Adaptation of a eukaryote-like ProRS to a prokaryote-like tRNAPro.

Prolyl-tRNA synthetases (ProRSs) are unique among aminoacyl-tRNA synthetases (aaRSs) in having two distinct structural architectures across different organisms: prokaryote-like (P-type) and eukaryote/archaeon-like (E-type). Interestingly, Bacillus thuringiensis harbors both types, with P-type (BtProRS1) and E-type ProRS (BtProRS2) coexisting. Despite their differences, both enzymes are constitutively expressed and functional in vivo. Similar to BtProRS1, BtProRS2 selectively charges the P-type tRNAPro and displays higher halofuginone tolerance than canonical E-type ProRS. However, these two isozymes recognize the primary identity elements of the P-type tRNAPro-G72 and A73 in the acceptor stem-through distinct mechanisms. Moreover, BtProRS2 exhibits significantly higher tolerance to stresses (such as heat, hydrogen peroxide, and dithiothreitol) than BtProRS1 does. This study underscores how an E-type ProRS adapts to a P-type tRNAPro and how it may contribute to the bacterium's survival under stress conditions.

Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate.

Prolyl-tRNA synthetase (ProRS), belonging to the family of aminoacyl-tRNA synthetases responsible for pairing specific amino acids with their respective tRNAs, is categorized into two distinct types: the eukaryote/archaeon-like type (E-type) and the prokaryote-like type (P-type). Notably, these types are specific to their corresponding cognate tRNAs. In an intriguing paradox, Thermus thermophilus ProRS (TtProRS) aligns with the E-type ProRS but selectively charges the P-type tRNAPro, featuring the bacterium-specific acceptor-stem elements G72 and A73. This investigation reveals TtProRS's notable resilience to the inhibitor halofuginone, a synthetic derivative of febrifugine emulating Pro-A76, resembling the characteristics of the P-type ProRS. Furthermore, akin to the P-type ProRS, TtProRS identifies its cognate tRNA through recognition of the acceptor-stem elements G72/A73, along with the anticodon elements G35/G36. However, in contrast to the P-type ProRS, which relies on a strictly conserved R residue within the bacterium-like motif 2 loop for recognizing G72/A73, TtProRS achieves this through a non-conserved sequence, RTR, within the otherwise non-interacting eukaryote-like motif 2 loop. This investigation sheds light on the adaptive capacity of a typically conserved housekeeping enzyme to accommodate a novel substrate.

Sequence-specific targeting of Caenorhabditis elegans C-Ala to the D-loop of tRNAAla.

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.

A naturally occurring mini-alanyl-tRNA synthetase.

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology, consisting of catalytic, tRNA-recognition, editing, and C-Ala domains. The catalytic and tRNA-recognition domains catalyze aminoacylation, the editing domain hydrolyzes mischarged tRNAAla, and C-Ala-the major tRNA-binding module-targets the elbow of the L-shaped tRNAAla. Interestingly, a mini-AlaRS lacking the editing and C-Ala domains is recovered from the Tupanvirus of the amoeba Acanthamoeba castellanii. Here we show that Tupanvirus AlaRS (TuAlaRS) is phylogenetically related to its host's AlaRS. Despite lacking the conserved amino acid residues responsible for recognition of the identity element of tRNAAla (G3:U70), TuAlaRS still specifically recognized G3:U70-containing tRNAAla. In addition, despite lacking C-Ala, TuAlaRS robustly binds and charges microAla (an RNA substrate corresponding to the acceptor stem of tRNAAla) as well as tRNAAla, indicating that TuAlaRS exclusively targets the acceptor stem. Moreover, this mini-AlaRS could functionally substitute for yeast AlaRS in vivo. This study suggests that TuAlaRS has developed a new tRNA-binding mode to compensate for the loss of C-Ala.

Recognition of tRNAHis in an RNase P-Free Nanoarchaeum.

The 5' extra guanosine with 5'-monophosphate at position -1 (G-1) of tRNAHis (p-tRNAHis) is a nearly universal feature that establishes tRNAHis identity. G-1 is either genome encoded and retained after processing by RNase P (RNase P) or posttranscriptionally incorporated by tRNAHis guanylyltransferase (Thg1) after RNase P cleavage. However, RNase P is not found in the hyperthermophilic archaeum Nanoarchaeum equitans; instead, all of its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 5'-triphosphate (ppp-tRNAs). How N. equitans histidyl-tRNA synthetase (NeHisRS) recognizes its cognate tRNA (NetRNAHis) is of particular interest. In this paper, we show that G-1 serves as the major identity element of NetRNAHis, with its anticodon performing a similar role, though to a lesser extent. Moreover, NeHisRS distinctly preferred p-tRNAHis over ppp-tRNAHis (~5-fold difference). Unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme could charge tRNAsHis with A73 and C73 with nearly equal efficiency. As a result, mutation at the C73-recognition amino acid residue Q112 had only a minor effect (<2-fold reduction). This study suggests that NeHisRS has evolved to disregard C73, but it still maintains its evolutionarily preserved preference toward tRNAHis with 5'-monophosphate. IMPORTANCE Mature tRNAHis has, at its 5'-terminus, an extra guanosine with 5'-monophosphate, designated G-1. G-1 is the major recognition element for histidyl-tRNA synthetase (HisRS), regardless of whether it is of eukaryotic or prokaryotic origin. However, in the hyperthermophilic archaeum Nanoarchaeum equitans, all its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 5'-triphosphate. This piqued our curiosity about whether N. equitans histidyl-tRNA synthetase (NeHisRS) prefers tRNAHis with 5'-triphosphate. We show herein that G-1 is still the major recognition element for NeHisRS. However, unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme shows almost the same preference for C73 and A73. Most intriguingly, NeHisRS still prefers 5'-monophosphate over 5'-triphosphate. It thus appears that the preference of HisRS for tRNAHis with 5'-monophosphate emerged very early in evolution.

Gain of C-Ala enables AlaRS to target the L-shaped tRNAAla.

Unlike many other aminoacyl-tRNA synthetases, alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout biology. While Caenorhabditis elegans cytoplasmic AlaRS (CeAlaRSc) retains the prototype structure, its mitochondrial counterpart (CeAlaRSm) contains only a residual C-terminal domain (C-Ala). We demonstrated herein that the C-Ala domain from CeAlaRSc robustly binds both tRNA and DNA. It bound different tRNAs but preferred tRNAAla. Deletion of this domain from CeAlaRSc sharply reduced its aminoacylation activity, while fusion of this domain to CeAlaRSm selectively and distinctly enhanced its aminoacylation activity toward the elbow-containing (or L-shaped) tRNAAla. Phylogenetic analysis showed that CeAlaRSm once possessed the C-Ala domain but later lost most of it during evolution, perhaps in response to the deletion of the T-arm (part of the elbow) from its cognate tRNA. This study underscores the evolutionary gain of C-Ala for docking AlaRS to the L-shaped tRNAAla.

Low concentrations of 4-ABP promote liver carcinogenesis in human liver cells and a zebrafish model.

4-Aminobiphenyl (4-ABP) is a human bladder cancer carcinogen found in the manufacture of azo dyes and the composition of cigarette smoke in the environment. To determine whether low concentrations of 4-ABP induced or promote liver carcinogenesis and investigate the underlying mechanism, we have established the liver cell carcinogenesis model in human liver cell lines and zebrafish to evaluate liver cancer development associated with long-term exposure to low concentrations of 4-ABP. Results show that repeated 4-ABP exposure promoted cellular proliferation and migration via the involvement of ROS in Ras/MEK/ERK pathway in vitro. Also, 4-ABP (1, 10, and 100 nM) induces hepatocellular carcinoma (HCC) formation in HBx, Src (p53-/-) transgenic zebrafish at four months of age and in wild-type zebrafish at seven months of age. In addition, we observed a correlation between the Ras-ERK pathway and 4-ABP-induced HCC in vitro and in vivo. Our finding suggests low concentrations of 4-ABP repeated exposure is a potential risk factor for liver cancer. To our knowledge, this is the first report on the promotion of liver carcinogenesis in human liver cells and zebrafish following 4-ABP exposure.

Study design for a three-arm equivalence clinical trial with binomially distributed outcomes.

Equivalence in a clinical trial may be assessed through a three-arm trial (test drug, reference drug, and placebo). A three-arm equivalence trial consists of three hypothesis tests in practice, with two hypothesis tests demonstrating the superiority of the test and reference drugs against placebo, and the other one demonstrating the equivalence of the test and reference drugs. When designing a three-arm equivalence clinical trial, the practitioner should minimize the chances that a test drug will be found to be equivalent to the reference drug but non-superior to the placebo. One way to minimize these chances at the design stage, for a three-arm equivalence trial with a binary primary outcome, is to test the equivalence through hypotheses based upon the ratio of the differences of the proportions. In this article, we derived the test statistics and the power functions based on maximum likelihood estimate (MLE) and restricted MLE for the proposed hypotheses. The required sample size for achieving the desired power at the given significance level can be obtained by solving the power function. We illustrated the proposed design through an example and investigated the required sample sizes for various conditions.

4-Aminobiphenyl inhibits the DNA homologous recombination repair in human liver cells: The role of miR-630 in downregulating RAD18 and MCM8.

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.

Naturally occurring dual recognition of tRNAHis substrates with and without a universal identity element.

The extra 5' guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis (CetRNAnHis) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNAHis (CetRNAmHis) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNAmHis strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNAnHis without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNAHis identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNAHis effectively converted a non-cognate tRNA to a tRNAHis-like substrate. Our study suggests that a new balance between identity elements of tRNAHis relieves HisRS from the absolute requirement for G-1.

Modulating the Structure and Function of an Aminoacyl-tRNA Synthetase Cofactor by Biotinylation.

Arc1p is a yeast-specific tRNA-binding protein that forms a ternary complex with glutamyl-tRNA synthetase (GluRSc) and methionyl-tRNA synthetase (MetRS) in the cytoplasm to regulate their catalytic activities and subcellular distributions. Despite Arc1p not being involved in any known biotin-dependent reaction, it is a natural target of biotin modification. Results presented herein show that biotin modification had no obvious effect on the growth-supporting activity, subcellular distribution, tRNA binding, or interactions of Arc1p with GluRSc and MetRS. Nevertheless, biotinylation of Arc1p was temperature dependent; raising the growth temperature from 30 to 37 °C drastically reduced its biotinylation level. As a result, Arc1p purified from a yeast culture that had been grown overnight at 37 °C was essentially biotin free. Non-biotinylated Arc1p was more heat stable, more flexible in structure, and more effective than its biotinylated counterpart in promoting glutamylation activity of the otherwise inactive GluRSc at 37 °C in vitro Our study suggests that the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes.

The change of microbial community from chlorinated solvent-contaminated groundwater after biostimulation using the metagenome analysis.

The compositions of bacterial community in one site contaminated with PCE/TCE after the slow polycolloid-releasing substrate (SPRS) (contained vegetable oil, cane molasses, and surfactants) addition were analyzed. Results show that SPRS caused a rapid enhancement of reductive dechlorination of TCE. The transformation of PCE/TCE into ethene was observed after 20 days of operation. To compare the change of bacterial communities before and after SPRS addition, 16S rRNA amplicon sequencing using the metagenome analysis was performed. Results demonstrated the detection of the increased amounts of Dehalogenimonas by 2.2-fold, Pseudomonas by 3.4-fold and Sulfuricurvum by 4-fold with the analysis of the ribosomal database project (RDP). Metagenomic DNA was extracted from PCE/TCE-contaminated groundwater after SPRS addition, and subjected to sequencing. Results obtained from metagenomic sequencing indicate that genes from Dehalococcoides mccartyi was ranked as the second abundant bacteria among all of the detected bacteria via the analysis of the lowest common ancestor (LCA). Abundance of these bacterial groups, as shown above suggests their role in TCE biodegradation. Functional analysis of the metagenome, with the specific reference to chloroalkane and chloroalkene degradation, revealed the presence of some genes responsible for TCE biodegradation. Overall, results of this study provided new insights for a better understanding of the potential of biostimulation on TCE-contaminated sites.

Joint model of multiple longitudinal measures and a binary outcome: An application to predict orthostatic hypertension for subacute stroke patients.

Stroke patients with orthostatic hypertensive responses that are one of the blood pressure regulation problems can easily fall down while doing rehabilitation, which may result in prolonged hospitalization and delayed treatment and recovery. This may result in increasing the medical cost and burden. In turn, developing a diagnostic test for the orthostatic hypertension (OH) is clinically important for patients who are suffering from stroke. Clinically, a patient needs to have a tilt testing that requires measuring the change of blood pressures and heart rate at all angles to determine whether a stroke patient has OH. It takes lots of time and effort to perform the test. Assuming there exist measurement errors when obtaining the blood pressures and heart rate at all angles, this paper proposes using multiple mixed-effect models to obtain the true trajectories of these measurements, which take into account the measurement error and the possible correlation among multiple measurements, and a logistic regression uses these true trajectories at a given time and other fixed-effect covariates as predictors to predict the status of OH. The joint likelihood function is derived to estimate parameters and the area under the receiver operating characteristics curve is used to estimate the predictive power of the model. Monte Carlo simulations are performed to evaluate the feasibility of the proposed methods. Also, the proposed model is implemented in the real data and provides an acceptable predictive power.

Molecular mechanisms of 3,3'-dichlorobenzidine-mediated toxicity in HepG2 cells.

3,3'-Dichlorobenzidine (DCB) (CAS 91-94-1), a synthetic, chlorinated, primary aromatic amine, is typically used as an intermediate in the manufacturing of pigments for printing inks, textiles, paints, and plastics. In this study, we found that DCB could significantly inhibit the cell viability of HepG2 cells in a concentration-dependent manner. Flow cytometry revealed that DCB induced G2/M-phase arrest and apoptosis in HepG2 cells. DCB treatment dramatically induced the dissipation of mitochondrial membrane potential (Δψm ) and enhanced the enzymatic activities of caspase-9 and caspase-3 whilst hardly affecting caspase-8 activity. Furthermore, Western blotting indicated that DCB-induced apoptosis was accompanied by the down-regulation of Bcl-2/Bax ratio. These results suggested that DCB led to cytotoxicity involving activation of mitochondrial-dependent apoptosis through Bax/Bcl-2 pathways in HepG2 cells. Furthermore, HepG2 cells treated with DCB showed significant DNA damage as supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by concentration- and time-dependent increase in histone H2AX phosphorylation (γ-H2AX). Two-dimensional-difference gel electrophoresis (2D-DIGE), combined with mass spectrometry (MS), was used to unveil the differences in protein expression between cells exposed to 25 µM or 100 µM of DCB for 24 hr and the control cells. Twenty-seven differentially expressed proteins involved in DNA repair, unfolded protein response, metabolism, cell signaling, and apoptosis were identified. Among these, 14-3-3 theta, CGI-46, and heat-shock 70 protein 4 were confirmed using Western blot assay. Taken together, these data suggest that DCB is capable of inducing DNA damage and some cellular stress responses in HepG2 cells, thus eventually leading to cell death by apoptosis.

Risk of dementia after anaesthesia and surgery.

BACKGROUND: The potential relationship between anaesthesia, surgery and onset of dementia remains elusive. AIMS: To determine whether the risk of dementia increases after surgery with anaesthesia, and to evaluate possible associations among age, mode of anaesthesia, type of surgery and risk of dementia. METHOD: The study cohort comprised patients aged 50 years and older who were anaesthetised for the first time since 1995 between 1 January 2004 and 31 December 2007, and a control group of randomly selected patients matched for age and gender. Patients were followed until 31 December 2010 to identify the emergence of dementia. RESULTS: Relative to the control group, patients who underwent anaesthesia and surgery exhibited an increased risk of dementia (hazard ratio = 1.99) and a reduced mean interval to dementia diagnosis. The risk of dementia increased in patients who received intravenous or intramuscular anaesthesia, regional anaesthesia and general anaesthesia. CONCLUSIONS: The results of our nationwide, population-based study suggest that patients who undergo anaesthesia and surgery may be at increased risk of dementia.

An insertion peptide in yeast glycyl-tRNA synthetase facilitates both productive docking and catalysis of cognate tRNAs.

The yeast Saccharomyces cerevisiae possesses two distinct glycyl-tRNA synthetase (GlyRS) genes: GRS1 and GRS2. GRS1 is dually functional, encoding both cytoplasmic and mitochondrial activities, while GRS2 is dysfunctional and not required for growth. The protein products of these two genes, GlyRS1 and GlyRS2, are much alike but are distinguished by an insertion peptide of GlyRS1, which is absent from GlyRS2 and other eukaryotic homologues. We show that deletion or mutation of the insertion peptide modestly impaired the enzyme's catalytic efficiency in vitro (with a 2- to 3-fold increase in Km and a 5- to 8-fold decrease in kcat). Consistently, GRS2 can be conveniently converted to a functional gene via codon optimization, and the insertion peptide is dispensable for protein stability and the rescue activity of GRS1 at 30°C in vivo. A phylogenetic analysis further showed that GRS1 and GRS2 are paralogues that arose from a gene duplication event relatively recently, with GRS1 being the predecessor. These results indicate that GlyRS2 is an active enzyme essentially resembling the insertion peptide-deleted form of GlyRS1. Our study suggests that the insertion peptide represents a novel auxiliary domain, which facilitates both productive docking and catalysis of cognate tRNAs.

Alanyl-tRNA synthetase genes of Vanderwaltozyma polyspora arose from duplication of a dual-functional predecessor of mitochondrial origin.

In eukaryotes, the cytoplasmic and mitochondrial forms of a given aminoacyl-tRNA synthetase (aaRS) are typically encoded by two orthologous nuclear genes, one of eukaryotic origin and the other of mitochondrial origin. We herein report a novel scenario of aaRS evolution in yeast. While all other yeast species studied possess a single nuclear gene encoding both forms of alanyl-tRNA synthetase (AlaRS), Vanderwaltozyma polyspora, a yeast species descended from the same whole-genome duplication event as Saccharomyces cerevisiae, contains two distinct nuclear AlaRS genes, one specifying the cytoplasmic form and the other its mitochondrial counterpart. The protein sequences of these two isoforms are very similar to each other. The isoforms are actively expressed in vivo and are exclusively localized in their respective cellular compartments. Despite the presence of a promising AUG initiator candidate, the gene encoding the mitochondrial form is actually initiated from upstream non-AUG codons. A phylogenetic analysis further revealed that all yeast AlaRS genes, including those in V. polyspora, are of mitochondrial origin. These findings underscore the possibility that contemporary AlaRS genes in V. polyspora arose relatively recently from duplication of a dual-functional predecessor of mitochondrial origin.

Joint modeling of survival and longitudinal data: likelihood approach revisited.

The maximum likelihood approach to jointly model the survival time and its longitudinal covariates has been successful to model both processes in longitudinal studies. Random effects in the longitudinal process are often used to model the survival times through a proportional hazards model, and this invokes an EM algorithm to search for the maximum likelihood estimates (MLEs). Several intriguing issues are examined here, including the robustness of the MLEs against departure from the normal random effects assumption, and difficulties with the profile likelihood approach to provide reliable estimates for the standard error of the MLEs. We provide insights into the robustness property and suggest to overcome the difficulty of reliable estimates for the standard errors by using bootstrap procedures. Numerical studies and data analysis illustrate our points.

Age-specific and lifetime behavior patterns in Drosophila melanogaster and the Mediterranean fruit fly, Ceratitis capitata.

Patterns of behavior were recorded every 10 min during a 2-h period each day from eclosion to death for individual Drosophila melanogaster (both sexes) and Ceratitis capitata (males-only) including walking, preening, feeding, flying, and resting for the former species, and walking, calling (signaling), supine (upside-down), and resting in the latter. Results reveal that, with the exception of preening in D. melanogaster, behavioral patterns are age-specific and the frequency of several behaviors (e.g. supine in medfly; walking and resting in D. melanogaster) are correlated with time-to-death. This is the first set of studies to report the age patterns over a range of behavioral categories throughout the lives of individuals and thus the first that systematically documents the behavior of individuals at advanced ages. We suggest that the new and unique behaviors (e.g. supine) that emerge from the aging process be referred to as degenerative behaviors, not only to distinguish them from the conventional behavioral classifications (innate, learned), but also to reflect their emergent nature.