was similar when comparing the colostomy group with the ileostomy group (3.6% vs 2.2%, P = .38). Emergency stoma surgeries (40.2%) were preoperatively sited compared with 95.9% of elective cases. CONCLUSIONS: Our prospective multicenter study demonstrated that stoma-related complications are similar irrespective of whether the stoma was formed via an elective surgery or emergency surgery. Nighttime symptoms of leakage, soiling, and emptying were high post-stoma formation, particularly in the ileostomy group, and worsened over the 2-year period. Findings from our study highlight the presence and persistence of complications. We believe our work highlights the importance of having frequent discussions with patients about making decisions about approaches to reduce complications to enhance patient outcomes.
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Estomas Cirúrgicos/efeitos adversos , Adulto , Idoso , Estudos de Coortes , Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Procedimentos Cirúrgicos do Sistema Digestório/métodos , Procedimentos Cirúrgicos do Sistema Digestório/estatística & dados numéricos , Feminino , Hospitais/estatística & dados numéricos , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/classificação , Complicações Pós-Operatórias/epidemiologia , Estudos Prospectivos , Escócia/epidemiologiaRESUMO
The interplay between micromorphology and electronic properties is an important theme in organic electronic materials. Here, we show that a spirofluorene-functionalized boron-dipyrromethene (BODIPY) with an alkyl norbornyl tail self-assembles into nanoparticles with qualitatively different properties as compared to the polymerized species. Further, the nanoparticles exhibit a host of unique emissive properties, including photobrightening, a blue satellite peak, and spectral diffusion. Extensive photophysical characterization, including single-particle imaging and spectroscopy, and time-resolved fluorescence, coupled with electronic structure calculations based on an experimentally determined crystal structure, allow a mechanism to be developed. Specifically, BODIPY chromophores are observed to form quasi-two-dimensional layers, where stacking of unit cells adds either J-aggregate character or H-aggregate character depending on the direction of the stacking. Particularly strongly H-coupled domains show the rare process of emission from an upper exciton state, in violation of Kasha's rule, and result in the blue satellite peak. The spatial heterogeneity of structure thus maps onto a gradient of photophysical behavior as seen in single-particle imaging, and the temporal evolution of structure maps onto fluctuating emissive behavior, as seen in single-particle spectroscopy. Taken together, this system provides a striking example of how physical structure and electronic properties are intertwined, and a rare opportunity to use one to chart the other.
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While there is great interest in understanding the fate and transport of nanomaterials in the environment and in biological systems, the detection of nanomaterials in complex matrices by fluorescence methods is complicated by photodegradation, blinking, and the presence of natural organic material and other fluorescent background signals that hamper detection of fluorescent nanomaterials of interest. Optically detected magnetic resonance (ODMR) of nitrogen-vacancy (NV) centers in diamond nanoparticles provides a pathway toward background-free fluorescence measurements, as the application of a resonant microwave field can selectively modulate the intensity from NV centers in nanodiamonds of various diameters in complex materials systems using on-resonance and off-resonance microwave fields. This work represents the first investigation showing how nanoparticle diameter impacts the NV center lifetime and thereby directly impacts the accessible contrast and signal-to-noise ratio when using ODMR to achieve background-free imaging of NV-nanodiamonds in the presence of interfering fluorophores. These results provide new insights that will guide the choice of optimum nanoparticle size and methodology for background-free imaging and sensing applications, while also providing a model system to explore the fate and transport of nanomaterials in the environment.
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Nanodiamantes/química , Fluorescência , Espectroscopia de Ressonância Magnética/métodos , Nitrogênio/química , Tamanho da PartículaRESUMO
BACKGROUND: The non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses. METHODS: The terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5' and 3' rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis. RESULTS: Among the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5' nt variants of T36-CA contained the consensus nts "AATTTCAAA" in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5' terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5' terminal nts, those at the extreme 3' termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3' end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes. CONCLUSIONS: The extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.
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Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Closterovirus/genética , Variação Genética , Genoma Viral , RNA Viral/genética , California , Closterovirus/classificação , Closterovirus/isolamento & purificação , Genótipo , Análise de Sequência de DNARESUMO
The action of molecular catalysts comprises multiple microscopic kinetic steps whose nature is of central importance in determining catalyst activity and selectivity. Single-molecule microscopy enables the direct examination of these steps, including elucidation of molecule-to-molecule variability. Such molecular diversity is particularly important for the behavior of molecular catalysts supported at surfaces. We present the first combined investigation of the initiation dynamics of an operational palladium cross-coupling catalyst at the bulk and single-molecule levels, including under turnover conditions. Base-initiated kinetics reveal highly heterogeneous behavior indicative of diverse catalyst population. Unexpectedly, this distribution becomes more heterogeneous at increasing base concentration. We model this behavior with a two-step saturation mechanism and identify specific microscopic steps where chemical variability must exist in order to yield observed behavior. Critically, we reveal how structural diversity at a surface translates into heterogeneity in catalyst behavior, while demonstrating how single-molecule experiments can contribute to understanding of molecular catalysts.
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A virus with a large genome was identified in the transcriptome of the potato aphid (Macrosiphum euphorbiae) and was named Macrosiphum euphorbiae virus 1 (MeV-1). The MeV-1 genome is 22â780ânt in size, including 3' and 5' non-coding regions, with a single large ORF encoding a putative polyprotein of 7333 aa. The C-terminal region of the predicted MeV-1 polyprotein contained sequences with similarities to helicase, methyltransferase and RNA-dependent RNA polymerase (RdRp) motifs, while the N-terminal region lacked any motifs including structural proteins. Phylogenetic analysis of the helicase placed MeV-1 close to pestiviruses, while the RdRp region placed it close to pestiviruses and flaviviruses, suggesting MeV-1 has a positive-polarity ssRNA genome and is a member of the family Flaviviridae. Since the MeV-1 genome is predicted to contain a methyltransferase, a gene present typically in flaviviruses but not pestiviruses, MeV-1 is likely a member of the genus Flavivirus. MeV-1 was present in nymphal and adult stages of the aphid, aphid saliva and plant tissues fed upon by aphids. However, the virus was unable to multiply and spread in tomato plants. In addition, dsRNA, the replication intermediate of RNA viruses, was isolated from virus-infected M. euphorbiae and not from tomato plants infested with the aphid. Furthermore, nymphs laid without exposure to infected plants harboured the virus, indicating that MeV-1 is an aphid-infecting virus likely transmitted transovarially. The virus was present in M. euphorbiae populations from Europe but not from North America and was absent in all other aphid species tested.
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Afídeos/virologia , Vírus de Insetos/genética , Vírus de Insetos/isolamento & purificação , Animais , Larva , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação , Replicação Viral/fisiologiaRESUMO
The nature of silica surfaces is relevant to many chemical systems, including heterogeneous catalysis and chromatographies utilizing functionalized-silica stationary phases. Surface linkages must be robust to achieve wide and reliable applicability. However, silyl ether-silica support linkages are known to be susceptible to detachment when exposed to basic conditions. We use single-molecule spectroscopy to examine the rate of surface linkage failure upon exposure to base at a variety of deposition conditions. Kinetic analysis elucidates the role of thermal annealing and addition of blocking layers in increasing stability. Critically, it was found that successful surface modification strategies alter the rate at which base molecules approach the silica surface as opposed to reducing surface linkage reactivity. Our results also demonstrate that the innate structural diversity of the silica surface is likely the cause of observed heterogeneity in surface-linkage disruption kinetics.
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RNA silencing functions as an antivirus defense strategy in plants, one that plant viruses counter by producing viral suppressors of RNA silencing (VSRs). VSRs have been identified in three members of the genus Crinivirus but they do not all share identical suppression mechanisms. Here, we used Agrobacterium co-infiltration assays to investigate the suppressor activity of proteins encoded by Lettuce chlorosis virus (LCV). Of 7 LCV proteins (1b, P23, HSP70 homolog, P60, CP, CPm, and P27) tested for the suppression of silencing of green fluorescent protein (GFP) expression in wild-type Nicotiana benthamiana plants, only P23 suppressed the onset of local silencing. Small-interfering (si)RNA accumulation was reduced in leaves co-infiltrated with P23, suggesting that P23 inhibited the accumulation or enhanced the degradation of siRNA. P23 also inhibited the cell-to-cell and systemic movement of RNA silencing in GFP-expressing transgenic N. benthamiana plants. Expression of P23 via agroinfiltration of N. benthamiana leaves induced local necrosis that increased in severity at elevated temperatures, a novelty given that a direct temperature effect on necrosis severity has not been reported for the other crinivirus VSRs. These results further affirm the sophistication of crinivirus VSRs in mediating the evasion of host's antiviral defenses and in symptom modulation.