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Vivek R, Montemayor EJ, Butcher SE, Petersen RJ, Bingman CA, Tonelli M, Wickens M, Nomura Y, Yan J, Roschdi S, Kennedy SG, Escobar CA
[
Nat Struct Mol Biol,
2022]
The addition of poly(UG) ('pUG') repeats to 3' termini of mRNAs drives gene silencing and transgenerational epigenetic inheritance in the metazoan Caenorhabditis elegans. pUG tails promote silencing by recruiting an RNA-dependent RNA polymerase (RdRP) that synthesizes small interfering RNAs. Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4) structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. The compact pUG fold binds six potassium ions and brings the RNA ends into close proximity. The biological importance of the pUG fold is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific 7-deaza substitutions that disrupt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA structural motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in noncoding regions of human RNAs, suggesting that the fold probably has biological roles beyond gene silencing.
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[
Environ Sci Pollut Res Int,
2019]
The adverse effects of heavy metals, such as cadmium, zinc, and copper, occur due to the generation of reactive oxygen species (ROS). The use of Caenorhabditis elegans for the purposes of conservation and biomonitoring is of great interest. In the present study, ROS, malondialdehyde (MDA), and citric acid levels and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in a model organism were tested to study toxicity. C. elegans was exposed to three different concentrations of cadmium (CdCl<sub>2</sub>, 5, 10, 50 M), zinc (ZnSO<sub>4</sub>, 10, 100, 500 M), and copper (CuSO<sub>4</sub>, 10, 100, 500 M) for 3 days. ROS levels increased by 1.3- to 2.1-fold with increasing metal concentrations. The MDA content increased by approximately 7-, 5-, 2-fold after exposure to high concentrations of cadmium, zinc, and copper, respectively. Furthermore, the citric acid content increased by approximately 3-fold in the cadmium (Cd, 5 M), zinc (Zn, 10 M), and copper (Cu, 100 M) treatment groups compared to that in untreated C. elegans. Therefore, citric acid may play an important role in heavy metal detoxification. Excess citric acid also slightly increased the LC<sub>50</sub> by 1.3- to 2.0-fold, basic movements by 1.0- to 1.5-fold, decreased the ROS content by 2.4- to 2.1-fold, the MDA content by 4- to 2-fold, the SOD activity by 9- to 3-fold, the GPx activity by 4.0- to 3.0-fold, and the mRNA expression levels of GPxs by 3.2- to 1.8-fold after metals treatment. And it is most significantly in the alleviation of citric acid to cadmium. This study not only provides information to further understand the effects of heavy metal exposure on ROS, MDA, GPx, SOD, and citric acid in worms but also indicates that supplemental citric acid can protect animals from heavy metal stress and has broad application prospects in decreasing oxidative damage caused by heavy metals.
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[
PLoS One,
2012]
BACKGROUND: The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. Recently we demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpC(KP), to mediate antimicrobial resistance in K. pneumoniae. PRINCIPAL FINDINGS: In this study, functional characterization of the putative porin OmpC(KP) (denoted kpnO) with respect to antimicrobial susceptibility and virulence was evaluated by generating an isogenic mutant, kpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that kpnO produced 2.0 fold lesser capsular polysaccharide than the wild-type. The kpnO displayed higher sensitivity to hyper osmotic and bile conditions. Disruption of kpnO increased the susceptibility of K. pneumoniae to oxidative and nitrostative stress by 1.6 fold and >7 fold respectively. The loss of the Klebsiella porin led to an increase in the minimum inhibitory concentration of tetracycline (3-fold), nalidixic acid (4-fold), tobramycin (4-fold), streptomycin (10-fold), and spectinomycin (10-fold), which could be restored following complementation. The single deletion of kpnO reduced the survival of the pathogen by 50% when exposed to disinfectants. In Caenorhabditis elegans model, the kpnO mutant exhibited significantly (P<0.01) lower virulence. To dissect the role of PhoBR signaling system in regulating the expression of the kpnO, a phoB(KP) isogenic mutant was constructed. The phoB(KP) mutant exhibited impaired gastrointestinal stress response and decreased antimicrobial susceptibility. The mRNA levels of kpnO were found to be 4-fold less in phoB(KP) mutant compared to wild type. A regulatory role of PhoB(KP) for the expression of kpnO was further supported by the specific binding of PhoB(KP) to the putative promoter of kpnO. CONCLUSIONS AND SIGNIFICANCE: Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.
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[
Microbiology,
2013]
Klebsiella pneumoniae is a Gram-negative bacillus that causes serious infections in immunocompromised human hosts and exhibits significant multidrug resistance. In this study, we identified a novel lysR-family regulator (designated oxyR(KP)) in the genome of K. pneumoniae NTUH-K2044 whose functions have remained enigmatic so far. Functional characterization of the putative lysR regulator oxyR(KP) with respect to cellular physiology and antimicrobial susceptibility was performed by generating an isogenic mutant, oxyR(KP) in a hypervirulent clinical isolate of K. pneumoniae. The K. pneumoniae oxyR(KP) mutant was sensitive to hyperosmotic and bile conditions. Disruption of oxyR(KP) increased the susceptibility of K. pneumoniae to oxidative (0.78947 mM hydrogen peroxide) and nitrosative (30 mM acidified nitrite) stress by ~1.4-fold and ~10-fold, respectively. Loss of the Klebsiella regulator led to a decrease in the minimum inhibitory concentrations for chloramphenicol (10-fold), erythromycin (6-fold), nalidixic acid (>50-fold) and trimethoprim (10-fold), which could be restored following complementation. The relative change in expression of resistance-nodulation-cell division super family (RND) efflux gene acrB was decreased by approximately fivefold in the oxyR(KP) mutant as evidenced by qRT-PCR. In a Caenorhabditis elegans model, the oxyR(KP) mutant exhibited significantly (P<0.01) lower virulence. Overall, results detailed in this report reflect the pleiotropic role of the oxyR(KP) signalling system and diversity of the resistance determinants in hypervirulent K1 serotype K. pneumoniae NTUH-K2044.
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Tian L, Svoboda K, Mao T, Petreanu L, Looger LL, Huber D, Chiappe ME, Chalasani SH, Hires SA, Bargmann CI, McKinney SA, Schreiter ER, Jayaraman V, Akerboom J
[
Nat Methods,
2009]
Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.
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[
Mol Cell Proteomics,
2003]
Although nematodes like Caenorhabditis elegans are incapable of the de novo cholesterol biosynthesis, they can utilize nonfunctional sterols by converting them into cholesterol and other sterols for cellular function. The results reported previously and presented here suggest that blocking of sterol conversion to cholesterol in C. elegans by 25-azacoprostane-HCL (azacoprostane) treatment causes a serious defect in germ cell development, growth, cuticle development, and motility behavior. To establish a biochemical basis for these physiological abnormalities, we performed proteomic analysis of mixed stage worms that had been treated with the drug. Our results from a differential display proteomic analysis revealed significant decreases in the levels of proteins involved in collagen and cytoskeleton organization such as protein disulfide isomerase (6.7-fold), beta-tubulin (5.41-fold), and NEX-1 protein (>30-fold). Also reduced were enzymes involved in energy production such as phosphoglycerate kinase (4.8-fold) and phosphoenolypyruvate carboxykinase (8.5-fold), a target for antifilarial drugs such as azacoprostane. In particular, reductions in the expression of lipoprotein familie4s such as vetellogenin-2 (7.7-fold) and vitellogenin-6 (5.4-fold) were prominent in the drug-treated worms, indicating that sterol metabolism disturbance caused by azacoprostane treatment is tightly coupled with suppression of the lipid transfer-related proteins at the protein level. However, competitive quantitative reverse transcriptase polymerase chain reaction showed that the transcriptional levels of
vit-2,
vit-6, and their receptors (e.g.
rme-2 and
lrp-1) in drug-treated worms were 3- to 5-fold higher than those in the untreated group, suggesting a presence of a sterol regulatory element-binding protein (SREBP)-like pathway in these genes. In fact, multiple predicted sterol regulatory elements or related regulatory sequences responding to sterols were found to be located at the 5'-flanking regions in
vit-2 and
lrp-1 genes, and their transcriptional activities fluctuated highly in response to changes in sterol concentration. Thus, many physiological abnormalities caused by azacoprostane-mediated sterol metabolism disturbance appear to be exerted at least in
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[
Cancer Treat Res Commun,
2021]
Early cancer detection is critical for effective treatment. N-NOSE (Nematode-NOSE) is a simple, inexpensive, and highly sensitive cancer screening method based on the chemotaxis of the nematode Caenorhabditis elegans, which shows evasive action from the urine of healthy individuals while being attracted to the urine of cancer patients. Initially, N-NOSE relied on chemotaxis indexes obtained with 10-fold dilutions of urine samples. However, cancer tissue size and concentrations of cancer odors differ among cancer patients. In this study, we examined the accuracy improvement of N-NOSE method by using two types of dilutions, 10-fold and 100-fold. We have conducted N-NOSE tests with urine samples from 32 cancer patients (esophageal, gastric, colorectal, gallbladder, cholangiocarcinoma, breast, malignant lymphoma, and acute myeloid leukemia) along with 143 healthy subjects. Our data showed a significant difference in the N-NOSE at 10-fold dilution between the two groups (p < 0.0001), with an area under the ROC curve (AUC) of 0.9188 based on receiver operating characteristic (ROC) analysis. N-NOSE index at 100-fold dilutions was also significantly different between the two groups (p < 0.0001), with an AUC of 0.9032 based on ROC analysis. In this clinical study, we further improve N-NOSE with a combined method of two dilutions (10-fold and 100-fold) of urine samples, which results in a markedly improvement in cancer detection sensitivity of 87.5%. N-NOSE sensitivity improvement was significantly high even for early-stage cancer detection, which is in stark contrast with the sensitivity of detection using blood tumor markers (CEA, CA19-9 and CA15-3). These results strongly suggest that the N-NOSE test by this new combined method strikes a good balance between sensitivity and specificity.
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[
Mech Ageing Dev,
1983]
In an attempt to provide additional quantitative markers of senescence in the nematode Caenorhabditis elegans, we have identified age-dependent increases in four lysosomal enzymes: acid phosphatase, beta-N-acetyl-D-glucosaminidase, beta-D-glucosidase, and alpha-D- mannosidase. These enzymes were judged to be lysosomal on the basis of their resemblance to analogous mammalian lysosomal enzymes with regard to subcellular fractionation, lectin binding, Km, molecular weights, inhibitor sensitivities, and pH optima. In nematode populations which had a median lifespan of 8.9 +/- 0.7 days and a maximum lifespan of 14-16 days, we observed the following increases in acid hydrolase activities per animal from day 3 (early adulthood) to day 10: (1) up to 2.5-fold for acid phosphatase; (2) 8-fold for beta-N-acetyl-D-glucosaminidase; (3) 9-fold for beta-D-glucosidase; and (4) 4-fold for alpha-D-mannosidase. Three forms of acid phosphatase and two forms of beta-D-glucosidase were separated by ion- exchange chromatography, but in each case only one form of the enzyme was primarily responsible for the age-dependent increase in total activity: acid phosphatase I increased 18-fold, while beta-D- glucosidase I increased 100-fold. By contrast, there were only slight age-dependent changes in choline acetyltransferase, acetylcholinesterase, or alpha-D-glucosidase activities after early adulthood. The age-dependent increases in acid phosphatase, beta-N- acetyl-D-glucosaminidase, beta-D-glucosidase, and alpha-D-mannosidase activities are sufficiently large and reproducible to be useful quantitative markers of senescence in C. elegans.
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[
Protein Eng,
2000]
The quality of three-dimensional homology models derived from protein sequences provides an independent measure of the suitability of a protein sequence for a certain fold. We have used automated homology modeling and model assessment tools to identify putative nuclear hormone receptor ligand-binding domains in the genome of Caenorhabditis elegans. Our results indicate that the availability of multiple crystal structures is crucial to obtaining useful models in this receptor family. The majority of annotated mammalian nuclear hormone receptors could be assigned to a ligand-binding domain fold by using the best model derived from any of four template structures. This strategy also assigned the ligand-binding domain fold to a number of C.elegans. sequences without prior annotation. Interestingly, the retinoic acid receptor crystal structure contributed most to the number of sequences that could be assigned to a ligand-binding domain fold. Several causes for this can be suggested, including the high quality of this protein structure in terms of our assessment tools, similarity between the biological function or ligand of this receptor and the modeled genes and gene duplication in C.elegans.
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[
Mech Ageing Dev,
1988]
The activities of 3 lysosomal proteases in the nematode Caenorhabditis elegans are markedly lower in older animals. The aspartyl protease cathepsin D declines about 10-fold from day 3 (early adulthood) to day 11 (near the mean lifespan); this reflects a net decline in the amount of cathepsin D antigen. The specific activity of the thiol protease cathepsin Ce1 declines about 2.5-fold over the same period, and the specific activity of the thiol protease cathepsin Ce2 declines about 8-fold. The activity of a new non- lysosomal protease, designated cathepsin CeX, is invariant with age. The data are consistent with the hypothesis that reduced protease activity in older animals may cause a decline in the rate of protein turnover with age, but do not prove this hypothesis.