The terminal uridylyltransferase, TUT1, builds or repairs the 3'-oligo-uridylylated tail of U6 snRNA. The 3'-oligo-uridylylated tail is the Lsm-binding site for U4/U6 di-snRNP formation and U6 snRNA ...recycling for pre-mRNA splicing. Here, we report crystallographic and biochemical analyses of human TUT1, which revealed the mechanisms for the specific uridylylation of the 3'-end of U6 snRNA by TUT1. The O
and O
atoms of the UTP base form hydrogen bonds with the conserved His and Asn in the catalytic pocket, respectively, and TUT1 preferentially incorporates UMP onto the 3'-end of RNAs. TUT1 recognizes the entire U6 snRNA molecule by its catalytic domains, N-terminal RNA-recognition motifs and a previously unidentified C-terminal RNA-binding domain. Each domain recognizes specific regions within U6 snRNA, and the recognition is coupled with the domain movements and U6 snRNA structural changes. Hence, TUT1 functions as the U6 snRNA-specific terminal uridylyltransferase required for pre-mRNA splicing.
Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has ...clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Automated cell counters that utilize still images of sample cells are widely used. However, they are not well suited to counting slender, aggregate-prone microorganisms such as Trypanosoma cruzi. ...Here, we developed a motion-based cell-counting system, using an image-recognition method based on a cubic higher-order local auto-correlation feature. The software successfully estimated the cell density of dispersed, aggregated, as well as fluorescent parasites by motion pattern recognition. Loss of parasites activeness due to drug treatment could also be detected as a reduction in apparent cell count, which potentially increases the sensitivity of drug screening assays. Moreover, the motion-based approach enabled estimation of the number of parasites in a co-culture with host mammalian cells, by disregarding the presence of the host cells as a static background.
Atmospheric Scanning Electron Microscopy (ASEM) is a powerful tool to observe a wet specimen at high resolution under atmospheric pressure. Here, we visualized a protozoan parasite Trypanosoma cruzi ...over the course of its infection cycle in the host mammalian cell. This is the first observation of intracellular parasite using a liquid-phase EM. Unlike regular SEM, aldehyde-fixed cell body of T. cruzi appears translucent, allowing the visualization of internal structures such as kinetoplast of trypomastigote and nucleus of amastigote. Plasma membrane of the host mammalian cell also appears translucent, which enabled direct observation of differentiating intracellular parasites and dynamic change of host cellular structures in their near-natural states. Various water-rich structures including micro- and macro- vesicles were visualized around T. cruzi. In addition, Correlative Light and Electron Microscopy exploiting open sample dish of ASEM allowed identification of parasite nucleus and transfected fluorescence-labeled parasites soon after internalization, while location of this morphological intermediate was otherwise obscure. Successful visualization of the differentiation of T. cruzi within the host cell demonstrated here opens up the possibility of using ASEM for observation of variety of intracellular parasites.
Using Atmospheric Scanning Electron Microscopy (ASEM), we visualized interaction between infectious stage of Trypanosoma cruzi and completely intact host mammalian cell. Plasma membrane appears translucent under ASEM, which not only enables direct observation of T. cruzi within its host cell, but also reveals internal structures of the parasite itself. Sample deformation is minimal, since the specimen remains hydrated under atmospheric pressure at all times. This nature of ASEM, along with the open structure of ASEM sample dish, is suited for correlative light-electron microscopy, which can further be exploited in identification of fluorescent protein in the intracellular parasites.
Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary ...tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The 5′ terminus of trypanosome mRNA is protected by a hypermethylated cap 4 derived from spliced leader (SL) RNA. Trypanosoma brucei nuclear capping enzyme with cap guanylyltransferase and ...methyltransferase activities (TbCgm1) modifies the 5′-diphosphate RNA (ppRNA) end to generate an m7G SL RNA cap. Here we show that T. brucei cytoplasmic capping enzyme (TbCe1) is a bifunctional 5′-RNA kinase and guanylyltransferase that transfers a γ-phosphate from ATP to pRNA to form ppRNA, which is then capped by transfer of GMP from GTP to the RNA β-phosphate. A Walker A-box motif in the N-terminal domain is essential for the RNA kinase activity and is targeted preferentially to a SL RNA sequence with a 5′-terminal methylated nucleoside. Silencing of TbCe1 leads to accumulation of uncapped mRNAs, consistent with selective capping of mRNA that has undergone trans -splicing and decapping. We identify T. brucei mRNA decapping enzyme (TbDcp2) that cleaves m7GDP from capped RNA to generate pRNA, a substrate for TbCe1. TbDcp2 can also remove GDP from unmethylated capped RNA but is less active at a mature cap 4 end and thus may function in RNA cap quality surveillance. Our results establish the enzymology and relevant protein catalysts of a cytoplasmic recapping pathway that has broad implications for the functional reactivation of processed mRNA ends.
Significance The 5′ end of eukaryotic mRNA is capped and methylated to protect mRNA from degradation and enhance protein synthesis. However, the cap can be removed from mRNA by decapping. We identified a recapping enzyme with 5′-monophosphate RNA kinase activity from trypanosome and provide evidence that decapped transcripts can be recapped to regenerate translatable mRNA. The kinase activity is dependent on mRNA leader sequence and is stimulated by hypermethylation found in the trypanosome mRNA. We also identify a trypanosome decapping enzyme that removes cap structure from the mRNA, but is less active on hypermethylated capped mRNA. These results suggest that hypermethylated cap structure can influence certain transcripts to be preferentially decapped or recapped during the parasite life cycle.
Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of ...detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients’ homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE²RD), which addresses all these impediments on a single platform. The NE²RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE²RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE²RD’s broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the pointof-care or primary care settings and at patients’ homes.
In this study, we simultaneously determined three antioxidants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), using HPLC equipped both with a ...photodiode array detector and a fluorescence detector in 25 minutes per sample. Due to the combined use of the two detectors, we could achieve improved target selectivity. Further, quantification at the specific wavelengths for each target substance particularly increased BHT detection sensitivity. This approach enabled us to avoid repeated measurements during daily inspections. Furthermore, detections were performed using LC-MS/MS instead of GC-MS to overcome the problem of helium gas shortage.In addition, we investigated antioxidant stability in standard solutions during storage. Although TBHQ was stable in methanol with ascorbic acid at −20℃, ascorbic acid storage has possibility to lead to decrease in BHT and BHA concentrations. We recognized that the mixture of BHT and BHA dissolved in methanol at 4℃ and that of BHT, BHA and TBHQ dissolved in methanol with ascorbic acid at −20℃ were suitable for about one year.
Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and ...PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)(4)-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4.
PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity.
Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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