Regulatory mechanisms of stem cell differentiation: Biotechnological applications for neurogenesis.

The world population's life expectancy is growing, and neurodegenerative disorders common in old age require more efficient therapies. In this context, neural stem cells (NSCs) are imperative for the development and maintenance of the functioning of the nervous system and have broad therapeutic applicability for neurodegenerative diseases. Therefore, knowing all the mechanisms that govern the self-renewal, differentiation, and cell signaling of NSC is necessary. This review will address some of these aspects, including the role of growth and transcription factors, epigenetic modulators, microRNAs, and extracellular matrix components. Furthermore, differentiation and transdifferentiation processes will be addressed as therapeutic strategies showing their significance for stem cell-based therapy.

Organoids as a novel tool in modelling infectious diseases.

Infectious diseases worldwide affect human health and have important societal impacts. A better understanding of infectious diseases is urgently needed. In vitro and in vivo infection models have brought notable contributions to the current knowledge of these diseases. Organoids are multicellular culture systems resembling tissue architecture and function, recapitulating many characteristics of human disease and elucidating mechanisms of host-infectious agent interactions in the respiratory and gastrointestinal systems, the central nervous system and the skin. Here, we discuss the applicability of the organoid technology for modeling pathogenesis, host response and features, which can be explored for the development of preventive and therapeutic treatments.

Strategic use of organoids and organs-on-chip as biomimetic tools.

Organoid development and organ-on-a-chip are technologies based on differentiating stem cells, forming 3D multicellular structures resembling organs and tissues in vivo. Hence, both can be strategically used for disease modeling, drug screening, and host-pathogen studies. In this context, this review highlights the significant advancements in the area, providing technical approaches to organoids and organ-on-a-chip that best imitate in vivo physiology.

A Novel PD-L1 Antibody Promotes Antitumor Function of Peripheral Cytotoxic Lymphocytes after Radical Nephrectomy in Patients with Renal Cell Carcinoma.

The intrinsic and acquired resistance to PD-1/PD-L1 immune checkpoint blockade is an important challenge for patients and clinicians because no reliable tool has been developed to predict individualized response to immunotherapy. In this study, we demonstrate the translational relevance of an ex vivo functional assay that measures the tumor cell killing ability of patient-derived CD8 T and NK cells (referred to as "cytotoxic lymphocytes," or CLs) isolated from the peripheral blood of patients with renal cell carcinoma. Patient-derived PBMCs were isolated before and after nephrectomy from patients with renal cell carcinoma. We compared the efficacy of U.S. Food and Drug Administration (FDA)-approved PD-1/PD-L1 inhibitors (pembrolizumab, nivolumab, atezolizumab) and a newly developed PD-L1 inhibitor (H1A Ab) in eliciting cytotoxic function. CL activity was improved at 3 mo after radical nephrectomy compared with baseline, and it was associated with higher circulating levels of tumor-reactive effector CD8 T cells (CD11ahighCX3CR1+GZMB+). Treatment of PBMCs with FDA-approved PD-1/PD-L1 inhibitors enhanced tumor cell killing activity of CLs, but a differential response was observed at the individual-patient level. H1A demonstrated superior efficacy in promoting CL activity compared with FDA-approved PD-1/PD-L1 inhibitors. PBMC immunophenotyping by mass cytometry revealed enrichment of effector CD8 T and NK cells in H1A-treated PBMCs and immunosuppressive regulatory T cells in atezolizumab-treated samples. Our study lays the ground for future investigation of the therapeutic value of H1A as a next-generation immune checkpoint inhibitor and the potential of measuring CTL activity in PBMCs as a tool to predict individual response to immune checkpoint inhibitors in patients with advanced renal cell carcinoma.

Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size.

The cell-adhesion proteins neuroligin-3 and neuroligin-4X (NLGN3/4X) have well described roles in synapse formation. NLGN3/4X are also expressed highly during neurodevelopment. However, the role these proteins play during this period is unknown. Here we show that NLGN3/4X localized to the leading edge of growth cones where it promoted neuritogenesis in immature human neurons. Super-resolution microscopy revealed that NLGN3/4X clustering induced growth cone enlargement and influenced actin filament organization. Critically, these morphological effects were not induced by autism spectrum disorder (ASD)-associated NLGN3/4X variants. Finally, actin regulators p21-activated kinase 1 and cofilin were found to be activated by NLGN3/4X and involved in mediating the effects of these adhesion proteins on actin filaments, growth cones and neuritogenesis. These data reveal a novel role for NLGN3 and NLGN4X in the development of neuronal architecture, which may be altered in the presence of ASD-associated variants.

Schizophrenia risk proteins ZNF804A and NT5C2 interact in cortical neurons.

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes are amongst the first schizophrenia susceptibility genes to have been identified in large-scale genome-wide association studies. ZNF804A has been implicated in the regulation of neuronal morphology and is required for activity-dependent changes to dendritic spines. Conversely, NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and has been implicated in protein synthesis in human neural progenitor cells. Schizophrenia risk genotype is associated with reduced levels of both NT5C2 and ZNF804A in the developing brain, and a yeast two-hybrid screening suggests that their encoded proteins physically interact. However, it remains unknown whether this interaction also occurs in cortical neurons and whether they could jointly regulate neuronal function. Here, we show that ZNF804A and NT5C2 colocalise and interact in HEK293T cells and that their rodent homologues, ZFP804A and NT5C2, colocalise and form a protein complex in cortical neurons. Knockdown of the Zfp804a or Nt5c2 genes resulted in a redistribution of both proteins, suggesting that both proteins influence the subcellular targeting of each other. The identified interaction between ZNF804A/ZFP804A and NT5C2 suggests a shared biological pathway pertinent to schizophrenia susceptibility within a neuronal cell type thought to be central to the neurobiology of the disorder, providing a better understanding of its genetic landscape.

AGHmatrix: genetic relationship matrices in R.

MOTIVATION: The resemble between relatives computed from pedigree and genomic data is an important resource for geneticists and ecologists, who are interested in understanding how genes influence phenotypic variation, fitness adaptation, and population dynamics. RESULTS: The AGHmatrix software is an R package focused on the construction of pedigree (A matrix) and/or molecular markers (G matrix), with the possibility of building a combined matrix of pedigree corrected by molecular markers (H matrix). Designed to estimate the relationships for any ploidy level, the software also includes auxiliary functions related to filtering molecular markers, and checks pedigree errors in large data sets. After computing the relationship matrices, results from the AGHmatrix can be used in different contexts, including on prediction of (genomic) estimated breeding values and genome-wide association studies. AVAILABILITY AND IMPLEMENTATION: AGHmatrix v2.1.0 is available under GPL-3 license in CRAN at https://cran.r-project.org/web/packages/AGHmatrix/index.html and also in GitHub at https://github.com/rramadeu/AGHmatrix. It has a comprehensive tutorial, and it follows with real data examples.

Human lower leg muscles grow asynchronously.

Muscle volume must increase substantially during childhood growth to generate the power required to propel the growing body. One unresolved but fundamental question about childhood muscle growth is whether muscles grow at equal rates; that is, if muscles grow in synchrony with each other. In this study, we used magnetic resonance imaging (MRI) and advances in artificial intelligence methods (deep learning) for medical image segmentation to investigate whether human lower leg muscles grow in synchrony. Muscle volumes were measured in 10 lower leg muscles in 208 typically developing children (eight infants aged less than 3 months and 200 children aged 5 to 15 years). We tested the hypothesis that human lower leg muscles grow synchronously by investigating whether the volume of individual lower leg muscles, expressed as a proportion of total lower leg muscle volume, remains constant with age. There were substantial age-related changes in the relative volume of most muscles in both boys and girls (p < 0.001). This was most evident between birth and five years of age but was still evident after five years. The medial gastrocnemius and soleus muscles, the largest muscles in infancy, grew faster than other muscles in the first five years. The findings demonstrate that muscles in the human lower leg grow asynchronously. This finding may assist early detection of atypical growth and allow targeted muscle-specific interventions to improve the quality of life, particularly for children with neuromotor conditions such as cerebral palsy.

Molecular breeding of flower load related traits in dioecious autotetraploid Actinidia arguta.

Flowering plants exhibit a wide range of sexual reproduction systems, with the majority being hermaphroditic. However, some plants, such as Actinidia arguta (kiwiberry), have evolved into dioecious species with distinct female and male vines. In this study, we investigated the flower load and growth habits of female kiwiberry genotypes to identify the genetic basis of high yield with low maintenance requirements. Owing to the different selection approaches between female and male genotypes, we further extended our study to male kiwiberry genotypes. By combining both investigations, we present a novel breeding tool for dioecious crops. A population of A. arguta seedlings was phenotyped for flower load traits, in particular the proportion of non-floral shoots, proportion of floral shoots, and average number of flowers per floral shoot. Quantitative trait locus (QTL) mapping was used to analyse the genetic basis of these traits. We identified putative QTLs on chromosome 3 associated with flower-load traits. A pleiotropic effect of the male-specific region of the Y chromosome (MSY) on chromosome 3 affecting flower load-related traits between female and male vines was observed in an A. arguta breeding population. Furthermore, we utilized Genomic Best Linear Unbiased Prediction (GBLUP) to predict breeding values for the quantitative traits by leveraging genomic data. This approach allowed us to identify and select superior genotypes. Our findings contribute to the understanding of flowering and fruiting dynamics in Actinidia species, providing insights for kiwiberry breeding programs aiming to improve yield through the utilization of genomic methods and trait mapping. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01476-7.

Effects of Two Different Low-Volume Resistance Training Programs Applied During the Off-Season Period on the Speed-Power Performance of Elite Youth Soccer Players.

ABSTRACT: Loturco, I, Nunes, RFH, Lampert, RR, Silva, RLP, Hespanhol, JE, Novack, LF, Conde, JHS, Pereira, LA, and McGuigan, MR. Effects of two different low-volume resistance training programs applied during the off-season period on the speed-power performance of elite youth soccer players. . J Strength Cond Res 38(3): 571-576, 2024-The aim of this study was to analyze the changes in the speed-power performance of elite youth soccer players submitted to 2 different low-volume resistance training programs during the off-season period. Twenty under-17 players were randomly allocated to "traditional nonballistic" or "ballistic training" groups. Countermovement jump (CMJ), 20-m sprinting speed, and half-squat (HS) power tests were performed after the final match of the season (pretesting session) and at the beginning of the subsequent season (post-testing session), after 4 weeks of detraining. Between-group differences were assessed using a 2-way ANOVA with repeated measures followed by the Tukey's post hoc test. Performance variations were individually analyzed with the use of the "true changes" calculation. At post-tests, CMJ height and HS power remained unchanged ( p > 0.05) but similar and significant improvements in sprint speed were observed in both groups ( p < 0.05). However, notably, a larger number of players in the ballistic group exhibited "true changes" in HS power (i.e., 55 vs. 33%, compared with the traditional group, respectively). In conclusion, either low-volume ballistic or traditional resistance training schemes were able to increase sprint speed and maintain power output during a short interseason break in youth soccer players. Despite this apparent similarity, at the individual level, ballistic movements were more efficient at improving lower-body power. Practitioners can use the strategies described here to improve the sprint and power performance of soccer players during short periods of soccer-specific training cessation.

Unraveling the Metabolic Alterations Induced by Zika Infection in Prostate Epithelial (PNT1a) and Adenocarcinoma (PC-3) Cell Lines.

The outbreak of Zika virus infection in 2016 led to the identification of its presence in several types of biofluids, including semen. Later discoveries associated Zika infection with sexual transmission and persistent replication in cells of the male reproductive tract. Prostate epithelial and carcinoma cells are favorable to virus replication, with studies pointing to transcriptomics alterations of immune and inflammation genes upon persistence. However, metabolome alterations promoted by the Zika virus in prostate cells are unknown. Given its chronic effects and oncolytic potential, we aim to investigate the metabolic alterations induced by the Zika virus in prostate epithelial (PNT1a) and adenocarcinoma (PC-3) cells using an untargeted metabolomics approach and high-resolution mass spectrometry. PNT1a cells were viable up to 15 days post ZIKV infection, in contrast to its antiproliferative effect in the PC-3 cell lineage. Remarkable alterations in the PNT1a cell metabolism were observed upon infection, especially regarding glycerolipids, fatty acids, and acylcarnitines, which could be related to viral cellular resource exploitation, in addition to the over-time increase in oxidative stress metabolites associated with carcinogenesis. The upregulation of FA20:5 at 5 dpi in PC-3 cells corroborates the antiproliferative effect observed since this metabolite was previously reported to induce PC-3 cell death. Overall, Zika virus promotes extensive lipid alterations on both PNT1a and PC-3 cells, promoting different outcomes based on the cellular metabolic state.

Exogenous succinate impacts mouse brown adipose tissue mitochondrial proteome and potentiates body mass reduction induced by liraglutide.

Obesity is one of the leading noncommunicable diseases in the world. Despite intense efforts to develop strategies to prevent and treat obesity, its prevalence continues to rise worldwide. A recent study has shown that the tricarboxylic acid intermediate succinate increases body energy expenditure by promoting brown adipose tissue thermogenesis through the activation of uncoupling protein-1; this has generated interest surrounding its potential usefulness as an approach to treat obesity. It is currently unknown how succinate impacts brown adipose tissue protein expression, and how exogenous succinate impacts body mass reduction promoted by a drug approved to treat human obesity, the glucagon-like-1 receptor agonist, liraglutide. In the first part of this study, we used bottom-up shotgun proteomics to determine the acute impact of exogenous succinate on the brown adipose tissue. We show that succinate rapidly affects the expression of 177 brown adipose tissue proteins, which are mostly associated with mitochondrial structure and function. In the second part of this study, we performed a short-term preclinical pharmacological intervention, treating diet-induced obese mice with a combination of exogenous succinate and liraglutide. We show that the combination was more efficient than liraglutide alone in promoting body mass reduction, food energy efficiency reduction, food intake reduction, and an increase in body temperature. Using serum metabolomics analysis, we showed that succinate, but not liraglutide, promoted a significant increase in the blood levels of several medium and long-chain fatty acids. In conclusion, exogenous succinate promotes rapid changes in brown adipose tissue mitochondrial proteins, and when used in association with liraglutide, increases body mass reduction.NEW & NOTEWORTHY Exogenous succinate induces major changes in brown adipose tissue protein expression affecting particularly mitochondrial respiration and structural proteins. When given exogenously in drinking water, succinate mitigates body mass gain in a rodent model of diet-induced obesity; in addition, when given in association with the glucagon-like peptide-1 receptor agonist, liraglutide, succinate increases body mass reduction promoted by liraglutide alone.

Tissue-resident glial cells associate with tumoral vasculature and promote cancer progression.

Cancer cells are embedded within the tissue and interact dynamically with its components during cancer progression. Understanding the contribution of cellular components within the tumor microenvironment is crucial for the success of therapeutic applications. Here, we reveal the presence of perivascular GFAP+/Plp1+ cells within the tumor microenvironment. Using in vivo inducible Cre/loxP mediated systems, we demonstrated that these cells derive from tissue-resident Schwann cells. Genetic ablation of endogenous Schwann cells slowed down tumor growth and angiogenesis. Schwann cell-specific depletion also induced a boost in the immune surveillance by increasing tumor-infiltrating anti-tumor lymphocytes, while reducing immune-suppressor cells. In humans, a retrospective in silico analysis of tumor biopsies revealed that increased expression of Schwann cell-related genes within melanoma was associated with improved survival. Collectively, our study suggests that Schwann cells regulate tumor progression, indicating that manipulation of Schwann cells may provide a valuable tool to improve cancer patients' outcomes.

Sex differences in weight gain, blood pressure control, and responses to melanocortin-4 receptor antagonism in offspring from lean and obese parents.

We examined potential sex differences in appetite and blood pressure (BP) responses to melanocortin-4 receptor (MC4R) blockade in offspring from lean and obese parents. Offspring from normal (N) diet-fed parents were fed N (NN) or high-fat (H) diets (NH) from weaning until adulthood. Offspring from obese H diet-fed parents were also fed N (HN) or H diets (HH). Adult male and female offspring were implanted with BP telemetry probes and intracerebroventricular cannulas to infuse MC4R antagonist or vehicle. Infusion of the MC4R antagonist SHU-9119 (1 nmol/h) for 7 days caused larger increases in calorie intake and body weight in obese compared with lean offspring. In male offspring, HH and HN groups exhibited higher baseline BP compared with NN and NH, and HH showed a greater reduction in BP during SHU-9119 infusion. In female offspring, HH also showed higher baseline BP and greater reduction in BP during MC4R blockade. SHU-9119 reduced heart rate in all groups, but reductions were more pronounced in offspring from lean parents. Combined α and ß-adrenergic blockade reduced BP more in male HH offspring compared with NN controls. Losartan reduced BP more in male NH, HN, and HH offspring compared with NN controls. Losartan and α- and ß-adrenergic blockade reduced BP similarly in all female groups. These results suggest that endogenous MC4R activity contributes to elevated BP in obese offspring from obese parents. Our findings also indicate important sex differences in the mechanisms of BP control in male and female offspring of obese parents.

Nitrogen enrichment and foliar fungal pathogens affect the mechanisms of multispecies plant coexistence.

Changes in resources (e.g. nitrogen) and enemies (e.g. foliar pathogens) are key drivers of plant diversity and composition. However, their effects have not been connected to the niche and fitness differences that determine multispecies coexistence. Here, we combined a structuralist theoretical approach with a detailed grassland experiment factorially applying nitrogen addition and foliar fungal pathogen suppression to evaluate the joint effect of nitrogen and pathogens on niche and fitness differences, across a gradient from two to six interacting species. Nitrogen addition and pathogen suppression modified species interaction strengths and intrinsic growth rates, leading to reduced multispecies fitness differences. However, contrary to expected, we also observed that they promote stabilising niche differences. Although these modifications did not substantially alter species richness, they predicted major changes in community composition. Indirect interactions between species explained these community changes in smaller assemblages (three and four species) but lost importance in favour of direct pairwise interactions when more species were involved (five and six). Altogether, our work shows that explicitly considering the number of interacting species is critical for better understanding the direct and indirect processes by which nitrogen enrichment and pathogen communities shape coexistence in grasslands.

Acute IL-6 exposure triggers canonical IL6Ra signaling in hiPSC microglia, but not neural progenitor cells.

BACKGROUND: Prenatal exposure to elevated interleukin (IL)-6 levels is associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin, such as schizophrenia (SZ), autism spectrum condition (ASC) and bipolar disorder (BD). Although rodent models provide causal evidence for this association, we lack a detailed understanding of the cellular and molecular mechanisms in human model systems. To close this gap, we characterized the response of human induced pluripotent stem cell (hiPSC-)derived microglia-like cells (MGL) and neural progenitor cells (NPCs) to IL-6 in monoculture. RESULTS: We observed that human forebrain NPCs did not respond to acute IL-6 exposure in monoculture at both protein and transcript levels due to the absence of IL6R expression and soluble (s)IL6Ra secretion. By contrast, acute IL-6 exposure resulted in STAT3 phosphorylation and increased IL6, JMJD3 and IL10 expression in MGL, confirming activation of canonical IL6Ra signaling. Bulk RNAseq identified 156 up-regulated genes (FDR < 0.05) in MGL following acute IL-6 exposure, including IRF8, REL, HSPA1A/B and OXTR, which significantly overlapped with an up-regulated gene set from human post-mortem brain tissue from individuals with schizophrenia. Acute IL-6 stimulation significantly increased MGL motility, consistent with gene ontology pathways highlighted from the RNAseq data and replicating rodent model indications that IRF8 regulates microglial motility. Finally, IL-6 induces MGLs to secrete CCL1, CXCL1, MIP-1α/ß, IL-8, IL-13, IL-16, IL-18, MIF and Serpin-E1 after 3 h and 24 h. CONCLUSION: Our data provide evidence for cell specific effects of acute IL-6 exposure in a human model system, ultimately suggesting that microglia-NPC co-culture models are required to study how IL-6 influences human cortical neural progenitor cell development in vitro.

Probabilistic survival modeling in health research: an assessment using cohort data from hospitalized patients with COVID-19 in a Latin American city.

Probabilistic survival methods have been used in health research to analyze risk factors and adverse health outcomes associated with COVID-19. The aim of this study was to employ a probabilistic model selected among three distributions (exponential, Weibull, and lognormal) to investigate the time from hospitalization to death and determine the mortality risks among hospitalized patients with COVID-19. A retrospective cohort study was conducted for patients hospitalized due to COVID-19 within 30 days in Londrina, Brazil, between January 2021 and February 2022, registered in the database for severe acute respiratory infections (SIVEP-Gripe). Graphical and Akaike Information Criterion (AIC) methods were used to compare the efficiency of the three probabilistic models. The results from the final model were presented as hazard and event time ratios. Our study comprised of 7,684 individuals, with an overall case fatality rate of 32.78%. Data suggested that older age, male sex, severe comorbidity score, intensive care unit admission, and invasive ventilation significantly increased risks for in-hospital mortality. Our study highlights the conditions that confer higher risks for adverse clinical outcomes attributed to COVID-19. The step-by-step process for selecting appropriate probabilistic models may be extended to other investigations in health research to provide more reliable evidence on this topic.

Design and methodology of the SARCopenia trajectories and associations with adverse clinical outcomes in patients on HemoDialysis: the SARC-HD study.

BACKGROUND: Sarcopenia has been associated with adverse outcomes in patients with chronic kidney disease (CKD), particularly in those undergoing hemodialysis (HD). However, the trajectories across sarcopenia stages, their determinants, and associations with adverse clinical outcomes have yet to be comprehensively examined. METHODS: The SARC-HD is a multicenter, observational prospective cohort study designed to comprehensively investigate sarcopenia in patients on HD. Eligibility criteria include adult patients undergoing HD for ≥ 3 months. The primary objective is to investigate the trajectories of sarcopenia stages and their potential determinants. Secondary objectives include evaluating the association between sarcopenia and adverse clinical outcomes (i.e., falls, hospitalization, and mortality). Sarcopenia risk will be assessed by the SARC-F and SARC-CalF questionnaire. Sarcopenia traits (i.e., low muscle strength, low muscle mass, and low physical performance) will be defined according to the revised European Working Group on Sarcopenia in Older People and will be assessed at baseline and after 12 follow-up months. Patients will be followed-up at 3 monthly intervals for adverse clinical outcomes during 24 months. DISCUSSION: Collectively, we expect to provide relevant clinical findings for healthcare professionals from nephrology on the association between sarcopenia screening tools (i.e., SARC-F and SARC-CalF) with objective sarcopenia measurements, as well as to investigate predictors of trajectories across sarcopenia stages, and the impact of sarcopenia on adverse clinical outcomes. Hence, our ambition is that the data acquired from SARC-HD study will provide novel and valuable evidence to support an adequate screening and management of sarcopenia in patients on HD.

Redox Status in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy characterized by the progressive loss of vision. It is a rare disease. Despite being a genetic disease, its progression is influenced by oxidative damage and chemokines and cytokines released by the activated immune cells (e.g., macrophages or microglia). The role of oxidative stress is very important in the retina. Rods are the main consumers of oxygen (O2), so they are constantly exposed to oxidative stress and lipid peroxidation. According to the oxidative hypothesis, after rod death in the early stages of the disease, O2 would accumulate in large quantities in the retina, producing hyperoxia and favoring the accumulation of reactive oxygen species and reactive nitrogen species that would cause oxidative damage to lipids, proteins, and DNA, exacerbating the process of retinal degeneration. Evidence shows alterations in the antioxidant-oxidant state in patients and in animal models of RP. In recent years, therapeutic approaches aimed at reducing oxidative stress have emerged as useful therapies to slow down the progression of RP. We focus this review on oxidative stress and its relationship with the progression of RP.

To flat or not to flat? Exploring the impact of flat-side design on rotary instruments using a comprehensive multimethod investigation.

AIM: To assess the influence of a flat-side design on the geometry, metallurgy, mechanical performance and shaping ability of a novel nickel-titanium rotary instrument. METHODOLOGY: Sixty-five new 25-mm flat-side rotary instruments (size 25, taper 0.04) and their nonflat-side prototypes (n = 65) were assessed for major deformations and examined regarding macroscopic and microscopic design, determination of nickel and titanium elements ratio, measurement of phase transformation temperature and evaluation of mechanical performance parameters including time/cycles to fracture, maximum torque, angle of rotation, maximum bending and buckling strengths and cutting ability. Additionally, unprepared canal areas, volume of hard tissue debris and percentage reduction of dentine thickness were calculated for each tested instrument after preparing mesial canals of mandibular molars (n = 12), using micro-CT imaging. Statistical analyses were performed using the U-Mann-Whitney test and independent Student t-test (α = 5%). RESULTS: The number of spirals (n = 8) and blade direction (clockwise) were similar between both flat and nonflat instruments, whilst the helical angles were equivalent (⁓25°). Flat-instruments showed inconsistencies in the homogeneity of the gold colour on the flat-side surface, blade discontinuity, and incomplete and variable S-shaped cross-sections. The titanium-to-nickel ratios were equivalent, but significant differences in the R-phase finish and austenitic start phase transformation temperatures were observed between the flat and nonflat-side instruments. The flat-side instruments demonstrated superior cutting ability compared to the nonflat instruments, as well as, significantly lower values for time to fracture, rotation to fracture and maximum torque to fracture (p < .001). No statistical difference was observed between tested instruments regarding angle of rotation (p = .437), maximum bending (p = .152) and buckling load (p = .411). Preparation protocols using flat and nonflat instruments did not show any statistically significant differences (p > .05). All flat-side instruments exhibited deformation after shaping procedures. CONCLUSIONS: The flat-side instrument showcased enhanced cutting ability compared to its nonflat counterpart. However, it exhibited inferior performance in terms of time, rotation and maximum torque to fracture, along with distinct phase transformation temperatures. No differences were observed in the titanium-to-nickel ratios, angle of rotation, maximum bending, buckling load, preparation time, percentage of untouched canal walls, volume of hard tissue debris and percentage reduction of dentine thickness.