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Mitochondrial activity directs neuronal differentiation dynamics during brain development. In this context, the long-established metabolic coupling of mitochondria and the eukaryotic host falls short of a satisfactory mechanistic explanation, hinting at an undisclosed facet of mitochondrial function. Here, we reveal an RNA-based inter-organellar communication mode that complements metabolic coupling of host-mitochondria and underpins neuronal differentiation. We show that within minutes of exposure to differentiation cues and activation of the electron transport chain, the mitochondrial outer membrane transiently fuses with the nuclear membrane of neural progenitors, leading to efflux of nuclear-encoded RNAs (neRNA) into the positively charged mitochondrial intermembrane space. Subsequent degradation of mitochondrial neRNAs by Polynucleotide phosphorylase 1 (PNPase) located in the intermembrane space curbs the transcriptomic memory of progenitor cells. Further, acquisition of neRNA by mitochondria leads to a collapse of proton motive force, suppression of ATP production, and a resultant amplification of autophagic flux that attenuates proteomic memory. Collectively, these events force the progenitor cells towards a "tipping point" characterised by emergence of a competing neuronal differentiation program. It appears that neuronal differentiation is a consequence of reprogrammed coupling of metabolomic and transcriptomic landscapes of progenitor cells, with mitochondria emerging as key "reprogrammers" that operate by acquiring and metabolising neRNAs. However, the documented role of mitochondria as "reprogrammers" of differentiation remains to be validated in other neuronal lineages and in vivo., (© 2024. The Author(s).)

Objective: Scrotal hyperthermia poses a significant threat to spermatogenesis and fertility in mammalian species. This study investigated the effects of vitamin B12 and vitamin C on spermatogenesis in adult male mice subjected to long-term scrotal hyperthermia. The rationale is based on the sensitivity of germ cells and epididymal sperm to increased scrotal temperatures. While various factors, both internal and external, can raise the testicular temperature, this study focused on the potential therapeutic roles of vitamins B12 and C., Methods: After inducing scrotal hyperthermia in mice, vitamin B12 and vitamin C were administered for 35 days. We assessed sperm parameters, serum testosterone levels, stereological parameters, the percentage of apoptotic cells, reactive oxygen species (ROS) levels, and glutathione (GSH) levels. Additionally, real-time polymerase chain reaction was used to analyze the expression of the c-kit, stimulated by retinoic acid gene 8 (Stra8), and proliferating cell nuclear antigen (Pcna) genes., Results: Vitamin C was more effective than vitamin B12 in improving sperm parameters and enhancing stereological parameters. The study showed a significant decrease in apoptotic cells and a beneficial modulation of ROS and GSH levels following vitamin administration. Moreover, both vitamins positively affected the expression levels of the c-kit, Stra8, and Pcna genes., Conclusion: This research deepens our understanding of the combined impact of vitamins B12 and C in mitigating the effects of scrotal hyperthermia, providing insights into potential therapeutic strategies for heat stress-related infertility. The findings highlight the importance of considering vitamin supplementation as a practical approach to counter the detrimental effects of elevated scrotal temperatures on male reproductive health.

Metazoan signalling pathways can be rewired to dampen or amplify the rate of events, such as those that occur in development and aging. Given that a linear network topology restricts the capacity to rewire signalling pathways, such scalability of the pace of biological events suggests the existence of programmable non-linear elements in the underlying signalling pathways. Here, we review the network topology of key signalling pathways with a focus on redox-sensitive proteins, including PTEN and Ras GTPase, that reshape the connectivity profile of signalling pathways in response to an altered redox state. While this network-level impact of redox is achieved by the modulation of individual redox-sensitive proteins, it is the population by these proteins of critical nodes in a network topology of signal transduction pathways that amplifies the impact of redox-mediated reprogramming. We propose that redox-mediated rewiring is essential to regulate the rate of transmission of biological signals, giving rise to a programmable cellular clock that orchestrates the pace of biological phenomena such as development and aging. We further review the evidence that an aberrant redox-mediated modulation of output of the cellular clock contributes to the emergence of pathological conditions affecting the human brain.

During the early Cambrian period metazoan life forms diverged at an accelerated rate to occupy multiple ecological niches on earth. A variety of explanations have been proposed to address this major evolutionary phenomenon termed the "Cambrian explosion." While most hypotheses address environmental, developmental, and ecological factors that facilitated evolutionary innovations, the biological basis for accelerated emergence of species diversity in the Cambrian period remains largely conjectural. Herein, we posit that morphogenesis by self-organization enables the uncoupling of genomic mutational landscape from phenotypic diversification. Evidence is provided for a two-tiered interpretation of genomic changes in metazoan animals wherein mutations not only impact upon function of individual cells, but also alter the self-organization outcome during developmental morphogenesis. We provide evidence that the morphological impacts of mutations on self-organization could remain repressed if associated with an unmet negative energetic cost. We posit that accelerated morphological diversification in transition to the Cambrian period has occurred by emergence of dormant (i.e., reserved) morphological novelties whose molecular underpinnings were seeded in the Precambrian period., (© 2022 The Authors. BioEssays published by Wiley Periodicals LLC.)

We assessed the impact of photobiomodulation (PBM) plus adipose-derived stem cells (ASCs) during the anabolic and catabolic stages of bone healing in a rat model of a critical size femoral defect (CSFD) that was filled with a decellularized bone matrix (DBM). Stereological analysis and gene expression levels of bone morphogenetic protein 4 (BMP4), Runt-related transcription factor 2 (RUNX2), and stromal cell-derived factor 1 (SDF1) were determined. There were six groups of rats. Group 1 was the untreated control or DBM. Study groups 2-6 were treated as follows: ASC (ASC transplanted into DBM, then implanted in the CSFD); PBM (CSFD treated with PBM); irradiated ASC (iASC) (ASCs preconditioned with PBM, then transplanted into DBM, and implanted in the CSFD); ASC + PBM (ASCs transplanted into DBM, then implanted in the CSFD, followed by PBM administration); and iASC + PBM (the same as iASC, except CSFDs were exposed to PBM). At the anabolic step, all treatment groups had significantly increased trabecular bone volume (TBV) (24.22%) and osteoblasts (83.2%) compared to the control group (all, p = .000). However, TBV in group iASC + PBM groups were superior to the other groups (97.48% for osteoblast and 58.8% for trabecular bone volume) (all, p = .000). The numbers of osteocytes in ASC (78.2%) and iASC + PBM (30%) groups were remarkably higher compared to group control (both, p = .000). There were significantly higher SDF (1.5-fold), RUNX2 (1.3-fold), and BMP4 (1.9-fold) mRNA levels in the iASC + PBM group compared to the control and some of the treatment groups. At the catabolic step of bone healing, TBV increased significantly in PBM (30.77%), ASC + PBM (32.27%), and iASC + PBM (35.93%) groups compared to the control group (all, p = .000). There were significantly more osteoblasts and osteocytes in ASC (71.7%, 62.02%) (p = .002, p = .000); PBM (82.54%, 156%), iASC (179%, 23%), and ASC + PBM (108%, 110%) (all, p = .000), and iASC + PBM (79%, 100.6%) (p = .001, p = .000) groups compared to control group. ASC preconditioned with PBM in vitro plus PBM in vivo significantly increased stereological parameters and SDF1, RUNX2, and BMP4 mRNA expressions during bone healing in a CSFD model in rats., (© 2022. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)

Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notch on to Notch off state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notch off state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in β-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notch off state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notch on state via the ankyrin domain and is eventually switched off in a temperature-dependent manner. Video abstract., (© 2022. The Author(s).)

Accumulating evidence suggests that mural pericytes, apart from stabilizing the associated microvessels, play additional roles in regeneration of local cellular elements. Herein, the mechanistic basis for such diverse and at times contradictory roles adopted by pericytes in the brain is reviewed. Core concepts of an emerging model are discussed wherein mural pericytes reside in a metastable "archival state" that conceals a neural progenitor identity. Upon angiogenic remodeling, a selected subpopulation of pericytes reclaim the progenitor state during transdifferentiation and contribute to neural regeneration. The genomic basis for neural transdifferentiation of pericytes is reviewed with reference to the extant literature., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Transdifferentiation describes transformation in vivo of specialized cells from one lineage into another. While there is extensive literature on forced induction of lineage reprogramming in vitro, endogenous mechanisms that govern transdifferentiation remain largely unknown. The observation that human microvascular pericytes transdifferentiate into neurons provided an opportunity to explore the endogenous molecular basis for lineage reprogramming., Results: We show that abrupt destabilization of the higher-order chromatin topology that chaperones lineage memory of pericytes is driven by transient global transcriptional arrest. This leads within minutes to localized decompression of the repressed competing higher-order chromatin topology and expression of pro-neural genes. Transition to neural lineage is completed by probabilistic induction of R-loops in key myogenic loci upon re-initiation of RNA polymerase activity, leading to depletion of the myogenic transcriptome and emergence of the neurogenic transcriptome., Conclusions: These findings suggest that the global transcriptional landscape not only shapes the functional cellular identity of pericytes, but also stabilizes lineage memory by silencing the competing neural program within a repressed chromatin state., (© 2021. The Author(s).)

A core imprint of metazoan life is that perturbations of cell cycle are offset by compensatory changes in successive cellular generations. This trait enhances robustness of multicellular growth and requires transmission of signaling cues within a cell lineage. Notably, the identity and mode of activity of transgenerational signals remain largely unknown. Here we report the discovery of a natural antisense transcript encoded in exon 25 of notch-1 locus (nAS25) by which mother cells control the fate of notch-1 transcript in daughter cells to buffer against perturbations of cell cycle. The antisense transcript is transcribed at G1 phase of cell cycle from a bi-directional E2F1-dependent promoter in the mother cell where the titer of nAS25 is calibrated to the length of G1. Transmission of the antisense transcript from mother to daughter cells stabilizes notch-1 sense transcript in G0 phase of daughter cells by masking it from RNA editing and resultant nonsense-mediated degradation. In consequence, nAS25-mediated amplification of notch-1 signaling reprograms G1 phase in daughter cells to compensate for the altered dynamics of the mother cell. The function of nAS25/notch-1 in integrating G1 phase history of the mother cell into that of daughter cells is compatible with the predicted activity of a molecular oscillator, slower than cyclins, that coordinates cell cycle within cell lineage., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tramadol is a centrally acting synthetic opioid analgesic and SNRI (serotonin/norepinephrine reuptake-inhibitor) that structurally resembles codeine and morphine. Given the tramadol neurotoxic effect and the body of studies on the effect of tramadol on the cerebellum, this study aims to provide deeper insights into molecular and histological alterations in the cerebellar cortex related to tramadol administration. In this study, twenty-four adult male albino rats were randomly and equally divided into two groups: control and tramadol groups. The tramadol group received 50 mg/kg of tramadol daily for 3 weeks via oral gavage. The functional and structural change of the cerebellum under chronic exposure of tramadol were measured. Our data revealed that treating rats with tramadol not only lead to cerebellum atrophy but also resulted in the actuation of microgliosis, neuroinflammatoin, and apoptotic biomarkers. Our results illustrated a significant drop in VEGF (vascular endothelial growth factor) level in the tramadol group. Additionally, tramadol impaired motor coordination and neuromuscular activity. We also identified several signaling cascades chiefly related to neurodegenerative disease and energy metabolism that considerably deregulated in the cerebellum of tramadol-treated rats. Overall, the outcomes of this study suggest that tramadol administration has a neurodegeneration effect on the cerebellar cortex via several pathways consisting of microgliosis, apoptosis, necroptosis, and neuroinflammatoin., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Metabolic support was long considered to be the only developmental function of hematopoiesis, a view that is gradually changing. Here, we disclose a mechanism triggered during neurulation that programs brain development by donation of sacrificial yolk sac erythroblasts to neuroepithelial cells. At embryonic day (E) 8.5, neuroepithelial cells transiently integrate with the endothelium of yolk sac blood vessels and cannibalize intravascular erythroblasts as transient heme-rich endosymbionts. This cannibalistic behavior instructs precocious neuronal differentiation of neuroepithelial cells in the proximity of blood vessels. By experiments in vitro, we show that access to erythroblastic heme accelerates the pace of neurogenesis by induction of a truncated neurogenic differentiation program from a poised state. Mechanistically, the poised state is invoked by activation of the mitochondrial electron transport chain that leads to amplified production of reactive oxygen species in addition to omnipresent guanosine triphosphate (GTP) with consequential upregulation of pro-differentiation β-catenin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Methamphetamine (METH) is a high addictive psychostimulant drug which triggers brain atrophy via neuronal degeneration. Striatum is the main part of the brain that is regarded as a key target for drug-induced damages. MiRNAs as small regulatory molecules at the post-transcriptional level play a major role in biological pathways. In this study, initially we performed behavioral assessment in METH-treated rats. Then, we examined striatal volume and dendritic length, and also the levels of tyrosine hydroxylase (TH), caspase-3 and glial fibrillary acidic protein (GFAP) were immunohistochemically assessed. Moreover, we investigated miRNA expression profiling using high-throughput small RNA-seq technology. Based on our data, METH provoked declined motor coordination, decreases in striatal volume and dendritic length along with over-activation of astrogliosis. In addition, METH treatment down-regulated TH level while it induced up-regulation of caspase-3 in the striatal region. Furthermore, according to miR-seq analysis, we found 167 deregulated miRNAs in the striatum upon METH treatment, that among them rno-let-7b-5p, rno-miR-485-5p, rno-miR-326-3p, rno-miR-34a-5p, rno-miR-3068-5p showed high miRNA-target gene interaction. Pathway analysis revealed that miRNAs and their target genes may be involved in cell apoptosis, growth, differentiation as well as synaptic plasticity associated pathways. Altogether, we can conclude that METH noticeably elicited neuro-degeneration in the dorsal striatum., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The genomic platform that informs evolution of microRNA cascades remains unknown. Here we capitalised on the recent evolutionary trajectory of hominin-specific miRNA-4673, encoded in intron 4 of notch-1, to uncover the identity of one such precursor genomic element and the selective forces acting upon it. The miRNA targets genes that regulate Wnt/β-catenin signalling cascade. Primary sequence of the microRNA and its target region in Wnt modulating genes evolved from homologous signatures mapped to homotypic cis-clusters recognised by TCF3/4 and TFAP2A/B/C families. Integration of homologous TFAP2A/B/C cis-clusters (short range inhibitor of β-catenin) into the transcriptional landscape of Wnt cascade genes can reduce noise in gene expression. Probabilistic adoption of miRNA secondary structure by one such cis-signature in notch-1 reflected selection for superhelical curvature symmetry of precursor DNA to localise a nucleosome that overlapped the latter cis-cluster. By replicating the cis-cluster signature, non-random interactions of the miRNA with key Wnt modulator genes expanded the transcriptional noise buffering capacity via a coherent feed-forward loop mechanism. In consequence, an autonomous transcriptional noise dampener (the cis-cluster/nucleosome) evolved into a post-transcriptional one (the miRNA). The findings suggest a latent potential for remodelling of transcriptional landscape by miRNAs that capitalise on non-random distribution of genomic cis-signatures.

The current study evaluates potential applications of Sertoli cell (SC)-conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28-30 g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10 μl), (3) busulfan (40 mg/kg single dose) and (4) busulfan/CM (10 μl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10 μl of CM was injected over 3-5 min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8 weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell-conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.

Notch signalling pathway is central to development of metazoans. The pathway codes a binary fate switch. Upon activation, downstream signals contribute to resolution of fate dichotomies such as proliferation/differentiation or sub-lineage differentiation outcome. There is, however, an interesting paradox in the Notch signalling pathway. Despite remarkable predictability of fate outcomes instructed by the Notch pathway, the associated transcriptome is versatile and plastic. This inconsistency suggests the presence of an interface that compiles input from the plastic transcriptome of the Notch pathway but communicates only a binary output in biological decisions. Herein, we address the interface that determines fate outcomes. We provide an alternative hypothesis for the Notch pathway as a biological master switch that operates by induction of genetic noise and bistability in order to facilitate resolution of dichotomous fate outcomes in development.

Molecular noise refers to fluctuations of biological signals that facilitate phenotypic heterogeneity in a population. While endogenous mechanisms exist to limit genetic noise in biological systems, such restrictions are sometimes removed to propel phenotypic variability as an adaptive strategy. Herein, we review evidence for the potential role of β-catenin in restricting gene expression noise by transcriptional and post-transcriptional mechanisms. We discuss mechanisms that restrict intrinsic noise subsequent to nuclear mobilization of β-catenin. Nuclear β-catenin promotes initiation of transcription but buffers against the resultant noise by restraining transcription elongation. Acceleration of cell cycle, mediated via Wnt/β-catenin downstream signals, further diminishes intrinsic noise by curtailing the efficiency of protein synthesis. Extrinsic noise, on the other hand, is restricted by β-catenin-mediated regulation of major cellular stress pathways., (Copyright © 2019 Rezaei-Lotfi, Hunter and Farahani.)

Self-organization is central to the morphogenesis of multicellular organisms. However, the molecular platform that coordinates the robust emergence of complex morphological patterns from local interactions between cells remains unresolved. Here we demonstrate that neural self- organization is driven by coupled cycling of progenitor cells. In a coupled cycling mode, intercellular contacts relay extrinsic cues to override the intrinsic cycling rhythm of an individual cell and synchronize the population. The stringency of coupling and hence the synchronicity of the population is programmed by recruitment of a key coupler, β-catenin, into junctional complexes. As such, multicellular self-organization is driven by the same basic mathematical principle that governs synchronized behavior of macro-scale biological systems as diverse as the synchronized chirping of crickets, flashing of fireflies and schooling of fish; that is synchronization by coupling. It is proposed that coupled cycling foreshadows a fundamental adaptive change that facilitated evolution and diversification of multicellular life forms.

Objective: Polycystic ovary syndrome (PCOS) is a common and multifactorial disease associated with female factor infertility. Ulmus minor bark (UMB) is one of the medicinal plants used in Persian folklore as a fertility enhancer. In the current study, we aimed to elucidate the effect of UMB hydro-alcoholic extract on histological parameters and testosterone condition in an experimental model of PCOS rats., Methods: Thirty female rats were randomly divided into five groups: (1) control, (2) vehicle, (3) PCOS/50 mg [6 mg/kg dehydroepiandrosterone (DHEA) + 50 mg/kg UMB hydro-alcoholic extract], (4) PCOS/150 mg (6 mg/kg DHEA + 150 mg/kg UMB hydro-alcoholic extract), and (5) PCOS (6 mg/kg DHEA). All interventions were performed for 21 days. Afterwards, stereological analysis was done for determination of ovarian volume and follicle number. The serum level of testosterone was measured by ELISA kit., Results: UMB hydro-alcoholic extract improved the total number of the corpus luteum in the treatment groups when compared to the PCOS group (p<0.05). PCOS/150 mg and PCOS/50 mg groups showed significantly lower total number of the primordial, primary, and secondary follicles as well as testosterone level compared to the PCOS group (p<0.05). The total number of antral follicles and volume of ovary did not differ significantly between groups., Conclusion: UMB extract may be an effective and good alternative in improving PCOS histo-logical and testosterone disturbances although further studies are warranted to confirm the safety of UMB plant in human.

Resistance of neoplastic cells to therapy is considered a key challenge in the treatment of cancer. Emergence of resistance is commonly attributed to the gradual mutational evolution of neoplastic cells. However, accumulating evidence suggests that exogenous stressors could significantly accelerate the emergence of resistant clones during the course of treatment. Herein, we review molecular mechanisms that regulate the evolution of resistance in a tumor with particular emphasis on the role of cell cycle.

Consistent adult neurogenic activity in humans is observed in specific niches within the central nervous system. However, the notion of an adult neurogenic niche is challenged by accumulating evidence for ectopic neurogenic activity in other cerebral locations. Herein we interface precision of ultrastructural resolution and anatomical simplicity of accessible human dental pulp neurogenic zone to address this conflict. We disclose a basal level of adult neurogenic activity characterized by glial invasion of terminal microvasculature followed by release of individual platelet-derived growth factor receptor-β mural pericytes and subsequent reprogramming into NeuN + local interneurons. Concomitant angiogenesis, a signature of adult neurogenic niches, accelerates the rate of neurogenesis by amplifying release and proliferation of the mural pericyte population by ≈10-fold. Subsequent in vitro and in vivo experiments confirmed gliogenic and neurogenic capacities of human neural pericytes. Findings foreshadow the bimodal nature of the glio-vascular assembly where pericytes, under instruction from glial cells, can stabilize the quiescent microvasculature or enrich local neuronal microcircuits upon differentiation., (© 2018 Wiley Periodicals, Inc.)

We have an interest in the cellular response to mechanical stimuli, and here describe an in-vitro method to examine the response of cells cultured in a three-dimensional matrix to mechanical compressive and tensile stress. Synthetic aliphatic polyester scaffolds coated with 45S5 bioactive glass were seeded with human dental follicular cells (HDFC), and attached to well inserts and magnetic endplates in six well palates. Scaffolds were subjected to either cyclic 10% tensile deformation, or 8% compression, at 1 Hz and 2 Hz respectively for 6, 24 or 48 h, by uniaxial motion of magnetically-coupled endplates. It was possible to isolate high quality mRNA from cells in these scaffolds, as demonstrated by high RNA integrity numbers scores, and ability to perform meaningful cRNA microarray analysis, in which 669 and 727 genes were consistently upregulated, and 662 and 518 genes down regulated at all times studied under tensile and compressive loading conditions respectively. MetaCore analysis revealed the most regulated gene ontogenies under both loading conditions to be for: cytoskeletal remodelling; cell adhesion-chemokines and adhesion; cytoskeleton remodelling-TGF WNT and cytoskeletal remodelling pathways. We believe the method here described will be of value for analysis of the cellular response to cyclic loading., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: In patients with breast cancer, HER2 gene expression is of a great importance in reacting to Herceptin treatment. To evaluate this event, immunohistochemistry (IHC) has been done routinely on the basis of scoring it and so the patients were divided into 4 groups. Lately, as there have been disagreements about how to treat score 2 patients, chromogenic in situ hybridization (CISH) and florescence in situ hybridization (FISH) are introduced. Since CISH method is more convenient than FISH for gene amplification study, FISH has been substituted by CISH., Aim: The current study is conducted in order to investigate whether using CISH is a better method comparison to IHC method for determines HER2 expression in patients with breast cancer in., Methods: In this cross-sectional descriptive analytical study, information of 44 female patients with invasive ductal breast cancer were gathered from Imam Reza and Omid Hospital in Mashhad. IHC staining was done for all patients in order to determine the level of HER2 expression, and after scoring them into 4 groups of 0, +1, +2 and +3, CISH staining was carried out for all 4 groups. At the end, results from both methods were statistically evaluated using SPSS software V.22.0., Results: The average age of patients was 50.2 with the standard deviation of 10.96. Using IHC method was observed that 2.6% (1 patient), 26.3% (10 patients), 65.8% (25 patients) and 5.3% (2 patients) percentage of patients had scores of 0, +1, +2 and +3. On the other hand, CISH method showed 36 patients (90%) with no amplifications and 4 (10%) with sever amplifications. In a comparative study using Fisher's exact test (p = 0.000), we found a significant relation between IHC method and CISH method indicating that all patients showing severe amplifications in CISH method, owned scores of +2 and +3 in IHC method., Conclusion: According to the present study and comparing the results with similar previous studies, it can be concluded that CISH method works highly effective in determining HER2 expression level in patients with breast cancer. This method is also able to determine the status of patients with score +2 in IHC for their treatment with herceptin.

Therapeutic resistance of neoplasms is mainly attributed to gradual evolution of mutational profile 1 . Here, we demonstrate a microRNA-mediated mechanism that effectively improves fitness of SKBR3 mammary carcinoma cells by cytoplasmic reprogramming. The reprogramming is triggered by endogenous miR4673 transcribed from notch-1 locus. The miRNA downregulates cdk-18, a cyclin-dependent kinase that regulates M-G1 transition in cycling cells 2,3 . Suppression of cdk-18 triggers mitophagy and autophagy. Due to high autophagic flux, oestrogen receptor-1 + /progesterone receptor + /p53 + (Esr1 + /Pr + /p53 + ) SKBR3 cells are coerced into an Esr1 - /Pr low /p53 - profile. Increased mitophagy in combination with proteasomal degradation of p53 transiently arrests the cycling cells at G0 and enhances radio-resistance of the SKBR3 population. These findings highlight the impact on cancer therapy of non-encoded neoplastic resistance, arising as a consequence of miRNA-mediated autophagic reprogramming that uncouples phenotype and genotype.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation., (Copyright © 2018. Published by Elsevier Inc.)

Cerebellar ataxia (CA) is regarded as a miscellaneous cluster of brain disorders related to the cerebellum. Resveratrol is a naturally occurring polyphenolic compound. Previous reports suggest that resveratrol confers neuroprotection in various animal models of brain damage. Indeed, we considered it invaluable to investigate whether a treatment with resveratrol has a therapeutic role against CA induced by 3-acetylpyridine (3-AP) in rats. In addition, no investigation has examined neuroprotective effect of resveratrol in rat model of CA. Initially, 3-AP administration generated CA rat models followed by intraperitoneal injection with resveratrol. Then, motor performance and muscle electromyography (EMG) activity were assessed. Moreover, the anti-apoptotic role of resveratrol in CA and its relationship to protection of Purkinje cells were explored. According to what we have found, resveratrol administration improved the muscle activity and movement coordination in 3-AP-lesioned rats. Also under resveratrol treatment, the total number of the Purkinje neurons increased whereas a reduction in apoptotic bodies was observed. In conclusion, post-treatment with resveratrol evidently ameliorated motor performance as well as muscle activity accompanied by a protection of Purkinje cells in ataxic rats.

Busulfan is an anticancer drug, which causes the apoptosis germ cells and azoospermia in humans and animals. Abnormal morphology of spermatozoa related to the male infertility. The sperm morphology is evaluation of sperm size, shape and appearance characteristics should be assessed by carefully observing a stained sperm sample under the microscope. Evaluation of sperm morphology has been considered as one of the most important factors for a successful fertilization and determining sperm quality. The mice were assigned to tow experimental groups: control and busulfan. Each group included six mice that were housed under standard conditions. The volume was estimated using the nucleator method. The sperm's flegellum and mid-piece length was estimated by counting the number of intersections between the tails and Merz grid test line in an unbiased counting frame, superimposed on live images of sperms. Our results demonstrated a significant different in the volume and surface of the sperm's head and the length of the sperm's flagellum in the control and busulfan groups. Busulfan can effect on the volume of the sperm's head and the length of the sperm's flagellum in rat.

Currently, natural polymer based hydrogels has attracted great attention of orthopedic surgeons for application in bone tissue engineering. With this aim, osteoinductive capacity of Gum Tragacanth (GT) based hydrogel was compared to collagen hydrogel and tissue culture plate (TCPS). For this purpose, adipose-derived mesenchymal stem cells (AT-MSCs) was cultured on the hydrogels and TCPS and after investigating the biocompatibility of hydrogels using MTT assay, osteoinductivity of hydrogels were evaluated using pan osteogenic markers such as Alizarin red staining, alkaline phosphatase (ALP) activity, calcium content and osteo-related genes. Increasing proliferation trend of AT-MSCs on GT hydrogel demonstrated that TG has no-cytotoxicity and can even be better than the other groups i.e., highest proliferation at day 5. GT hydrogel displayed highest ALP activity and mineralization when compared to the collagen hydrogel and TCPS. Relative gene expression levels have demonstrated that highest expression of Runx2, osteonectin and osteocalcin in the cells cultured GT hydrogel but the expression of collagen type-1 remains constant in hydrogels. Above results demonstrate that GT hydrogel could be an appropriate scaffold for accelerating and supporting the adhesion, proliferation and osteogenic differentiation of stem cells which further can be used for orthopedic applications., (Copyright © 2016. Published by Elsevier Ltd.)

Early life stress can disrupt development and negatively impact long-term health trajectories. Reconstructing histories of early life exposure to external stressors is hampered by the absence of retrospective time-specific biomarkers. Defects in tooth enamel have been used to reconstruct stress but the methods used are subjective and do not identify the specific biological systems impacted by external stressors. Here we show that external physical and social stressors impart biochemical signatures in primate teeth that can be retrieved to objectively reconstruct the timing of early life developmental disruptions. Using teeth from captive macaques, we uncovered elemental imprints specific to disruptions of skeletal growth, including major disruptions in body weight trajectory and moderate to severe illnesses. Discrete increases in heat shock protein-70 expression in dentine coincided with elemental signatures, confirming that elemental signals were associated with activation of stress-related pathways. To overcome limitations of conventional light-microscopic analysis, we used high resolution Raman microspectral imaging to identify structural and compositional alterations in enamel and dentine that coincided with elemental signatures and with detailed medical and behavioural data. Integrating these objective biochemical markers with temporal mapping of teeth enables the retrospective study of early life developmental disruptions and their ensuing health sequelae.

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.

Three decades on, the mesenchymal stem cells (MSCs) have been intensively researched on the bench top and used clinically. However, ambiguity still exists in regard to their anatomical locations, identities, functions, and extent of their differentiative abilities. One of the major impediments in the quest of the MSC research has been lack of appropriate in vivo markers. In recent years, this obstacle has been resolved to some degree as PDGFRα emerges as an important mesenchymal stem cell marker. Accumulating lines of evidence are showing that the PDGFRα (+) cells reside in the perivascular locations of many adult interstitium and fulfil the classic concepts of MSCs in vitro and in vivo. PDGFRα has long been recognised for its roles in the mesoderm formation and connective tissue development during the embryogenesis. Current review describes the lines of evidence regarding the role of PDGFRα in morphogenesis and differentiation and its implications for MSC biology.

Diabetes mellitus (DM) is associated with poor wound healing. Studies have shown accelerated wound healing following pulsed low-level laser therapy (LLLT) in non-diabetic animals. The present study aims to evaluate the effect of pulsed LLLT on wound healing in streptozotocin-induced diabetic (STZ-D) rats. We divided 48 rats into two groups of non-diabetic and diabetic. Type 1 DM was induced in the diabetic rat group by injections of STZ. Two, full-thickness skin incisions were made on the dorsal region of each rat. One month after the STZ injection, wounds of the non-diabetic and diabetic rats were submitted to a pulsed, infrared 890-nm laser with an 80-Hz frequency and 0.2 J/cm(2) for each wound point. Control wounds did not receive LLLT. Animals were sacrificed on days 4, 7, and 15 post-injury for histomorphometry and reverse transcription polymerase chain reaction (RT-PCR) analyses of basic fibroblast growth factor (bFGF) gene expression. Pulsed LLLT significantly increased the numbers of macrophages, fibroblasts, and blood vessel sections compared to the corresponding control groups. Semi-quantitative analysis of bFGF gene expression at 48 h post-injury revealed a significant increase in gene expression in both non-diabetic and diabetic rats following LLLT (the ANOVA test). Pulsed LLLT at 0.2 J/cm(2) accelerated the wound healing process in both non-diabetic and diabetic rats as measured by histological characteristics and semi-quantitative bFGF gene expression.

Women with tubal ectopic pregnancies have high levels of circulating interleukin 6 (IL-6). IL-6 treatment in vitro significantly reduces the ciliary activity of tubal epithelium. The effects of IL-6 on target cells occur via the formation of a high-affinity complex with its receptors IL-6Rα and glycoprotein 130 (Gp130). IL-6Rα is specifically expressed in the cilia of the epithelial cells. In this study, we performed a quantitative reverse transcriptase polymerase chain reaction to determine the mRNA expression of IL-6Rα and Gp130 in the fallopian tubes obtained from 12 women with ectopic pregnancies, 12 women with normal pregnancies, and 12 healthy nonpregnant women in the luteal phase of their menstrual cycle. Fallopian tubes were evaluated from specimens taken during tubal ligation in normal pregnancies and nonpregnant fertile women or during tubal surgery in ectopic pregnancies. We observed that IL-6Rα mRNA expression in fallopian tubes was increased in ectopic pregnancy compared with that in the midluteal phase. We also found that the Gp130 mRNA expression was significantly lower in fallopian tubes from ectopic pregnancies than in those from nonpregnant women during the midluteal phase of their menstrual cycle, although its expression was noticeably high in fallopian tubes in the midluteal phase, which suggests that high Gp130 levels may possibly contribute to embryo transport into the uterus.

The specialized tightly controlled microcirculation of craniofacial neurosensory organs is an essential evolutionary adaptation and yet a dilemma where angiogenic remodeling occurs. Despite extreme plasticity of neurosensory structures, the capacity to reconcile barrier phenotype of the microcirculation with an angiogenic cascade is not known. Here we provide primary evidence for such a response in an elemental neurosensory structure, human dental pulp, following chronic carious insult. In response to hypoxic challenge neurosensory odontoblasts express hypoxia-inducible factor-1α and notch-1. Associated radial rearrangement of astrocyte-like telacytes that communicate through a cell-poor zone with the microvasculature is observed. Activated pericytes characterized by expression of α-smooth muscle actin are located adjacent to the telacyte attachment to the vasculature. In this location, endothelial expression of sonic hedgehog parallels expression of notch-1 by pericytes. The angiogenic response is initiated by pericyte contraction and altered endothelial polarity and proliferation leading to intussusception of endothelial cells and extensive remodeling of basement membrane with upregulation of laminin-8 and laminin-5. These responses guide intravascular loop formation that maintains both intact basement membrane and tight junctions. This initial phase is followed by formation of anastomoses that enhance the hemodynamic capacity of the intravascular loops. The formation of anastomoses is mediated by extension of cytonemes from pericytes guided by MHC-II(+)/CD-163(+) microglia aligned with the telacytes. The cytonemes seek out pericytes on adjacent intravascular loops to initiate migration of endothelial cells. These findings support a new paradigm for understanding angiogenic capacity of neurosensory structures and aberrations of this response manifest as neurovasculopathies., (Copyright © 2012 Wiley Periodicals, Inc.)

Bacteria within endocarditis vegetations are encased in fibrin matrix that is resistant to resolution. We have previously shown that FSS2 Challisin, a serine protease from Streptococcus gordonii, is able to hydrolyse the Aα and Bβ chains of fibrinogen and has potent angiotensin converting enzyme (ACE) activity. The alteration in the structure of fibrin formed from FSS2 Challisin-degraded fibrinogen may therefore contribute to the resistant fibrin matrix. To this end, we have investigated the specific interactions of FSS2 Challisin with fibrinogen. FSS2 Challisin extensively degrades the αC region of fibrinogen Aα chains, hydrolysing both the αC-domain and αC-connnector. Additionally, the N-terminal region of the Bβ chains is cleaved twice, at Leu19 and Ser28, removing the B fibrinopeptides and 'B' knobs. Substrate analysis indicates FSS2 Challisin has specific requirement for proline two residues before the cleavage point and a neutral or basic un-branched amino acid preceding the cleavage point. Fibrin formation by thrombin was modified and the initiation of fibrinolysis extended, in FSS2 Challisin-treated plasma clots. Digestion of fibrinogen by FSS2 Challisin prior to thrombin action increased fiber density and fiber branch point density. The velocity of fibrinolysis was significantly slower for fibrin formed from FSS2 Challisin-treated fibrinogen but was faster when data was normalised for the increased fibrin density. Thromboelastography of whole blood treated with FSS2 Challisin indicated reduced clot coagulation time and increased shear resistance. Combined ACE and fibrinogenase activities of FSS2 Challisin suggest a pro-coagulant effect of this virulence factor which is conserved in the viridans streptococci.

Odontoblast synthesis of dentin proceeds through discrete but overlapping phases characterized by formation of a patterned organic matrix followed by remodelling and active mineralization. Microbial invasion of dentin in caries triggers an adaptive response by odontoblasts, culminating in formation of a structurally altered reactionary dentin, marked by biochemical and architectonic modifications including diminished tubularity. Scanning electron microscopy of the collagen framework in reactionary dentin revealed a radically modified yet highly organized meshwork as indicated by fractal and lacunarity analyses. Immuno-gold labelling demonstrated increased density and regular spatial distribution of dentin sialoprotein (DSP) in reactionary dentin. DSP contributes putative hydroxyapatite nucleation sites on the collagen scaffold. To further dissect the formation of this altered dentin matrix, the associated enzymatic machinery was investigated. Analysis of extracted dentin matrix indicated increased activity of matrix metalloproteinase-2 (MMP-2) in the reactionary zone referenced to physiologic dentin. Likewise, gene expression analysis of micro-dissected odontoblast layer revealed up-regulation of MMP-2. Parallel up-regulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane type 1- matrix metalloproteinase (MT1-MMP) was observed in response to caries. Next, modulation of odontoblastic dentinogenic enzyme repertoire was addressed. In the odontoblast layer expression of Toll-like receptors was markedly altered in response to bacterial invasion. In carious teeth TLR-2 and the gene encoding the corresponding adaptor protein MyD88 were down-regulated whereas genes encoding TLR-4 and adaptor proteins TRAM and Mal/TIRAP were up-regulated. TLR-4 signalling mediated by binding of bacterial products has been linked to up-regulation of MMP-2. Further, increased expression of genes encoding components of the TGF-β signalling pathway, namely SMAD-2 and SMAD-4, may explain the increased synthesis of collagen by odontoblasts in caries. These findings indicate a radical adaptive response of odontoblasts to microbial invasion of dentin with resultant synthesis of modified mineralized matrix., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Sensory function of human dental pulp has long been known. A composite role has been suggested for odontoblasts as sensory cells in addition to the synthesis of dentinal matrix. However, the neural basis for such a composite sensory activity remains enigmatic. Here, we aimed to probe the question by pursuing an evolutionary logic; if dental pulp is a vestigial sensory organ co-opted to a function of synthesis of mineralized matrix, essential elements of neurosensory organs may persist in dental pulp. Through structural analysis by confocal laser scanning microscopy, three distinct cell populations adjacent to odontoblasts, glial fibrillary acidic protein (GFAP)(+) seracytes, S100(+) telacytes, and HLA-II(+) alacytes were identified in peripheral human dental pulp. Subsequent molecular fingerprinting by quantitative reverse transcriptase-polymerase chain reaction established these cells as analogous to radial glia (GFAP(+) cells), astrocytes (S100(+) cells), and microglia (HLA-II(+) cells) of central nervous system organs. In the cell-rich zone of the pulp, S100(+) cells formed a network, ensheathed unmyelinated axons, and extended end-feet around the capillaries. The microcirculation adjacent to the glial cells in the cell-rich zone possessed ultrastructural features and a gene expression profile typical of the blood-brain barrier system. These novel findings support a new paradigm for understanding sensory functionality of dental pulp by the demonstration of a sophisticated neural structure in the human dental pulp that is analogous to other central sensory organs. Further, the structure that is revealed informs the concept of the evolutionary origin of the dental pulp, suggesting that a neurosensory organ was the precursor structure of teeth., (Copyright © 2011 Wiley-Liss, Inc.)

Purpose: To evaluate the clinical efficiency of the coronally advanced flap (CAF) with and without plasma rich in growth factors (PRGF) in the management of gingival recession defects., Methods: 20 bilateral similar recession defects in six healthy non-smoker subjects were included in the present study. The test sites received PRGF as an adjunct to the coronally advanced flap (CAF) while the control sites were treated with CAF only. The clinical parameters including recession depth (RD), percentage of root coverage (RC), recession width (RW), mucogingival junction position (MGJ), width of keratinized tissue (WKT), clinical attachment level (CAL), and probing depth (PD) were measured at the baseline, 1, and 3 months post-operatively. The data were analyzed using Wilcoxon signed rank and Mann-Whitney U tests., Results: After 3 months, the mean root coverage was 43 +/- 34.9% in the CAF group and 61 +/- 23.5%) in the CAF + PRGF. Both treatment protocols led to a significant improvement in all measured variables compared to the baseline values, except the width of keratinized tissue. While PRGF enhanced the outcomes of CAF especially throughout the first month post-operatively, it offered no clinical advantage over CAF alone during the subsequent 2 months.

We report evidence for anatomical and functional changes of dental pulp in response to bacterial invasion through dentin that parallel responses to noxious stimuli reported in neural crest-derived sensory tissues. Sections of resin-embedded carious adult molar teeth were prepared for immunohistochemistry, in situ hybridization, ultrastructural analysis, and microdissection to extract mRNA for quantitative analyses. In odontoblasts adjacent to the leading edge of bacterial invasion in carious teeth, expression levels of the gene encoding dentin sialo-protein were 16-fold greater than in odontoblasts of healthy teeth, reducing progressively with distance from this site of the carious lesion. In contrast, gene expression for dentin matrix protein-1 by odontoblasts was completely suppressed in carious teeth relative to healthy teeth. These changes in gene expression were related to a gradient of deposited reactionary dentin that displayed a highly modified structure. In carious teeth, interodontoblastic dentin sialo-protein(-) cells expressing glutamine synthetase (GS) showed up-regulation of glial fibrillary acidic protein (GFAP). These cells extended processes that associated with odontoblasts. Furthermore, connexin 43 established a linkage between adjacent GFAP(+)/GS(+) cells in carious teeth only. These findings indicate an adaptive pulpal response to encroaching caries that includes the deposition of modified, calcified, dentin matrix associated with networks of GFAP(+)/GS(+) interodontoblastic cells. A regulatory role for the networks of GFAP(+)/GS(+) cells is proposed, mediated by the secretion of glutamate to modulate odontoblastic response.

Aim: The aim of this study was to evaluate the age-specific effect of treatment duration on pediatric patient behavior., Methods and Materials: A total of 450 children 3 to 9 years of age were allocated into six consecutive age groups (n=75 for each group). All children received dental treatment procedures which included the fabrication of a Type 1 composite resin restoration, then a dental prophylaxis followed by fluoride therapy with each procedure requiring an average of 20 minutes. The children's behavior was evaluated at the end of each treatment period using the sound, eye, and motor (SEM) scale., Results: The results of a mixed analysis of variance (ANOVA) indicated a significant main effect of treatment duration, F (1.69, 255.01) = 188.29, P < 0.001, and of chronological age, F (1, 144) = 115.82, P < 0.001. Moreover, the main effects of time and chronological age was qualified by a significant interaction between time and chronological age, F (8.86, 255.01) = 115.82, P < 0.001. The beta weights (0.64 for age versus 0.44 for time) suggest chronological age contributes the most to predicting the behavior of children during dental treatment followed by the duration of treatment., Conclusion: Treatment duration may affect the behavior of pediatric patients parallel with chronological age and, thus, should be considered in the arrangement of the treatment plan., Clinical Significance: The findings of this study suggest appropriate pediatric behavior management should include thoughtful scheduling of appointments according to a treatment plan formulated with consideration of the effects of age and appointment length.

Objectives: The restoration of the severely damaged anterior teeth is considered a serious challenge in contemporary dental practice. The aim of the present study was the clinical and radiographic evaluation of a modified omega loop technique for the restoration of the severely damaged primary anterior teeth., Study Design: A total of 144 anterior teeth in 60 healthy children (male: 32, female: 28) aged 3-4 years, severely damaged by dental caries, were included in the present study. The root canal therapy was performed for the selected teeth. After the construction of the modified omega loop, coronal 4 mm of intracanal ZOE was removed. A thin layer of polycarboxylate cement was placed over ZOE followed by the insertion of the modified omega loop and subsequent restoration of the crown with an internal compomer core and an external composite restoration. The patients were followed at the intervals of 6, 12, and 24 months postoperatively., Results: The partial loss of the restorative material after 6 months occurred in 5.9% of the teeth. The failure rates after 12 and 24 months were 10.8% and 18.5%, respectively. The primary canines exhibited minimum loss of the restorative material. Two teeth exhibited pathological mobility after two years. There were not any signs of root fracture or recurrent caries in any of the restored teeth., Conclusion: It may be concluded that the modified omega loop is an efficient technique for the restoration of the severely damaged anterior teeth. The ease of manipulation and short chair-side time are further advantages of the technique.

A total of 455 children (242 males and 213 females) aged 5-6 years were investigated for their pain reactions to maxillary and mandibular infiltration anesthesia, middle superior alveolar nerve block, posterior superior alveolar nerve block, greater palatine nerve block, nasopalatine nerve block, and inferior alveolar nerve block, and the responses were quantified using the sound, eye and motor (SEM) scale. Administration of nasopalatine nerve block produced maximum pain (median SEM score: 10) while that of posterior superior alveolar nerve block and inferior alveolar nerve block was accompanied by minimum pain (3 and 4, respectively). There was no significant gender-specific difference in pain reactions (P = 0.39). Administration of local anesthesia in the maxilla was more painful than injections into the mandible (7 versus 5, P < 0.05). Furthermore, infiltration into the anterior and posterior segments of the maxilla produced maximum and minimum pain reactions, respectively (8 versus 3, P < 0.001). It seems that the anatomical location of an injection is one of the most important determinants of pediatric pain reaction, and that mandibular injections are generally less painful. Prioritization of treatment by consideration of site-dependent variability in pain sensitivity may help to achieve optimal behavioral control during dental treatment in young children.

Aim: The aim of the study was to evaluate the effect of cooling the soft tissue of injection sites on the pain perceptions of pediatric patients during the administration of local anesthesia for routine dental procedures., Methods and Materials: A total of 160 children aged 5-6 years were included in the present study. On a random basis, the subjects were allocated to the without ice pretreatment (WIP) group (topical anesthesia + counterstimulation + distraction) or the ice pretreatment (IP) group (cooling + topical anesthesia + counterstimulation + distraction). During the administration of an inferior alveolar nerve block, the children's behavior was assessed using the sound, eye, and motor (SEM) scale. The statistical analysis of data was performed based on the analysis of variance (ANOVA)., Results: There were no significant differences within the groups between the values of the sound, eye, and motor components for either the WIP or the IP groups (P>0.05). All three components of the SEM in the IP group were consistently lower than the WIP group (P<0.05). Moreover, the SEM value for the WIP group surpassed the IP group (P<0.05)., Conclusions: Cooling the site of infiltration block prior to the injection of local anesthesia significantly reduced the pain perceived during injection of local anesthetic agent in pediatric patients., Clinical Significance: Pre-cooling of the soft tissues of an injection site prior to the administration of a local anesthetic can minimize the discomfort and anxiety associated with the injection procedure and facilitates the management of pediatric patients during this phase of a dental procedure.