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1.
Plant Biotechnol J ; 22(5): 1299-1311, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38124291

RESUMO

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (Oryza glaberrima), however, introgression into Oryza sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single and double knockout mutants showed neither substantial growth defects nor symptoms indicative lesion mimic phenotypes, possibly reflecting functional differentiation. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.


Assuntos
Arabidopsis , Oryza , Vírus de Plantas , Oryza/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Arabidopsis/genética , Edição de Genes
2.
Plant Cell ; 33(3): 511-530, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33955487

RESUMO

The leaf vasculature plays a key role in solute translocation. Veins consist of at least seven distinct cell types, with specific roles in transport, metabolism, and signaling. Little is known about leaf vascular cells, in particular the phloem parenchyma (PP). PP effluxes sucrose into the apoplasm as a basis for phloem loading, yet PP has been characterized only microscopically. Here, we enriched vascular cells from Arabidopsis leaves to generate a single-cell transcriptome atlas of leaf vasculature. We identified at least 19 cell clusters, encompassing epidermis, guard cells, hydathodes, mesophyll, and all vascular cell types, and used metabolic pathway analysis to define their roles. Clusters comprising PP cells were enriched for transporters, including SWEET11 and SWEET12 sucrose and UmamiT amino acid efflux carriers. We provide evidence that PP development occurs independently from ALTERED PHLOEM DEVELOPMENT, a transcription factor required for phloem differentiation. PP cells have a unique pattern of amino acid metabolism activity distinct from companion cells (CCs), explaining differential distribution/metabolism of amino acids in veins. The kinship relation of the vascular clusters is strikingly similar to the vein morphology, except for a clear separation of CC from the other vascular cells including PP. In summary, our single-cell RNA-sequencing analysis provides a wide range of information into the leaf vasculature and the role and relationship of the leaf cell types.


Assuntos
Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Transcriptoma/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Floema/metabolismo , Folhas de Planta/genética , Proteínas de Plantas/genética
3.
New Phytol ; 238(2): 637-653, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36636779

RESUMO

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.


Assuntos
Plasmodesmos , Proteoma , Proteoma/metabolismo , Plasmodesmos/metabolismo , Filogenia , Reprodutibilidade dos Testes , Parede Celular/metabolismo
4.
Int J Mol Sci ; 23(20)2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36293360

RESUMO

Most plant viruses lack the 5'-cap and 3'-poly(A) structures, which are common in their host mRNAs, and are crucial for translation initiation. Thus, alternative translation initiation mechanisms were identified for viral mRNAs, one of these being controlled by an RNA element in their 3'-ends that is able to enhance mRNA cap-independent translation (3'-CITE). The 3'-CITEs are modular and transferable RNA elements. In the case of poleroviruses, the mechanism of translation initiation of their RNAs in the host cell is still unclear; thus, it was studied for one of its members, cucurbit aphid-borne yellows virus (CABYV). We determined that efficient CABYV RNA translation requires the presence of a 3'-CITE in its 3'-UTR. We showed that this 3'-CITE requires the presence of the 5'-UTR in cis for its eIF4E-independent activity. Efficient virus multiplication depended on 3'-CITE activity. In CABYV isolates belonging to the three phylogenetic groups identified so far, the 3'-CITEs differ, and recombination prediction analyses suggest that these 3'-CITEs have been acquired through recombination with an unknown donor. Since these isolates have evolved in different geographical regions, this may suggest that their respective 3'-CITEs are possibly better adapted to each region. We propose that translation of other polerovirus genomes may also be 3'-CITE-dependent.


Assuntos
Luteoviridae , Luteoviridae/genética , Fator de Iniciação 4E em Eucariotos/genética , Filogenia , RNA Viral/metabolismo , Regiões 5' não Traduzidas , Regiões 3' não Traduzidas , Proteínas Virais/metabolismo , Biossíntese de Proteínas
5.
Int J Mol Sci ; 23(20)2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36293011

RESUMO

Chronic liver rejection (CR) represents a complex clinical situation because many patients do not respond to increased immunosuppression. Killer cell immunoglobulin-like receptors/Class I Human Leukocyte Antigens (KIR/HLA-I) interactions allow for predicting Natural Killer (NK) cell alloreactivity and influence the acute rejection of liver allograft. However, its meaning in CR liver graft remains controversial. KIR and HLA genotypes were studied in 513 liver transplants using sequence-specific oligonucleotides (PCR-SSO) methods. KIRs, human leucocyte antigen C (HLA-C) genotypes, KIR gene mismatches, and the KIR/HLA-ligand were analyzed and compared in overall transplants with CR (n = 35) and no-chronic rejection (NCR = 478). Activating KIR (aKIR) genes in recipients (rKIR2DS2+ and rKIR2DS3+) increased CR compared with NCR groups (p = 0.013 and p = 0.038). The inhibitory KIR (iKIR) genes in recipients rKIR2DL2+ significantly increased the CR rate compared with their absence (9.1% vs. 3.7%, p = 0.020). KIR2DL3 significantly increases CR (13.1% vs. 5.2%; p = 0.008). There was no influence on NCR. CR was observed in HLA-I mismatches (MM). The absence of donor (d) HLA-C2 ligand (dC2-) ligand increases CR concerning their presence (13.1% vs. 5.6%; p = 0.018). A significant increase of CR was observed in rKIR2DL3+/dC1- (p = 0.015), rKIR2DS4/dC1- (p = 0.014) and rKIR2DL3+/rKIR2DS4+/dC1- (p = 0.006). Long-term patient survival was significantly lower in rKIR2DS1+rKIR2DS4+/dC1- at 5-10 years post-transplant. This study shows the influence of rKIR/dHLA-C combinations and aKIR gene-gene mismatches in increasing CR and KIR2DS1+/C1-ligands and the influence of KIR2DS4+/C1-ligands in long-term graft survival.


Assuntos
Doença Enxerto-Hospedeiro , Hepatopatias , Humanos , Antígenos HLA-C/genética , Rejeição de Enxerto/genética , Ligantes , Receptores KIR/genética , Genótipo , Oligonucleotídeos
6.
Phytopathology ; 109(5): 895-903, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30620690

RESUMO

Moroccan watermelon mosaic virus (MWMV) represents an emerging threat to cucurbit production in the Mediterranean Basin. We sequenced the near complete genome of MWMV-SQ10_1.1, a cloned Spanish isolate. MWMV-SQ10_1.1 has the typical potyvirus genomic structure, and phylogenetic analysis showed that it shared a common ancestor with other Mediterranean MWMV isolates. We used MWMV SQ10_1.1 to inoculate plants in a collection of commercial squash cultivars, including some described as potyvirus resistant. All inoculated plants from all cultivars showed severe infection symptoms. Twenty-four Cucurbita spp. accessions were then tested for their susceptibility to MWMV-SQ10_1.1. Plants of the C. ecuadorensis PI 432441 accession showed no symptoms and their enzyme-linked immunosorbent assay readings were similar to uninfected controls. Progeny analysis of F1 and F2 populations suggested that two recessive genes control PI 432441 resistance to MWMV. We hypothesized that this resistance could be associated with alleles of genes encoding the eukaryotic translation initiation factor 4E (eIF4E), particularly after determination of its recessive nature. A multiple sequence alignment including the two eIF4E ortholog sequences from PI 432441 (CeeIF4E1 and CeeIF4E2) identified three amino acid substitutions in CeeIF4E1 and two amino acid substitutions in CeeIF4E2 potentially involved in potyvirus resistance. Polymerase chain reaction markers for CeeIF4E1 and CeeIF4E2 were developed and used to genotype 156 F2 individuals already phenotyped; this analysis did not support an association of either CeeIF4E2 or CeeIF4E1 with MWMV resistance.


Assuntos
Cucurbita/genética , Resistência à Doença/genética , Doenças das Plantas/genética , Potyvirus/patogenicidade , Cucurbita/virologia , Genes de Plantas , Genes Recessivos , Filogenia , Doenças das Plantas/virologia
7.
Plant Physiol ; 174(3): 1476-1491, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28522457

RESUMO

The association-dissociation of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) with eIF4G is a key control step in eukaryotic translation. The paradigm on the eIF4E-eIF4G interaction states that eIF4G binds to the dorsal surface of eIF4E through a single canonical alpha-helical motif, while metazoan eIF4E-binding proteins (m4E-BPs) advantageously compete against eIF4G via bimodal interactions involving this canonical motif and a second noncanonical motif of the eIF4E surface. Metazoan eIF4Gs share this extended binding interface with m4E-BPs, with significant implications on the understanding of translation regulation and the design of therapeutic molecules. Here we show the high-resolution structure of melon (Cucumis melo) eIF4E in complex with a melon eIF4G peptide and propose the first eIF4E-eIF4G structural model for plants. Our structural data together with functional analyses demonstrate that plant eIF4G binds to eIF4E through both the canonical and noncanonical motifs, similarly to metazoan eIF4E-eIF4G complexes. As in the case of metazoan eIF4E-eIF4G, this may have very important practical implications, as plant eIF4E-eIF4G is also involved in a significant number of plant diseases. In light of our results, a universal eukaryotic bipartite mode of binding to eIF4E is proposed.


Assuntos
Cucumis melo/metabolismo , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/química , Fator de Iniciação Eucariótico 4G/metabolismo , Peptídeos/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Substituição de Aminoácidos , Cristalografia por Raios X , Resistência à Doença/genética , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutação/genética , Ligação Proteica , Domínios Proteicos , Alinhamento de Sequência
8.
Phytopathology ; 107(9): 1080-1086, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28545349

RESUMO

Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus (family Virgaviridae) that causes serious economic losses in cucurbit crops. A possibility for CGMMV control is the use of cross-protection, for which stable attenuated isolates are required. In this study, an infectious clone was constructed for the hn isolate of CGMMV. Unexpectedly, this clone carried a nonconserved mutation involving a single nucleotide change resulting in the replacement of Arg by Cys at residue 284 of the replicase protein; this mutation correlated with delayed symptom induction and RNA accumulation, as shown in time-course experiments. Sequencing of the viral progeny showed that restoration of wild-type symptoms and increased RNA accumulation correlated with reversion of the mutation to the wild-type sequence, a phenomenon that occurred at approximately 7 to 10 days postinoculation. Thus, Arg284 seems to be crucial but not strictly necessary for virus infection. Subsequently, four other mutants in the triplet encoding Arg284 were constructed and assayed. Results showed that symptoms and their timing were diverse for the different mutants, with enhanced pathogenicity and RNA accumulation always correlating with reversion to Arg284. Therefore, the nature of the mutation strongly influenced the genetic stability of the mutant. At least two mutants were identified for which reversion did not occur by 30 days postinoculation, and these were defined as good candidates to attain stable symptom attenuation that could be useful in cross-protection.


Assuntos
Cucumis sativus/virologia , Doenças das Plantas/virologia , Vírus de Plantas/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Substituição de Aminoácidos , Regulação Enzimológica da Expressão Gênica , Regulação Viral da Expressão Gênica , Modelos Moleculares , Mutação , Vírus de Plantas/genética , Conformação Proteica , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
Alcohol Clin Exp Res ; 40(4): 794-805, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27012317

RESUMO

BACKGROUND: Alcoholic cirrhosis (AC) is a common cause of death among individuals abusing alcohol. In the last resort, liver transplantation (LT) is considered the only solution to save the patient's life, generating socioeconomic and public health problems. Clinical and sociodemographic characteristics, rejection frequency, and short- and long-term graft survival are not well known in end-term AC patients undergoing LT. The aim was to determine the sociodemographic and clinical characteristics, their incidence in LT, main pre- and posttransplant complications, and short- and long-term post-transplant graft survival in AC patients in southeastern Spain. METHODS: The medical records of 1,026 patients who underwent LT over the last 23 years were retrospectively reviewed, and demographic data and posttransplant survivals were analyzed and compared. Biochemical characteristics, major pre- and posttransplant complications and short- and long-term survivals were analyzed in a total of 398 male patients with AC undergoing LT. RESULTS: AC and viral cirrhosis are the main indications for LT in our study. Mostly represented in our study are AC men without associated viral infections with a mean age of 53.06 years. Main pretransplant complications in AC patients are ascites (78.3%) and encephalopathy (43.5%), while acute graft rejection is the most common liver posttransplant complication (26.6%), nevertheless with low graft loss frequency (1.1%). AC and autoimmune cirrhosis show the best posttransplant survival in both the short and long term. Patients with AC included on the waiting list for LT were Child-Pugh class B (52.1%) and Model for End-Stage Liver Disease score of 10 to 19 (71.2%). The highest percentage of AC patient survival was observed at 1 year posttransplant (81.2%) and progressively decreased over time up to 10 years posttransplant (69.6%). Pretransplant complications such as ascites and encephalopathy did not have an influence on the percentage of posttransplant survivals, although better survival rates were observed in nonviral AC patients. CONCLUSIONS: AC without viral infections is the main indication for LT in southeastern Spain although its frequency has decreased in last decade. AC is a good indication for LT for its high survival rate and few posttransplant complications. Despite having a high percentage of pretransplant complications (ascites and encephalopathy) but does not appear to influence survivals being observed posttransplant survival rates above those expected. Conversely, viral infections in the patient with AC decrease patient survivals. The main future goals are design new strategies to detect, treat, and reduce AC frequency in our population and know alcoholic recidivism rate posttransplant in our population.


Assuntos
Cirrose Hepática Alcoólica/mortalidade , Cirrose Hepática Alcoólica/cirurgia , Transplante de Fígado/mortalidade , Transplante de Fígado/tendências , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Espanha/epidemiologia , Taxa de Sobrevida/tendências
10.
Liver Transpl ; 21(8): 1056-65, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25990257

RESUMO

A national, multicenter, retrospective study was conducted to assess the results obtained for liver transplant recipients with conversion to everolimus in daily practice. The study included 477 recipients (481 transplantations). Indications for conversion to everolimus were renal dysfunction (32.6% of cases), hepatocellular carcinoma (HCC; 30.2%; prophylactic treatment for 68.9%), and de novo malignancy (29.7%). The median time from transplantation to conversion to everolimus was 68.7 months for de novo malignancy, 23.8 months for renal dysfunction, and 7.1 months for HCC and other indications. During the first year of treatment, mean everolimus trough levels were 5.4 (standard deviation [SD], 2.7) ng/mL and doses remained stable (1.5 mg/day) from the first month after conversion. An everolimus monotherapy regimen was followed by 28.5% of patients at 12 months. Patients with renal dysfunction showed a glomerular filtration rate (4-variable Modification of Diet in Renal Disease) increase of 10.9 mL (baseline mean, 45.8 [SD, 25.3] versus 57.6 [SD, 27.6] mL/minute/1.73 m(2) ) at 3 months after everolimus initiation (P < 0.001), and 6.8 mL at 12 months. Improvement in renal function was higher in patients with early conversion (<1 year). Adverse events were the primary reason for discontinuation in 11.2% of cases. The probability of survival at 3 years after conversion to everolimus was 83.0%, 71.1%, and 59.5% for the renal dysfunction, de novo malignancy, and HCC groups, respectively. Everolimus is a viable option for the treatment of renal dysfunction, and earlier conversion is associated with better recovery of renal function. Prospective studies are needed to confirm advantages in patients with malignancy.


Assuntos
Substituição de Medicamentos , Everolimo/administração & dosagem , Rejeição de Enxerto/prevenção & controle , Imunossupressores/administração & dosagem , Rim/efeitos dos fármacos , Transplante de Fígado , Adolescente , Adulto , Idoso , Criança , Monitoramento de Medicamentos , Everolimo/efeitos adversos , Everolimo/sangue , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/sangue , Rim/fisiopatologia , Transplante de Fígado/efeitos adversos , Transplante de Fígado/mortalidade , Masculino , Pessoa de Meia-Idade , Recuperação de Função Fisiológica , Estudos Retrospectivos , Fatores de Risco , Espanha , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
11.
Hepatol Res ; 45(4): 423-31, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24906075

RESUMO

AIM: The aim of our study was develop and validate an algorithm system based on morphological features for finding the differences between recurrent hepatitis C virus (HCV) and acute cellular rejection (ACR) in liver biopsies of HCV-transplanted patients. METHODS: Two hundred and eighty-eight liver biopsies were analyzed from 121 patients transplanted for HCV. A diagnostic consensus was reached between clinicians and pathologists in 214 biopsies for the diagnosis of recurrent HCV or ACR. A random sample of 114 liver biopsies (derivation cohort) was taken to generate the diagnostic tree and was subsequently evaluated using the validation cohort in 100 liver biopsies by recursive partitioning analysis of morphological variables and time since transplantation. RESULTS: The presence of endotheliitis together with a time of less than 6 weeks since LT definitely excluded recurrent HCV. After obtaining the regression tree, diagnostic accuracy was 96% and 93% in the derivation and validation cohort, respectively. Both cases surpassed the pathologist's original diagnosis, which had a diagnostic accuracy of 91% (P < 0.05, for both comparisons). CONCLUSION: A recursive partitioning analysis of the morphological features in liver biopsies from HCV-transplanted patients may be useful for easily distinguishing between recurrent HCV and ACR.

12.
New Phytol ; 202(1): 233-246, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24372390

RESUMO

Many plant viruses depend on functional RNA elements, called 3'-UTR cap-independent translation enhancers (3'-CITEs), for translation of their RNAs. In this manuscript we provide direct proof for the existing hypothesis that 3'-CITEs are modular and transferable by recombination in nature, and that this is associated with an advantage for the created virus. By characterizing a newly identified Melon necrotic spot virus (MNSV; Tombusviridae) isolate, which is able to overcome eukaryotic translation initiation factor 4E (eIF4E)-mediated resistance, we found that it contains a 55 nucleotide insertion in its 3'-UTR. We provide strong evidence that this insertion was acquired by interfamilial recombination with the 3'-UTR of an Asiatic Cucurbit aphid-borne yellows virus (CABYV; Luteoviridae). By constructing chimeric viruses, we showed that this recombined sequence is responsible for resistance breaking. Analysis of the translational efficiency of reporter constructs showed that this sequence functions as a novel 3'-CITE in both resistant and susceptible plants, being essential for translation control in resistant plants. In conclusion, we showed that a recombination event between two clearly identified viruses from different families led to the transfer of exactly the sequence corresponding to a functional RNA element, giving rise to a new isolate with the capacity to infect an otherwise nonsusceptible host.


Assuntos
Resistência à Doença/imunologia , Luteoviridae/genética , Doenças das Plantas/virologia , Biossíntese de Proteínas/genética , RNA Viral/genética , Recombinação Genética , Tombusviridae/genética , Sequência de Bases , Elementos Facilitadores Genéticos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Especificidade de Hospedeiro , Luteoviridae/fisiologia , Dados de Sequência Molecular , Mutagênese Insercional/genética , Conformação de Ácido Nucleico , Doenças das Plantas/imunologia , Capuzes de RNA/metabolismo , RNA Viral/química , Tombusviridae/isolamento & purificação , Tombusviridae/patogenicidade , Tombusviridae/fisiologia , Virulência
13.
J Plant Physiol ; 270: 153633, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35151953

RESUMO

During multicellularization, plants evolved unique cell-cell connections, the plasmodesmata (PD). PD of angiosperms are complex cellular domains, embedded in the cell wall and consisting of multiple membranes and a large number of proteins. From the beginning, it had been assumed that PD provide passage for a wide range of molecules, from ions to metabolites and hormones, to RNAs and even proteins. In the context of assimilate allocation, it has been hypothesized that sucrose produced in mesophyll cells is transported via PD from cell to cell down a concentration gradient towards the phloem. Entry into the sieve element companion cell complex (SECCC) is then mediated on three potential routes, depending on the species and conditions, - either via diffusion across PD, after conversion to raffinose via PD using a polymer trap mechanism, or via a set of transporters which secrete sucrose from one cell and secondary active uptake into the SECCC. Multiple loading mechanisms can likely coexist. We here review the current knowledge regarding photoassimilate transport across PD between cells as a prerequisite for translocation from leaves to recipient organs, in particular roots and developing seeds. We summarize the state-of-the-art in protein composition, structure, transport mechanism and regulation of PD to apprehend their functions in carbohydrate allocation. Since many aspects of PD biology remain elusive, we highlight areas that require new approaches and technologies to advance our understanding of these enigmatic and important cell-cell connections.

14.
Arch Med Sci ; 17(3): 764-774, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025847

RESUMO

INTRODUCTION: The molecular mechanisms underlying alcoholic liver fibrosis and cirrhosis are not completely understood. Hepatic fibrosis involves the interplay of diverse cells and factors, including hepatic stellate cells (HSCs), Kupffer, NK cells, and T-lymphocyte subsets. Killer-cell immunoglobulin-like receptors (KIR) are membrane receptors involved in mediation between NK and activated HSCs, regulating NK cell function through their interaction with HLA-I molecules. The aim of this study was to analyse the genetic association between KIR genes and the susceptibility to or protection from alcoholic cirrhosis (AC) in a cohort of male AC patients undergoing liver transplantation (LT) with and without concomitant viral infections. MATERIAL AND METHODS: KIR genotyping was performed in nuclear DNA extracted from 281 AC patients and compared with 319 male controls. RESULTS: Significant differences between total AC patients and healthy controls were only found in the case of KIR2DL2 and KIR2DS5. KIR2DL2 was significantly underrepresented in non-viral AC patients (52.6% vs. 63.3%; p = 0.015), while patients heterozygous for KIR2DL2 were also underrepresented in the non-viral AC group compared with controls (p = 0.034). KIR2DS5 was overrepresented in this group compared with healthy controls (p = 0.002). All these observations were only evident in AC patients older than 54 years old. CONCLUSIONS: Our data suggest a contrary effect of KIR2DL2 and KIR2DS5 in AC patients older than 54 years, in whom the presence of KIR2DL2 appears to be protective against AC, whereas the presence of KIR2DS5 seems to promote the fibrotic process, particularly in patients with no associated viral infection.

15.
J Virol Methods ; 278: 113837, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32061591

RESUMO

Melon necrotic spot virus (MNSV) is endemic in cucurbit crops worldwide, causing epidemic outbreaks from time to time. MNSV is transmitted in nature by a soil-inhabiting fungus and also through seeds, making its detection in seed certification programs a necessity. Polyclonal antisera and RT-PCR-based detection assays have been developed for MNSV, but up to now no monoclonal antibodies (mAbs) have been described for this virus. In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10-3-10-4 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as IgG2a and kappa light-chain. Western-blot analyses showed that mAb 2D4H4 provided sensitive and specific detection of MNSV. A TAS-ELISA protocol was developed for mAb 2D4H4. Using this protocol, limits of detection of 1:20,480 and 1:10,240 (g/mL, w/v) were attained for the homologous isolate and a heterologous MNSV isolate, respectively. Moreover, mAb 2D4H4 was used successfully to localize the MNSV CP in infected cells by immunocytochemistry/transmission electron microscopy, illustrating the usefulness of this mAb for advanced cellular studies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Tombusviridae/imunologia , Animais , Linhagem Celular , Hibridomas , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Doenças das Plantas/virologia , Folhas de Planta/virologia
16.
Front Plant Sci ; 10: 1583, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31850047

RESUMO

Cucumber vein yellowing virus (CVYV) causes severe yield losses in cucurbit crops across Mediterranean countries. The control of this virus is based on cultural practices to prevent the presence of its vector (Bemisia tabaci) and breeding for natural resistance, which requires the identification of the loci involved and the development of molecular markers for linkage analysis. In this work, we mapped a monogenic locus for resistance to CVYV in cucumber by using a Bulked Segregant Analysis (BSA) strategy coupled with whole-genome resequencing. We phenotyped 135 F3 families from a segregating population between a susceptible pickling cucumber and a resistant Long Dutch type cucumber for CVYV resistance. Phenotypic analysis determined the monogenic and incomplete dominance inheritance of the resistance. We named the locus CsCvy-1. For mapping this locus, 15 resistant and 15 susceptible homozygous F2 individuals were selected for whole genome resequencing. By using a customized bioinformatics pipeline, we identified a unique region in chromosome 5 associated to resistance to CVYV, explaining more than 80% of the variability. The resequencing data provided us with additional SNP markers to decrease the interval of CsCvy-1 to 625 kb, containing 24 annotated genes. Markers flanking CsCvy-1 in a 5.3 cM interval were developed for marker-assisted selection (MAS) in breeding programs and will be useful for the identification of the target gene in future studies.

17.
Exp Clin Transplant ; 17(3): 355-362, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-29957165

RESUMO

OBJECTIVES: The aim of this study was to analyze alcoholic cirrhosis in women who were to undergo liver transplant, including their biochemical and clinical characteristics, main complications, survival rates, and main causes of death compared with men with alcoholic cirrhosis. MATERIALS AND METHODS: Our study included 400 patients with alcoholic cirrhosis, which we divided according to sex and viral infections. Biochemical parameters and the presence and degree of ascites and encephalopathy, liver function status, and liver rejection and survival rates were analyzed from 1 to 10 years and the main cause of death at 10 years. RESULTS: Patients with nonviral alcoholic cirrhosis and liver transplant had significantly better survival rates (84.1%) at 1 year versus those with viral alcoholic cirrhosis (74.5%; P = .036). Men with nonviral alcoholic cirrhosis (14%) and women with hepatitis C virus (29%) had the lowest short-term survival rates. In long-term survival analysis, the lowest rate was observed in women with nonviral alcoholic cirrhosis (26.1%), and the highest rate was observed in women with hepatitis C virus (42.9%). Liver graft failure was one of the main causes of death in male patients (19.5%). CONCLUSIONS: Women with alcoholic cirrhosis showed a higher rate of ascites and encephalopathy but lower liver graft rejection than men with alcoholic cirrhosis. Survival rates were similar between men and women, although slightly lower in women who had hepatitis C virus.


Assuntos
Cirrose Hepática Alcoólica/cirurgia , Transplante de Fígado , Causas de Morte , Feminino , Humanos , Cirrose Hepática Alcoólica/complicações , Cirrose Hepática Alcoólica/diagnóstico , Cirrose Hepática Alcoólica/mortalidade , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores Sexuais , Taxa de Sobrevida
18.
Transpl Immunol ; 18(3): 246-9, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18047932

RESUMO

The CTLA4 molecule is a homolog for CD28, and both molecules and their common ligands (CD80 and CD86) constitute the B7/CD28-CTLA4 co-stimulatory pathway for T cells activation. The CTLA4-ligand interaction has an inhibitory effect on T cell activation and might contribute to peripheral tolerance. A recently described CT60 A/G polymorphism affects the production of soluble CTLA4 and is strongly associated with immune-mediated diseases and with allogenic stem cell transplantation outcome. Thus, we examined this marker on liver transplant outcome by a PCR-RFLP method. The CT60 G allele was significantly associated with acute rejection (Pc=0.038; OR=1.49; AR vs. NAR). Patients who lacked this allele had the lowest risk of acute rejection development. Allograft survival data did not show statistical differences between genotypes. In conclusion, CT60 A/G dimorphism within the 3'-UTR of CTLA4 gene, which encodes for reduced sCTLA4 production, influence acute rejection development in liver transplantation.


Assuntos
Regiões 3' não Traduzidas , Antígenos CD/genética , Antígenos de Diferenciação/genética , Rejeição de Enxerto , Transplante de Fígado , Polimorfismo de Nucleotídeo Único , Adulto , Antígeno CTLA-4 , Feminino , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto , Humanos , Imunossupressores/metabolismo , Estimativa de Kaplan-Meier , Transplante de Fígado/imunologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição
19.
Front Plant Sci ; 9: 625, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29868081

RESUMO

In eukaryotes, the formation of a 5'-cap and 3'-poly(A) dependent protein-protein bridge is required for translation of its mRNAs. In contrast, several plant virus RNA genomes lack both of these mRNA features, but instead have a 3'-CITE (for cap-independent translation enhancer), a RNA element present in their 3'-untranslated region that recruits translation initiation factors and is able to control its cap-independent translation. For several 3'-CITEs, direct RNA-RNA long-distance interactions based on sequence complementarity between the 5'- and 3'-ends are required for efficient translation, as they bring the translation initiation factors bound to the 3'-CITE to the 5'-end. For the carmovirus melon necrotic spot virus (MNSV), a 3'-CITE has been identified, and the presence of its 5'-end in cis has been shown to be required for its activity. Here, we analyze the secondary structure of the 5'-end of the MNSV RNA genome and identify two highly conserved nucleotide sequence stretches that are complementary to the apical loop of its 3'-CITE. In in vivo cap-independent translation assays with mutant constructs, by disrupting and restoring sequence complementarity, we show that the interaction between the 3'-CITE and at least one complementary sequence in the 5'-end is essential for virus RNA translation, although efficient virus translation and multiplication requires both connections. The complementary sequence stretches are invariant in all MNSV isolates, suggesting that the dual 5'-3' RNA:RNA interactions are required for optimal MNSV cap-independent translation and multiplication.

20.
Transplantation ; 84(5): 650-4, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17876280

RESUMO

Lamivudine combined with hepatitis B immune globulin (HBIg) is the standard of care for preventing the recurrence hepatitis B virus after liver transplant. To determine the risk of hepatitis B virus (HBV) recurrence after early withdrawal of HBIg in patients receiving lamivudine maintenance therapy, 20 patients receiving a course of HBIg and lamivudine after transplantation and long-term maintenance therapy with lamivudine and 9 patients receiving HBIg and lamivudine indefinitely were analyzed. The survival rate was 90% after a mean follow-up of 83 months. The HBV recurrence rate was 14% with a mean period of 91 months free from HBV recurrence. Both groups had similar HBV recurrence rates, 15% for the combination and 11% for lamivudine alone. Four patients, 3 of whom were noncompliant with therapy, experienced posttransplant HBV recurrence. Patients who adhere to long-term prophylaxis with lamivudine after early withdrawal of HBIg have a low risk of HBV recurrence, similar to those who receive combination prophylaxis.


Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatite B/prevenção & controle , Imunoglobulinas/administração & dosagem , Lamivudina/farmacologia , Transplante de Fígado , Intervalo Livre de Doença , Feminino , Seguimentos , Hepatite B/imunologia , Hepatite B/virologia , Humanos , Imunoglobulinas/imunologia , Lamivudina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Prevenção Secundária , Fatores de Tempo
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