Primordial aminoacyl-tRNA synthetases preferred minihelices to full-length tRNA.

Aminoacyl-tRNA synthetases (AARS) and tRNAs translate the genetic code in all living cells. Little is known about how their molecular ancestors began to enforce the coding rules for the expression of their own genes. Schimmel et al. proposed in 1993 that AARS catalytic domains began by reading an 'operational' code in the acceptor stems of tRNA minihelices. We show here that the enzymology of an AARS urzyme•TΨC-minihelix cognate pair is a rich in vitro realization of that idea. The TΨC-minihelixLeu is a very poor substrate for full-length Leucyl-tRNA synthetase. It is a superior RNA substrate for the corresponding urzyme, LeuAC. LeuAC active-site mutations shift the choice of both amino acid and RNA substrates. AARS urzyme•minihelix cognate pairs are thus small, pliant models for the ancestral decoding hardware. They are thus an ideal platform for detailed experimental study of the operational RNA code.

Corrigendum: Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.

[This corrects the article DOI: 10.3389/fimmu.2023.1213920.].

Genomic prediction of pig growth traits based on machine learning.

This study aimed to assess and compare the performance of different machine learning models in predicting selected pig growth traits and genomic estimated breeding values (GEBV) using automated machine learning, with the goal of optimizing whole-genome evaluation methods in pig breeding. The research employed genomic information, pedigree matrices, fixed effects, and phenotype data from 9968 pigs across multiple companies to derive four optimal machine learning models: deep learning (DL), random forest (RF), gradient boosting machine (GBM), and extreme gradient boosting (XGB). Through 10-fold cross-validation, predictions were made for GEBV and phenotypes of pigs reaching weight milestones (100 kg and 115 kg) with adjustments for backfat and days to weight. The findings indicated that machine learning models exhibited higher accuracy in predicting GEBV compared to phenotypic traits. Notably, GBM demonstrated superior GEBV prediction accuracy, with values of 0.683, 0.710, 0.866, and 0.871 for B100, B115, D100, and D115, respectively, slightly outperforming other methods. In phenotype prediction, GBM emerged as the best-performing model for pigs with B100, B115, D100, and D115 traits, achieving prediction accuracies of 0.547, followed by DL at 0.547, and then XGB with accuracies of 0.672 and 0.670. In terms of model training time, RF required the most time, while GBM and DL fell in between, and XGB demonstrated the shortest training time. In summary, machine learning models obtained through automated techniques exhibited higher GEBV prediction accuracy compared to phenotypic traits. GBM emerged as the overall top performer in terms of prediction accuracy and training time efficiency, while XGB demonstrated the ability to train accurate prediction models within a short timeframe. RF, on the other hand, had longer training times and insufficient accuracy, rendering it unsuitable for predicting pig growth traits and GEBV.

Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.

Introduction: The complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885. Methods: The interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers. Results: Zilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies. Conclusion: Our findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.

Domain acquisition by class I aminoacyl-tRNA synthetase urzymes coordinated the catalytic functions of HVGH and KMSKS motifs.

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that synthesizes leucyl-tRNAleu for codon-directed protein synthesis. Two signature sequences, HxGH and KMSKS help stabilize transition-states for amino acid activation and tRNA aminoacylation by all Class I aaRS. Separate alanine mutants of each signature, together with the double mutant, behave in opposite ways in Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC). Free energy coupling terms, Δ(ΔG‡), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with 32Pα-ATP show correspondingly different internal products. These results implicate domain motion in catalysis by full-length LeuRS. The distributed thermodynamic cycle of mutational changes authenticates LeuAC urzyme catalysis far more convincingly than do single point mutations. Most importantly, the evolutionary gain of function induced by acquiring the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain appears to be to coordinate the catalytic function of the HxGH and KMSKS signature sequences. The implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains.

Computational investigation of the impact of core sequence on immobile DNA four-way junction structure and dynamics.

Immobile four-way junctions (4WJs) are core structural motifs employed in the design of programmed DNA assemblies. Understanding the impact of sequence on their equilibrium structure and flexibility is important to informing the design of complex DNA architectures. While core junction sequence is known to impact the preferences for the two possible isomeric states that junctions reside in, previous investigations have not quantified these preferences based on molecular-level interactions. Here, we use all-atom molecular dynamics simulations to investigate base-pair level structure and dynamics of four-way junctions, using the canonical Seeman J1 junction as a reference. Comparison of J1 with equivalent single-crossover topologies and isolated nicked duplexes reveal conformational impact of the double-crossover motif. We additionally contrast J1 with a second junction core sequence termed J24, with equal thermodynamic preference for each isomeric configuration. Analyses of the base-pair degrees of freedom for each system, free energy calculations, and reduced-coordinate sampling of the 4WJ isomers reveal the significant impact base sequence has on local structure, isomer bias, and global junction dynamics.

Comparison of Outcomes between Robot-Assisted Minimally Invasive Transforaminal Lumbar Interbody Fusion and Oblique Lumbar Interbody Fusion in Single-Level Lumbar Spondylolisthesis.

OBJECTIVE: To compare the safety and effectiveness of robot-assisted minimally invasive transforaminal lumbar interbody fusion (Mis-TLIF) and oblique lumbar interbody fusion (OLIF) for the treatment of single-level lumbar degenerative spondylolisthesis (LDS). METHODS: This is a retrospective study. Between April 2018 and April 2020, a total of 61 patients with single-level lumbar degenerative spondylolisthesis and treated with robot-assisted OLIF (28 cases, 16 females, 12 males, mean age 50.4 years) or robot-assisted Mis-TLIF (33 cases, 18 females, 15 males, mean age 53.6 years) were enrolled and evaluated. All the pedicle screws were implanted percutaneously assisted by the TiRobot system. Surgical data included the operation time, blood loss, and length of postoperative hospital stay. The clinical and functional outcomes included Oswestry Disability Index (ODI), Visual Analog scores (VAS) for back and leg pain, complication, and patient's satisfaction. Radiographic outcomes include pedicle screw accuracy, fusion status, and disc height. These data were collected before surgery, at 1 week, 3 months, 6 months, and 12 months postoperatively. RESULTS: There were no significantly different results in preoperative measurement between the two groups. There was significantly less blood loss (142.4 ± 89.4 vs 291.5 ± 72.3 mL, P < 0.01), shorter hospital stays (3.2 ± 1.8 vs 4.2 ± 2.5 days, P < 0.01), and longer operative time (164.9 ± 56.0 vs 121.5 ± 48.2 min, P < 0.01) in OLIF group compared with Mis-TLIF group. The postoperative VAS scores and ODI scores in both groups were significantly improved compared with preoperative data (P < 0.05). VAS scores for back pain were significantly lower in OLIF group than Mis-TLIF group at 1 week (2.8 ± 1.2 vs 3.5 ± 1.6, P < 0.05) and 3 months postoperatively (1.6 ± 1.0 vs 2.1 ± 1.1, P < 0.05), but there was no significant difference at further follow-ups. ODI score was also significantly lower in OLIF group than Mis-TLIF group at 3 months postoperatively (22.3 ± 10.0 vs 26.1 ± 12.8, P < 0.05). There was no significant difference in the proportion of clinically acceptable screws between the two groups (97.3% vs 96.2%, P = 0.90). At 1 year, the OLIF group had a higher interbody fusion rate compared with Mis-TLIF group (96.0% vs 87%, P < 0.01). Disc height was significantly higher in the OLIF group than Mis-TLIF group (12.4 ± 3.2 vs 11.2 ± 1.3 mm, P < 0.01). Satisfaction rates at 1 year exceeded 90% in both groups and there was no significant difference (92.6% for OLIF vs 91.2% for Mis-TLIF, P = 0.263). CONCLUSION: Robot-assisted OLIF and Mis-TLIF both have similar good clinical outcomes, but OLIF has the additional benefits of less blood loss, less postoperative hospital stays, higher disc height, and higher fusion rates. Robots are an effective tool for minimally invasive spine surgery.

Correction to: Intrathecal Epigallocatechin Gallate Treatment Improves Functional Recovery After Spinal Cord Injury by Upregulating the Expression of BDNF and GDNF.

Since the publication of our article [1] it has come to our attention that there was an error in Figure 4 in which the bottom left immunochemistry panel Control/Bax was a duplication of the bottom right immunohistochemistry panel EGCG/GDNF in Figure 3.

Correlating Transcription Initiation and Conformational Changes by a Single-Subunit RNA Polymerase with Near Base-Pair Resolution.

We provide a comprehensive analysis of transcription in real time by T7 RNA Polymerase (RNAP) using single-molecule fluorescence resonance energy transfer by monitoring the entire life history of transcription initiation, including stepwise RNA synthesis with near base-pair resolution, abortive cycling, and transition into elongation. Kinetically branching pathways were observed for abortive initiation with an RNAP either recycling on the same promoter or exchanging with another RNAP from solution. We detected fast and slow populations of RNAP in their transition into elongation, consistent with the efficient and delayed promoter release, respectively, observed in ensemble studies. Real-time monitoring of abortive cycling using three-probe analysis showed that the initiation events are stochastically branched into productive and failed transcription. The abortive products are generated primarily from initiation events that fail to progress to elongation, and a majority of the productive events transit to elongation without making abortive products.

[Effects of gap and growth substrate on nitrogen and phosphorus contents of bryophytes in an alpine forest]. / 高山森林林窗和生长基质对苔藓植物氮和磷含量的影响.

Bryophyte plays an important role in nutrient enrichment and cycling in the forest ecosystems. The role of bryophyte in nitrogen (N) and phosphorus (P) cycles might be affected by forest regeneration and growth substrate. To understand the role of bryophyte in N and P cycling in the forest ecosystem, we measured the contents of N and P in the bryophytes that grew on different positions (gap center, gap edge, and closed canopy) and growth substrates (standing tree, fallen log, snag, large dead branch, stump and forest floor) in an alpine forest ecosystem. The results showed that the N content in the bryophyte on the forest floor was 3.12 mg·g-1, which was significantly lower than those on other growth substrates. Although N content in the bryophyte on the snag reached up to 17.41 mg·g-1, no significant differences of N contents in the bryophytes were observed among standing tree, fallen log, large dead branch and snag. The highest and lowest P contents was 1.09 mg·g-1 in the bryophyte on the forest floor and 0.61 mg·g-1 in the bryophytes on the snag, respectively. Furthermore, P content in the bryophyte on the forest floor was significantly higher than that on other growth substrates, but no significant differences of P contents in the bryophytes were detected among standing tree, fallen log, large dead branch and stump. The gap position significantly affected N and P contents in the bryophytes, with the N and P contents in the bryophytes on fallen log and large dead branch at gap center being significantly higher than those at the gap edge. The effects of coarse woody debris (CWD) on the N and P contents in the bryophyte depended on its types and decay classes, with their interaction having much stronger effects on N and P contents in the bryophytes. The N contents in the epiphytic bryophytes on fallen logs with V decay class were significantly higher than those with other decay classes. Similarly, the N contents in the epiphytic bryophytes on large dead branches with III decay class were significantly higher than those with other decay classes. Meanwhile, the P contents in the bryophytes on fallen logs with 2 decay class were significantly higher than those with other decay classes. Moreover, the P contents in the epiphytic bryophytes on the snags with 4 decay class were significantly higher than those with other decay classes. In conclusion, both forest gap regeneration and CWD decay process can affect the N and P contents in the bryophytes, and thereafter manipulate the nutrient cycles in the forest ecosystems.

The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.

An RNA Hybridization Assay for Screening Influenza A Virus Polymerase Inhibitors Using the Entire Ribonucleoprotein Complex.

Novel antiviral drugs, which are less prone to resistance development, are desirable alternatives to the currently approved drugs for the treatment of potentially serious influenza virus infections. The viral polymerase is highly conserved and serves as an attractive target for antiviral drugs since potent inhibitors would directly stop viral replication at an early stage. Recent structural studies on the functional domains of the heterotrimeric influenza polymerase, which comprises subunits PA, PB1, and PB2, opened the way to a structure-based approach for optimizing inhibitors of viral replication. These strategies, however, are limited by the use of isolated protein fragments instead of employing the entire ribonucleoprotein complex (RNP), which represents the functional form of the influenza polymerase in infected cells. In this study, we have established a screening assay for efficient and reliable analysis of potential influenza polymerase inhibitors of various molecular targets such as monoselective polymerase inhibitors targeting the endonuclease site, the cap-binding domain, and the polymerase active site, respectively. By utilizing whole viral RNPs and a radioactivity-free endpoint detection with the capability for efficient compound screening while offering high-content information on potential inhibitors to drive medicinal chemistry program in a reliable manner, this biochemical assay provides significant advantages over the currently available conventional assays. We propose that this assay can eventually be adapted for coinstantaneous analysis and subsequent optimization of two or more different chemical scaffold classes targeting multiple active sites within the polymerase complex, thus enabling the evaluation of drug combinations and characterization of molecules with dual functionality.

Relaxed rotational and scrunching changes in P266L mutant of T7 RNA polymerase reduce short abortive RNAs while delaying transition into elongation.

Abortive cycling is a universal feature of transcription initiation catalyzed by DNA-dependent RNA polymerases (RNAP). In bacteriophage T7 RNAP, mutation of proline 266 to leucine (P266L) in the C-linker region connecting the N-terminal promoter binding domain with the C-terminal catalytic domain drastically reduces short abortive products (4-7 nt) while marginally increasing long abortives (9-11 nt). Here we have investigated the transcription initiation pathway of P266L with the goal of understanding the mechanistic basis for short and long abortive synthesis. We show that the P266L mutation does not alter the affinity for the promoter, mildly affects promoter opening, and increases the +1/+2 GTP K(d) by 2-fold. However, unlike wild-type T7 RNAP that undergoes stepwise rotation of the promoter binding domain and DNA scrunching during initial transcription, the P266L mutant does not undergo coupled rotational/scrunching movements until 7 nt RNA synthesis. The lack of rotation/scrunching correlates with greater stabilities of the initiation complexes of the P266L and decreased short abortive products. The results indicate that the increased flexibility in the C-linker due to P266L mutation enables T7 RNAP to absorb the stress from the growing RNA:DNA hybrid thereby decreasing short abortive products. Increased C-linker flexibility, however, has an adverse effect of delaying the transition into elongation by 1-2 nt, which gives rise to long abortive products. However, a mutation in the upstream promoter region greatly decreases long abortive products in P266L reactions, rendering the combination of P266L and A-15C promoter a desirable pair for efficient in vitro transcription for RNA production. We conclude that the conformational rigidity in the C-linker region conferred by the proline at position 266 is responsible for the undesirable short abortive products, but the rigidity is critical for efficient promoter clearance and transition into elongation.

Intrathecal epigallocatechin gallate treatment improves functional recovery after spinal cord injury by upregulating the expression of BDNF and GDNF.

This study aimed to investigate the therapeutic effects of epigallocatechin-3-gallate (EGCG) administered by subarachnoid injection following spinal cord injury (SCI) in rats and to explore the underlying mechanism. Sprague-Dawley rats were randomly divided into four groups of 12 as follows: a sham group (laminectomy only); a control group; a 10 mg/kg EGCG-treated group; and a 20 mg/kg EGCG-treated group. SCI was induced in the rats using the modified weight-drop method (10 g × 4 cm) at the T10 (10th thoracic vertebral) level. EGCG (10 or 20 mg/kg) or vehicle as control was administered by subarachnoid injection at lumbar level 4 immediately after SCI. Locomotor functional recovery was assessed during the four weeks post-operation using open-field locomotor tests and inclined-plane tests. At the end of the study, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemical and Western blot analyses were performed to observe the expression of: the B cell CLL/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The results showed that the EGCG-treated animals had significantly better recovery of locomotor function, less myelin loss, greater Bcl-2 expression and attenuated Bax expression. In addition, the EGCG treatment significantly increased the expression of BDNF and GDNF after SCI. These findings suggest that EGCG treatment can significantly improve locomotor recovery, and this neuroprotective effect may be related to the up-regulation of BDNF and GDNF, and the inhibition of apoptosis-related proteins. Therefore, EGCG may be a promising therapeutic agent for SCI.

Elucidation of the binding properties of a photosensitizer to salmon sperm DNA and its photobleaching processes by spectroscopic methods.

Methylene blue (MB) is a tricyclic heteroaromatic photosensitizer with a promising application in the photodynamic therapy (PDT) for anticancer treatment. The binding properties of MB to salmon sperm DNA have been investigated by the measurements of absorption spectra, quenching experiments and the photobleaching processes. Remarkable hypochromic and bathochromic effects of MB in the presence of increasing amounts of DNA have been observed in the absorption spectra. The quenching of MB by the DNA bases obeys the Stern-Volmer equation and ferrocyanide quenching of MB in the absence and presence of DNA is also measured as extended experiments. Results from the above spectral measurements are all consistent with the intercalative binding mode of MB to DNA with the K b value of 5.6 × 10(3) M(-1). The photobleaching processes of MB and its DNA complex have also been studied, which indicate that the photobleaching of MB and its DNA complex proceed with different mechanisms and the reactive oxygen species are responsible for the self-sensitized photooxidation of MB.

[Endophytic bacterial diversity in Codonopsis pilosula, Ephedra sinica, and Lamiophlomis rotate: a study with LH-PCR].

To investigate the endophytic bacterial diversity in the three medicinal plant species Codonopsis pilosula, Ephedra sinica, and Lamiophlomis rotata in Ganzi of Sichuan, Southwest China, the total DNA of the three species were extracted by stringent surface sterilization, and studied with length heterogeneity-PCR (LH-PCR) method. For the same plant species, their root-, stem-, and leaf LH-PCR profiles were in a high level of similarity, with little differences in band richness. However, there existed great differences in the LH-PCR profiles among different plant species. C. pilosula had the biggest band richness, followed by E. sinica, and L. rotata. In the three plant species, the endophytic bacteria with an approximately 474 bp DNA length were dominant. The endophytic bacterial diversity of the plants was negatively correlated with rhizosphere soil available phosphorus content, but positively correlated with rhizosphere soil pH. Elevation and rhizosphere soil total nitrogen content were the important environmental factors affecting the distribution of enophytic bacteria in these plant species. The information of population diversity obtained from LH-PCR could more intuitively reflect the differences of bacterial diversity among different plant species, and thus, LH-PCR would be available to be used for analyzing the endophytic bacterial diversity in medicinal plants, providing information and guidance for the further isolation of microbial resources.

Spectral discrimination between normal and leukemic human sera using delayed luminescence.

In this work, photoinduced delayed luminescence (DL) was used to distinguish serum samples of patients with acute lymphoblastic leukemia from those of healthy volunteers. DL decay kinetics of human serum samples was measured using a homebuilt ultraweak luminescence detection system. It was found a significant difference in the weight distribution of the decay rate between normal and leukemic serum samples. A comparison of the DL kinetics parameters including the initial intensity, the peak decay rate, and the peak weight value was used in making discrimination between normal and leukemic human sera. Results in this work contribute to the development of a novel optical method for the early diagnosis of leukemia.

Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase.

TWINKLE is a nucleus-encoded human mitochondrial (mt)DNA helicase. Point mutations in TWINKLE are associated with heritable neuromuscular diseases characterized by deletions in the mtDNA. To understand the biochemical basis of these diseases, it is important to define the roles of TWINKLE in mtDNA metabolism by studying its enzymatic activities. To this end, we purified native TWINKLE from Escherichia coli. The recombinant TWINKLE assembles into hexamers and higher oligomers, and addition of MgUTP stabilizes hexamers over higher oligomers. Probing into the DNA unwinding activity, we discovered that the efficiency of unwinding is greatly enhanced in the presence of a heterologous single strand-binding protein or a single-stranded (ss) DNA that is complementary to the unwound strand. We show that TWINKLE, although a helicase, has an antagonistic activity of annealing two complementary ssDNAs that interferes with unwinding in the absence of gp2.5 or ssDNA trap. Furthermore, only ssDNA and not double-stranded (ds)DNA competitively inhibits the annealing activity, although both DNAs bind with high affinities. This implies that dsDNA binds to a site that is distinct from the ssDNA-binding site that promotes annealing. Fluorescence anisotropy competition binding experiments suggest that TWINKLE has more than one ssDNA-binding sites, and we speculate that a surface-exposed ssDNA-specific site is involved in catalyzing DNA annealing. We propose that the strand annealing activity of TWINKLE may play a role in recombination-mediated replication initiation found in the mitochondria of mammalian brain and heart or in replication fork regression during repair of damaged DNA replication forks.

Arsenic trioxide inhibits Ewing's sarcoma cell invasiveness by targeting p38(MAPK) and c-Jun N-terminal kinase.

Ewing's sarcoma is the second most frequent primary malignant bone tumor, mainly affecting children and young adults. The notorious metastatic capability of this tumor aggravates patient mortality and remains a problem to be overcome. We investigated the effect of arsenic trioxide (As2O3) on the metastasis capability of Ewing's sarcoma cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide assays to choose appropriate concentrations of As2O3 for the experiments. Migration, invasion, and adhesion assays were performed to assess the effect of As2O3 on the metastasis of Ewing's sarcoma. Immunofluorescent staining was used to observe cytoskeleton reorganization in Ewing's sarcoma cells treated with As2O3. Changes in matrix metalloproteinase-9 expression and the mitogen-activated protein kinase (MAPK) pathway were investigated using western blot. Inhibitors of p38(MAPK) (sb202190) and c-Jun NH2-terminal kinase (JNK, sp600125) were used in invasion assays to determine the effect of p38(MAPK) and JNK. We found that As2O3 may markedly inhibit the migration and invasion capacity of Ewing's sarcoma cells with structural rearrangements of the actin cytoskeleton. The expressions of matrix metalloproteinase-9, phosphor-p38(MAPK), and phosphor-JNK were suppressed by As2O3 treatment in a dose-dependent manner. The inhibitors of p38(MAPK) (sb202190) and JNK (sp600125) enhanced the inhibition induced by As2O3, which was counteracted by anisomycin, an activating agent of p38(MAPK) and JNK. Taken together, our results demonstrate that As2O3 can inhibit the metastasis capability of RD-ES and A-673 cells and may have new therapeutic value for Ewing's sarcoma.

Opening-closing dynamics of the mitochondrial transcription pre-initiation complex.

Promoter recognition and local melting of DNA are key steps of transcription initiation catalyzed by RNA polymerase and initiation factors. From single molecule fluorescence resonance energy transfer studies of the yeast (Saccharomyces cerevisiae) mitochondrial RNA polymerase Rpo41 and its transcription factor Mtf1, we show that the pre-initiation complex is highly dynamic and undergoes repetitive opening-closing transitions that are modulated by Mtf1 and ATP. We found that Rpo41 alone has the intrinsic ability to bend the promoter but only very briefly. Mtf1 enhances bending/opening transition and suppresses closing transition, indicating its dual roles of nucleating promoter opening and stabilizing the open state. The cognate initiating ATP prolongs the lifetime of the open state, plausibly explaining the 'ATP sensing mechanism' suggested for the system. We discovered short-lived opening trials upon initial binding of Rpo41-Mtf1 before the establishment of the opening/closing equilibrium, which may aid in promoter selection before the formation of stable pre-initiation complex. The dynamics of open complex formation provides unique insights into the interplay between RNA polymerase and transcription factors in regulating initiation.