DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis.

Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2-but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought

Clinical Features, Cardiovascular Risk Profile, and Therapeutic Trajectories of Patients with Type 2 Diabetes Candidate for Oral Semaglutide Therapy in the Italian Specialist Care

Introduction: This study aimed to address therapeutic inertia in the management of type 2 diabetes (T2D) by investigating the potential of early treatment with oral semaglutide. Methods: A cross-sectional survey was conducted between October 2021 and April 2022 among specialists treating individuals with T2D. A scientific committee designed a data collection form covering demographics, cardiovascular risk, glucose control metrics, ongoing therapies, and physician judgments on treatment appropriateness. Participants completed anonymous patient questionnaires reflecting routine clinical encounters. The preferred therapeutic regimen for each patient was also identified. Results: The analysis was conducted on 4449 patients initiating oral semaglutide. The population had a relatively short disease duration (42%  60% of patients, and more often than sitagliptin or empagliflozin. Conclusion: The study supports the potential of early implementation of oral semaglutide as a strategy to overcome therapeutic inertia and enhance T2D management

A role for post-transcriptional gene silencing in vegetative phase change in Arabidopsis

Higher plants undergo a transition from a juvenile to an adult phase of vegetative development prior to flowering. Screens for mutants that undergo this transition precociously resulted in the identification of a number of complementation groups with loss-of-function phenotypes similar to that of mutations in the Argonaute gene ZIPPY (ZIP). Two groups correspond to mutations in the SGS2/SDE1/RDR6 and SGS3 loci, genes originally identified in screens for mutants deficient in posttranscriptional gene silencing (PTGS). By positional cloning, another group was found to correspond to mutations in the previously uncharacterized At5g20320 (DCL4), one of the four Dicer-like family members in Arabidopsis. Double and triple mutant analyses suggest that ZIP, RDR6, SGS3, and DCL4 act in the same pathway, along with HST, the Arabidopsis exportin-5 homologue, a protein that has been shown to regulate the export of miRNAs in mammals, and which probably has the same function in Arabidopsis. Such observations suggested that RDR6 and SGS3 are important for both transgene silencing and the regulation of endogenous genes, and we hypothesized that DCL4 might provide the dicer activity necessary for the production of such a class of small regulatory RNAs. Indeed, genetic and molecular analysis showed that these genes operate as core components of a PTGS pathway in Arabidopsis where DCL4 is required for the biogenesis and function of a novel class of endogenous siRNAs, termed tans-acting siRNAs (ta-siRNAs). The work presented here, demonstrates a role for endogenous siRNAs in the normal regulation of gene expression, and suggest that this system plays a central role in the temporal control of shoot development in Arabidopsis

A role for post-transcriptional gene silencing in vegetative phase change in Arabidopsis

Higher plants undergo a transition from a juvenile to an adult phase of vegetative development prior to flowering. Screens for mutants that undergo this transition precociously resulted in the identification of a number of complementation groups with loss-of-function phenotypes similar to that of mutations in the Argonaute gene ZIPPY (ZIP). Two groups correspond to mutations in the SGS2/SDE1/RDR6 and SGS3 loci, genes originally identified in screens for mutants deficient in posttranscriptional gene silencing (PTGS). By positional cloning, another group was found to correspond to mutations in the previously uncharacterized At5g20320 (DCL4), one of the four Dicer-like family members in Arabidopsis. Double and triple mutant analyses suggest that ZIP, RDR6, SGS3, and DCL4 act in the same pathway, along with HST, the Arabidopsis exportin-5 homologue, a protein that has been shown to regulate the export of miRNAs in mammals, and which probably has the same function in Arabidopsis. Such observations suggested that RDR6 and SGS3 are important for both transgene silencing and the regulation of endogenous genes, and we hypothesized that DCL4 might provide the dicer activity necessary for the production of such a class of small regulatory RNAs. Indeed, genetic and molecular analysis showed that these genes operate as core components of a PTGS pathway in Arabidopsis where DCL4 is required for the biogenesis and function of a novel class of endogenous siRNAs, termed tans-acting siRNAs (ta-siRNAs). The work presented here, demonstrates a role for endogenous siRNAs in the normal regulation of gene expression, and suggest that this system plays a central role in the temporal control of shoot development in Arabidopsis

A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis

The Arabidopsis genes, TAS2 and TAS1a, produce structurally similar noncoding transcripts that are transformed into short (21-nucleotide [nt]) and long (24-nt) siRNAs by RNA silencing pathways. Some of these short siRNAs direct the cleavage of protein-coding transcripts, and thus function as trans-acting siRNAs (ta-siRNAs). Using genetic analysis, we defined the pathway by which ta-siRNAs and other short siRNAs are generated from these loci. This process is initiated by the miR173-directed cleavage of a primary poly(A) transcript. The 3′ fragment is then transformed into short siRNAs by the sequential activity of SGS3, RDR6, and DCL4: SGS3 stabilizes the fragment, RDR6 produces a complementary strand, and DCL4 cleaves the resulting double-stranded molecule into short siRNAs, starting at the end with the miR173 cleavage site and proceeding in 21-nt increments from this point. The 5′ cleavage fragment is also processed by this pathway, but less efficiently. The DCL3-dependent pathway that generates long siRNAs does not require miRNA-directed cleavage and plays a minor role in the silencing of these loci. Our results define the core components of a post-transcriptional gene silencing pathway in Arabidopsis and reveal some of the features that direct transcripts to this pathway

SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis

Higher plants undergo a transition from a juvenile to an adult phase of vegetative development prior to flowering. Screens for mutants that undergo this transition precociously produced alleles of two genes required for posttranscriptional gene silencing (PTGS)—SUPPRESSOR OF GENE SILENCING3 (SGS3) and SUPPRESSOR OF GENE SILENCING2(SGS2)/SILENCING DEFECTIVE1 (SDE1)/RNA-DEPENDENT POLYMERASE6 (RDR6). Loss-of-function mutations in these genes have a phenotype similar to that of mutations in the Argonaute gene ZIPPY (ZIP). Epistasis analysis suggests that ZIP, SGS3, SGS2/SDE1/RDR6, and the putative miRNA export receptor, HASTY (HST), operate in the same pathway(s). Microarray analysis revealed a small number of genes whose mRNA is increased in ZIP, SGS3, and SGS2/SDE1/RDR6 mutants, as well as genes that are up-regulated in SGS3 and SGS2/SDE1/RDR6 mutants, but not in ZIP mutants. One of these latter genes (At5g18040) is silenced posttranscriptionally in trans by the sRNA255 family of endogenous, noncoding, small interfering RNAs (siRNAs). The increase in At5g18040 mRNA in SGS3 and SGS2/SDE1/RDR6 mutants is attributable to the absence of sRNA255-like siRNAs in these mutants. These results demonstrate a role for endogenous siRNAs in the regulation of gene expression, and suggest that PTGS plays a central role in the temporal control of shoot development in plants

Recognition of adult and pediatric acute lymphoblastic leukemia blasts by natural killer cells.

In this study we aimed at investigating the pathways of recognition of acute lymphoblastic leukemia blasts by natural killer cells and at verifying whether differences in natural killer cell activating receptor ligands’ expression among groups defined by age of patients or presence of cytogenetic/molecular aberrations correlate with the susceptibility to recognition and killing. We analyzed 103 newly diagnosed acute lymphoblastic leukemia patients (46 adults and 57 children). Pediatric blasts showed a significantly higher expression of Nec-2 (p=.03), ULBP-1 (p=.01) and ULBP-3 (p=.04) compared to adult cells. The differential expression of these ligands between adults and children was confined to B-lineage acute lymphoblastic leukemia with no known molecular alterations. Within molecularly defined subgroups of patients, a high surface expression of NKG2D and DNAM1 ligands was found on BCR-ABL+ blasts, regardless of patient age. Accordingly, BCR-ABL+ blasts proved to be significantly more susceptible to natural killerdependent lysis than B-lineage blasts without molecular aberrations (p=.03). Cytotoxic tests performed in the presence of neutralizing antibodies indicated a pathway of acute lymphoblastic leukemia cell recognition in the setting of the Nec-2/DNAM-1 interaction. These data provide a biologic explanation to the different role played by alloreactive natural killer cells in pediatric versus adult acute lymphoblastic leukemia and suggest that new natural killer-based strategies targeting specific subgroups of patients, particularly those BCR-ABL+, are worthy of being further pursued