Disruption of the standard kinetochore in holocentric Cuscuta species.

The segregation of chromosomes depends on the centromere. Most species are monocentric, with the centromere restricted to a single region per chromosome. In some organisms, the monocentric organization changed to holocentric, in which the centromere activity is distributed over the entire chromosome length. However, the causes and consequences of this transition are poorly understood. Here, we show that the transition in the genus Cuscuta was associated with dramatic changes in the kinetochore, a protein complex that mediates the attachment of chromosomes to microtubules. We found that in holocentric Cuscuta species, the KNL2 genes were lost; the CENP-C, KNL1, and ZWINT1 genes were truncated; the centromeric localization of CENH3, CENP-C, KNL1, MIS12, and NDC80 proteins was disrupted; and the spindle assembly checkpoint (SAC) degenerated. Our results demonstrate that holocentric Cuscuta species lost the ability to form a standard kinetochore and do not employ SAC to control the attachment of microtubules to chromosomes.

KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants.

Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

Cytomolecular trends in Chamaecrista Moench (Caesalpinioideae, Leguminosae) diversification.

Chamaecrista is a Pantropical legume genus of the tribe Cassieae, which includes six other genera. In contrast to most of the other Cassieae genera, Chamaecrista shows significant variability in chromosome number (from 2n = 14 to 2n = 56), with small and morphologically similar chromosomes. Here, we performed a new cytomolecular analysis on chromosome number, genome size, and rDNA site distribution in a molecular phylogenetic perspective to interpret the karyotype trends of Chamaecrista and other two genera of Cassieae, seeking to understand their systematics and evolution. Our phylogenetic analysis revealed that Chamaecrista is monophyletic and can be divided into four major clades corresponding to the four sections of the genus. Chromosome numbers ranged from 2n = 14, 16 (section Chamaecrista) to 2n = 28 (sections Absus, Apoucouita, and Baseophyllum). The number of 5S and 35S rDNA sites varied between one and three pairs per karyotype, distributed on different chromosomes or in synteny, with no obvious phylogenetic significance. Our data allowed us to propose x = 7 as the basic chromosome number of Cassieae, which was changed by polyploidy generating x = 14 (sections Absus, Apoucouita, and Baseophyllum) and by ascending dysploidy to x = 8 (section Chamaecrista). The DNA content values supported this hypothesis, with the genomes of the putative tetraploids being larger than those of the putative diploids. We hypothesized that ascending dysploidy, polyploidy, and rDNA amplification/deamplification are the major events in the karyotypic diversification of Chamaecrista. The chromosomal marks characterized here may have cytotaxonomic potential in future studies.

Supplementary use of the XP-endo Finisher on postoperative pain in posterior teeth with periapical lesions: a randomized clinical trial.

OBJECTIVE: This study evaluated the effect of the supplementary use of the XP-endo Finisher on postoperative endodontic pain. METHODS: This study was a randomized clinical trial with a parallel design. Ninety-two posterior teeth with necrotic pulps and apical periodontitis were instrumented with a single file (Reciproc) in a reciprocating movement followed or not (control) by additional instrumentation with XP-endo Finisher. Postoperative pain was assessed 24 h, 48 h, 72 h, and 7 days after the endodontic treatment (single session) using a universal pain assessment tool. The pain level was scored as absent, mild, moderate, or severe. The occurrence of sealer extrusion and flare-up was also recorded. Data on postoperative pain were analyzed through chi-square analysis, and the odds ratio was adjusted using a logistic regression model (α = 0.05). RESULTS: Similar levels and risks of postoperative pain were observed for both interventions, regardless of the assessment time. Approximately half of the participants presented any postoperative pain in the first 24 h after the endodontic treatment, and this occurrence reduced by less than 20% after 72 h. The extrusion of root filling material was observed in 36% of cases, and no participant presented flared-up. CONCLUSIONS: The supplementary use of the XP-endo Finisher file did not affect the incidence or level of postoperative pain reported after the endodontic treatment of posterior teeth with periapical lesions. CLINICAL RELEVANCE: The supplementary use of the XP-endo Finisher did not affect postoperative pain following the endodontic treatment of posterior teeth using a single-file reciprocating system. CLINICAL TRIAL REGISTRATION: The study protocol was registered in the Brazilian Clinical Trials Registry under identification number RBR-76w7cj (June 19, 2018).

Impact XP-endo finisher on the 1-year follow-up success of posterior root canal treatments: a randomized clinical trial.

OBJECTIVE: To evaluate the clinical relevance of using the XP-endo Finisher as a supplementary tool to improve the success of root canal treatment of posterior teeth with apical periodontitis, as assessed by 1-year follow-up. METHODS: A randomized clinical trial was conducted with 92 posterior teeth with apical periodontitis. Root canal treatment was performed using a single reciprocating file, with or without the supplementary use of the XP-endo Finisher. The status of apical periodontitis was assessed using the periapical index (PAI) at baseline and 1 year follow-up. Changes on PAI indicated that the lesions were healed, healing, or not healed. Successful treatments were defined as healed or healing lesions without clinical symptoms. Chi-square analysis and logistic regression were used for data analysis (α = 0.05). RESULTS: There was no significant difference in the distribution of healing status between the XP-endo Finisher group and the control group (p = 0.690). The success rates were also similar, with 81% in the XP-endo Finisher group and 78% in the control group. However, gender had a significant impact on success rates, with higher rates observed in females. CONCLUSIONS: The use of the XP-endo Finisher file as a supplementary tool did not affect the success rate of root canal treatment in posterior teeth with apical periodontitis. The findings indicate that the XP-endo Finisher file has limited clinical relevance in improving treatment outcomes for root canal treatment in posterior teeth with apical periodontitis. CLINICAL TRIAL REGISTRATION: The study protocol was registered in the Brazilian Clinical Trials Registry under identification number RBR-76w7cj (June 19, 2018).

Impact of parasitic lifestyle and different types of centromere organization on chromosome and genome evolution in the plant genus Cuscuta.

The parasitic genus Cuscuta (Convolvulaceae) is exceptional among plants with respect to centromere organization, including both monocentric and holocentric chromosomes, and substantial variation in genome size and chromosome number. We investigated 12 species representing the diversity of the genus in a phylogenetic context to reveal the molecular and evolutionary processes leading to diversification of their genomes. We measured genome sizes and investigated karyotypes and centromere organization using molecular cytogenetic techniques. We also performed low-pass whole genome sequencing and comparative analysis of repetitive DNA composition. A remarkable 102-fold variation in genome sizes (342-34 734 Mbp/1C) was detected for monocentric Cuscuta species, while genomes of holocentric species were of moderate sizes (533-1545 Mbp/1C). The genome size variation was primarily driven by the differential accumulation of LTR-retrotransposons and satellite DNA. The transition to holocentric chromosomes in the subgenus Cuscuta was associated with loss of histone H2A phosphorylation and elimination of centromeric retrotransposons. In addition, basic chromosome number of holocentric species (x = 7) was smaller than in monocentrics (x = 15 or 16). We demonstrated that the transition to holocentricity in Cuscuta was accompanied by significant changes in epigenetic marks, chromosome number and the repetitive DNA sequence composition.

Effectiveness of a reciprocating single file, single cone endodontic treatment approach: a randomized controlled pragmatic clinical trial.

OBJECTIVES: To compare the root filling quality, the sealer extrusion, and the healing rates of apical lesions addressed via two endodontic treatment approaches. The hypothesis tested was that both techniques present similar apical periodontitis healing results. MATERIALS AND METHODS: This study was a parallel-design, pragmatic, and randomized clinical trial. One hundred twenty anterior teeth with necrotic pulps and apical periodontitis were randomly allocated to be either instrumented with hand files and obturated with the lateral compaction technique or instrumented with a single file in a reciprocating movement and obturated with a single-cone technique. The root canal filling quality, the occurrence of sealer extrusion, and apical periodontitis healing were the outcomes of interest. Data were analyzed through chi-square analysis, and the odds ratio for healing was adjusted using a logistic regression model (α = 0.05). RESULTS: No significant differences were observed between the endodontic techniques regarding the root filling quality or sealer extrusion. Six months after treatment, both endodontic techniques presented equivalent results regarding apical periodontitis healing. The healing rate was affected only by the periapical status at baseline. CONCLUSIONS: The obturation of the root canal of the anterior teeth using reciprocating file-matched single cones presented a similar quality to that obtained with manual instrumentation followed by the lateral condensation technique. Similar healing rates of apical lesions were also observed. CLINICAL RELEVANCE: For treating anterior teeth with apical periodontitis, a reciprocating single-file, single-treatment protocol was as effective as a traditional protocol combining hand instrumentation and the lateral compaction obturation technique.

Plant centromeres: genetics, epigenetics and evolution.

The centromere is a functional locus of the chromosome responsible for chromatid cohesion and segregation in cell division. Usually, the centromeres can be distinguished from the remaining chromosomal regions either in structure, as they are heterochromatic constrictions that divide the chromosome in two arms, or in molecular constitution, as they have an exclusive H3 histone variant and specific DNA sequences. Besides being essential for genetic stability of eukaryotes, centromere is particularly interesting since it plays conserved roles but show high variability on organization and DNA composition. Centromeres are usually composed by satellite repeats and retrotransposons and the sequences can differ even among closely related species. Some unusual configurations containing single copy DNA were also described, including even some transcribed genes. In this review, we discuss molecular constitution, epigenetics and different types of centromere, with emphasis on plant centromeres. We also present recent advances about evolutionary processes involved in origin and differentiation of centromeres.

Effects of adhesive used as modeling liquid on the stability of the color and opacity of composites.

OBJECTIVE: This study evaluated the effects of adhesive type used as modeling liquid on the stability of the color and opacity of composites submitted to thermal cycling in staining solutions followed by a bleaching procedure. METHODS: Thirty cylinder-shaped composite specimens (10 mm diameter × 1.5 mm thickness) were built using or not using (control) an adhesive (Adper Universal or Scotchbond Multipurpose) as the modeling liquid. After polishing procedures, the color and opacity were measured, and the specimens were submitted to 200 thermal cycles with 10 s of dwell time in baths of grape juice at 5°C, water at 37°C, and coffee at 55°C. Changes in opacity and color caused by the staining solutions were measured. Then, the specimens were bleached with 35% hydrogen peroxide for 45 minutes followed by color/opacity measurements. Data were analyzed by ANOVA and Tukey's test (α = 0.05). RESULTS: The use of modeling liquids did not affect the initial color and opacity of composite. Reduced changes to color (E00 = 3.44) and opacity (+2.67%) were observed for specimens modeled using Adper Universal. Bleaching procedures reduced the color (E00 = 1.9-3.8) and opacity (-2.3 to 3.1%) alterations caused by staining solutions but were unable to restore the values observed at baseline. CONCLUSION: The use of universal adhesive as modeling liquid significantly reduced the color and opacity changes caused by staining solutions, and the bleaching procedure partially re-established the opacity and color of the composites. CLINICAL SIGNIFICANCE: This study evaluates whether using adhesive systems for modeling a composite affects the color and opacity changes caused by staining solutions followed by a bleaching procedure. The findings suggest that the use of a universal adhesive as modeling liquid can reduce the alterations in optical properties caused by staining solutions, and the application of high-concentrated hydrogen peroxide over the composite reduce the color changes without fully recover the initial color.

Boom-Bust Turnovers of Megabase-Sized Centromeric DNA in Solanum Species: Rapid Evolution of DNA Sequences Associated with Centromeres.

Centromeres are composed of long arrays of satellite repeats in most multicellular eukaryotes investigated to date. The satellite repeat-based centromeres are believed to have evolved from "neocentromeres" that originally contained only single- or low-copy sequences. However, the emergence and evolution of the satellite repeats in centromeres has been elusive. Potato (Solanum tuberosum) provides a model system for studying centromere evolution because each of its 12 centromeres contains distinct DNA sequences, allowing comparative analysis of homoeologous centromeres from related species. We conducted genome-wide analysis of the centromeric sequences in Solanum verrucosum, a wild species closely related to potato. Unambiguous homoeologous centromeric sequences were detected in only a single centromere (Cen9) between the two species. Four centromeres (Cen2, Cen4, Cen7, and Cen10) in S. verrucosum contained distinct satellite repeats that were amplified from retrotransposon-related sequences. Strikingly, the same four centromeres in potato contain either different satellite repeats (Cen2 and Cen7) or exclusively single- and low-copy sequences (Cen4 and Cen10). Our sequence comparison of five homoeologous centromeres in two Solanum species reveals rapid divergence of centromeric sequences among closely related species. We propose that centromeric satellite repeats undergo boom-bust cycles before a favorable repeat is fixed in the population.

A chromosome-scale reference genome of grasspea (Lathyrus sativus).

Grasspea (Lathyrus sativus L.) is an underutilised but promising legume crop with tolerance to a wide range of abiotic and biotic stress factors, and potential for climate-resilient agriculture. Despite a long history and wide geographical distribution of cultivation, only limited breeding resources are available. This paper reports a 5.96 Gbp genome assembly of grasspea genotype LS007, of which 5.03 Gbp is scaffolded into 7 pseudo-chromosomes. The assembly has a BUSCO completeness score of 99.1% and is annotated with 31719 gene models and repeat elements. This represents the most contiguous and accurate assembly of the grasspea genome to date.

Generalized anxiety disorder and associated factors in primary health care workers in Minas Gerais, Brazil.

BACKGROUND: The COVID-19 pandemic had direct and indirect impacts on public health, also affecting the mental health of the population. OBJECTIVE: To analyze symptoms of generalized anxiety disorder (GAD) and associated factors, in primary health care (PHC) professionals. METHODS: Cross-sectional and analytical study conducted with professionals who work in the Primary Health Care (PHC) of the Northern health macro-region of the state of Minas Gerais, Brazil. Data collection was carried out through an online, self-administered questionnaire, made available from August 27, 2021, to October 30, 2021 using the Google Forms tool. The questionnaire included sociodemographic questions as well as questions related to the history of anxiety and work during the pandemic. To assess anxiety symptoms, the General Anxiety Disorder-7 (GAD-7) instrument was used. Data treatment consisted of descriptive analysis of the variables, bivariate analysis followed by multivariate Poisson Regression with robust variance. RESULTS: 702 health professionals participated in the study and the general prevalence of GAD was 32.2%. Current anxiety symptoms were self-reported by 37.6% of the participants. In the final model, the associated factors identified were: female sex (PR = 1.82; 95% CI = 1.17-2.84; p = 0.007), previous symptoms (PR = 1.58; 95% CI = 1.19-2.10; p = 0.002) and (PR = 2.68; 95% CI = 2.00-3.62; p < 0.001) current self-reported anxiety. CONCLUSION: The results show the need to implement actions in mental health promotion and anxiety prevention, through the dissemination of information about mental health care, health education activities, encouraging the adoption of healthy habits and professional monitoring when necessary.

Facial profile evaluation of isolated Pierre Robin sequence.

OBJECTIVE: To evaluate numerically the facial profile of children with isolated Pierre Robin sequence (PRS) and to compare them with a control group that has no pathologies and exhibits regular and balanced facial growth, with no skeletal alterations. PATIENTS: Eighty-three children aged 5 to 10 years (PRS group, n  =  60; control group, n  =  23) were selected. SETTING: Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo (HRAC-USP). Children from the control group were taken from the program of Interceptive Orthodontics at HRAC-USP. DESIGN: Angular and ratio analyses of the facial profiles in both groups were realized through digital photographs. The PRS group was subdivided into two groups--complete and incomplete--according to the sagittal extension of the cleft palate, to investigate the possible influence of cleft extension on the face. RESULTS: The facial convexity angle and the facial inferior third angle were considerably higher in the PRS groups than in the control group and were not significantly different between PRS groups. Nasolabial angle did not differ between groups. CONCLUSION: The facial profile was more convex in individuals with PRS than in those with regular facial growth and with no pathology. The mandible was responsible for the convexity of the profile in PRS because of its lack off anterior projection. An important relationship between the extension of the cleft palate and alterations in facial profile in PRS was not observed.

Antimony(V) and bismuth(V) complexes of lapachol: synthesis, crystal structure and cytotoxic activity.

Antimony(V) and bismuth(V) complexes of lapachol have been synthesized by the reaction of Ph3SbCl2 or Ph3BiCl2 with lapachol (Lp) and characterized by several physicochemical techniques such as IR, and NMR spectroscopy and X-ray crystallography. The compounds contain six-coordinated antimony and bismuth atoms. The antimony(V) complex is a monomeric derivative, (Lp)(Ph3Sb)OH, and the bismuth(V) complex is a dinuclear compound bridged by an oxygen atom, (Lp)2(Ph3Bi)2O. Both compounds inhibited the growth of a chronic myelogenous leukemia cell line and the complex of Bi(V) was about five times more active than free lapachol. This work provides a rare example of an organo-Bi(V) complex showing significant cytotoxic activity.

Complex sequence organization of heterochromatin in the holocentric plant Cuscuta europaea elucidated by the computational analysis of nanopore reads.

Repeat-rich regions of higher plant genomes are usually associated with constitutive heterochromatin, a specific type of chromatin that forms tightly packed nuclear chromocenters and chromosome bands. There is a large body of cytogenetic evidence that these chromosome regions are often composed of tandemly organized satellite DNA. However, comparatively little is known about the sequence arrangement within heterochromatic regions, which are difficult to assemble due to their repeated nature. Here, we explore long-range sequence organization of heterochromatin regions containing the major satellite repeat CUS-TR24 in the holocentric plant Cuscuta europaea. Using a combination of ultra-long read sequencing with assembly-free sequence analysis, we reveal the complex structure of these loci, which are composed of short arrays of CUS-TR24 interrupted frequently by emerging simple sequence repeats and targeted insertions of a specific lineage of LINE retrotransposons. These data suggest that the organization of satellite repeats constituting heterochromatic chromosome bands can be more complex than previously envisioned, and demonstrate that heterochromatin organization can be efficiently investigated without the need for genome assembly.

The effectiveness of the supplementary use of the XP-endo Finisher on bacteria content reduction: a systematic review and meta-analysis.

OBJECTIVES: This systematic review evaluated the efficacy of the supplementary use of the XP-endo Finisher on bacteria content reduction in the root canal system. MATERIALS AND METHODS: In-vitro studies evaluating the use of the XP-endo Finisher on bacteria content were searched in four databases in July 2020. Two authors independently screened the studies for eligibility. Data were extracted, and risk of bias was assessed. Data were meta-analyzed by using random-effects model to compare the effect of the supplementary use (experimental) or not (control) of the XP-endo Finisher on bacteria counting reduction, and results from different endodontic protocols were combined. Four studies met the inclusion criteria while 1 study was excluded from the meta-analysis due to its high risk of bias and outlier data. The 3 studies that made it to the meta-analysis had an unclear risk of bias for at least one criterion. RESULTS: No heterogeneity was observed among the results of the studies included in the meta-analysis. The study excluded from the meta-analysis assessing the bacteria counting deep in the dentin demonstrated further bacteria reduction upon the use of the XP-endo Finisher. CONCLUSIONS: This systematic review found no evidence supporting the supplementary use of the XP-endo Finisher on further bacteria counting the reduction in the root canal.

Correction: Apical periodontitis healing and postoperative pain following endodontic treatment with a reciprocating single-file, single-cone approach: A randomized controlled pragmatic clinical trial.

[This corrects the article DOI: 10.1371/journal.pone.0227347.].

Apical periodontitis healing and postoperative pain following endodontic treatment with a reciprocating single-file, single-cone approach: A randomized controlled pragmatic clinical trial.

This trial assessed post-operative pain and healing of apical periodontitis following endodontic therapy with a reciprocating system compared to a crown-down technique with hand files and lateral compaction filling. One-hundred and twenty nonvital anterior teeth with apical periodontitis were randomly treated using either a reciprocating single file followed by matching-taper single-cone filling or a hand file and lateral compaction filling. Postoperative pain was assessed during the 7 days after the treatment, using a visual analogue scale and a verbal rating scale. Apical healing was assessed using the periapical index score after a 12-month follow-up. The hypothesis tested was that both protocols were equivalent and present similar effectiveness in healing periapical lesions. Data were analyzed through two one-sided tests, t-tests, as well as Mann-Whitney and Chi-squared tests (α = 0.05). Logistic regression was used to investigate the association of clinical and demographic factors with the success of treatment. Regardless of the assessment time, no difference in incidence (38%-43% at first 24h), intensity of postoperative pain, and incidence of flare-up (≈ 3%) was observed between the two endodontic protocols. Both protocols resulted in a similar healing rate of apical periodontitis. After 12 months, the success rate ranged from 73% to 78% and the difference between the treatments fell within the pre-established equivalence margin (-0.1; -0.41 to 0.2). Endodontic treatment combining a reciprocating single file with matching-taper single cone showed similar clinical effectiveness to the treatment using hand-file instrumentation and the lateral compaction filling.

Mitotic Spindle Attachment to the Holocentric Chromosomes of Cuscuta europaea Does Not Correlate With the Distribution of CENH3 Chromatin.

The centromere is the region on a chromosome where the kinetochore assembles and spindle microtubules attach during mitosis and meiosis. In the vast majority of eukaryotes, the centromere position is determined epigenetically by the presence of the centromere-specific histone H3 variant CENH3. In species with monocentric chromosomes, CENH3 is confined to a single chromosomal region corresponding to the primary constriction on metaphase chromosomes. By contrast, in holocentrics, CENH3 (and thus centromere activity) is distributed along the entire chromosome length. Here, we report a unique pattern of CENH3 distribution in the holocentric plant Cuscuta europaea. This species expressed two major variants of CENH3, both of which were deposited into one to three discrete regions per chromosome, whereas the rest of the chromatin appeared to be devoid of CENH3. The two CENH3 variants fully co-localized, and their immunodetection signals overlapped with the positions of DAPI-positive heterochromatic bands containing the highly amplified satellite repeat CUS-TR24. This CENH3 distribution pattern contrasted with the distribution of the mitotic spindle microtubules, which attached at uniform density along the entire chromosome length. This distribution of spindle attachment sites proves the holocentric nature of C. europaea chromosomes and also suggests that, in this species, CENH3 either lost its function or acts in parallel to an additional CENH3-free mechanism of kinetochore positioning.

In vitro evaluation of tooth discoloration induced by regenerative endodontic therapy and the effectiveness of the walking bleach technique.

BACKGROUND: The present study evaluated the coronal discoloration induced by different materials or blood as well as the effect of internal bleaching on discoloration. METHODS: After tooth color measurement at baseline with a sphere spectrophotometer (CIE L*a*b*), the root canals of molars were partially filled with white mineral trioxide aggregate (MTA), triple antibiotic paste (TAP), or a cotton pellet with bovine blood (control) was placed into the pulp chamber. The color changes were assessed at 30, 60, and 180 days after the procedure. Following the discoloration, internal bleaching was performed with a mixture of sodium perborate and hydrogen peroxide inserted into the pulp chamber, and this mixture was replaced weekly for 3 weeks. At baseline, after staining and after bleaching, the data of each color parameter were individually analyzed by one-way ANOVA, while differences in pooled color changes (∆E00) were assessed by two-way repeated measures ANOVA (α = 0.05). RESULTS: After the staining period, the TAP-stained specimens were darker and greener than the other specimens, and no difference was observed between the teeth stained with blood and those stained with MTA. The walking bleach technique resulted in an improved tooth color without a difference in the color changes among the different groups. CONCLUSION: The TAP-stained specimens had the greatest tooth discoloration, and the discoloration remained the highest after the walking bleach technique. PRACTICAL IMPLICATIONS: This in vitro study demonstrated that TAP yielded higher tooth discoloration, and this color might hinder a satisfactory tooth color from being obtained with the walking bleach technique.