Substrate-dependent effects of quaternary structure on RNase E activity.

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2.

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.

Endoribonuclease-mediated control of hns mRNA stability constitutes a key regulatory pathway for Salmonella Typhimurium pathogenicity island 1 expression.

Bacteria utilize endoribonuclease-mediated RNA processing and decay to rapidly adapt to environmental changes. Here, we report that the modulation of hns mRNA stability by the endoribonuclease RNase G plays a key role in Salmonella Typhimurium pathogenicity. We found that RNase G determines the half-life of hns mRNA by cleaving its 5' untranslated region and that altering its cleavage sites by genome editing stabilizes hns mRNA, thus decreasing S. Typhimurium virulence in mice. Under anaerobic conditions, the FNR-mediated transcriptional repression of rnc encoding RNase III, which degrades rng mRNA, and simultaneous induction of rng transcription resulted in rapid hns mRNA degradation, leading to the derepression of genes involved in the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS). Together, our findings show that RNase III and RNase G levels-mediated control of hns mRNA abundance acts as a regulatory pathway upstream of a complex feed-forward loop for SPI-1 expression.

Regulator of RNase E activity modulates the pathogenicity of Salmonella Typhimurium.

RNase E-mediated RNA processing and degradation are involved in bacterial adaptation to environmental changes. The RraA regulatory protein, which is highly conserved in γ-proteobacteria, differentially modulates RNase E activity. Recent studies have revealed the association of Salmonella enterica serovar Typhimurium RNase E (STRNase E) with bacterial pathogenicity; however, the molecular mechanisms are unknown. Here, we show that the expression levels of STRraA, a protein regulator of STRNase E activity, affect S. Typhimurium pathogenicity. RNA-sequencing and RT-PCR analyses indicated positive effects of STRraA levels on the abundance of mRNA species from class II flagellar operons. Primer extension analysis further identified STRraA-regulated STRNase E cleavage in the 5' untranslated region of fliDST mRNA. The cleavage affected the stability of this polycistronic mRNA, suggesting that STRraA protects fliDST mRNA from STRNase E cleavage, leading to enhanced flagellar assembly. Accordingly, STRraA positively regulated flagellar assembly and motility. In addition, STrraA-deleted cells showed decreased invasion ability and cytotoxicity in infection of human cervical epithelial carcinoma cells and reduced mortality in a mouse infection model compared to wild-type cells. These results support an active role of STRraA in RNase E-mediated modulation of pathogenesis in S. Typhimurium.

Specialised ribosomes as versatile regulators of gene expression.

The ribosome has long been thought to be a homogeneous cellular machine that constitutively and globally synthesises proteins from mRNA. However, recent studies have revealed that ribosomes are highly heterogeneous, dynamic macromolecular complexes with specialised roles in translational regulation in many organisms across the kingdoms. In this review, we summarise the current understanding of ribosome heterogeneity and the specialised functions of heterogeneous ribosomes. We also discuss specialised translation systems that utilise orthogonal ribosomes.

Characteristics and Fitness Analysis through Interspecific Hybrid Progenies of Transgenic Brassica napus and B. rapa L. ssp.

Interspecific hybridization between transgenic crops and their wild relatives is a major concern for transgene dispersal in the environment. Under controlled conditions, artificial hand pollination experiments were performed in order to assess the hybridization potential and the fitness of interspecific hybrids between Brassica rapa and genetically modified (GM) Brassica napus. Initially, six subspecies of B. rapa were hybridized with GM B. napus through hand pollination. In the resulting F1 hybrids, the combination of B. rapa ssp. narinosa (♀) × GM B. napus (♂) had the highest crossability index (16.9 ± 2.6). However, the F1 selfing progenies of B. rapa ssp. rapa (♀) × GM B. napus were found to be more effective in producing viable future generations with the highest crossability index (1.6 ± 0.69) compared to other subspecies. Consequently, they were used for the generation of F2 and F3 progenies. The 18 different morphological characteristics among the parental cross-combinations and F1 hybrid progenies were measured and visualized through hierarchical clustering. Different generations were found to be grouped based on their different morphological characteristics. The chromosome numbers among the interspecific hybrids ranged from 2n = 29 to 2n = 40. Furthermore, the SSR markers revealed the presence of genomic portions in the hybrids in comparison with their parental lines. There is a high possibility of transgene flow between GM B. napus and B. rapa. The study concluded that the interspecific hybrids between B. napus and B. rapa can be viable and can actively hybridize up to F3 generations and more. This suggests that the GM B. napus can disperse the transgene into B. rapa, and that it can pass through for several generations by hand pollination in a greenhouse environment.

Discrimination of Brassica juncea Varieties Using Visible Near-Infrared (Vis-NIR) Spectroscopy and Chemometrics Methods.

Brown mustard (Brassica juncea (L.) is an important oilseed crop that is mostly used to produce edible oils, industrial oils, modified lipids and biofuels in subtropical nations. Due to its higher level of commercial use, the species has a huge array of varieties/cultivars. The purpose of this study is to evaluate the use of visible near-infrared (Vis-NIR) spectroscopy in combination with multiple chemometric approaches for distinguishing four B. juncea varieties in Korea. The spectra from the leaves of four different growth stages of four B. juncea varieties were measured in the Vis-NIR range of 325-1075 nm with a stepping of 1.5 nm in reflectance mode. For effective discrimination, the spectral data were preprocessed using three distinct approaches, and eight different chemometric analyses were utilized. After the detection of outliers, the samples were split into two groups, one serving as a calibration set and the other as a validation set. When numerous preprocessing and chemometric approaches were applied for discriminating, the combination of standard normal variate and deep learning had the highest classification accuracy in all the growth stages achieved up to 100%. Similarly, few other chemometrics also yielded 100% classification accuracy, namely, support vector machine, generalized linear model, and the random forest. Of all the chemometric preprocessing methods, Savitzky-Golay filter smoothing provided the best and most convincing discrimination. The findings imply that chemometric methods combined with handheld Vis-NIR spectroscopy can be utilized as an efficient tool for differentiating B. juncea varieties in the field in all the growth stages.

Dynamics of Bacterial Community Structure in the Rhizosphere and Root Nodule of Soybean: Impacts of Growth Stages and Varieties.

Bacterial communities in rhizosphere and root nodules have significant contributions to the growth and productivity of the soybean (Glycine max (L.) Merr.). In this report, we analyzed the physiological properties and dynamics of bacterial community structure in rhizosphere and root nodules at different growth stages using BioLog EcoPlate and high-throughput sequencing technology, respectively. The BioLog assay found that the metabolic capability of rhizosphere is in increasing trend in the growth of soybeans as compared to the bulk soil. As a result of the Illumina sequencing analysis, the microbial community structure of rhizosphere and root nodules was found to be influenced by the variety and growth stage of the soybean. At the phylum level, Actinobacteria were the most abundant in rhizosphere at all growth stages, followed by Alphaproteobacteria and Acidobacteria, and the phylum Bacteroidetes showed the greatest change. But, in the root nodules Alphaproteobacteria were dominant. The results of the OTU analysis exhibited the dominance of Bradyrhizobium during the entire stage of growth, but the ratio of non-rhizobial bacteria showed an increasing trend as the soybean growth progressed. These findings revealed that bacterial community in the rhizosphere and root nodules changed according to both the variety and growth stages of soybean in the field.

An Overview of Near Infrared Spectroscopy and Its Applications in the Detection of Genetically Modified Organisms.

Near-infrared spectroscopy (NIRS) has become a more popular approach for quantitative and qualitative analysis of feeds, foods and medicine in conjunction with an arsenal of chemometric tools. This was the foundation for the increased importance of NIRS in other fields, like genetics and transgenic monitoring. A considerable number of studies have utilized NIRS for the effective identification and discrimination of plants and foods, especially for the identification of genetically modified crops. Few previous reviews have elaborated on the applications of NIRS in agriculture and food, but there is no comprehensive review that compares the use of NIRS in the detection of genetically modified organisms (GMOs). This is particularly important because, in comparison to previous technologies such as PCR and ELISA, NIRS offers several advantages, such as speed (eliminating time-consuming procedures), non-destructive/non-invasive analysis, and is inexpensive in terms of cost and maintenance. More importantly, this technique has the potential to measure multiple quality components in GMOs with reliable accuracy. In this review, we brief about the fundamentals and versatile applications of NIRS for the effective identification of GMOs in the agricultural and food systems.

Discrimination of Transgenic Canola (Brassica napus L.) and their Hybrids with B. rapa using Vis-NIR Spectroscopy and Machine Learning Methods.

In recent years, the rapid development of genetically modified (GM) technology has raised concerns about the safety of GM crops and foods for human health and the ecological environment. Gene flow from GM crops to other crops, especially in the Brassicaceae family, might pose a threat to the environment due to their weediness. Hence, finding reliable, quick, and low-cost methods to detect and monitor the presence of GM crops and crop products is important. In this study, we used visible near-infrared (Vis-NIR) spectroscopy for the effective discrimination of GM and non-GM Brassica napus, B. rapa, and F1 hybrids (B. rapa X GM B. napus). Initially, Vis-NIR spectra were collected from the plants, and the spectra were preprocessed. A combination of different preprocessing methods (four methods) and various modeling approaches (eight methods) was used for effective discrimination. Among the different combinations, the Savitzky-Golay and Support Vector Machine combination was found to be an optimal model in the discrimination of GM, non-GM, and hybrid plants with the highest accuracy rate (100%). The use of a Convolutional Neural Network with Normalization resulted in 98.9%. The same higher accuracy was found in the use of Gradient Boosted Trees and Fast Large Margin approaches. Later, phenolic acid concentration among the different plants was assessed using GC-MS analysis. Partial least squares regression analysis of Vis-NIR spectra and biochemical characteristics showed significant correlations in their respective changes. The results showed that handheld Vis-NIR spectroscopy combined with chemometric analyses could be used for the effective discrimination of GM and non-GM B. napus, B. rapa, and F1 hybrids. Biochemical composition analysis can also be combined with the Vis-NIR spectra for efficient discrimination.

BAX is an essential key mediator of AP5M1-induced apoptosis in cervical carcinoma cells.

The adaptor-related protein complex 5 subunit mu 1 (AP5M1) is an evolutionally conserved protein with ubiquitous expression in human tissues. However, the major function of AP5M1 in living organisms is unclear owing to few published studies. Here, we demonstrate that AP5M1 is a potent apoptosis-inducing molecule in cervical cancer cells. We also found that AP5M1 upregulated the level of BAX protein, a key pro-apoptotic B cell lymphoma (BCL)-2 family member regulating mitochondrial apoptotic cell death pathway. Moreover, AP5M1 completely lost its apoptotic activity in BAX-knockout or -knockdown cells, indicative of its functional dependence on BAX. Comparative analysis of cervical tissues from patients with cervical carcinoma and non-cancer control revealed a prominent downregulation in AP5M1 expression with a concomitant downregulation in BAX expression; AP5M1 and BAX mRNA expression levels in cervical tissues exhibited a strong positive correlation (r = 0.97). Thus, we identified AP5M1 as a previously unrecognized apoptotic protein that governs BAX expression and revealed the association between AP5M1 and malignancy.

EGR2 is a gonadotropin-induced survival factor that controls the expression of IER3 in ovarian granulosa cells.

Pituitary gonadotropins are key hormones that orchestrate the growth and development of ovarian follicles. However, limited information is available on intra-ovarian factors that mediate the actions of gonadotropins. In this study, we identified that the early growth response 2 gene (EGR2) is a gonadotropin-inducible gene in granulosa cells of rats and humans. Analysis of consensus EGR-binding elements (EBEs) showed that the immediate early response 3 gene (IER3) is a novel transcriptional target gene of EGR2 as confirmed by the luciferase assay, electrophoretic mobility-shift assay (EMSA), chromatin immunoprecipitation (ChIP), and western blot analysis. Overexpression of EGR2 promoted survival of KGN human granulosa-derived cells in which IER3 acts as a mediator; knockdown of EGR2 induced death in KGN cells. Additionally, EGR2 was found to regulate the expression of myeloid cell leukemia 1 (MCL-1), which belongs to the BCL-2 family of proteins regulating cell survival. Thus, this study identified a novel signaling axis, comprised of gonadotropins-EGR2-IER3, which is important for the survival of granulosa cells during folliculogenesis.

Identification and Validation of Differential Phosphorylation Sites of the Nuclear FOXL2 Protein as Potential Novel Biomarkers for Adult-Type Granulosa Cell Tumors.

Granulosa cell tumor (GCT) is a rare form of ovarian cancer classified as a sex cord-stromal tumor. The c.402C→G missense mutation in the FOXL2 gene that changes cysteine 134 to tryptophan (C134W) is found in more than 97% of adult-type GCTs, and the C134W FOXL2 mutant is hyperphosphorylated. We identified three differential phosphorylation sites, at serine 33 (S33), tyrosine 186 (Y186), and serine 238 (S238), of the C134W mutant by tandem mass spectrometry. Among these sites, antibodies were raised against the pS33 and pY186 epitopes using specific peptides, and they were tested by immunostaining tissue microarrays of archival adult-type GCT specimens, other tumors, and normal tissues. The pS33 antibody showed greater sensitivity and specificity for the detection of adult-type GCTs than that of the other phospho and nonphospho antibodies. The specificity of the pS33 antibody to the pS33 epitope was further confirmed by enriching the pS33 peptide by affinity chromatography using the immobilized antibody, followed by mass spectrometric and western blot analyses from whole cell lysates of the adult-type GCT cell line, KGN. pS33 FOXL2 immunostaining levels were significantly higher in adult-type GCTs than those in other tumors and tissues. The receiver operating characteristic curve analysis of pS33 FOXL2 showed high sensitivity (1.0) and specificity (0.76) to adult-type GCTs with a cutoff score of >30% positive cells, and the area under the curve was 0.96. This suggests the potential of pS33 FOXL2 to serve as a new biomarker for the diagnosis of adult-type GCT.

Oestrogen up-regulates interleukin-21 production by CD4(+) T lymphocytes in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which abnormal immune responses are mediated by tissue-binding autoantibodies and immune complex deposition. Because most SLE patients are women of child-bearing age, oestrogen has been suggested to play an important role in SLE pathogenesis. One proposed role is to induce B-cell activation, culminating in increased autoantibody production. Interleukin-21 (IL-21) has been shown to be crucial in the differentiation of activated B cells into plasma cells. We therefore hypothesized that oestrogen up-regulates IL-21 production and induces subsequent B-cell activation in SLE patients. Peripheral blood was obtained from 22 SLE patients and 16 healthy controls. Expression levels of IL-21 and its receptor in serum, peripheral blood mononuclear cells, and CD4(+) T cells were higher in SLE patients than in healthy controls. Exposure of CD4(+) T cells from SLE patients to 17ß-oestradiol led to a dose- and time-dependent increase in IL-21 expression, which was abolished in the presence of mitogen-activated protein kinase (MAPK) (MAPK kinase, p38, Jun N-terminal kinase) inhibitors. B cells from healthy controls showed increased antibody production when they were co-cultured with oestrogen-treated CD4(+) T cells from SLE patients. Treatment with IL-21 antibody abrogated the increased antibody production of the co-culture systems. This study revealed the association between oestrogen and IL-21 in SLE patients. Oestrogen up-regulates IL-21 expression of CD4(+) T cells via MAPK-dependent pathways in SLE patients, which in turn induces increased antibody production by B cells.

Psychometric properties of scale constructed from the International Classification of Functioning, Disability and Health (ICF) core set for breast cancer based on Rasch analysis.

OBJECTIVES: The purpose of this study was to construct a clinical instrument to measure functioning in breast cancer survivors using the International Classification of Functioning, Disability and Health (ICF) categories for body functions, activity and participation, and environmental factors, based on a Rasch analysis. METHODS: Items were generated from the brief ICF core set for breast cancer and in-depth interviews from eight oncologists. Psychometric properties were evaluated in 158 female Korean patients with breast cancer using Rasch analysis, such as fit of the ICF categories, targeting between the ICF categories and a person's abilities, unidimensionality, and reliability. RESULTS: The Rasch refinement led to a change from the original 43-item, 5-level scale to a 30-item, 3- or 4-level scale. Rasch reliabilities were 0.89 (body function scale), 0.96 (activity and participation scale), and 0.93 (environmental scale). The item-difficulty hierarchy was stable across age (<50 or ≥50 years) and had no non-fitting items or gaps (all information weighted fit (infit)/outlier sensitive fit (outfit) mean square error of 0.7-1.3, n = 140). CONCLUSION: The Brief Core Set Breast Cancer Questionnaire for Screening is a reliable and valid 30-item questionnaire based on the brief ICF core set. It allows measurement of functioning as a unidimensional construct in patients with breast cancer.

Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I.

RNase E is an essential enzyme in Escherichia coli. The cleavage site of this single-stranded specific endoribonuclease is well-characterized in many RNA substrates. Here, we report that the upregulation of RNase E cleavage activity by a mutation that affects either RNA binding (Q36R) or enzyme multimerization (E429G) was accompanied by relaxed cleavage specificity. Both mutations led to enhanced RNase E cleavage in RNA I, an antisense RNA of ColE1-type plasmid replication, at a major site and other cryptic sites. Expression of a truncated RNA I with a major RNase E cleavage site deletion at the 5'-end (RNA I-5) resulted in an approximately twofold increase in the steady-state levels of RNA I-5 and the copy number of ColE1-type plasmid in E. coli cells expressing wild-type or variant RNase E compared to those expressing RNA I. These results indicate that RNA I-5 does not efficiently function as an antisense RNA despite having a triphosphate group at the 5'-end, which protects the RNA from ribonuclease attack. Our study suggests that increased cleavage rates of RNase E lead to relaxed cleavage specificity on RNA I and the inability of the cleavage product of RNA I as an antisense regulator in vivo does not stem from its instability by having 5'-monophosphorylated end.

Functional conservation of specialized ribosomes bearing genome-encoded variant rRNAs in Vibrio species.

Heterogeneity of ribosomal RNA (rRNA) sequences has recently emerged as a mechanism that can lead to subpopulations of specialized ribosomes. Our previous study showed that ribosomes containing highly divergent rRNAs expressed from the rrnI operon (I-ribosomes) can preferentially translate a subset of mRNAs such as hspA and tpiA in the Vibrio vulnificus CMCP6 strain. Here, we explored the functional conservation of I-ribosomes across Vibrio species. Exogenous expression of the rrnI operon in another V. vulnificus strain, MO6-24/O, and in another Vibrio species, V. fischeri (strain MJ11), decreased heat shock susceptibility by upregulating HspA expression. In addition, we provide direct evidence for the preferential synthesis of HspA by I-ribosomes in the V. vulnificus MO6-24/O strain. Furthermore, exogenous expression of rrnI in V. vulnificus MO6-24/O cells led to higher mortality of infected mice when compared to the wild-type (WT) strain and a strain expressing exogenous rrnG, a redundant rRNA gene in the V. vulnificus CMCP6 strain. Our findings suggest that specialized ribosomes bearing heterogeneous rRNAs play a conserved role in translational regulation among Vibrio species. This study shows the functional importance of rRNA heterogeneity in gene expression control by preferential translation of specific mRNAs, providing another layer of specialized ribosome system.

Gold nanoparticle-DNA aptamer-assisted delivery of antimicrobial peptide effectively inhibits Acinetobacter baumannii infection in mice.

Acinetobacter baumannii causes multidrug resistance, leading to fatal infections in humans. In this study, we showed that Lys AB2 P3-His-a hexahistidine-tagged form of an antimicrobial peptide (AMP) loaded onto DNA aptamer-functionalized gold nanoparticles (AuNP-Apt)-can effectively inhibit A. baumannii infection in mice. When A. baumannii-infected mice were intraperitoneally injected with AuNP-Apt loaded with Lys AB2 P3-His, a marked reduction in A. baumannii colonization was observed in the mouse organs, leading to prominently increased survival time and rate of the mice compared to those of the control mice treated with AuNP-Apt or Lys AB2 P3-His only. This study shows that AMPs loaded onto AuNP-Apt could be an effective therapeutic tool against infections caused by multidrug-resistant pathogenic bacteria in humans.