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Current Issues in Molecular Biology (ISSN 1467-3045) was launched in 1999 and has published international and multidisciplinary articles on all aspects of molecular biology spanning from basic mechanisms to applications in fields primarily, but not exclusively, relevant to microbiology and virology [...].

The 35-amino acid C2 protein of Cotton leaf curl Kokhran virus, Burewala, implicated in resistance breaking in cotton, retains some activities of the full-length protein.

With one exception, all the begomoviruses characterized so far encode an ~134-amino acid (aa) (A)C2 protein. The exception is the "Burewala" strain of Cotton leaf curl Kokhran virus (CLCuKoV-Bu), associated with resistance breaking in cotton across Pakistan and northwestern India, that encodes a truncated 35-aa C2. The C2 protein encoded by begomoviruses performs multiple functions including suppression of post-transcriptional gene silencing (PTGS), modulating microRNA (miRNA) expression and may be a pathogenicity determinant. The study described here was designed to investigate whether the CLCuKoV-Bu 35-aa C2 retains the activities of the full-length C2 protein. The results showed the 35-aa C2 of CLCuKoV-Bu acts as a pathogenicity determinant, suppresses PTGS and upregulates miRNA expression when expressed from a Potato virus X vector in Nicotiana benthamiana. The symptoms induced by expression of full-length C2 were more severe than those induced by the 35-aa C2. The accumulation of most developmental miRNAs decreases with the full-length C2 protein and increases with the 35-aa peptide of CLCuKoV-Bu. The study also revealed that 35-aa peptide of CLCuKoV-Bu maintains suppressor of silencing activity at a level equal to that of full-length C2. The significance of the results with respect to virus fitness and resistance breaking is discussed.

Introns of plant pri-miRNAs enhance miRNA biogenesis.

Plant MIR genes are independent transcription units that encode long primary miRNA precursors, which usually contain introns. For two miRNA genes, MIR163 and MIR161, we show that introns are crucial for the accumulation of proper levels of mature miRNA. Removal of the intron in both cases led to a drop-off in the level of mature miRNAs. We demonstrate that the stimulating effects of the intron mostly reside in the 5'ss rather than on a genuine splicing event. Our findings are biologically significant as the presence of functional splice sites in the MIR163 gene appears mandatory for pathogen-triggered accumulation of miR163 and proper regulation of at least one of its targets.

Primary and secondary siRNAs in geminivirus-induced gene silencing.

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3'-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5'-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.

Modification of the increased accumulation of plants microRNAs during the infection by tobacco mosaic virus.

RNA silencing is central to the struggle between plants and viral pathogens. To counteract RNA silencing, viruses have evolved suppressor proteins able to block the mechanism at different stages. Virus-infected plants generally develop viral symptoms characterized by morphological changes. Recent works have shown that viral symptoms have at least two different origins related to sRNAs: these can be either due to the targeting of host genes by siRNAs of viral origins (vsiRNAs) or to alteration of the normal functioning of certain host sRNAs (endogenous sRNAs) important for plant development. Although Tobacco mosaic virus (TMV) is one of the most studied plant virus worldwide, the origin of its viral symptoms as well as that of the increased accumulation of microRNAs and other sRNAs remain to be clarified. The most recent data are summarized and discussed in the present review.

RNA silencing pathways of plants: silencing and its suppression by plant DNA viruses.

RNA silencing refers to processes that depend on small (s)RNAs to regulate the expression of eukaryotic genomes. In plants, these processes play critical roles in development, in responses to a wide array of stresses, in maintaining genome integrity and in defense against viral and bacterial pathogens. We provide here an updated view on the array of endogenous sRNA pathways, including microRNAs (miRNAs), discovered in the model plant Arabidopsis, which are also the basis for antiviral silencing. We emphasize the current knowledge as well as the recent advances made on understanding the defense and counter-defense strategies evolved in the arms race between plants and DNA viruses on both the nuclear and the cytoplasmic front. This article is part of a Special Issue entitled: MicroRNA's in viral gene regulation.

miR393 and secondary siRNAs regulate expression of the TIR1/AFB2 auxin receptor clade and auxin-related development of Arabidopsis leaves.

The phytohormone auxin is a key regulator of plant growth and development that exerts its functions through F-box receptors. Arabidopsis (Arabidopsis thaliana) has four partially redundant of these receptors that comprise the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX1 auxin receptor (TAAR) clade. Recent studies have shown that the microRNA miR393 regulates the expression of different sets of TAAR genes following pathogen infection or nitrate treatment. Here we report that miR393 helps regulate auxin-related development of leaves. We found that AtMIR393B is the predominant source for miR393 in all aerial organs and that miR393 down-regulates all four TAAR genes by guiding the cleavage of their mRNAs. A mutant unable to produce miR393 shows developmental abnormalities of leaves and cotyledons reminiscent of enhanced auxin perception by TAARs. Interestingly, miR393 initiates the biogenesis of secondary siRNAs from the transcripts of at least two of the four TAAR genes. Our results indicate that these siRNAs, which we call siTAARs, help regulate the expression of TAAR genes as well as several unrelated genes by guiding the cleavage of their mRNAs. Thus, miR393 and possibly siTAARs regulate auxin perception and certain auxin-related aspects of leaf development.

Transcript mapping of Cotton leaf curl Burewala virus and its cognate betasatellite, Cotton leaf curl Multan betasatellite.

BACKGROUND: Whitefly-transmitted geminiviruses (family Geminiviridae, genus Begomovirus) are major limiting factors for the production of numerous dicotyledonous crops throughout the warmer regions of the world. In the Old World a small number of begomoviruses have genomes consisting of two components whereas the majority have single-component genomes. Most of the monopartite begomoviruses associate with satellite DNA molecules, the most important of which are the betasatellites. Cotton leaf curl disease (CLCuD) is one of the major problems for cotton production on the Indian sub-continent. Across Pakistan, CLCuD is currently associated with a single begomovirus (Cotton leaf curl Burewala virus [CLCuBuV]) and the cotton-specific betasatellite Cotton leaf curl Multan betasatellite (CLCuMuB), both of which have recombinant origins. Surprisingly, CLCuBuV lacks C2, one of the genes present in all previously characterized begomoviruses. Virus-specific transcripts have only been mapped for few begomoviruses, including one monopartite begomovirus that does not associate with betasatellites. Similarly, the transcripts of only two betasatellites have been mapped so far. The study described has investigated whether the recombination/mutation events involved in the evolution of CLCuBuV and its associated CLCuMuB have affected their transcription strategies. RESULTS: The major transcripts of CLCuBuV and its associated betasatellite (CLCuMuB) from infected Nicotiana benthamiana plants have been determined. Two complementary-sense transcripts of ~1.7 and ~0.7 kb were identified for CLCuBuV. The ~1.7 kb transcript appears similar in position and size to that of several begomoviruses and likely directs the translation of C1 and C4 proteins. Both complementary-sense transcripts can potentially direct the translation of C2 and C3 proteins. A single virion-sense transcript of ~1 kb, suitable for translation of the V1 and V2 genes was identified. A predominant complementary-sense transcript was also confirmed for the betasatellite. CONCLUSIONS: Overall, the transcription of CLCuBuV and the recombinant CLCuMuB is equivalent to earlier mapped begomoviruses/betasatellites. The recombination events that featured in the origins of these components had no detectable effects on transcription. The transcripts spanning the mutated C2 gene showed no evidence for involvement of splicing in restoring the ability to express intact C2 protein.

siRNAs from miRNA sites mediate DNA methylation of target genes.

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants.

Genome-wide survey of alternative splicing in the grass Brachypodium distachyon: a emerging model biosystem for plant functional genomics.

A draft sequence of the genome of Brachypodium distachyon, the emerging grass model, was recently released. This represents a unique opportunity to determine its functional diversity compared to the genomes of other model species. Using homology mapping of assembled expressed sequence tags with chromosome scale pseudomolecules, we identified 128 alternative splicing events in B. distachyon. Our study identified that retention of introns is the major type of alternative splicing events (53%) in this plant and highlights the prevalence of splicing site recognition for definition of introns in plants. We have analyzed the compositional profiles of exon-intron junctions by base-pairing nucleotides with U1 snRNA which serves as a model for describing the possibility of sequence conservation. The alternative splicing isoforms identified in this study are novel and represent one of the potentially biologically significant means by which B. distachyon controls the function of its genes. Our observations serve as a basis to understand alternative splicing events of cereal crops with more complex genomes, like wheat or barley.

Publisher's Note: Continued Publication of Tomography by MDPI.

Tomography was launched in 2015 and has published international and multidisciplinary research on all aspects of imaging science, spanning from basic research to clinical trials [...].

Evolution of Arabidopsis MIR genes generates novel microRNA classes.

In Arabidopsis, canonical 21-nt miRNAs are generated by Dicer-like (DCL) 1 from hairpin precursors. We have identified a novel class of functional 23- to 25-nt long-miRNAs that is generated independently from the same miRNA precursors by DCL3. Long-miRNAs are developmentally regulated and in some cases have been conserved during evolution implying that they have biological functions. Plant microRNA genes (MIR) have been proposed to evolve by inverted duplication of the target gene. We found that recently evolved MIR genes consistently give rise to long-miRNAs, while ancient MIR genes give rise predominantly to canonical miRNAs. Transcripts from inverted repeats representing evolving proto-MIR genes were processed by DCL3 into long-miRNAs and also by DCL1, DCL2 or DCL4 depending on hairpin stem length to produce different sizes of miRNAs. Our results suggest that evolution of MIR genes is associated with gradual, overlapping changes in DCL usage resulting in specific size classes of miRNAs.

Publisher's Note: Continued Publication of Pathophysiology, the Official Journal of the International Society of Pathophysiology, by MDPI.

Pathophysiology (ISSN 0928-4680) was launched in 1994 and has been published during the past 26 years by Elsevier [...].

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Clinics and Practice was launched in 2011 and it has been published over the past nine years by PAGEPress Publications [...].

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Infectious Disease Reports was launched in 2009 and it has been published over the past eleven years by PAGEPress Publications [...].

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Neurology International was launched in 2009 and it has been published over the past eleven years by PAGEPress Publications [...].

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Audiology Research (ISSN 2039-4349) was launched in 2011 and published over the past nine years by PAGEPress Publications [...].

Publisher's Note: Continued Publication of Pediatric Reports by MDPI.

Pediatric Reports was launched in 2009 and it has been published over the past eleven years by PAGEPress Publications [...].

Medicine 4.0: New Technologies as Tools for a Society 5.0.

Are new technologies in the medicine sector a driver to support the development of a society 5.0? Innovation pushes the artisan to become smart and lean, customer-oriented but within a standardized environment of production, maintaining and ensuring the quality of the product. An artisan is a user and innovator, as an essential part of the industrial chain. In the healthcare sector, the doctor is the industrial artisan, and medicine can be considered as an example of a smart tool, strongly tailored, that embeds the innovation of materials, nano-devices, and smart technology (e.g., sensors and controllers). But how much of society is ready to host smart technology "on board", becoming "on life", constantly connected with remote controls that allow us to monitor, gather data, and, in any case, act, with preventive healthcare solutions? After a short overview of the medicine sector, a preliminary, tentative link between technological innovation and the healthcare sector allows us to adopt several outlooks on how to change research, always more transdisciplinary, combining science with social science in order to remain human-centered.