Physical Peculiarity of Two Sites in Human Promoters: Universality and Diverse Usage in Gene Function.

Since the discovery of physical peculiarities around transcription start sites (TSSs) and a site corresponding to the TATA box, research has revealed only the average features of these sites. Unsettled enigmas include the individual genes with these features and whether they relate to gene function. Herein, using 10 physical properties of DNA, including duplex DNA free energy, base stacking energy, protein-induced deformability, and stabilizing energy of Z-DNA, we clarified for the first time that approximately 97% of the promoters of 21,056 human protein-coding genes have distinctive physical properties around the TSS and/or position -27; of these, nearly 65% exhibited such properties at both sites. Furthermore, about 55% of the 21,056 genes had a minimum value of regional duplex DNA free energy within TSS-centered ±300 bp regions. Notably, distinctive physical properties within the promoters and free energies of the surrounding regions separated human protein-coding genes into five groups; each contained specific gene ontology (GO) terms. The group represented by immune response genes differed distinctly from the other four regarding the parameter of the free energies of the surrounding regions. A vital suggestion from this study is that physical-feature-based analyses of genomes may reveal new aspects of the organization and regulation of genes.

Expression and function of an S1-type nuclease in the digestive fluid of a sundew, Drosera adelae.

BACKGROUND AND AIMS: Carnivorous plants trap and digest insects and similar-sized animals. Many studies have examined enzymes in the digestive fluids of these plants and have gradually unveiled the origins and gene expression of these enzymes. However, only a few attempts have been made at characterization of nucleases. This study aimed to reveal gene expression and the structural, functional and evolutionary characteristics of an S1-type nuclease (DAN1) in the digestive fluid of an Australian sundew, Drosera adelae, whose trap organ shows unique gene expression and related epigenetic regulation. METHODS: Organ-specificity in Dan1 expression was examined using glandular tentacles, laminas, roots and inflorescences, and real-time PCR. The methylation status of the Dan1 promoter in each organ was clarified by bisulphite sequencing. The structural characteristics of DAN1 were studied by a comparison of primary structures of S1-type nucleases of three carnivorous and seven non-carnivorous plants. DAN1 was prepared using a cell-free protein synthesis system. Requirements for metal ions, optimum pH and temperature, and substrate preference were examined using conventional methods. KEY RESULTS: Dan1 is exclusively expressed in the glandular tentacles and its promoter is almost completely unmethylated in all organs. This is in contrast to the S-like RNase gene da-I of Dr. adelae, which shows similar organ-specific expression, but is controlled by a promoter that is specifically unmethylated in the glandular tentacles. Comparison of amino acid sequences of S1-type nucleases identifies seven and three positions where amino acid residues are conserved only among the carnivorous plants and only among the non-carnivorous plants, respectively. DAN1 prefers a substrate RNA over DNA in the presence of Zn2+, Mn2+ or Ca2+ at an optimum pH of 4.0. CONCLUSIONS: Uptake of phosphates from prey is suggested to be the main function of DAN1, which is very different from the known functions of S1-type nucleases. Evolution has modified the structure and expression of Dan1 to specifically function in the digestive fluid.

Organ-specific expression and epigenetic traits of genes encoding digestive enzymes in the lance-leaf sundew (Drosera adelae).

Over the last two decades, extensive studies have been performed at the molecular level to understand the evolution of carnivorous plants. As fruits, the repertoire of protein components in the digestive fluids of several carnivorous plants have gradually become clear. However, the quantitative aspects of these proteins and the expression mechanisms of the genes that encode them are still poorly understood. In this study, using the Australian sundew Drosera adelae, we identified and quantified the digestive fluid proteins. We examined the expression and methylation status of the genes corresponding to major hydrolytic enzymes in various organs; these included thaumatin-like protein, S-like RNase, cysteine protease, class I chitinase, ß-1, 3-glucanase, and hevein-like protein. The genes encoding these proteins were exclusively expressed in the glandular tentacles. Furthermore, the promoters of the ß-1, 3-glucanase and cysteine protease genes were demethylated only in the glandular tentacles, similar to the previously reported case of the S-like RNase gene da-I. This phenomenon correlated with high expression of the DNA demethylase DEMETER in the glandular tentacles, strongly suggesting that it performs glandular tentacle-specific demethylation of the genes. The current study strengthens and generalizes the relevance of epigenetics to trap organ-specific gene expression in D. adelae. We also suggest similarities between the trap organs of carnivorous plants and the roots of non-carnivorous plants.

Cruciform Formable Sequences within Pou5f1 Enhancer Are Indispensable for Mouse ES Cell Integrity.

DNA can adopt various structures besides the B-form. Among them, cruciform structures are formed on inverted repeat (IR) sequences. While cruciform formable IRs (CFIRs) are sometimes found in regulatory regions of transcription, their function in transcription remains elusive, especially in eukaryotes. We found a cluster of CFIRs within the mouse Pou5f1 enhancer. Here, we demonstrate that this cluster or some member(s) plays an active role in the transcriptional regulation of not only Pou5f1, but also Sox2, Nanog, Klf4 and Esrrb. To clarify in vivo function of the cluster, we performed genome editing using mouse ES cells, in which each of the CFIRs was altered to the corresponding mirror repeat sequence. The alterations reduced the level of the Pou5f1 transcript in the genome-edited cell lines, and elevated those of Sox2, Nanog, Klf4 and Esrrb. Furthermore, transcription of non-coding RNAs (ncRNAs) within the enhancer was also upregulated in the genome-edited cell lines, in a similar manner to Sox2, Nanog, Klf4 and Esrrb. These ncRNAs are hypothesized to control the expression of these four pluripotency genes. The CFIRs present in the Pou5f1 enhancer seem to be important to maintain the integrity of ES cells.

A strong structural correlation between short inverted repeat sequences and the polyadenylation signal in yeast and nucleosome exclusion by these inverted repeats.

DNA sequences that read the same from 5' to 3' in either strand are called inverted repeat sequences or simply IRs. They are found throughout a wide variety of genomes, from prokaryotes to eukaryotes. Despite extensive research, their in vivo functions, if any, remain unclear. Using Saccharomyces cerevisiae, we performed genome-wide analyses for the distribution, occurrence frequency, sequence characteristics and relevance to chromatin structure, for the IRs that reportedly have a cruciform-forming potential. Here, we provide the first comprehensive map of these IRs in the S. cerevisiae genome. The statistically significant enrichment of the IRs was found in the close vicinity of the DNA positions corresponding to polyadenylation [poly(A)] sites and ~ 30 to ~ 60 bp downstream of start codon-coding sites (referred to as 'start codons'). In the former, ApT- or TpA-rich IRs and A-tract- or T-tract-rich IRs are enriched, while in the latter, different IRs are enriched. Furthermore, we found a strong structural correlation between the former IRs and the poly(A) signal. In the chromatin formed on the gene end regions, the majority of the IRs causes low nucleosome occupancy. The IRs in the region ~ 30 to ~ 60 bp downstream of start codons are located in the + 1 nucleosomes. In contrast, fewer IRs are present in the adjacent region downstream of start codons. The current study suggests that the IRs play similar roles in Escherichia coli and S. cerevisiae to regulate or complete transcription at the RNA level.

New Aspects of Magnesium Function: A Key Regulator in Nucleosome Self-Assembly, Chromatin Folding and Phase Separation.

Metal cations are associated with many biological processes. The effects of these cations on nucleic acids and chromatin were extensively studied in the early stages of nucleic acid and chromatin research. The results revealed that some monovalent and divalent metal cations, including Mg2+, profoundly affect the conformations and stabilities of nucleic acids, the folding of chromatin fibers, and the extent of chromosome condensation. Apart from these effects, there have only been a few reports on the functions of these cations. In 2007 and 2013, however, Mg2+-implicated novel phenomena were found: Mg2+ facilitates or enables both self-assembly of identical double-stranded (ds) DNA molecules and self-assembly of identical nucleosomes in vitro. These phenomena may be deeply implicated in the heterochromatin domain formation and chromatin-based phase separation. Furthermore, a recent study showed that elevation of the intranuclear Mg2+ concentration causes unusual differentiation of mouse ES (embryonic stem) cells. All of these phenomena seem to be closely related to one another. Mg2+ seems to be a key regulator of chromatin dynamics and chromatin-based biological processes.

Correction to: Requirement or exclusion of inverted repeat sequences with cruciform-forming potential in Escherichia coli revealed by genome-wide analyses.

The article 'Requirement or exclusion of inverted repeat sequences with cruciform-forming potential in Escherichia coli revealed by genome-wide analyses' written by Osamu Miura, Toshihiro Ogake, Takashi Ohyama was originally published online on SpringerLink with open access.

Requirement or exclusion of inverted repeat sequences with cruciform-forming potential in Escherichia coli revealed by genome-wide analyses.

Inverted repeat (IR) sequences are DNA sequences that read the same from 5' to 3' in each strand. Some IRs can form cruciforms under the stress of negative supercoiling, and these IRs are widely found in genomes. However, their biological significance remains unclear. The aim of the current study is to explore this issue further. We constructed the first Escherichia coli genome-wide comprehensive map of IRs with cruciform-forming potential. Based on the map, we performed detailed and quantitative analyses. Here, we report that IRs with cruciform-forming potential are statistically enriched in the following five regions: the adjacent regions downstream of the stop codon-coding sites (referred to as the stop codons), on and around the positions corresponding to mRNA ends (referred to as the gene ends), ~ 20 to ~45 bp upstream of the start codon-coding sites (referred to as the start codons) within the 5'-UTR (untranslated region), ~ 25 to ~ 60 bp downstream of the start codons, and promoter regions. For the adjacent regions downstream of the stop codons and on and around the gene ends, most of the IRs with a repeat unit length of ≥ 8 bp and a spacer size of ≤ 8 bp were parts of the intrinsic terminators, regardless of the location, and presumably used for Rho-independent transcription termination. In contrast, fewer IRs were present in the small region preceding the start codons. In E. coli, IRs with cruciform-forming potential are actively placed or excluded in the regulatory regions for the initiation and termination of transcription and translation, indicating their deep involvement or influence in these processes.

Magnesium chloride and polyamine can differentiate mouse embryonic stem cells into trophectoderm or endoderm.

Magnesium chloride and polyamines stabilize DNA and chromatin. Furthermore, they can induce nucleosome aggregation and chromatin condensation in vitro. To determine the effects of elevating the cation concentrations in the nucleus of a living cell, we microinjected various concentrations of mono-, di- and polyvalent cation solutions into the nuclei of mouse embryonic stem (ES) cells and traced their fates. Here, we show that an elevation of either MgCl2, spermidine or spermine concentration in the nucleus exerts a significant effect on mouse ES cells, and can differentiate a certain population of the cells into trophectoderm, a lineage that mouse ES cells do not normally generate, or endoderm. It is hypothesized that the cell differentiation was most probably caused by the condensation of chromatin including the Oct3/4 locus, which was induced by the elevated concentrations of these cations.

Higher Frequency of Reflux Symptoms and Acid-Related Dyspepsia in Women than Men Regardless of Endoscopic Esophagitis: Analysis of 3,505 Japanese Subjects Undergoing Medical Health Checkups.

BACKGROUND/AIMS: This study aimed at (i) clarifying the factors associated with high scores on the modified frequency scale for the symptoms of gastroesophageal reflux disease (FSSG) among 3,505 relatively healthy subjects undergoing routine medical health checkups with gastrointestinal endoscopy and (ii) comparing risk factors for high FSSG scores between subjects with and without reflux esophagitis. METHODS: In total, 3,505 subjects (male/female: 1,922/1,583) who underwent upper gastrointestinal endoscopy during health medical checkups at 5 hospitals in Saga, Japan from January 2013 to December 2013 were enrolled. All subjects completed a modified FSSG questionnaire, which comprised 7 questions regarding reflux symptoms and 7 questions regarding acid-related dyspepsia. Each question was assigned a score based on the frequency of symptoms. RESULTS: Younger age, female gender, hiatal herniation, and endoscopic reflux esophagitis were risk factors for a FSSG score with a high total. Subjects with high scores but without esophagitis were women, and hiatal herniation and Barrett's esophagus were frequently seen in patients with reflux esophagitis. CONCLUSION: Younger age, female gender, hiatal hernia, and endoscopic esophagitis were risk factors for a high FSSG score, and women tended to complain of upper gastrointestinal symptoms more frequently than did men among subjects without endoscopic esophagitis.

Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation.

Selective association between nucleosomes with identical DNA sequences.

Self-assembly is the autonomous organization of constituents into higher order structures or assemblages and is a fundamental mechanism in biological systems. There has been an unfounded idea that self-assembly may be used in the sensing and pairing of homologous chromosomes or chromatin, including meiotic chromosome pairing, polytene chromosome formation in Diptera and transvection. Recent studies proved that double-stranded DNA molecules have a sequence-sensing property and can self-assemble, which may play a role in the above phenomena. However, to explain these processes in terms of self-assembly, it first must be proved that nucleosomes retain a DNA sequence-sensing property and can self-assemble. Here, using atomic force microscopy (AFM)-based analyses and a quantitative interaction assay, we show that nucleosomes with identical DNA sequences preferentially associate with each other in the presence of Mg(2+) ions. Using Xenopus borealis 5S rDNA nucleosome-positioning sequence and 601 and 603 sequences, homomeric or heteromeric octa- or tetranucleosomes were reconstituted in vitro and induced to form weak intracondensates by MgCl(2). AFM clearly showed that DNA sequence-based selective association occurs between nucleosomes with identical DNA sequences. Selective association was also detected between mononucleosomes. We propose that nucleosome self-assembly and DNA self-assembly constitute the mechanism underlying sensing and pairing of homologous chromosomes or chromatin.

Structural and functional characteristics of S-like ribonucleases from carnivorous plants.

Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k cat/K m of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k cat/K m of the recombinant S. leucophylla enzyme was the lowest, and was ~1/30 of that for recombinant RNS1. The magnitudes of the k cat/K m values or k cat values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.

Distinctive physical properties of DNA shared by RNA polymerase II gene promoters and 5'-flanking regions of tRNA genes.

Numerous noncoding (nc)RNAs have been identified. Similar to the transcription of protein-coding (mRNA) genes, long noncoding (lnc)RNA genes and most of micro (mi)RNA genes are transcribed by RNA polymerase II (Pol II). In the transcription of mRNA genes, core promoters play an indispensable role; they support the assembly of the preinitiation complex (PIC). However, the structural and/or physical properties of the core promoters of lncRNA and miRNA genes remain largely unexplored, in contrast with those of mRNA genes. Using the core promoters of human genes, we analyzed the repertoire and population ratios of residing core promoter elements (CPEs) and calculated the following five DNA physical properties (DPPs): duplex DNA free energy, base stacking energy, protein-induced deformability, rigidity and stabilizing energy of Z-DNA. Here, we show that their CPE and DPP profiles are similar to those of mRNA gene promoters. Importantly, the core promoters of these three classes of genes have two highly distinctive sites in their DPP profiles around the TSS and position -27. Similar characteristics in DPPs are also found in the 5'-flanking regions of tRNA genes, indicating their common essential roles in transcription initiation over the kingdom of RNA polymerases.

Most methylation-susceptible DNA sequences in human embryonic stem cells undergo a change in conformation or flexibility upon methylation.

DNA methylation in eukaryotes occurs on the cytosine bases in CG, CHG, and CHH (where H indicates non-G nucleotides) contexts and provides an important epigenetic mark in various biological processes. However, the structural and physical properties of methylated DNA are poorly understood. Using nondenaturing polyacrylamide gel electrophoresis, we performed a systematic study of the influence of DNA methylation on the conformation and physical properties of DNA for all CG, CHG, and CHH contexts. In the CG context, methylated multimers of the CG/CG-containing unit fragment migrated in gels slightly faster than their unmethylated counterparts. In the CHG context, both homo- and hemimethylation caused retarded migration of multimers of the CAG/CTG-containing fragment. In the CHH context, methylation caused or enhanced retarded migration of the multimers of CAA/TTG-, CAT/ATG-, CAC/GTG-, CTA/TAG-, or CTT/AAG-containing fragments. These results suggest that methylation increases DNA rigidity in the CG context and introduces distortions into several CHG and CHH sequences. More interestingly, we found that nearly all of the methylation repertoires in the CHG context and 98% of those in the CHH context in human embryonic stem cells were species that undergo conformational changes upon methylation. Similarly, most of the methylation repertoires in the Arabidopsis CHG and CHH contexts were sequences with methylation-induced distortion. We hypothesize that the methylation-induced properties or conformational changes in DNA may facilitate nucleosome formation, which provides the essential mechanism for alterations of chromatin density.

S-like ribonuclease gene expression in carnivorous plants.

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

Regions of unusually high flexibility occur frequently in human genomic DNA.

Remarkable progress has been made in genome science during the past decade, but understanding of genomes of eukaryotes is far from complete. We have created DNA flexibility maps of the human, mouse, fruit fly, and nematode chromosomes. The maps revealed that all of these chromosomes have markedly flexible DNA regions (We named them SPIKEs). SPIKEs occur more frequently in the human chromosomes than in the mouse, fruit fly, and nematode chromosomes. Markedly rigid DNA regions (rSPIKEs) are also present in these chromosomes. The ratio of the number of SPIKEs to the total number of SPIKEs and rSPIKEs correlated positively with evolutionary stage among the organisms. Repetitive DNA sequences with flexible and rigid properties contribute to the formation of SPIKEs and rSPIKEs respectively. However, non-repetitive flexible and rigid sequences appear to play a major role in SPIKE and rSPIKE formation respectively. They might be involved in the genome-folding mechanism of eukaryotes.

Prosthodontic treatment can improve the ingestible food profile in Japanese adult outpatients.

PURPOSE: To investigate the effect of prosthodontic treatment on the ingestible food profile in adult Japanese outpatients, and to identify the related risk factors that can deteriorate the profile. METHODS: The participants were 277 outpatients who visited university-based specialty clinics in Japan for prosthodontic treatment. The demographic data, number of present teeth assessed via intraoral examination, and oral health-related quality of life assessed by the total Oral Health Impact Profile (OHIP-J54) scores of all participants were recorded before treatment. Ingestible food profile score (IFS) was recorded using a validated food intake questionnaire. Eligible participants who answered the questionnaire before and after treatment were categorized into five groups based on the prosthodontic treatments they received (i.e., crowns, bridges, removable partial dentures, removable complete dentures, and removable complete and partial dentures). RESULTS: Multivariate analysis of covariance revealed a statistically significant main effect of prosthodontic intervention (time course: before and after treatment) on mean IFS (P=0.035, F=4.526), even after adjusting for covariates (age, number of present teeth, and treatment modality). Multiple linear regression analysis revealed that the low number of present teeth (r=0.427, P<0.001) and a high OHIP-J54 total score (r=-0.519, P<0.001) of the patients at the baseline were significantly associated with their baseline IFSs, even after adjusting for confounding variables. CONCLUSIONS: The findings of this multicenter follow-up study indicate the importance of prosthodontic rehabilitation in improving patients' ingestible food profiles.

Competence of an artificial bent DNA as a transcriptional activator in mouse ES cells.

Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.

Development of Innovative Contra-angle Handpiece Device with Piston Movement for Root Canal Preparation.

INTRODUCTION: The purpose of this study was to assess the optimal amplitude and weight of the newly developed contra-angle handpiece. The handpiece uses piston movement without using an endodontic motor and enables a safe, quick, and reliable canal preparation. METHODS: A prototype handpiece was designed. Instrumentation was performed on root canal resin blocks by 20 operators in 3 groups: the prototype handpiece with an H file (a stainless steel #25 manual H file, the piston group), a manually standardized technique with a K file (stainless steel #15-25 K files, the manual group), and a nickel-titanium (NiTi) reciprocating file with an endodontic motor (Reciproc Blue R25 [VDW, Munich, Germany], the NiTi group). Transportation of the canal center line and the time required for preparation were measured and statistically analyzed. RESULTS: The optimal condition was an amplitude of 1.35 mm and a weight of 61.0 g. Transportation of the canal center was observed in all groups. A statistically significant difference was found at 2.0-3.0 mm from the apical foramen between the piston or NiTi group and the manual group, but no significant difference was found between the piston and NiTi groups. The least transportation was found in the NiTi and piston groups. The handpiece with a #25 H file demonstrated a good centering ability, similar to the NiTi file, which enabled speedy preparation. The time required for preparation between the piston or NiTi group and the manual group was statistically different. No significant difference was observed between the piston and NiTi groups (P < .05). CONCLUSIONS: We concluded that the newly designed handpiece achieved efficient canal preparation and negotiation. The handpiece could avoid endodontic accidents, including ledge formation, instrument separation, and perforation.