Enhancement of the Evoked Excitatory Transmission in the Nucleus Tractus Solitarius Neurons after Sustained Hypoxia in Mice Depends on A2A Receptors.

The first synapses of the afferents of peripheral chemoreceptors are located in the Nucleus Tractus Solitarius (NTS) and there is evidence that short-term sustained hypoxia (SH - 24 h, FiO2 0.1) facilitates glutamatergic transmission in NTS neurons of rats. Adenosine is an important neuromodulator of synaptic transmission and hypoxia contributes to increase its extracellular concentration. The A2A receptors mediate the excitatory actions of adenosine and are active players in the modulation of neuronal networks in the NTS. Herein, we used knockout mice for A2A receptors (A2AKO) and electrophysiological recordings of NTS neurons were performed to evaluate the contribution of these receptors in the changes in synaptic transmission in NTS neurons of mice submitted to SH. The membrane passive properties and excitability of NTS neurons were not affected by SH and were similar between A2AKO and wild-type mice. The overall amplitude of spontaneous glutamatergic currents in NTS neurons of A2AKO mice was lower than in Balb/c WT mice. SH increased the amplitude of evoked glutamatergic currents of NTS neurons from WT mice by a non-presynaptic mechanism, but this enhancement was not observed in NTS neurons of A2AKO mice. Under normoxia, the amplitude of evoked glutamatergic currents was similar between WT and A2AKO mice. The data indicate that A2A receptors (a) modulate spontaneous glutamatergic currents, (b) do not modulate the evoked glutamatergic transmission in the NTS neurons under control conditions, and (c) are required for the enhancement of glutamatergic transmission observed in the NTS neurons of mice submitted to SH.

Nanoscale hybrid implant surfaces and Osterix-mediated osseointegration.

Endosseous implant surface topography directly affects adherent cell responses following implantation. The aim of this study was to examine the impact of nanoscale topographic modification of titanium implants on Osterix gene expression since this gene has been reported as key factor for bone formation. Titanium implants with smooth and nanoscale topographies were implanted in the femurs of Osterix-Cherry mice for 1-21 days. Implant integration was evaluated using scanning electron microscopy (SEM) to evaluate cell adhesion on implant surfaces, histology, and nanotomography (NanoCT) to observe and quantify the formed bone-to-implant interface, flow cytometry to quantify of Osterix expressing cells in adjacent tissues, and real-time PCR (qPCR) to quantify the osteoinductive and osteogenic gene expression of the implant-adherent cells. SEM revealed topography-dependent adhesion of cells at early timepoints. NanoCT demonstrated greater bone formation at nanoscale implants and interfacial osteogenesis was confirmed histologically at 7 and 14 days for both smooth and nanosurface implants. Flow cytometry revealed greater numbers of Osterix positive cells in femurs implanted with nanoscale versus smooth implants. Compared to smooth surface implants, nanoscale surface adherent cells expressed higher levels of Osterix (Osx), Alkaline phosphatase (Alp), Paired related homeobox (Prx1), Dentin matrix protein 1 (Dmp1), Bone sialoprotein (Bsp), and Osteocalcin (Ocn). In conclusion, nanoscale surface implants demonstrated greater bone formation associated with higher levels of Osterix expression over the 21-day healing period with direct evidence of surface-associated gene regulation involving a nanoscale-mediated osteoinductive pathway that utilizes Osterix to direct adherent cell osteoinduction.

Analysis In Vivo of Bone Modulation in Hydrophilic and Hydrophobic Titanium Implant Surfaces.

PURPOSE: To evaluate whether a hydrophilic surface treatment compared with a hydrophobic implant surface can enhance osseointegration by analysis of calcium deposition, bone-to-implant contact (BIC), bone volume (BV), and upregulation of genes involved in bone formation. MATERIALS AND METHODS: Sixty implants (n = 60) with a hydrophobic (SAE) or hydrophilic (SAE-HD) surface treatment were placed bilaterally in each femur of 3-month-old male mice (n = 30). Scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDS) were utilized for quantifying the presence of calcium on the implant surface 7 days after implant placement. The specimens were analyzed after 14 and 21 days for BIC and BV by Nano CT scanning followed by histologic evaluation. Additionally, 1, 3, and 7 days postsurgery, femurs were harvested, implants were explanted, and gene expression (Sp7, Bsp, Sost, IL-1α, and IL-10) by quantitative real-time polymerase chain reaction was studied. Moreover, implants were characterized for surface roughness area. Data were statistically analyzed with two-way analysis of variance (ANOVA) followed by the Tukey test (P < .05). RESULTS: The amount of calcium on the surface was higher for SAE-HD after 7 days. Nano CT revealed significantly more BV in the SAE-HD than the SAE surface. The histologic assessment showed increased BIC in SAE-HD in comparison to SAE. SAEHD showed significantly increased levels of genes involved in bone formation. CONCLUSION: Both surfaces were able to modulate bone responses toward osteoblast differentiation. SAE-HD presented a better response compared with SAE.

Chondrocyte Tsc1 controls cranial base bone development by restraining the premature differentiation of synchondroses.

Cranial base bones are formed through endochondral ossification. Synchondroses are growth plates located between cranial base bones that facilitate anterior-posterior growth of the skull. Coordinated proliferation and differentiation of chondrocytes in cranial base synchondroses is essential for cranial base bone growth. Herein, we report that constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling via Tsc1 (Tuberous sclerosis 1) deletion in chondrocytes causes abnormal skull development with decreased size and rounded shape. In contrast to decreased anterior-posterior growth of the cranial base, mutant mice also exhibited significant expansion of cranial base synchondroses including the intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS). Cranial base synchondrosis expansion in TSC1-deficient mice was accounted for by an expansion of the resting zone due to increased cell number and size without alteration in cell proliferation. Furthermore, our data showed that mTORC1 activity is inhibited in the resting and proliferating zone chondrocytes of wild type mice, and Tsc1 deletion activated mTORC1 signaling of the chondrocytes in the resting zone area. Consequently, the chondrocytes in the resting zone of TSC1-deficient mice acquired characteristics generally attributed to pre-hypertrophic chondrocytes including high mTORC1 activity, increased cell size, and increased expression level of PTH1R (Parathyroid hormone 1 receptor) and IHH (Indian hedgehog). Lastly, treatment with rapamycin, an inhibitor of mTORC1, rescued the abnormality in synchondroses. Our results established an important role for TSC1-mTORC1 signaling in regulating cranial base bone development and showed that chondrocytes in the resting zone of synchondroses are maintained in an mTORC1-inhibitory environment.

Sustained Hypoxia Reduces GABAergic Modulation on NTS Neurons Sending Projections to Ventral Medulla of Rats.

Peripheral chemoreflex is activated during short-term sustained hypoxia (SH), and the first synapse of these afferents is located in Nucleus Tractus Solitarius(NTS). NTS neurons projecting to the ventral lateral medulla (NTS-VLM) are part of the respiratory pathways of the chemoreflex. SH increases the magnitude of basal respiratory parameters in rats from Wistar-Hannover strain. In this study, we hypothesized that the observed changes in the respiratory pattern in response to SH were due to changes in the GABAergic modulation of the synaptic transmission of NTS-VLM neurons. We used an electrophysiological approach to record the synaptic activity of NTS neurons labeled with a retrograde tracer previously microinjected into VLM of Wistar-Hannover rats submitted to 24 h SH. The data are showing that: (a) the amplitude of evoked inhibitory currents in NTS-VLM neurons of SH rats was reduced and not accompanied by any change in rise-time and decay-time; (b) the 1/CV2 and the number of failures in response to evoked currents were also affected by SH; (c) the frequency of spontaneous inhibitory currents was reduced by SH without changes in amplitude and half-width. These effects of SH were observed in NTS-VLM neurons located in caudal and intermediate NTS, but not in NTS-VLM neurons located in the rostral NTS. We conclude that SH causes a reduction in inhibitory modulation onto NTS-VLM neurons by pre-synaptic mechanisms, which may contribute to the observed changes in the respiratory pattern of Wistar-Hannover rats submitted to SH.

Hydrophilic titanium surface modulates early stages of osseointegration in osteoporosis.

OBJECTIVE: Using a mouse osteoporotic model, this study aimed to determine the influence of hydrophilic titanium surfaces on gene expression and bone formation during the osseointegration process. BACKGROUND: Based on the previous evidence, it is plausible to assume that osteoporotic bone has a different potential of bone healing. Therefore, implant surface modification study that aims at enhancing bone formation to further improve short- and long-term clinical outcomes in osteoporosis is necessary. MATERIAL AND METHODS: Fifty female, 3-month-old mice were included in this study. Osteoporosis was induced by ovariectomy (OVX, test group) in 25 mice. The further 25 mice had ovaries exposed but not removed (SHAM, control group). Seven weeks following the ovariectomy procedures, one customized implant (0.7 × 8 mm) of each surface was placed in each femur for both groups. Implants had either a hydrophobic surface (SAE) or a hydrophilic treatment surface (SAE-HD). Calcium (Ca) and phosphorus (P) content was measured by energy-dispersive X-ray spectroscopy (EDS) after 7 days. The femurs were analyzed for bone-to-implant contact (BIC) and bone volume fraction (BV) by nano-computed tomography (nano-CT) after 14 and 28 days. Same specimens were further submitted to histological analysis. Additionally, after 3 and 7 days, implants were removed and cells were collected around the implant to access gene expression profile of key osteogenic (Runx2, Alp, Sp7, Bsp, Sost, Ocn) and inflammatory genes (IL-1ß, IL-10, Tnf-α, and Nos2) by qRT-PCR assay. Statistical analysis was performed by ANOVA and paired t test with significance at P < .05. RESULTS: The amount of Ca and P deposited on the surface due to the mineralization process was higher for SAE-HD compared to SAE on the intra-group analysis. Nano-CT and histology revealed more BV and BIC for SAE-HD in SHAM and OVX groups compared to SAE. Analysis in OVX group showed that most genes (ie, ALP, Runx2) involved in the bone morphogenetic protein (BMP) signaling were significantly activated in the hydrophilic treatment. CONCLUSION: Both surfaces were able to modulate bone responses toward osteoblast differentiation. SAE-HD presented a faster response in terms of bone formation and osteogenic gene expression compared to SAE. Hydrophilic surface in situations of osteoporosis seems to provide additional benefits in the early stages of osseointegration.

Electrical stimulation enhances early palatal wound healing in mice.

BACKGROUND/OBJECTIVE: Electrical stimulation (ES) has been used to treat chronic wound and other clinical applications, showing favorable results in wound closure. It was hypothesized that ES can present a positive effect on oral mucosa healing. The aim of this study was to investigate the effects of ES during the palatal mucosa early healing process in Swiss mice. METHODS: Ninety animals were divided into two groups: Control (C; n = 45), which received Sham ES applications, and Test (ES; n = 45), which received ES (100 µA; 9 kHz; 660 mVpp) once a day for 3 days. A full thickness wound was performed with a 1.5 mm diameter biopsy punch in the hard palate. Histologically, the following parameters were evaluated: palatal wound closure and epithelial and connective wound edge distance (EED and CED). Furthermore, IL-1ß, IL-6, IL-10 TNF-α, and VEGF cytokine levels were evaluated by multiplex assay. The percentage of collagen fibers was assessed using the polarization method and the Smad proteins using the immunofluorescence method. RESULTS: Palatal wound closure presented a significant reduction on day 5 in the ES group (p = 0.01). Additionally, both EED and CED were shorter for all time points in the ES group (p < 0.05), and the inflammatory markers IL-6, IL-10, TNF-α, and VEGF were reduced (p < 0.05). There were no differences in collagen fibers and phospho-Smad2 between the groups. CONCLUSION: ES had a positive effect on early palatal wound closure outcomes, as well as on inflammatory markers.

MicroRNA-1911-3p targets mEAK-7 to suppress mTOR signaling in human lung cancer cells.

Regulation of mTOR signaling depends on an intricate interplay of post-translational protein modification. Recently, mEAK-7 (mTOR associated protein, eak-7 homolog) was identified as a positive activator of mTOR signaling via an alternative mTOR complex. However, the upstream regulation of mEAK-7 in human cells is not known. Because microRNAs are capable of modulating protein translation of RNA in eukaryotes, we conducted a bioinformatic search for relevant mEAK-7 targeting microRNAs using the Exiqon miRSearch V3.0 algorithm. Based on the score obtained through miRSearch V3.0, the top predicted miRNA (miR-1911-3p) was studied. miR-1911-3p mimics decreased protein levels of both mEAK-7 and mTORC1 downstream effectors p-S6 and p-4E-BP1 in non-small cell lung carcinoma (NSCLC) cell lines H1975 and H1299. miR-1911-3p levels and MEAK7 mRNA/mEAK-7/mTOR signaling levels were negatively correlated between normal lung and NSCLC cells. miR-1911-3p directly interacted with MEAK7 mRNA at the 3'-UTR to negatively regulate mEAK-7 and significantly decreased mTOR localization to the lysosome. Furthermore, miR-1911-3p significantly decreased cell proliferation and migration in both H1975 and H1299 cells. Thus, miR-1911-3p functions as a suppressor of mTOR signaling through the regulation of MEAK7 mRNA translation in human cancer cells.

The influence of text messages and anxiety on pain perception and its impact on orthodontic patients routine.

OBJECTIVE: This prospective study aimed at assessing the effects of anxiety and a follow-up text message on pain perception after the installation of fixed orthodontic appliances and its impact on the patients' routine. METHODS: The sample of this study consisted of 103 orthodontic patients, 40 males and 63 females (mean age 20.5 years), distributed in two groups: G1 (n=51), including control patients that did not receive any post-procedure communication; and G2 (n=52), including patients that received a structured text message. In baseline phase, the patients completed a questionnaire to assess their level of anxiety prior to treatment. Pain was assessed by using 100-mm visual analog scale (VAS) in baseline and ten times prospectively in predetermined time points. VAS was also applied to assess the patient's routine alterations caused by the pain. All data were analyzed using ANOVA, Tukey, Mann-Whitney, t-test, chi-square and Spearman's correlation tests. All statistical tests were performed with significance level of 5%. RESULTS: Low-level and high-level anxiety was observed in 42.7% and 7.8% of the patients, respectively. Statistically significant correlation was observed between anxiety and pain (p< 0.05). Maximum mean pain intensity was detected in the second treatment day (G1=36.9mm and G2=26.2mm) and was significantly higher in G1. Nearly 53% of the patients in G1 reported alterations in the routine (18.8mm), while in G2 the percentage rate reached 28.8% (9.9mm) (p=0.013). CONCLUSIONS: Anxious patients report more pain after the installation of orthodontic appliances. Text messages were effective to reduce pain levels and to decrease the negative effects on patients' daily routine.

The influence of text messages and anxiety on pain perception and its impact on orthodontic patients routine

ABSTRACT Objective: This prospective study aimed at assessing the effects of anxiety and a follow-up text message on pain perception after the installation of fixed orthodontic appliances and its impact on the patients' routine. Methods: The sample of this study consisted of 103 orthodontic patients, 40 males and 63 females (mean age 20.5 years), distributed in two groups: G1 (n=51), including control patients that did not receive any post-procedure communication; and G2 (n=52), including patients that received a structured text message. In baseline phase, the patients completed a questionnaire to assess their level of anxiety prior to treatment. Pain was assessed by using 100-mm visual analog scale (VAS) in baseline and ten times prospectively in predetermined time points. VAS was also applied to assess the patient's routine alterations caused by the pain. All data were analyzed using ANOVA, Tukey, Mann-Whitney, t-test, chi-square and Spearman's correlation tests. All statistical tests were performed with significance level of 5%. Results: Low-level and high-level anxiety was observed in 42.7% and 7.8% of the patients, respectively. Statistically significant correlation was observed between anxiety and pain (p< 0.05). Maximum mean pain intensity was detected in the second treatment day (G1=36.9mm and G2=26.2mm) and was significantly higher in G1. Nearly 53% of the patients in G1 reported alterations in the routine (18.8mm), while in G2 the percentage rate reached 28.8% (9.9mm) (p=0.013). Conclusions: Anxious patients report more pain after the installation of orthodontic appliances. Text messages were effective to reduce pain levels and to decrease the negative effects on patients' daily routine.

RESUMO Objetivo: O presente estudo prospectivo teve como objetivo avaliar os efeitos da ansiedade e de mensagens de texto de acompanhamento na percepção da dor após a instalação de aparelhos ortodônticos fixos e seu impacto na rotina dos pacientes. Métodos: A amostra deste estudo foi composta por 103 pacientes ortodônticos, 40 homens e 63 mulheres (idade média de 20,5 anos), distribuídos em dois grupos: G1 (n = 51), que incluiu pacientes controle, que não receberam comunicação pós-procedimento; e G2 (n = 52), incluindo os pacientes que receberam mensagem de texto estruturada. Na fase inicial, os pacientes responderam a um questionário para avaliar seu nível de ansiedade antes do tratamento. A dor foi avaliada por meio de escala visual analógica (EVA) de 100 mm antes da instalação dos aparelhos e em 10 períodos consecutivos predeterminados. A EVA também foi aplicada para avaliar as alterações de rotina do paciente causadas pela dor. Os resultados foram analisados usando ANOVA, Tukey, Mann-Whitney, teste t, Qui-quadrado e testes de correlação de Spearman. Todos os testes estatísticos foram realizados com nível de significância de 5%. Resultados: Níveis baixos e altos de ansiedade foram observados em 42,7% e 7,8% dos pacientes, respectivamente. Foi observada correlação estatisticamente significativa entre ansiedade e dor (p< 0,05). A intensidade média máxima da dor foi detectada no segundo dia de tratamento (G1 = 36,9mm e G2 = 26,2mm) e foi significativamente maior no G1. Quase 53% dos pacientes do G1 relataram alterações na rotina (18,8mm), enquanto no G2 o percentual atingiu 28,8% (9,9mm) (p= 0,013). Conclusões: Pacientes ansiosos relatam mais dor após a instalação de aparelhos ortodônticos. As mensagens de texto foram eficazes para reduzir os níveis de dor e diminuir os efeitos negativos na rotina diária dos pacientes.

Influence of Titanium Alloy Scaffolds on Enzymatic Defense against Oxidative Stress and Bone Marrow Cell Differentiation.

Studies have been directed towards the production of new titanium alloys, aiming for the replacement of Ti-6 Aluminium-4 Vanadium (TiAlV) alloy in the future. Many mechanisms related to biocompatibility and chemical characteristics have been studied in the field of implantology, but enzymatic defenses against oxidative stress remain underexplored. Bone marrow stromal cells have been explored as source of cells, which have the potential to differentiate into osteoblasts and therefore could be used as cells-based therapy. The objective of this study was to evaluate the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in porous scaffolds of Ti-6 Aluminium-4 Vanadium (TiAlV), Ti-35 Niobium (TiNb), and Ti-35 Niobium-7 Zirconium-5 Tantalum (TiNbZrTa) on mouse bone marrow stromal cells. Porous titanium alloy scaffolds were prepared by powder metallurgy. After 24 hours, cells plated on the scaffolds were analyzed by scanning electron microscopy (SEM). The antioxidant enzyme activity was measured 72 hours after cell plating. Quantitative real time PCR (qRT-PCR) was performed after 3, 7, and 14 days, and Runx2 (Runt-related transcription factor2) expression was evaluated. The SEM images showed the presence of interconnected pores and growth, adhesion, and cell spreading in the 3 scaffolds. Although differences were noted for SOD and CAT activity for all scaffolds analyzed, no statistical differences were observed (p > 0.05). The osteogenic gene Runx2 presented high expression levels for TiNbZrTa at day 7, compared to the control group (TiAlV day 3). At day 14, all scaffolds had more than 2-fold induction for Runx2 mRNA levels, with statistically significant differences compared to the control group. Even though we were not able to confirm statistically significant differences to justify the replacement of TiAlV regarding antioxidant enzymes, TiNbZrTa was able to induce faster bone formation at early time points, making it a good choice for biomedical and tissue bioengineering applications.

mEAK-7 Forms an Alternative mTOR Complex with DNA-PKcs in Human Cancer.

MTOR associated protein, eak-7 homolog (mEAK-7), activates mechanistic target of rapamycin (mTOR) signaling in human cells through an alternative mTOR complex to regulate S6K2 and 4E-BP1. However, the role of mEAK-7 in human cancer has not yet been identified. We demonstrate that mEAK-7 and mTOR signaling are strongly elevated in tumor and metastatic lymph nodes of patients with non-small-cell lung carcinoma compared with those of patients with normal lung or lymph tissue. Cancer stem cells, CD44+/CD90+ cells, yield elevated mEAK-7 and activated mTOR signaling. mEAK-7 is required for clonogenic potential and spheroid formation. mEAK-7 associates with DNA-dependent protein kinase catalytic subunit isoform 1 (DNA-PKcs), and this interaction is increased in response to X-ray irradiation to regulate S6K2 signaling. DNA-PKcs pharmacologic inhibition or genetic knockout reduced S6K2, mEAK-7, and mTOR binding with DNA-PKcs, resulting in loss of S6K2 activity and mTOR signaling. Therefore, mEAK-7 forms an alternative mTOR complex with DNA-PKcs to regulate S6K2 in human cancer cells.

In vitro osteogenesis process induced by hybrid nanohydroxyapatite/graphene nanoribbons composites.

Carbon nanotubes combine high bend and mechanical strength, which is advantageous for many structural and biomedical purposes. Recently, some biomaterials, based on carbon nanostructures and nanohydroxyapatite (nHAp), have been investigated as bone substitutes in order to improve regeneration. The aim of this study was to access the expression of some RNA transcripts (involved in the process of osteoblast differentiation) by mesenchymal stem cells cultured over different nanocomposite surfaces. A multi-walled carbon nanotube (MWCNT) was firstly grown using chemical vapor deposition and then exfoliated using chemical and oxygen plasma treatments to obtain graphene nanoribbons (GNR). The hybrid composites nHAp/GNR were prepared using the wet method assisted by ultrasound irradiation with different amounts of GNR (1.0, 2.0 and 3.0 wt %). Five groups were tested in cell cultures. Group 1: synthesized nHAp; Group 2: synthesized GNR; Group 3: nHAp and 1.0% of GNR; Group 4: nHAp and 2.0% of GNR and group 5: nHAp and 3.0% of GNR. Real time reverse transcription polymerase chain reactions were performed, and all data was submitted to Kruskal Wallis and Dunn tests, at a significance level of 5%. As a result, three nanocomposites with different proportions of GNR were successfully produced. After cell culture, the expression of osteogenic genes demonstrated no significant differences among the groups and periods. However, bone morphogenetic protein II (BMP II), integrin binding sialoprotein (IBSP), and Osterix highest expressions were observed in the group containing 3.0% of GNR. In conclusion, our hybrid composites may be useful in bone interventions requiring mesenchymal stem cell differentiation into osteoblasts for healing.

Enhancement of excitatory transmission in NTS neurons projecting to ventral medulla of rats exposed to sustained hypoxia is blunted by minocycline.

KEY POINTS: Rats subjected to sustained hypoxia (SH) present increases in arterial pressure (AP) and in glutamatergic transmission in the nucleus tractus solitarius (NTS) neurons sending projections to ventrolateral medulla (VLM). Treatment with minocycline, a microglial inhibitor, attenuated the increase in AP in response to SH. The increase in the amplitude of glutamatergic postsynaptic currents in the NTS-VLM neurons, induced by postsynaptic mechanisms, was blunted by minocycline treatment. The number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS of minocycline-treated rats. The data show that microglial recruitment/proliferation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the observed increase in AP. ABSTRACT: Short-term sustained hypoxia (SH) produces significant autonomic and respiratory adjustments and triggers activation of microglia, the resident immune cells in the brain. SH also enhances glutamatergic neurotransmission in the NTS. Here we evaluated the role of microglial activation induced by SH on the cardiovascular changes and mainly on glutamatergic neurotransmission in NTS neurons sending projections to the ventrolateral medulla (NTS-VLM), using a microglia inhibitor (minocycline). Direct measurement of arterial pressure (AP) in freely moving rats showed that SH (24 h, fraction of inspired oxygen ( FI,O2 ) 0.1) in vehicle and minocycline (30 mg/kg i.p. for 3 days)-treated groups produced a significant increase in AP in relation to control groups under normoxic conditions, but this increase was significantly lower in minocycline-treated rats. Whole-cell patch-clamp recordings revealed that the active properties of the membrane were comparable among the groups. Nevertheless, the amplitudes of glutamatergic postsynaptic currents, evoked by tractus solitarius stimulation, were increased in NTS-VLM neurons of SH rats. Changes in asynchronous glutamatergic currents indicated that the observed increase in amplitude was due to postsynaptic mechanisms. These changes were blunted in the SH group previously treated with minocycline. Using immunofluorescence, we found that the number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS neurons of minocycline-treated rats. Our data support the concept that microglial activation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the increase in AP observed in this experimental model.

Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2.

The bone morphogenetic protein BMP2 plays a crucial role in the formation and regeneration of bone and cartilage, which is critical for maintaining skeletal integrity and bone fracture repair. Because of its important role in osteogenic properties it has been commercially produced for clinical use. Here we report attempts to express human BMP2 using two plant systems (lettuce chloroplast and soybean seeds). The rhBMP2 gene (coding for the 13 kDa active polypeptide) was introduced in two regions of the lettuce chloroplast genome. Two homoplasmic events were achieved and RT-PCR demonstrated that the BMP2 gene was transcribed. However, it was not possible to detect accumulation of rhBMP2 in leaves. Two soybean events were achieved to express a full-length hBMP2 gene (coding for the 45 kDa pro-BMP2) fused with the α-coixin signal peptide, under control of the ß-conglycinin promoter. Pro-BMP2 was expressed in the transgenic seeds at levels of up to 9.28% of the total soluble seed protein as determined by ELISA. It was demonstrated that this recombinant form was biologically active upon administration to C2C12 cell cultures, because it was able to induce an osteogenic cascade, as observed by the enhanced expression of SP7 (osterix) and ALPI (alkaline phosphatase) genes. Collectively, these results corroborated our previous observation that soybean seeds provide an effective strategy for achieving stable accumulation of functional therapeutic proteins, such as BMP2.

Astrocytic modulation of glutamatergic synaptic transmission is reduced in NTS of rats submitted to short-term sustained hypoxia.

Sustained hypoxia (SH) activates chemoreceptors to produce cardiovascular and respiratory responses to bring the arterial partial pressure of O2 back to the physiological range. We evaluated the effect of SH (fraction of inspired O2 = 0.10, 24 h) on glutamatergic synaptic transmission and the interaction neuron-astrocyte in neurons of the nucleus tractus solitarii (NTS). Tractus solitarius (TS) fiber stimulation induced glutamatergic currents in neurons and astrocytes. SH increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) [-183 ± 122 pA (n = 10) vs. -353 ± 101 pA (n = 10)] and N-methyl-d-aspartate (NMDA) current amplitude [61 ± 10 pA (n = 7) vs. 102 ± 37 pA (n = 10)]. To investigate the effects of SH, we used fluoroacetate (FAC), an astrocytic inhibitor, which revealed an excitatory modulation on AMPA/kainate current and an inhibitory modulation of NMDA current in control rats. SH blunted the astrocytic modulation of AMPA [artificial cerebrospinal fluid (aCSF): -353 ± 101 pA vs. aCSF + FAC: -369 ± 76 pA (n = 10)] and NMDA currents [aCSF: 102 ± 37 pA vs. aCSF + FAC: 108 ± 32 pA (n = 10)]. SH increased AMPA current density [control: -6 ± 3.5 pA/pF (n = 6) vs. SH: -20 ± 12 pA/pF (n = 7)], suggesting changes in density, conductance, or affinity of AMPA receptors. SH produced no effect on astrocytic resting membrane potential, input resistance, and AMPA/kainate current. We conclude that SH decreased the neuron-astrocyte interaction at the NTS level, facilitating the glutamatergic transmission, which may contribute to the enhancement of cardiovascular and respiratory responses to baro- and chemoreflexes activation in SH rats. NEW & NOTEWORTHY Using an electrophysiological approach, we have shown that in nucleus tractus solitarii (NTS) from control rats, astrocytes modulate the AMPA and NMDA currents in NTS neurons, changing their excitability. Sustained hypoxia (SH) increased both glutamatergic currents in NTS neurons due to 1) a reduction in the astrocytic modulation and 2) an increase in the density of AMPA receptors. These new findings show the importance of neuron-astrocyte modulation in the excitatory synaptic transmission in NTS of control and SH rats.

The Role of MicroRNAs in the Osseointegration Process.

Regulation of cellular function is key to bone formation at endosseous implant surfaces. Osseointegration was "discovered" prior to the discovery of genetic regulation of osteoinduction or characterization of mesenchymal stem cells. Understanding osseointegration in cellular and molecular terms has benefited from genome-wide characterization of this healing process at endosseous implants in vivo. These in vivo studies also demonstrate a role for osteoprogenitor cells and cells involved in immune regulation and osteoclastogenesis. The identification of noncoding RNAs, including microRNAs, as key factors controlling cell function has highlighted the role of microRNAs in cell differentiation control. This review summarizes emerging in vitro and in vivo investigations emphasizing the role of microRNAs in the osseointegration process. Many microRNAs influence key osteoinductive pathways controlling Osterix, runt-related transcription factor 2 (RUNX2), and bone morphogenetic protein (BMP)/SMAD function. Others influence the monocyte/macrophage lineage. While significant progress has been made in elucidating the mechanisms associated with the regulation of surface modulation of osteoblast differentiation by microRNAs, knowledge gaps are evident in the identification and characterization of microRNAs linked to osseointegration. Given existing knowledge regarding the varied expression of microRNAs and their role in inflammation, it is important to understand how microRNA expression may influence the process of bone accrual at implant surfaces during osseointegration.

Molecular Assessment of Human Peri-implant Mucosal Healing at Laser-Modified and Machined Titanium Abutments.

PURPOSE: To compare, by gene profiling analysis, the molecular events underscoring peri-implant mucosa formation at machined vs laser-microgrooved implant healing abutments. MATERIALS AND METHODS: Forty endosseous implants were placed by a one-stage approach in 20 healthy subjects in nonadjacent sites for single-tooth restorations. In a split-mouth design, machined smooth and laser-microgrooved healing abutments were randomly assigned in each subject. Peri-implant mucosa adjacent to healing abutments was harvested by tissue punch biopsy at either 1, 2, 4, or 8 weeks following abutment placement. Total RNA was isolated from the peri-implant transmucosal soft tissues. A whole genome microarray using the Affymetrix Human Gene 2.1 ST Array was performed to describe gene expression profiles in relation to abutment topography and healing time duration. Data analysis was completed using GeneSpring software v.12.6. RESULTS: Differential gene expression was revealed at all time points and among surfaces. Five hundred one genes were differentially expressed (fold change ≥ 2.0) at machined versus laser-modified abutments, and 459 of these were statistically significant (P ≤ .05). At 1 week, unique expression of IL-24 and MMP1 was observed in tissues from laser-treated surfaces. At 2, 4, and 8 weeks, mRNAs encoding keratins and protective proteins of cornified epithelium were upregulated in tissues from laser-modified abutments. At 4 weeks, upregulation (> 2-fold) of mRNAs encoding proteins associated with collagen fibril formation and function was observed in tissue from laser-modified abutments. In both tissues of machined and laser-modified abutments, mRNAs encoding junctional epithelium-specific proteins, ostogenic ameloblast associated protein (ODAM) and follicular dendritic cell secreted protein (FDCSP) were highly upregulated throughout weeks 2 to 8. CONCLUSION: Peri-implant abutment mucosal wound healing involves selective differentiation of epithelium and induction of the junctional epithelium. Laser-mediated alterations in abutment topography enhance collagen fibril-associated gene expression and alter epithelium/junctional epithelial gene expression. Clinically, shallower probing depths are measured at laser-mediated versus machined implant abutments.

Mammalian EAK-7 activates alternative mTOR signaling to regulate cell proliferation and migration.

Nematode EAK-7 (enhancer-of-akt-1-7) regulates dauer formation and controls life span; however, the function of the human ortholog mammalian EAK-7 (mEAK-7) is unknown. We report that mEAK-7 activates an alternative mechanistic/mammalian target of rapamycin (mTOR) signaling pathway in human cells, in which mEAK-7 interacts with mTOR at the lysosome to facilitate S6K2 activation and 4E-BP1 repression. Despite interacting with mTOR and mammalian lethal with SEC13 protein 8 (mLST8), mEAK-7 does not interact with other mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2) components; however, it is essential for mTOR signaling at the lysosome. This phenomenon is distinguished by S6 and 4E-BP1 activity in response to nutrient stimulation. Conventional S6K1 phosphorylation is uncoupled from S6 phosphorylation in response to mEAK-7 knockdown. mEAK-7 recruits mTOR to the lysosome, a crucial compartment for mTOR activation. Loss of mEAK-7 results in a marked decrease in lysosomal localization of mTOR, whereas overexpression of mEAK-7 results in enhanced lysosomal localization of mTOR. Deletion of the carboxyl terminus of mEAK-7 significantly decreases mTOR interaction. mEAK-7 knockdown decreases cell proliferation and migration, whereas overexpression of mEAK-7 enhances these cellular effects. Constitutively activated S6K rescues mTOR signaling in mEAK-7-knocked down cells. Thus, mEAK-7 activates an alternative mTOR signaling pathway through S6K2 and 4E-BP1 to regulate cell proliferation and migration.