Small RNA Gene Research Articles

Histone methyltransferases SDG33 and SDG34 regulate organ-specific nitrogen responses in tomato.

Histone posttranslational modifications shape the chromatin landscape of the plant genome and affect gene expression in response to developmental and environmental cues. To date, the role of histone modifications in regulating plant responses to environmental nutrient availability, especially in agriculturally important species, remains largely unknown. We describe the functions of two histone lysine methyltransferases, SET Domain Group 33 (SDG33) and SDG34, in mediating nitrogen (N) responses of shoots and roots in tomato. By comparing the transcriptomes of CRISPR edited tomato lines sdg33 and sdg34 with wild-type plants under N-supplied and N-starved conditions, we uncovered that SDG33 and SDG34 regulate overlapping yet distinct downstream gene targets. In response to N level changes, both SDG33 and SDG34 mediate gene regulation in an organ-specific manner: in roots, SDG33 and SDG34 regulate a gene network including Nitrate Transporter 1.1 (NRT1.1) and Small Auxin Up-regulated RNA (SAUR) genes. In agreement with this, mutations in sdg33 or sdg34 abolish the root growth response triggered by an N-supply; In shoots, SDG33 and SDG34 affect the expression of photosynthesis genes and photosynthetic parameters in response to N. Our analysis thus revealed that SDG33 and SDG34 regulate N-responsive gene expression and physiological changes in an organ-specific manner, thus presenting previously unknown candidate genes as targets for selection and engineering to improve N uptake and usage in crop plants.

Structural and Functional Analysis of Toxin and Small RNA Gene Promoter Regions in Bacillus anthracis.

It was previously demonstrated that anthrax toxin activator (AtxA) binds directly to the σA-like promoter region of pagA (encoding protective antigen, PA) immediately upstream of the RNA polymerase binding site. In this study, using electrophoretic mobility shift assays and in vivo analyses, we identified AtxA-binding sites in the promoter regions of the lef and cya genes (encoding lethal and edema factors, respectively) and of two Bacillus anthracis small RNAs (XrrA and XrrB). Activities of all four newly studied promoters were enhanced in the presence of CO2/bicarbonate and AtxA, as previously seen for the pagA promoter. Notably, the cya promoter was less activated by AtxA and CO2/bicarbonate conditions. The putative promoter of a recently described third small RNA, XrrC, showed a negligible response to AtxA and CO2/bicarbonate. RNA polymerase binding sites of the newly studied promoters show no consensus and differ from the σA-like promoter region of pagA. In silico analysis of the probable AtxA binding sites in the studied promoters revealed several palindromes. All the analyzed palindromes showed very little overlap with the σA-like pagA promoter. It remains unclear as to how AtxA and DNA-dependent RNA-polymerase identify such diverse DNA-sequences and differentially regulate promoter activation of the studied genes. IMPORTANCE Anthrax toxin activator (AtxA) is the major virulence regulator of Bacillus anthracis, the causative agent of anthrax. Understanding AtxA's mechanism of regulation could facilitate the development of therapeutics for B. anthracis infection. We provide evidence that AtxA binds to the promoters of the cya, lef, xrrA, and xrrB genes. In vivo assays confirmed the activities of all four promoters were enhanced in the presence of AtxA and CO2/bicarbonate, as previously seen for the pagA promoter. The cya and lef genes encode important toxin components. The xrrA and xrrB genes encode sRNAs with a suggested function as cell physiology regulators. Our data provides further evidence for the direct regulatory role of AtxA that was previously shown with the pagA promoter.

Modification of Gene Expression, DNA Methylation and Small RNAs Expression in Rice Plants under In Vitro Culture

Tissue culture is an important experimental technique widely used for plant transformation and can induce somaclonal variation that is shown to be associated with genetic and epigenetic changes. However, the molecular basis of somaclonal variation and plant cell response to tissue culture has yet to be fully understood. In this study, we investigated gene expression, DNA methylation, and small RNA changes in regenerated lines (RL) compared with the wild-type progenitor plants (WT) of rice cv. Hitomebore. Using microarray, we identified many genes that were differentially expressed in the shoot-tip tissue and showed that TEs were generally activated in RL. Methylation Sensitive Amplification Polymorphism (MSAP) analysis of 5′CCGG sites combined with bisulfite sequencing detected a generally reduced DNA methylation in the RL lines. Small RNA sequencing analysis detected widespread changes in small RNA accumulation between RL and WT. In particular, repeat and TE-associated 24-nt size class of small RNAs, the inducer of RNA-directed DNA methylation, was in general down-regulated in RL, consistent with reduced CHG and CHH methylation at some of the differentially methylated TE loci. A large number of differentially expressed miRNAs were identified in RL and WT lines, including known and novel miRNAs. The expression of some of these miRNAs exhibited inverse correlation with the predicted target genes, suggesting a regulatory function. The RL plants looked similar to WT plants under normal conditions but showed significant phenotypic alterations under abiotic stress conditions. The widespread changes in DNA methylation, small RNA accumulation and gene expression in regenerated plants supports the role of epigenetic changes in tissue culture-induced somaclonal variation.

Genome Sequence Resource of Bacillus velezensis Strain HC-8, a Native Bacterial Endophyte with Biocontrol Potential Against the Honeysuckle Powdery Mildew Causative Pathogen Erysiphe lonicerae var. lonicerae.

Genome Sequence Resource of Bacillus velezensis Strain HC-8, a Native Bacterial Endophyte with Biocontrol Potential Against the Honeysuckle Powdery Mildew Causative Pathogen Erysiphe lonicerae var. lonicerae.

First Characterization and Zoonotic Potential of Cryptosporidium spp. and Giardia duodenalis in Pigs in Hubei Province of China.

The zoonotic protozoa parasites Cryptosporidium spp. and Giardia duodenalis infect a wide range of hosts, including humans. Pigs are reservoir hosts for Cryptosporidium spp. and G. duodenalis, which can transmit cryptosporidiosis and giardiasis to humans and other animals. The current study sought to investigate the infection rates and species/genotypes of Cryptosporidium spp. and G. duodenalis in pigs in Hubei of China. The nested PCR and sequence analyses of the small ribosomal subunit RNA (SSU rRNA) gene for Cryptosporidium spp. and the β-giardin (bg) gene for G. duodenalis was utilized to screen for the infection of those parasites in a total of 826 fresh fecal samples. Both Cryptosporidium spp. and G. duodenalis infection rates were 0.97% (8/826). Of the genotyped positive isolates, 6/8 (75%) were C. scrofarum and 2/8 (25%) were C. suis, while two zoonotic species G. duodenalis assemblage E and assemblage A were also detected in 7/8 (87.5%) isolates and 1/8 (12.5%) isolates, respectively. The findings suggest that both of those parasites in pig in intensive farms of Hubei province, China, pose a potential public health risk.

SAUR8, a small auxin-up RNA gene in poplar, confers drought tolerance to transgenic Arabidopsis plants

SAUR (small auxin-up RNA) is an early auxin-responsive gene. In this study, a novel SAUR gene PtSAUR8 was cloned from poplar (Populus trichocarpa), and subcellular location analysis showed that it is targeted to the nuclear membrane. In addition, PtSAUR8 overexpression in Arabidopsis improved the plant resistance to drought stress. Meanwhile, the loss of function mutant saur53 showed more drought sensitivity compared to the WT. PtSAUR8 conferred drought tolerance in transgenic Arabidopsis, as determined through phenotypic and stress-associated physiological indicator analyses, namely, root length, germination rate, relative water content, proline content, CAT content, POD content, malondialdehyde content, hydrogen peroxide content, and relative conductivity. In addition, after the 1 μM abscisic acid (ABA) treatment, the PtSAUR8-OE lines promoted stomata closure. Quantitative fluorescence analysis of related genes induced by drought mutant stress further confirmed that overexpression of PtSAUR8 can improve drought resistance in transgenic Arabidopsis lines. Therefore, PtSAUR8 may play a role in plant drought resistance through ABA-mediated pathways; thus, providing new research materials for molecular breeding of poplar resistance.

PpSAUR43, an Auxin-Responsive Gene, Is Involved in the Post-Ripening and Softening of Peaches

Auxin’s role in the post-ripening of peaches is widely recognized as important. However, little is known about the processes by which auxin regulates fruit post-ripening. As one of the early auxin-responsive genes, it is critical to understand the role of small auxin-up RNA (SAUR) genes in fruit post-ripening and softening. Herein, we identified 72 PpSAUR auxin-responsive factors in the peach genome and divided them into eight subfamilies based on phylogenetic analysis. Subsequently, the members related to peach post-ripening in the PpSAUR gene family were screened, and we targeted PpSAUR43. The expression of PpSAUR43 was decreased with fruit post-ripening in melting flesh (MF) fruit and was high in non-melting flesh (NMF) fruit. The overexpression of PpSAUR43 showed a slower rate of firmness decline, reduced ethylene production, and a delayed fruit post-ripening process. The MADS-box gene family plays an important regulatory role in fruit ripening. In this study, we showed with yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BIFC) experiments that PpSAUR43 can interact with the MADS-box transcription factor PpCMB1(PpMADS2), which indicates that PpSAUR43 may inhibit fruit ripening by suppressing the function of the PpCMB1 protein. Together, these results indicate that PpSAUR43 acts as a negative regulator involved in the peach post-ripening process.

Small antisense RNA ThfR positively regulates Thf1 in Synechocystis sp. PCC 6803

Thylakoid formation1 (Thf1), encoded by sll1414 (thf1), is a multifunctional protein conserved in all photosynthetic organisms. thf1 expression is highly induced by high light in Synechocystis during photosynthesis-related stress. In this study, differential RNA sequencing analysis of the Synechocystis sp. PCC 6803 revealed a small antisense RNA (asRNA) gene located on the reverse-complementary strand of the thf1 gene. The full length of this asRNA (designated ThfR) was determined by 5′ and 3′ RACE analysis. The accumulation of thf1 mRNA was up-regulated synchronously with the ThfR level during survival after high-light stress or nitrogen starvation. Under nitrogen starvation or high-light stress, compared with the wild type, a ThfR overexpression mutant demonstrated relatively more Thf1 protein content, while a ThfR reduced-expression mutant accumulated less Thf1 protein. Furthermore, the overexpression of ThfR enhanced the electron transport rate and the proliferation of cyanobacteria under high-light stress. These results, which we confirmed further using an Escherichia coli sRNA expression platform, suggest that the thf1 gene is positively regulated by ThfR, possibly through protection of the RAUUW element at the RNase E cleavage site. This study represents the first report, to our knowledge, of a cis-transcript antisense RNA that targets thf1 in Synechocystis sp. PCC 6803 and provides evidence that ThfR regulates photosynthesis by positively modulating thf1 under high-light conditions.

Diversity and Evolution of DNA Transposons Targeting Multicopy Small RNA Genes from Actinopterygian Fish.

Simple SummaryDNA transposons are parasitic DNA segments that can move or duplicate themselves from one site to another in the genome. Dada is a unique group of DNA transposons, which specifically insert themselves into multicopy RNA genes such as transfer RNA (tRNA) genes or small nuclear RNA (snRNA) genes to avoid the disruption of single-copy functional genes. However, only a few Dada families have been characterized along with their target sequences. Here, vertebrate genomes were surveyed to characterize new Dada transposons, and over 120 Dada families were characterized from diverse fishes. They were classified into 12 groups with confirmed target specificities. Various tRNA genes, as well as 5S ribosomal RNA (rRNA) genes were inserted by Dada transposons. Phylogenetic analysis revealed that Dada transposons inserted in the same RNA genes are closely related. Phylogenetically related Dada transposons inserted in different RNA genes show the sequence similarity around their insertion sites, indicating Dada proteins recognize DNA nucleotide sequences to find their targets. Understanding how Dada discovers the targets would help develop target-specific insertions of foreign DNA segments.Dada is a unique superfamily of DNA transposons, inserted specifically in multicopy RNA genes. The zebrafish genome harbors five families of Dada transposons, whose targets are U6 and U1 snRNA genes, and tRNA-Ala and tRNA-Leu genes. Dada-U6, which is inserted specifically in U6 snRNA genes, is found in four animal phyla, but other target-specific lineages have been reported only from one or two species. Here, vertebrate genomes and transcriptomes were surveyed to characterize Dada families with new target specificities, and over 120 Dada families were characterized from the genomes of actinopterygian fish. They were classified into 12 groups with confirmed target specificities. Newly characterized Dada families target tRNA genes for Asp, Asn, Arg, Gly, Lys, Ser, Tyr, and Val, and 5S rRNA genes. Targeted positions inside of tRNA genes are concentrated in two regions: around the anticodon and the A box of RNA polymerase III promoter. Phylogenetic analysis revealed the relationships among actinopterygian Dada families, and one domestication event in the common ancestor of carps and minnows belonging to Cyprinoidei, Cypriniformes. Sequences targeted by phylogenetically related Dada families show sequence similarities, indicating that the target specificity of Dada is accomplished through the recognition of primary nucleotide sequences.

The SAUR gene family in coffee: genome-wide identification and gene expression analysis during somatic embryogenesis.

Small auxin-up RNA (SAUR) genes form a wide family supposedly involved in different physiological and developmental processes in plants such as leaf senescence, auxin signaling and transport, hypocotyl development and tolerance to abiotic stresses. The transcription of SAUR genes is quickly induced by auxins, a group of phytohormones of major importance on embryo development. To better understand the distribution and expression profile of such still not explored family in Coffea sp., especially during the development of somatic embryogenesis (SE), SAUR members were characterized in silico using the available Coffea canephora genome data and analyzed for gene expression by RT-qPCR in C. arabica embryogenic samples. Over C. canephora genome 31 CcSAURs were distributed by 11 chromosomes. Out of these 31 gene members, 5 SAURs were selected for gene expression analysis in C. arabica embryogenic materials. CaSAUR12 and CaSAUR18 were the members highly expressed through almost all plant materials. The other genes had more expression in at least one of the developing embryo stages or plantlets. The CaSAUR12 was the only member to exhibit an increased expression in both non-embryogenic calli and the developing embryo stages. The identification of SAUR family on C. canephora genome followed by the analysis of gene expression profile across coffee somatic embryogenesis process on C. arabica represents a further additional step towards a better comprehension of molecular components acting on SE. Along with new research about this gene family such knowledge may support studies about clonal propagation methods via somatic embryogenesis to help the scientific community towards improvements into coffee crop.

The QseEF Two-Component System-GlmY Small RNA Regulatory Pathway Controls Swarming in Uropathogenic Proteus mirabilis.

Bacterial sensing of environmental signals through the two-component system (TCS) plays a key role in modulating virulence. In the search for the host hormone-sensing TCS, we identified a conserved qseEGF locus following glmY, a small RNA (sRNA) gene in uropathogenic Proteus mirabilis. Genes of glmY-qseE-qseG-qseF constitute an operon, and QseF binding sites were found in the glmY promoter region. Deletion of glmY or qseF resulted in reduced swarming motility and swarming-related phenotypes relative to the wild-type and the respective complemented strains. The qseF mutant had decreased glmYqseEGF promoter activity. Both glmY and qseF mutants exhibited decreased flhDC promoter activity and mRNA level, while increased rcsB mRNA level was observed in both mutants. Prediction by TargetRNA2 revealed cheA as the target of GlmY. Then, construction of the translational fusions containing various lengths of cheA 5′UTR for reporter assay and site-directed mutagenesis were performed to investigate the cheA-GlmY interaction in cheA activation. Notably, loss of glmY reduced the cheA mRNA level, and urea could inhibit swarming in a QseF-dependent manner. Altogether, this is the first report elucidating the underlying mechanisms for modulation of swarming motility by a QseEF-regulated sRNA GlmY, involving expression of cheA, rcsB and flhDC in uropathogenic P. mirabilis.

MicroRNA biogenesis. Part 2. Formation of mature miRNAs. Maturation of non-canonical miRNAs

The scientific review presents the biogenesis of miRNAs. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka. The article shows the stages of formation of mature miRNAs. It is noted that duplex RNAs resulting from DICER-mediated cleavage interact with Argonaute (AGO) proteins to form an effector RNA-induced silencing complex (RISC). It is shown that the deficiency of AGO proteins leads to a significant decrease in the amount of miRs, and overexpression of AGO proteins is accompanied by an increase in the level of miRs. The main stages of assembling a fully functional RISC are presented. The first stage is the loading of duplex miRs on AGO proteins. The second stage is the promotion of duplex miRs. Human diseases associated with processing disorders in the cytoplasm of the cell are presented. Numerous alternative mechanisms involved in the formation of functionally active miRs are is characterized. There are three classes of mirtrons: typical mirtrons, 5’-tailed mirtrons and 3’-tailed mirtrons. Endogenous csRNAs resemble Drosha-independent synthetic csRNAs used to experimentally induce gene knockout. Chimeric hairpins of non-canonical miR genes are transcribed in tandem or as a part of another type of small RNA gene. Thus, the formation of mature miRs occurs due to the formation of the RISC complex. The core of the RISC complex consists of microRNA, AGO and protein with a trinucleotide repeat 6. Loading dsRNA on AGO proteins and subsequent promotion of duplex RNA are the main stages of assembly of a fully functional RISC. Disorders of processing in the cytoplasm of the cell are associated with the development of some human diseases. There are alternative mechanisms involved in the formation of functionally active miRs: mirtrons, endogenous short RNAs containing hairpins, chimeric hairpins.

Genome instability drives epistatic adaptation in the human pathogen Leishmania

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2'-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

MRNA and small RNA gene expression changes in peripheral blood to detect internal Ra-223 exposure

Purpose Excretion analysis is the established method for detection of incorporated alpha-emitting radionuclides, but it is laborious and time consuming. We sought a simplified method in which changes in gene expression might be measured in human peripheral blood to detect incorporated radionuclides. Such an approach could be used to quickly determine internal exposure in instances of a radiological dispersal device or a radiation accident. Materials and methods We evaluated whole blood samples from five patients with castration-resistant prostate cancer and multiple bone metastases (without visceral or nodal involvement), who underwent treatment with the alpha emitting isotope Radium-223 dichloride (Ra-223, Xofigo®). Patients received about 4 MBq per cycle and, depending on survival and treatment tolerance, were followed for six months. We collected 24 blood samples approximately monthly corresponding to treatment cycle. Results Firstly, we conducted whole genome screening of mRNAs (mRNA seq) and small RNAs (small RNA seq) using next generation sequencing in one patient at eight different time points during all six cycles of Ra-223-therapy. We identified 1900 mRNAs and 972 small RNAs (222 miRNAs) that were differentially up- or down-regulated during follow-up after the first treatment with Ra-223. Overall candidate RNA species inclusion criteria were a general (≥|2|-fold) change or with peaking profiles (≥|5|-fold) at specific points in time. Next we chose 72 candidate mRNAs and 101 small RNAs (comprising 29 miRNAs) for methodologic (n = 8 samples, one patient) and independent (n = 16 samples, four patients) validation by qRT-PCR. In total, 15 mRNAs (but no small RNAs) were validated by methodologic and independent testing. However, the deregulation occurred at different time points, showing a large inter-individual variability in response among patients. Conclusions This proof of concept provides support for the applicability of gene expression measurements to detect internalized alpha-emitting radionuclides, but further work is needed with a larger sample size. While our approach has merit for internal deposition monitoring, it was complicated by the severe clinical condition of the patients we studied.

Detection of Epstein-Barr virus encoded small RNA genes in oral squamous cell carcinoma and non-cancerous oral cavity samples

BackgroundEpstein-Barr Virus (EBV) is a human oncogenic virus that can lead to cancer in lymphoid and epithelial cells and is one of the hypothesized causes of oral cavity lesions including oral squamous cell carcinoma (OSCC), but the etiological association remains undetermined. The present investigation aimed to explore the EBV presence, viral load, and EBV-encoded small RNA (EBER) sequence variation in tissue samples of patients with OSCC and other oral cavity lesions including oral lichen planus (OLP), and oral irritation fibroma (OIF).MethodsIn total, 88 oral cavity samples (23 with OSCC, 29 with OLP, and 36 with OIF diagnosis) were examined by Real-Time PCR technique and some of them were sequenced.ResultsViral EBER sequence was detected in 6 out of the 23 OSCC (31.4%), 6 out of the 29 OLP (20.7%), and 3 out of the 36 OIF cases (8.3%). The mean EBV copy number was higher in OSCC samples (1.2 × 10−2 ± 1.3 × 10−2 copies/cell) compared to OLP (2.2 × 10−3 ± 2.6 × 10−3 copies/cell) and OIF (2.4 × 10−4 ± 2.0 × 10−4 copies/cell) samples, although this difference was not statistically significant (P = 0.318). The EBER gene was amplified and sequenced in 5 OSCC, 3 OLP, and 2 OIF samples with high EBV viral load. One OSCC, two OLP, and two OIF isolates showed different nucleotide variations compared with EBV-WT and AG876 prototype sequences: C6834T, C6870T, C6981T, C7085T, C7085G, and C7094T.ConclusionIn our study the presence of more than one genome copies per tumor cell indicates the possible role of EBV infection in oral cancers. However, more studies should be conducted to clarify the role of EBV in OSCC carcinogenesis.

Complete Genome of Marine Microalgae Associated Algicidal Bacterium Sulfitobacter pseudonitzschiae H46 with Quorum Sensing System.

Sulfitobacter pseudonitzschiae H46, a marine bacterium associated with microalgae, exhibits algicidal activity against harmful algal bloom microalgae Chattonella marina and Alexandrium tamarense. The complete genome of S. pseudonitzschiae H46 was sequenced to investigate its algicidal mechanism further. One chromosome, eight circular plasmids and add one not circularized plasmid (Plas II) were identified, which are 4.93Mb in length with 4871 open reading frames. The genome contains 43 transfer RNA (tRNA) genes; six ribosome RNA (rRNA) genes that can be classified into two 5S rRNA, two 16S rRNA, two 23S rRNA, and two small RNA (sRNA) genes. Genomic annotation revealed that S. pseudonitzschiae H46 has two pairs of LuxI/R-type Quorum-sensing (QS) systems. The QS systems can regulate the functions involved in host colonization and biofilm formation. Moreover, the existence of 49 Type-IV and Dddl genes in the chromosome indicated that Dimethylsulfoniopropionate (DMSP) catabolism of S. pseudonitzschiae H46 is the main interaction with marine microalgae. S. pseudonitzschiae H46 utilizes DMSP produced by microalgae as the carbon source, and this may induce algicidal activity. The complete genome sequence of S. pseudonitzschiae H46 can provide useful information on the underlying molecular mechanism between QS and algicidal activity.

Homology-Based Interactions between Small RNAs and Their Targets Control Dominance Hierarchy of Male Determinant Alleles of Self-Incompatibility in Arabidopsis lyrata

Self-incompatibility (SI) is conserved among members of the Brassicaceae plant family. This trait is controlled epigenetically by the dominance hierarchy of the male determinant alleles. We previously demonstrated that a single small RNA (sRNA) gene is sufficient to control the linear dominance hierarchy in Brassica rapa and proposed a model in which a homology-based interaction between sRNAs and target sites controls the complicated dominance hierarchy of male SI determinants. In Arabidopsis halleri, male dominance hierarchy is reported to have arisen from multiple networks of sRNA target gains and losses. Despite these findings, it remains unknown whether the molecular mechanism underlying the dominance hierarchy is conserved among Brassicaceae. Here, we identified sRNAs and their target sites that can explain the linear dominance hierarchy of Arabidopsis lyrata, a species closely related to A. halleri. We tested the model that we established in Brassica to explain the linear dominance hierarchy in A. lyrata. Our results suggest that the dominance hierarchy of A. lyrata is also controlled by a homology-based interaction between sRNAs and their targets.

Leishmania mexicana recombinant filamentous acid phosphatase as carrier for Toxoplasma gondii surface antigen 1 expression in Leishmania tarentolae.

Leishmania tarentolae has been used to produce recombinant intracellular and secreted proteins for their easy handling and posttranslational modifications. Filamentous acid phosphatase is a multimeric protein complex composed of many subunits assembled in a linear highly glycosylated filament, which is secreted in vast amounts into the culture supernatant via the flagellar pocket of Leishmania mexicana promastigotes. This suggested that the protein could be used as a carrier for the Surface Antigen1 protein of a Toxoplasma gondii (SAG1) for easy purification to generate a protein with multiple SAG1 subunits suitable for immunisation. SAG1 has an immunodominant structure that is involved in binding to host cells. Previous studies used this surface protein for vaccination for its immunological importance for triggering a type 1 immune response in the host. This study aims to determine the production of recombinant filamentous protein carried subunits of the surface protein of Toxoplasma gondii for vaccination purposes. Leishmania codon-optimised SAG1 was cloned as a fusion construct into pLEXSY-ble2.1 plasmid and introduced into Leishmania tarentolae to generate recombinant cell lines expressing a filamentous fusion protein called SAP2SAG1. PCR confirmed the correct integration into the small ribosomal subunit RNA gene locus of Leishmania tarentolae. Immunofluorescences and Immunoblot analyses were used to detect the fusion protein in the sediment of culture supernatants of recombinant L. tarentolae promastigotes after purification by ultracentrifugation. The yield of purified protein was low that suggested further investigations of other methods for scaling large production of secreted protein.

Argonaute proteins: structures and their endonuclease activity.

Argonaute proteins are highly conserved and widely expressed in almost all organisms. They not only play a critical role in the biogenesis of small RNAs but also defend against invading nucleic acids via small RNA or DNA-mediated gene silencing pathways. One functional mechanism of Argonaute proteins is acting as a nucleic-acid-guided endonuclease, which can cleave targets complementary to DNA or RNA guides. The cleavage then leads to translational silencing directly or indirectly by recruiting additional silencing proteins. Here, we summarized the latest research progress in structural and biological studies of Argonaute proteins and pointed out their potential applications in the field of gene editing.

Evaluation of a New Histoplasma spp. Quantitative RT-PCR Assay

Laboratory diagnosis of histoplasmosis is based on various methods, including microscopy, culture, antigen, and DNA detection of Histoplasma capsulatum var. capsulatum or Histoplasma capsulatum var. duboisii. To improve sensitivity of existing real-time quantitative PCR (qPCR) assays, we developed a new RT-qPCR assay that allows amplification of whole nucleic acids of Histoplasma spp. validated on suspected cases. The limit of detection was 20 copies, and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp. Whole nucleic acids of 1319 prospectively collected consecutive samples from 907 patients suspected of having histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. Forty-four had proven histoplasmosis attributable to H. capsulatum var. capsulatum (n = 40) or H.capsulatum var. duboisii (n = 4) infections. The results of RT-qPCR were positive in 43 of 44 patients (97.7% sensitivity) in at least one specimen. Nine of 863 cases (99% specificity) were RT-qPCR positive and therefore classified as possible cases. RT-qPCR was positive in 13 of 30 (43.3%) blood samples tested in proven cases. A positive RT-qPCR result in blood was significantly associated with H. capsulatum var. capsulatum progressively disseminated histoplasmosis with a positive RT-qPCR result in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RT-qPCR assay enabling amplification of H. capsulatum var. capsulatum and H. capsulatum var. duboisii is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and bronchoalveolar lavage fluid.