RAV1 mediates cytokinin signaling for regulating primary root growth in Arabidopsis.

Root growth dynamics is an outcome of complex hormonal crosstalk. The primary root meristem size, for example, is determined by antagonizing actions of cytokinin and auxin. Here we show that RAV1, a member of the AP2/ERF family of transcription factors, mediates cytokinin signaling in roots to regulate meristem size. The rav1 mutants have prominently longer primary roots, with a meristem that is significantly enlarged and contains higher cell numbers, compared with wild-type. The mutant phenotype could be restored on exogenous cytokinin application or by inhibiting auxin transport. At the transcript level, primary cytokinin-responsive genes like ARR1, ARR12 were significantly downregulated in the mutant root, indicating impaired cytokinin signaling. In concurrence, cytokinin induced regulation of SHY2, an Aux/IAA gene, and auxin efflux carrier PIN1 was hindered in rav1, leading to altered auxin transport and distribution. This effectively altered root meristem size in the mutant. Notably, CRF1, another member of the AP2/ERF family implicated in cytokinin signaling, is transcriptionally repressed by RAV1 to promote cytokinin response in roots. Further associating RAV1 with cytokinin signaling, our results demonstrate that cytokinin upregulates RAV1 expression through ARR1, during post-embryonic root development. Regulation of RAV1 expression is a part of secondary cytokinin response that eventually represses CRF1 to augment cytokinin signaling. To conclude, RAV1 functions in a branch pathway downstream to ARR1 that regulates CRF1 expression to enhance cytokinin action during primary root development in Arabidopsis.

Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress.

BACKGROUND: Despite its known significance in plant abiotic stress responses, the role of the RAV gene family in the response of Capsicum annuum to chilling stress remains largely unexplored. RESULTS: In this study, we identified and characterized six members of the CaRAV gene subfamily in pepper plants through genome-wide analysis. Subsequently, the CaRAV subfamily was classified into four branches based on homology with Arabidopsis thaliana, each exhibiting relatively conserved domains within the branch. We discovered that light response elements accounted for the majority of CaRAVs, whereas low-temperature response elements were specific to the NGA gene subfamily. After pepper plants were subjected to chilling stress, qRT‒PCR analysis revealed that CaRAV1, CaRAV2 and CaNGA1 were significantly induced in response to chilling stress, indicating that CaRAVs play a role in the response to chilling stress. Using virus-induced gene silencing (VIGS) vectors, we targeted key members of the CaRAV gene family. Under normal growth conditions, the MDA content and SOD enzyme activity of the silenced plants were slightly greater than those of the control plants, and the REC activity was significantly greater than that of the control plants. The levels of MDA and electrolyte leakage were greater in the silenced plants after they were exposed to chilling stress, and the POD and CAT enzyme activities were significantly lower than those in the control, which was particularly evident under repeated chilling stress. In addition, the relative expression of CaPOD and CaCAT was greater in V2 plants upon repeated chilling stress, especially CaCAT was significantly greater in V2 plants than in the other two silenced plants, with 3.29 and 1.10 increases within 12 and 24 h. These findings suggest that CaRAV1 and CaNGA1 positively regulate the response to chilling stress. CONCLUSIONS: Silencing of key members of the CaRAV gene family results in increased susceptibility to chilling damage and reduced antioxidant enzyme activity in plants, particularly under repeated chilling stress. This study provides valuable information for understanding the classification and putative functions of RAV transcription factors in pepper plants.

Molecular identification and functional characterization of a transcription factor GeRAV1 from Gelsemium elegans.

BACKGROUND: Gelsemium elegans is a traditional Chinese medicinal plant and temperature is one of the key factors affecting its growth. RAV (related to ABI3/VP1) transcription factor plays multiple roles in higher plants, including the regulation of plant growth, development, and stress response. However, RAV transcription factor in G. elegans has not been reported. RESULTS: In this study, three novel GeRAV genes (GeRAV1-GeRAV3) were identified from the transcriptome of G. elegans under low temperature stress. Phylogenetic analysis showed that GeRAV1-GeRAV3 proteins were clustered into groups II, IV, and V, respectively. RNA-sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR) analyses indicated that the expression of GeRAV1 and GeRAV2 was increased in response to cold stress. Furthermore, the GeRAV1 gene was successfully cloned from G. elegans leaf. It encoded a hydrophilic, unstable, and non-secretory protein that contained both AP2 and B3 domains. The amino acid sequence of GeRAV1 protein shared a high similarity of 81.97% with Camptotheca acuminata CaRAV. Subcellular localization and transcriptional self-activation experiments demonstrated that GeRAV1 was a nucleoprotein without self-activating activity. The GeRAV1 gene was constitutively expressed in the leaves, stems, and roots of the G. elegans, with the highest expression levels in roots. In addition, the expression of the GeRAV1 gene was rapidly up-regulated under abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) stresses, suggesting that it may be involved in hormonal signaling pathways. Moreover, GeRAV1 conferred improved cold and sodium chloride tolerance in Escherichia coli Rosetta cells. CONCLUSIONS: These findings provided a foundation for further understanding on the function and regulatory mechanism of the GeRAV1 gene in response to low-temperature stress in G. elegans.

Cyclopeptide RA-V from Rubia yunnanensis restores activity of Adagrasib against colorectal cancer by reducing the expression of Nrf2.

Adagrasib (MRTX849), an approved and promising KRAS G12C inhibitor, has shown the promising results for treating patients with advanced non-small cell lung cancer (NSCLC) or colorectal cancer (CRC) harboring KRAS-activating mutations. However, emergence of the acquired resistance limits its long-term efficacy and clinical application. Further understanding of the mechanism of the acquired resistance is crucial for developing more new effective therapeutic strategies. Herein, we firstly found a new connection between the acquired resistance to MRTX849 and nuclear factor erythroid 2-related factor 2 (Nrf2). The expression levels of Nrf2 and GLS1 proteins were substantially elevated in different CRC cell lines with the acquired resistance to MRTX849 in comparison with their corresponding parental cell lines. Next, we discovered that RA-V, one of natural cyclopeptides isolated from the roots of Rubia yunnanensis, could restore the response of resistant CRC cells to MRTX849. The results of molecular mechanisms showed that RA-V suppressed Nrf2 protein through the ubiquitin-proteasome-dependent degradation, leading to the induction of oxidative and ER stress, and DNA damage in CRC cell lines. Consequently, RA-V reverses the resistance to MRTX849 by inhibiting the Nrf2/GLS1 axis, which shows the potential for further developing into one of novel adjuvant therapies of MRTX849.

Reciprocal inhibition of expression between RAV1 and BES1 modulates plant growth and development in Arabidopsis.

RAV1 (Related to ABI3/VP1) is a plant-specific B3 and AP2 domain-containing transcription factor that acts as a negative regulator of growth in many plant species. The expression of RAV1 is downregulated by brassinosteroids (BRs); large-scale transcriptome analyses have shown that the expression of RAV1 was previously targeted by BRI1-EMS-SUPPRESOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1), which are critical transcription factors for the BR-signaling process. Using RAV1-overexpressing transgenic plants, we showed that RAV1 overexpression reduced the BR signaling capacity, resulting in the downregulation of BR biosynthetic genes and BES1 expression. Furthermore, we demonstrated that BES1, not BZR1, is directly bound to the RAV1 promoter and repressed RAV1 expression, and vice versa; RAV1 is also bound to the BES1 promoter and repressed BES1 expression. This mutual inhibition was specific to RAV1 and BES1 because RAV1 exhibited binding activity to the BZR1 promoter but did not repress BZR1 expression. We observed that constitutively activated BR signaling phenotypes in bes1-D were attenuated by the repression of endogenous BES1 expression in transgenic bes1-D plants overexpressing RAV1. RNA-sequencing analysis of RAV1-overexpressing transgenic plants and bes1-D mutant plants revealed differentially expressed genes by RAV1 and BES1 and genes that were oppositely co-regulated by RAV1 and BES1. RAV1 and BES1 regulated different transcriptomes but co-regulated a specific set of genes responsible for the balance between growth and defense. These results suggested that the mutual inhibitory transcriptional activities of RAV1 and BES1 provide fine regulatory mechanisms for plant growth and development.

The floral repressors TEMPRANILLO1 and 2 modulate salt tolerance by regulating hormonal components and photo-protection in Arabidopsis.

Members of the plant specific RAV family of transcription factors regulate several developmental and physiological processes. In the model plant Arabidopsis thaliana, the RAV TEMPRANILLO 1 (TEM1) and TEM2 control important phase changes such as the juvenile to adult and the vegetative to reproductive transitions. Besides their known regulatory function in plant development, a transcriptomics analysis of transgenic plants overexpressing TEM1 also revealed overrepresentation of Gene Ontology (GO) categories related to abiotic stress responses. Therefore, to investigate the biological relevance of these TEM-dependent transcriptomic changes and elucidate whether TEMs contribute to the modulation of plant growth in response to salinity, we analyzed the behavior of TEM gain and loss of function mutants subjected to mild and high salt stresses at different development stages. With respect to increasing salinity, TEM overexpressing plants were hypersensitive whereas the tem1 tem2 double mutants were more tolerant. Precisely, tem1 tem2 mutants germinated and flowered faster than the wild-type plants under salt stress conditions. Also, tem1 tem2 plants showed a delay in salt-induced leaf senescence, possibly as a consequence of downregulation of jasmonic acid biosynthesis genes. Besides a shorter life cycle and delayed senescence, tem1 tem2 mutants appeared to be better suited to withstand oxidative stress as they accumulated higher levels of α-tocopherol (an important antioxidant metabolite) and displayed a slower degradation of photosynthetic pigments. Taken together, our studies suggest novel and crucial roles for TEM in adaptive growth as they modulate plant development in response to environmental changes such as increasing soil salinity.

The co-modulation of RAV transcription factors in ROS burst and extensive transcriptional reprogramming underlies disease resistance in cassava.

KEY MESSAGE: MeRAVs positively regulate ROS burst and the expression of downstream disease resistance-related genes, which underlie improved disease resistance to Xam. Cassava (Manihot esculenta Crantz) is an important food crop and energy crop, but its yield is seriously affected by cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam). Related to ABI3/VP1 (RAV) transcription factor family belongs to the APETALA2/Ethylene-Responsive Factor (AP2/ERF) family, which plays an important role in plant growth, development and response to biotic and abiotic stresses. In this study, we found that MeRAVs positively co-regulates the resistance to Xam and stimulates the innate immune response by regulating reactive oxygen species (ROS) burst in cassava. Dual-luciferase assay showed that seven MeRAVs exhibited transcriptional activate activity by binding CAACA motif and CACCTG motif. A large number of differentially expressed genes (DEGs) were identified through RNA-seq analysis of MeRAVs-silenced lines, and the DEGs co-regulated by seven MeRAVs accounted for more than 45% of the total DEGs. In addition, seven MeRAVs positively regulate expression of disease resistance-related genes through directly binding to their promoters. In summary, MeRAVs co-regulate ROS burst and the expression of downstream disease resistance-related genes, which underlie improved disease resistance to Xam.

RAV transcription factor regulatory function in response to salt stress in two Iranian wheat landraces.

Amongst the transcription factor groups, the AP2/ERF (Apetala2/Ethylene Response Factor) superfamily is one of the main groups in plants and plays an essential role in tolerating abiotic and biotic stresses. The AP2/ERF superfamily consists of ERF, AP2, RAV, and Soloist families based on the AP2 domain number. The RAV (Related to ABI3/VP1) family members have been revealed to be stimulate by a number of biotic and abiotic environmental incentives; including pathogen infection, salicylic acid, osmotic stress, cold, high salinity, wounding, and exogenous hormone application. However, limited data are available on the contributions of RAV transcription factors in wheat (Triticum aestivum L.). In the present study, a total of 26 RAV genes were identified in wheat from a genome-wide search against the latest wheat genome data. Phylogenetic and sequence alignment analyses divided the wheat RAV genes into 4 clusters, I, II, III and IV. Chromosomal distribution, gene structure and motif composition were subsequently investigated. The 26 TaRAV genes were unevenly distributed on 21 chromosomes. After cloning and sequencing of 7 TaRAVs candidate genes the expression levels of two TaRAVs, TaRAV4 and TaRAV5, were validated through qPCR analyses in two salt-tolerant Iranian landraces of wheat. Our results showed that the TaRAV4 and TaRAV5 were co-expressed in wheat tissues and were highly correlated to salt tolerance indices such as the K+/Na+ ratio. Protein interaction revealed that the TaRAV4 and TaRAV5 were related to vital proteins such as PK4 and PP2C, and MYB and Zinc finger transcription factors, and Gigantea proteins. This study improved our knowledge of the RAV gene family function in wheat and the probable role of RAVs in salt tolerance mechanisms to improve crop production under changing environments. Also, the two relatively salt-tolerant landraces of wheat that were examined in this study could be suitable candidates for future breeding studies.

Genome-Wide Analysis of the RAV Gene Family in Wheat and Functional Identification of TaRAV1 in Salt Stress.

RAV transcription factors (TFs) are unique to higher plants and contain both B3 and APETALA2 (AP2) DNA binding domains. Although sets of RAV genes have been identified from several species, little is known about this family in wheat. In this study, 26 RAV genes were identified in the wheat genome. These wheat RAV TFs were phylogenetically clustered into three classes based on their amino acid sequences. A TaRAV gene located on chromosome 1D was cloned and named TaRAV1. TaRAV1 was expressed in roots, stems, leaves, and inflorescences, and its expression was up-regulated by heat while down-regulated by salt, ABA, and GA. Subcellular localization analysis revealed that the TaRAV1 protein was localized in the nucleus. The TaRAV1 protein showed DNA binding activity in the EMSA assay and transcriptional activation activity in yeast cells. Overexpressing TaRAV1 enhanced the salt tolerance of Arabidopsis and upregulated the expression of SOS genes and other stress response genes. Collectively, our data suggest that TaRAV1 functions as a transcription factor and is involved in the salt stress response by regulating gene expression in the SOS pathway.

Characterization of Chromatin Accessibility and Gene Expression upon Cold Stress Reveals that the RAV1 Transcription Factor Functions in Cold Response in Vitis Amurensis.

Cold tolerance is regulated by a variety of transcription factors (TFs) and their target genes. Except for the well-characterized C-repeat binding factors (CBFs)-dependent transcriptional cascade, the mechanisms of cold tolerance mediated by other transcriptional regulatory networks are still largely unknown. Here, we used the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq to identify cold responsive TFs in Vitis amurensis, a grape species with high cold hardiness. Nine TFs, including CBF4, RAV1 and ERF104, were identified after cold treatment. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) analysis revealed that these TFs may regulate cold response through different pathways. As a prime candidate TF, overexpression of VaRAV1 in grape cells improved its cold tolerance. The transgenic cells exhibited low electrolyte leakage and malondialdehyde content and high peroxidase activity. Moreover, the TF gene TCP8 and a gene involving in homogalacturonan biosynthesis were found to be regulated by VaRAV1, suggesting that the contribution of VaRAV1 to cold tolerance may be achieved by enhancing the stability of cell membrane and regulating the expression of target genes involved in plant cell wall composition. Our work provides novel insights into plant response to cold stress and demonstrates the utility of ATAC-seq and RNA-seq for the rapid identification of TFs in response to cold stress in grapevine. VaRAV1 may play an important role in adaption to cold stress.

Three AP2/ERF family members modulate flavonoid synthesis by regulating type IV chalcone isomerase in citrus.

Flavanones and flavones are excellent source of bioactive compounds but the molecular basis of their highly efficient production remains elusive. Chalcone isomerase (CHI) family proteins play essential roles in flavonoid biosynthesis but little are known about the transcription factors controlling their gene expression. Here, we identified a type IV CHI (designated as CitCHIL1) from citrus which enhances the accumulation of citrus flavanones and flavones (CFLs). CitCHIL1 participates in a CFL biosynthetic metabolon and assists the cyclization of naringenin chalcone to (2S)-naringenin, which leads to the efficient influx of substrates to chalcone synthase (CHS) and improves the catalytic efficiency of CHS. Overexpressing CitCHIL1 in Citrus and Arabidopsis significantly increased flavonoid content and RNA interference-induced silencing of CitCHIL1 in citrus led to a 43% reduction in CFL content. Three AP2/ERF transcription factors were identified as positive regulators of the CitCHIL1 expression. Of these, two dehydration-responsive element binding (DREB) proteins, CitERF32 and CitERF33, activated the transcription by directly binding to the CGCCGC motif in the promoter, while CitRAV1 (RAV: related to ABI3/VP1) formed a transcription complex with CitERF33 that strongly enhanced the activation efficiency and flavonoid accumulation. These results not only illustrate the specific function that CitCHIL1 executes in CFL biosynthesis but also reveal a new DREB-RAV transcriptional complex regulating flavonoid production.

Genome-Wide Analysis of RAV Transcription Factors and Functional Characterization of Anthocyanin-Biosynthesis-Related RAV Genes in Pear.

Related to ABSCISIC ACID INSENSITIVE3/VIVIPAROUS1 (ABI3/VP1, RAV), transcription factors (TFs) belonging to the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) TF family play critical roles in plant growth, development, and responses to abiotic and biotic stress. In this study, 11 novel RAV TFs were identified in pear (Pyrus bretschneideri Rehd). A phylogenetic analysis revealed that the TFs clustered into three groups with 10 conserved motifs, some of which were group- or subgroup-specific, implying that they are important for the functions of the RAVs in these clades. RAVs in Pyrus and Malus were closely related, and the former showed a collinear relationship. Analysis of their expression patterns in different tissues and at various growth stages and their responses to abiotic and biotic stress suggested that PbRAV6 and PbRAV7 play important roles in drought stress and salt stress, respectively. We investigated the function of RAVs in pear peel coloration using two red pear varieties with different color patterns and applying data from transcriptome analyses. We found that PbRAV6 participates in the regulation of pericarp color. These findings provide insight into a new TF family in pear and a basis for further studies on the response to drought stress and fruit coloration in this commercially important crop.

Letermovir Resistance Analysis in a Clinical Trial of Cytomegalovirus Prophylaxis for Hematopoietic Stem Cell Transplant Recipients.

BACKGROUND: Letermovir (LET), a cytomegalovirus (CMV) deoxyribonucleic acid (DNA) terminase inhibitor, was recently approved for prophylaxis of CMV infection in adult CMV-seropositive recipients of allogeneic hematopoietic stem cell transplantation. Cytomegalovirus genotyping was performed to identify LET-resistance-associated variants (RAVs) among subjects in a Phase 3 trial. METHODS: The CMV UL56 and UL89 genes, encoding subunits of CMV DNA terminase, were sequenced from plasma collected from subjects with clinically significant CMV infection (CS-CMVi). Novel variants were evaluated by recombinant phenotyping to assess their potential to confer resistance to LET. RESULTS: Genotyping was successful for 50 of 79 LET subjects with CS-CMVi. Resistance-associated variants (encoding pUL56 V236M and C325W) were detected independently in subjects 1 and 3 who experienced CS-CMVi while receiving LET prophylaxis, and 2 other variants (encoding pUL56 E237G and R369T) were detected >3 weeks after subjects 2 and 3, respectively, had discontinued LET prophylaxis and received preemptive therapy with ganciclovir. CONCLUSIONS: The detected incidence of CMV resistance among subjects who received LET as prophylaxis in this Phase 3 trial was low. The LET RAVs that were detected mapped to the CMV UL56 gene at positions associated with reduced susceptibility to LET based on resistance selections in cell culture.

The strawberry transcription factor FaRAV1 positively regulates anthocyanin accumulation by activation of FaMYB10 and anthocyanin pathway genes.

The RAV (related to ABI3/viviparous 1) group of transcription factors (TFs) play multifaceted roles in plant development and stress responses. Here, we show that strawberry (Fragaria × ananassa) FaRAV1 positively regulates anthocyanin accumulation during fruit ripening via a hierarchy of activation processes. Dual-luciferase assay screening of all fruit-expressed AP2/ERFs showed FaRAV1 had the highest transcriptional activation of the promoter of FaMYB10, a key activator of anthocyanin biosynthesis. Yeast one-hybrid and electrophoretic mobility shift assays indicated that FaRAV1 could directly bind to the promoter of FaMYB10. Transient overexpression of FaRAV1 in strawberry fruit increased FaMYB10 expression and anthocyanin production significantly. Correspondingly, transient RNA interference-induced silencing of FaRAV1 led to decreases in FaMYB10 expression and anthocyanin content. Transcriptome analysis of FaRAV1-overexpressing strawberry fruit revealed that transcripts of phenylpropanoid and flavonoid biosynthesis pathway genes were up-regulated. Luciferase assays showed that FaRAV1 could also activate the promoters of strawberry anthocyanin biosynthetic genes directly, revealing a second level of FaRAV1 action in promoting anthocyanin accumulation. These results show that FaRAV1 stimulates anthocyanin accumulation in strawberry both by direct activation of anthocyanin pathway gene promoters and by up-regulation of FaMYB10, which also positively regulates these genes.

Relative Alpha Variability Changes Precede Alpha-Delta Ratio Changes in Cerebral Ischemia.

The utility of quantitative EEG in early detection of cerebral ischemia is still underappreciated in clinical practice. We present a case of aneurysmal subarachnoid hemorrhage complicated by vasospasm as detected by the cerebral angiogram. The patient was being monitored on electroencephalogram. It showed early signs of cerebral ischemia represented by decline in the Alpha-Delta-Ratio (ADR) and the Relative-Alpha-Variability (RAV). Surprisingly, the RAV changes preceded the ADR changes. This is a significant finding that can also apply to early reocclusion or reperfusion injuries after mechanical thrombectomy.

The C-terminal WD40 repeats on the TOPLESS co-repressor function as a protein-protein interaction surface.

KEY MESSAGE: The two predicted WD40 propellers on TOPLESS function as protein-protein interaction domains. The 1st WD40 propeller mediates interaction with RAV1, and the 2nd WD40 propeller mediates interaction with VRN5. The TOPLESS/TOPLESS-RELATED (TPL/TPR) co-repressor family proteins are known to interact with a wide variety of proteins including transcription factors, Mediator subunits, histone deacetylases, and histone tails. Through these interactions, TPL/TPR act to repress transcription in an increasingly diverse array of plant pathways. Proteins that bind TPL/TPR typically contain one or more Repression Domains (RDs) that mediate the interaction. For example, the well-characterized Ethylene response factor-associated Amphiphilic Repression (EAR) motif is known to facilitate interaction by binding the TOPLESS Domain (TPD) located in the N-terminus. Here we show that in yeast two-hybrid assays, the non-EAR protein, Related to ABI3/VP1-1 (RAV1), binds a novel region located within the first nine WD40-repeats of TPL. Protein modeling and in silico analysis suggest that these nine WD40 repeats may form the first of two WD40 propellers located on C-terminus of TPL. The interaction between RAV1 and the 1st WD40 propeller is conserved with another RAV family member, TEMPRANILLO1 (TEM1) and is mediated by the B3 Repression Domain (BRD) located on both RAV1 and TEM1. Also, the predicted 2nd WD40 propeller was shown in yeast cells to bind Vernalization 5 (VRN5), which contains several unconfirmed partial RDs. Furthermore, we demonstrate that the 1st WD40 propeller of TPL can form a complex with RAV1 both in yeast and in Arabidopsis protoplasts.

Limitations of daclatasvir/asunaprevir plus beclabuvir treatment in cases of NS5A inhibitor treatment failure.

Combined daclatasvir (DCV)/asunaprevir (ASV) plus beclabuvir (BCV) treatment shows a high virological response for genotype 1b chronic hepatitis C patients. However, its efficacy for patients for whom previous direct-acting antiviral (DAA) therapy failed is not known. We analysed the efficacy of DCV/ASV/BCV treatment for HCV-infected mice and chronic hepatitis patients. Human hepatocyte chimaeric mice were injected with serum samples obtained from either a DAA-naïve patient or a DCV/ASV treatment failure and were then treated with DCV/ASV alone or in combination with BCV for 4 weeks. DCV/ASV treatment successfully eliminated the virus in DAA-naïve-patient HCV-infected mice. DCV/ASV treatment failure HCV-infected mice developed viral breakthrough during DCV/ASV treatment, with the emergence of NS5A-L31V/Y93H HCV resistance-associated variants (RAVs) being observed by direct sequencing. DCV/ASV/BCV treatment inhibited viral breakthrough in NS5A-L31V/Y93H-mutated HCV-infected mice, but HCV relapsed with the emergence of NS5B-P495S variants after the cessation of the treatment. The efficacy of the triple therapy was also analysed in HCV-infected patients; one DAA-naïve patient and four prior DAA treatment failures were treated with 12 weeks of DCV/ASV/BCV therapy. Sustained virological response was achieved in a DAA-naïve patient and one of the DCV/ASV treatment failures through DCV/ASV/BCV therapy; however, HCV relapse occurred in the other patients with prior DCV/ASV and/or sofosbuvir/ledipasvir treatment failures. DCV/ASV/BCV therapy seems to have limited efficacy for patients with NS5A RAVs for whom prior DAA treatment has failed.

Efficacy of generic oral directly acting agents in patients with hepatitis C virus infection.

Novel direct-acting antivirals (DAAs) are now the standard of care for the management of hepatitis C virus (HCV) infection. Branded DAAs are associated with high sustained virological response at 12 weeks post-completion of therapy (SVR12), but are costly. We aimed to assess the efficacy of generic oral DAAs in a real-life clinical scenario. Consecutive patients with known HCV infection who were treated with generic-oral DAA regimens (May 2015 to January 2017) were included. Demographic details, prior therapy and SVR12 were documented. Four hundred and ninety patients (mean age: 38.9 ± 12.7 years) were treated with generic DAAs in the study time period. Their clinical presentations included chronic hepatitis (CHC) in 339 (69.2%) of cases, compensated cirrhosis in 120 (24.48%) cases and decompensated cirrhosis in 31 (6.32%) cases. Genotype 3 was most common (n = 372, 75.9%) followed by genotype 1 (n = 97, 19.8%). Treatment naïve and treatment-experienced (defined as having previous treatment with peginterferon and ribavirin) were 432 (88.2%) and 58 (11.8%), respectively. Generic DAA treatment regimens included sofosbuvir in combination with ribavirin (n = 175), daclatasvir alone (n = 149), ribavirin and peginterferon (n = 80), ledipasvir alone (n = 43), daclatasvir and ribavirin (n = 37), and ledipasvir and ribavirin (n = 6). Overall SVR12 was 95.9% (470/490) for all treatment regimens. SVR12 for treatment naïve and experienced patients was 97.0% (419/432) and 87.9% (51/58), respectively, P = .005. High SVR12 was observed with various regimens, irrespective of genotype and underlying liver disease status. There were no differences in SVR12 with 12 or 24 weeks therapy. No major adverse event occurred requiring treatment stoppage. Generic oral DAAs are associated with high SVR rates in patients with HCV infection in a real-life clinical scenario.

RAV transcription factors are essential for disease resistance against cassava bacterial blight via activation of melatonin biosynthesis genes.

With 1 AP2 domain and 1 B3 domain, 7 MeRAVs in apetala2/ethylene response factor (AP2/ERF) gene family have been identified in cassava. However, the in vivo roles of these remain unknown. Gene expression assays showed that the transcripts of MeRAVs were commonly regulated after Xanthomonas axonopodis pv manihotis (Xam) and MeRAVs were specifically located in plant cell nuclei. Through virus-induced gene silencing (VIGS) in cassava, we found that MeRAV1 and MeRAV2 are essential for plant disease resistance against cassava bacterial blight, as shown by the bacterial propagation of Xam in plant leaves. Through VIGS in cassava leaves and overexpression in cassava leave protoplasts, we found that MeRAV1 and MeRAV2 positively regulated melatonin biosynthesis genes and the endogenous melatonin level. Further investigation showed that MeRAV1 and MeRAV2 are direct transcriptional activators of 3 melatonin biosynthesis genes in cassava, as evidenced by chromatin immunoprecipitation-PCR in cassava leaf protoplasts and electrophoretic mobility shift assay. Moreover, cassava melatonin biosynthesis genes also positively regulated plant disease resistance. Taken together, this study identified MeRAV1 and MeRAV2 as common and upstream transcription factors of melatonin synthesis genes in cassava and revealed a model of MeRAV1 and MeRAV2-melatonin biosynthesis genes-melatonin level in plant disease resistance against cassava bacterial blight.

Interferon-free treatment choice according to baseline RASs leads to high SVR rates in HCV genotype 1 infected patients.

AIM: Different combinations of direct antiviral agents (DAA) lead to high SVR rates in HCV genotype 1 infected patients. However, presence of baseline resistance-associated substitutions (RASs) represents a major risk factor for treatment failure. It is unknown whether choice of treatment based on RASs has the potential to decrease virologic failure rates. METHODS: Population-based sequencing of NS3 and NS5A genes was performed in HCV genotype 1 infected patients at a German university hospital. Treatment was individually selected based on resistance analyses. RESULTS: In total, 319 patients (50% treatment-experienced and 30% with cirrhosis) were included. With the treatment choice based on the baseline NS3 and NS5A resistance profile SVR rates between 96 and 100% were observed in all subgroups, including treatment-experienced patients with cirrhosis and HCV genotype 1a infected cirrhotic patients. CONCLUSIONS: The choice of treatment based on the RASs status at baseline may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure.