Disruption of TNF-α signaling improves osteoblastic differentiation of adipose-derived mesenchymal stem cells and bone repair.

Adipose tissue is an attractive source of mesenchymal stem cells (at-MSCs), but their low osteogenic potential limits their use in bone regeneration. Adipose tissue plays a role in pro-inflammatory diseases by releasing cytokines with a catabolic effect on bone, such as tumor necrosis factor-alpha (TNF-α). Thus, we hypothesized that endogenous TNF-α could have a negative effect on at-MSC differentiation into osteoblasts. Short interfering RNAs (siRNAs) targeting TNF-α receptors (siR1, siR2, and si1R/R2) were transfected into at-MSCs, and cell differentiation was assessed by measuring the expression of bone markers, ALP activity, and mineralized matrix. Scrambled was used as Control. Knockout at-MSCs (KOR1/R2) was injected in mice calvaria defects, and bone formation was evaluated by microtomography and histological analysis. Data were compared by Kruskal-Wallis or analysis of variance (5%). The expression of bone markers confirmed that at-MSCs differentiate less than bone marrow MSCs. In silenced cells, the expression of Alp, Runx2, and Opn was generally higher compared to Control. ALP, RUNX2, and OPN were expressed at elevated levels in silenced groups, most notably at-MSCs-siR1/R2. ALP was detected at high levels in at-MSCs-siR1/R2 and in-MSCs-siR1, followed by an increase in mineralized nodules in at-MSCs-siR1/R2. As the morphometric parameters increased, the groups treated with KOR1/R2 exhibited slight bone formation near the edges of the defects. Endogenous TNF-α inhibits osteoblast differentiation and activity in at-MSCs, and its disruption increases bone formation. While opening a path of investigation, that may lead to the development of new treatments for bone regeneration using at-MSC-based therapies.

Occupational Exposures and Risks of Non-Hodgkin Lymphoma: A Meta-Analysis.

Non-Hodgkin lymphoma (NHL) is a heterogeneous group with different types of diseases. It remains unclear as to what has led to an increase in incidences of NHL, however, chemical substance exposure is known to be one of the risk factors for the disease. Therefore, we performed a systematic review and meta-analysis including case-control, cohort, and cross-sectional observational epidemiological studies to verify the association between occupational exposure to carcinogens and NHL risk. Articles between the years 2000 and 2020 were collected. Two different reviewers performed a blind selection of the studies using the Rayyan QCRI web app. Post-completion, the selected articles were extracted and analyzed via the RedCap platform. Our review resulted in 2719 articles, of which 51 were included in the meta-analysis, resulting in an overall OR of 1.27 (95% CI 1.04-1.55). Furthermore, it was observed that the main occupation associated with the increased risk of NHL was that in which workers are exposed to pesticides. We therefore conclude that the evidence synthesis of the epidemiological literature supports an increased risk for NHL, regardless of subtype, considering occupational exposure to certain chemical compounds, mainly pesticides, benzene, and trichlorethylene, and certain classes of work, primarily in the field of agriculture.

Quality Assessment of Cryopreserved Human Biological Samples from the Biobank of Barretos Cancer Hospital.

Background: Biobanks process, store, and supply biological materials for research. Preanalytical factors, especially storage time and temperature, must be controlled and standardized at all stages when handling biospecimen samples, especially because the literature reports highly contradictory optimal parameters. As large-sample studies are required to better understand the influence of time and temperature on cryopreserved samples' quality for genomic research, this study evaluated the integrity and quality of cryopreserved samples stored for up to 9 years at the biobank of Barretos Cancer Hospital, one of the largest biobanks in Latin America. Methods: We randomly selected 447 samples with tumor tissue paired with buffy coat or peripheral blood mononuclear cells (PBMCs) that were stored from 2008 to 2016. The genetic material quality was evaluated based on RNA integrity (RIN) and DNA integrity (DIN) ≥7, which indicated undegraded samples, and compared with storage time, which means that for DNA storage time, samples <8.1 and ≥8.1 years and for RNA <4.5 and ≥4.5 were used. Results: A total of 190 tumor tissues were eligible for DNA and RNA extraction. Those stored for 8 years had lower DIN (68%) than those stored for a shorter period (92%). A similar pattern, based on storage time (<8.1 and ≥8.1 years), was observed in the buffy coat (74% and 95%, respectively) and PBMCs (54% and 96%, respectively). For RNA extracted from tumor tissues, we observed lower RIN in samples stored for 4.5 years (17%) than in samples stored for a shorter period (45%). Buffy coat and PBMC samples stored at -30°C exhibited greater degradation (26%) than those stored at -80°C (1%). The DIN (p = 0.15) and RNA (p = 0.18) were unrelated to topography type. Conclusion: The temperature, particularly cryopreservation methodology, and storage time were the main factors that affected nucleic acid integrity, especially RNA, during cryopreservation of biospecimens.

Superparamagnetic and highly bioactive SPIONS/bioactive glass nanocomposite and its potential application in magnetic hyperthermia.

Magnetic bioactive glass-ceramics are biomaterials applied for magnetic hyperthermia in bone cancer treatment, thereby treating the bone tumor besides regenerating the damaged bone. However, combining high bioactivity and high saturation magnetization remains a challenge since the thermal treatment step employed to grow magnetic phases is also related to loss of bioactivity. Here, we propose a new nanocomposite made of superparamagnetic iron oxide nanoparticles (SPIONs) dispersed in a sol-gel-derived bioactive glass matrix, which does not need any thermal treatment for crystallization of magnetic phases. The scanning and transmission electron microscopies, X-ray diffraction, and dynamic light scattering results confirm that the SPIONs are actually embedded in a nanosized glass matrix, thus forming a nanocomposite. Magnetic and calorimetric characterizations evidence their proper behavior for hyperthermia applications, besides evidencing inter-magnetic nanoparticle interactions within the nanocomposite. Bioactivity and in vitro characterizations show that such nanocomposites exhibit apatite-forming properties similar to the highly bioactive parent glass, besides being osteoinductive. This methodology is a new alternative to produce magnetic bioactive materials to which the magnetic properties only rely on the quality of the SPIONs used in the synthesis. Thereby, these nanocomposites can be recognized as a new class of bioactive materials for applications in bone cancer treatment by hyperthermia.

Interplay between epithelial and mesenchymal cells unveils essential proinflammatory and pro-resolutive mediators modulated by photobiomodulation therapy at 660 nm.

Photobiomodulation therapy (PBMT) has been widely used to promote tissue repair. However, PBMT's critical roles in the epithelial and mesenchymal tissues interactions are still barely known. Herein, we investigated light parameters on challenged keratinocytes (KC)-i.e., cultivated under oxidative stress-solely or associated with fibroblasts (FB) in a co-culture system. Cells were treated with PBMT at the wavelength of 660 nm, at 20 mW and 0.71 W/cm2 . Three different energy densities were primarily evaluated on KC: 1 (1.4 s), 5 (7 s), and 50 J/cm2 (70 s). Next, KC and FB were co-cultured and assessed at 5 J/cm2 . This energy density was also tested in ex vivo murine skin samples. Our main data suggest that PBMT can increase cellular proliferation at low doses and cell migration in a biphasic mode (1 and 50 J/cm2 ), both further confirmed by the epidermal growth factor receptor ligand-amphiregulin-upregulation. IL-1RA mRNA-the IL-1ß (interleukin-1ß) receptor antagonist recognized to fasten wound repair-was upregulated in the co-culture system. Upon PBMT, the ex vivo findings showed a progressive increase in the epidermal thickness, although presenting qualitatively less differentiated epithelium than the control group. In conclusion, PBMT effects are dependent on the cellular interactions with the surrounding microenvironment. Ultimately, PBMT is anti-inflammatory and contributes to the expression of critical mediators of wound repair.

Nestin and NG2 transgenes reveal two populations of perivascular cells stimulated by photobiomodulation.

Pericytes and glial cells are known to collaborate in dental pulp tissue repair. Cell-based therapies that stimulate these stromal components may be of therapeutic relevance for partially vital dental pulp conditions. This study aimed to examine the early effect of photobiomodulation (PBM) in pericytes from experimentally injured pulp tissue. To accomplish this, we used the Nestin-GFP/NG2-DsRed mice, which could allow the identification of distinct pericyte phenotypes. We discovered the presence of two pericytes subsets within the dental pulp, the Nestin + NG2+ (type-2) and Nestin- NG2+ (type-1). Upon injury, PBM treatment led to a significant increase in Nestin+ cells and pericytes. This boost was mainly conferred by the more committed pericyte subset (NestinNG2+ ). PBM also stimulated terminal blood vessels sprouting adjacent to the injury site while maintaining signs of pulp vitality. In vitro, PBM induced VEGF upregulation, improved dental pulp cells proliferation and migration, and favored their mineralization potential. Herein, different subsets of perivascular cells were unveiled in the pulp tissue. PBM enhanced not only NG2+ cells but nestin-expressing progenitors in the injured dental pulp.

The biobank of barretos cancer hospital: 14 years of experience in cancer research.

Despite the developments in cancer research over years, cancer is still one of the leading causes of death worldwide. In Brazil, the number of cancer cases for the several next years (2020-2022) is expected to increase up to 625,000. Thus, translational research has been vital to determine the potential risk, prognostic, and predictive biomarkers in cancer. Therefore, Barretos Cancer Hospital implemented a biobank (BB-BCH) in 2006, which is responsible for processing, storage, and provision of biological materials from cancer and non-cancer participants. Hence, this article aimed to describe BB-BCH's history, experiences, and outcomes and explore its impact on Brazilian translational oncology research scenario. BB-BCH has a multidisciplinary team who are responsible for guaranteeing the quality of all processes as recommended by international guidelines for biobanks. Furthermore, BB-BCH has ample equipment to ensure the quality of all material requested by researchers as genetic material (DNA and RNA) and/or entire biospecimens. From 2006 to 2019, BB-BCH contained 252,069 samples from 44,933 participants, the whole collection is represented by 15 different types of biospecimens collected from them. According to our data, the most collected and stored topography in men is head and neck (29%); in women is breast (28%); and in children is torso and limb (27%) samples. Finally, we supported national and international consortia and projects such as The Cancer Genome Atlas. BB-BCH is a vital knowledge source for scientific community, enabling the development of high-quality studies, with a wide variety of tumor categories and high national representativeness of Brazilian population.

MicroRNA Biomarkers of High-Grade Cervical Intraepithelial Neoplasia in Liquid Biopsy.

New prevention strategies are needed to detect cervical intraepithelial neoplasia (CIN). The microRNA expression analysis has already been reported as molecular biomarkers in the early detection of cervical cancer (CC) through minimally invasive samples, such as liquid biopsy, obtained through collection using liquid-based cytology (LBC). In this study, we aimed to identify molecular signatures of microRNAs in cervical precursor lesions from LBC cervical and the molecular pathways potentially associated with the CC progression. We analyzed 31 LBC cervical samples from women who underwent colposcopy. These samples were divided into two groups: the first group was composed of samples without precursor lesions of CC, considering the control group, referred to as healthy female subjects (HFS; n = 11). The second group corresponded to women diagnosed with cervical interepithelial neoplasia grade 3 (CIN 3; n = 20). We performed microRNA and gene expression profiling using the nCounter® miRNA Expression Assays (NanoString Technology) and PanCancer Pathways (NanoString Technology), respectively. A microRNA target prediction was performed by mirDIP, and molecular pathway interaction was constructed using Cytoscape. Bidirectional in silico analyses and Pearson's correlation were performed for associated the relation between genes, and miRNAs differentially expressed related cervical cancer progression were performed. We found that the expression of nine microRNAs was significantly higher, two were downregulated (miR-381-3p and miR-4531), and seven miRNAs were upregulated (miR-205-5p, miR-130a-3p, miR-3136-3p, miR-128-2-5p, let-7f-5p, miR-202-3p, and miR-323a-5p) in CIN 3 (fold change ≥ 2 and p ≤ 0.05). The miRNA expression patterns were independent of hr-HPV infection. We identified four miRNAs (miR-205-5p, miR-130a-3p, miR-4531, and miR-381-3p) that could be used as biomarkers for CIN 3 in LBC samples through multiple logistic regression analyses. We found 16 genes differentially expressed between CIN 3 and HSF samples (fold change ≥ 2 and p ≤ 0.05). We found the correlation between miR-130a-3p and CCND1(R = -0.52; p = 0.0029), miR-205-5p and EGFR (R = 0.53; p = 0.0021), and miR-4531 and SMAD2 (R = -0.54; p = 0.0016). In addition, we demonstrated the most significant pathways of the targets associated with cervical cancer progression (FDR-corrected p < 0.001). This study demonstrated that miRNA biomarkers may distinguish healthy cervix and CIN 3 and regulate important molecular pathways of carcinogenesis.

Integrated analysis of mRNA and miRNA profiles revealed the role of miR-193 and miR-210 as potential regulatory biomarkers in different molecular subtypes of breast cancer.

BACKGROUND: Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated. METHODS: The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively. RESULTS: The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3. CONCLUSIONS: In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.

BR-BCSC Signature: The Cancer Stem Cell Profile Enriched in Brain Metastases that Predicts a Worse Prognosis in Lymph Node-Positive Breast Cancer.

Brain metastases remain an unmet clinical need in breast oncology, being frequently found in HER2-overexpressing and triple-negative carcinomas. These tumors were reported to be highly cancer stem-like cell-enriched, suggesting that brain metastases probably arise by the seeding of cancer cells with stem features. Accordingly, we found that brain-tropic breast cancer cells show increased stem cell activity and tumorigenic capacity in the chick embryo choriallantoic membrane when compared to the parental cell line. These observations were supported by a significant increase in their stem cell frequency and by the enrichment for the breast cancer stem cell (BCSC) phenotype CD44+CD24-/low. Based on this data, the expression of BCSC markers (CD44, CD49f, P-cadherin, EpCAM, and ALDH1) was determined and found to be significantly enriched in breast cancer brain metastases when compared to primary tumors. Therefore, a brain (BR)-BCSC signature was defined (3-5 BCSC markers), which showed to be associated with decreased brain metastases-free and overall survival. Interestingly, this signature significantly predicted a worse prognosis in lymph node-positive patients, acting as an independent prognostic factor. Thus, an enrichment of a BCSC signature was found in brain metastases, which can be used as a new prognostic factor in clinically challenging breast cancer patients.

Deregulated microRNAs Are Associated with Patient Survival and Predicted to Target Genes That Modulate Lung Cancer Signaling Pathways.

(1) Background: Although the advances in diagnostic and treatment strategies, lung cancer remains the leading cause of cancer-related deaths, worldwide, with survival rates as low as 16% in developed countries. Low survival rates are mainly due to late diagnosis and the lack of effective treatment. Therefore, the identification of novel, clinically useful biomarkers is still needed for patients with advanced disease stage and poor survival. Micro(mi)RNAs are non-coding RNAs and potent regulators of gene expression with a possible role as diagnostic, prognostic and predictive biomarkers in cancer. (2) Methods: We applied global miRNA expression profiling analysis using TaqMan® arrays in paired tumor and normal lung tissues (n = 38) from treatment-naïve patients with lung adenocarcinoma (AD; n = 23) and lung squamous cell carcinoma (SCC; n = 15). miRNA target genes were validated using The Cancer Genome Atlas (TCGA) lung AD (n = 561) and lung SCC (n = 523) RNA-Seq datasets. (3) Results: We identified 33 significantly deregulated miRNAs (fold change, FC ≥ 2.0 and p < 0.05) in tumors relative to normal lung tissues, regardless of tumor histology. Enrichment analysis confirmed that genes targeted by the 33 miRNAs are aberrantly expressed in lung AD and SCC, and modulate known pathways in lung cancer. Additionally, high expression of miR-25-3p was significantly associated (p < 0.05) with poor patient survival, when considering both tumor histologies. (4) Conclusions: miR-25-3p may be a potential prognostic biomarker in non-small cell lung cancer. Genes targeted by miRNAs regulate EGFR and TGFß signaling, among other known pathways relevant to lung tumorigenesis.

Knowledge, attitudes, and practices of undergraduate students concerning Regenerative Endodontics.

BACKGROUND: Knowledge, attitudes, and beliefs of the students are important for the Tissue Engineering in Endodontic practice. The opinion of these future dentists would ultimately will decide the endurance of REPs as routine procedures in endodontic practice. The aim of this study was to perform a survey to identify the knowledge, attitudes, and practices of undergraduate students about regenerative endodontic procedures (REPs). METHODS: The questionnaire was obtained after cross-cultural adaptation of a questionnaire previously applied in USA and was applied to two hundred forty-eight undergraduates. Data were statistically analyzed. RESULTS: Most of the students (82.9%) agreed that regenerative therapy should be incorporated to dentistry and 87.5% of them believed that stem cells banks would be useful for the tissue regeneration. Most participants (58.1%) would like to obtain an internship/tutoring that addresses REPs and 80.8% of participants think that the major obstacle to a patient accepting a REP was the expected high cost of the treatment. The freshmen students were more optimistic about offering stem cell treatments to their patients (P≤0.05). CONCLUSIONS: The undergraduates were very optimistic about the future of REPs, stem cell banking, and tissue engineering. Although seniors demonstrated less enthusiasm towards REPs than the freshman, most students are willing to recommend these treatments to their patients.

Bioactive glass and high-intensity lasers as a promising treatment for dentin hypersensitivity: An in vitro study.

This in vitro study aimed to analyze the physical and chemical characteristics of the hypersensitive human dentin-like surface after application of a bioactive glass (BG) paste (BG/Ac) irradiated or not with high-power lasers. Dentin specimens were treated with 17% Ethylenediamine tetraacetic acid (EDTA) solution to mimic a hypersensitive dentin and then submitted to neodymium: yttrium-aluminum-garnet (Nd:YAG) laser or CO2 laser irradiation prior and after application of BG/Ac. Characterizations were performed by using X-ray diffraction, Fourier transformed infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy. The results suggested that application of BG/Ac by itself caused some obstructions of dentinal tubules. Nd:YAG laser irradiation reduced the opening of the dentinal tubules with no changes in the collagen structure. CO2 laser irradiation caused dentin melting and resolidification along with cracks and chemical changes in collagen fibers. However, when BG/Ac paste was irradiated with lasers, a sequence of surface reactions between glass and dentin interface led to the formation of an amorphous hydroxyapatite layer, similar to that of an inorganic component of the normal dentin. Moreover, BG/Ac was able to prevent the formation of cracks and degradation of collagen fibers caused by CO2 irradiation. Overall, this study supports that application of BG/Ac paste irradiated by high-power laser could represent an effective and long-lasting therapeutic approach for dentin hypersensitivity.

Identification of Cell-Free Circulating MicroRNAs for the Detection of Early Breast Cancer and Molecular Subtyping.

Early detection is crucial for achieving a reduction in breast cancer mortality. Analysis of circulating cell-free microRNAs present in the serum of cancer patients has emerged as a promising new noninvasive biomarker for early detection of tumors and for predicting their molecular classifications. The rationale for this study was to identify subtype-specific molecular profiles of cell-free microRNAs for early detection of breast cancer in serum. Fifty-four early-stage breast cancers with 27 age-matched controls were selected for circulating microRNAs evaluation in the serum. The 54 cases were molecularly classified (luminal A, luminal B, luminal B Her2 positive, Her-2, triple negative). NanoString platform was used for digital detection and quantitation of 800 tagged microRNA probes and comparing the overall differences in serum microRNA expression from breast cancer cases with controls. We identified the 42 most significant (P ≤ 0.05, 1.5-fold) differentially expressed circulating microRNAs in each molecular subtype for further study. Of these microRNAs, 19 were significantly differentially expressed in patients presenting with luminal A, eight in the luminal B, ten in luminal B HER 2 positive, and four in the HER2 enriched subtype. AUC is high with suitable sensitivity and specificity. For the triple negative subtype miR-25-3p had the best accuracy. Predictive analysis of the mRNA targets suggests they encode proteins involved in molecular pathways such as cell adhesion, migration, and proliferation. This study identified subtype-specific molecular profiles of cell-free microRNAs suitable for early detection of breast cancer selected by comparison to the microRNA profile in serum for female controls without apparent risk of breast cancer. This molecular profile should be validated using larger cohort studies to confirm the potential of these miRNA for future use as early detection biomarkers that could avoid unnecessary biopsy in patients with a suspicion of breast cancer.

Antibacterial effects and cytotoxicity of an adhesive containing low concentration of silver nanoparticles.

OBJECTIVES: To evaluate the antibacterial effects, cytotoxicity and microtensile bond strength of an adhesive containing low concentrations of silver nanoparticles (NAg). METHODS: Various concentrations of NAg (50, 100, 150, 200 and 250 ppm) were incorporated into the primer of the Scotchbond Multi-Purpose adhesive system (SBMP). Antibacterial activity was examined using a broth microdilution assay to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), agar diffusion assay and the MTT assay was used to examine the biofilm metabolic activity (S. mutans). The Microtensile Bond Test (µTBS) was performed after 24 h, followed by 6-months storage in distilled water. Cytotoxicity was assessed with an MTT reduction assay in human dental pulp stem cells viability after exposure to Nag-conditioned culture media during 0, 24, 48, and 72 h. The results were statistically analyzed (α ≤ 0.05). RESULTS: MIC was found between NAg 25 and 50 ppm MBC was determined at 50 ppm of NAg. Bacterial activity inhibition was higher than control in all NAg groups compared to control in agar diffusion assay. Biofilm inhibition was statistically higher in 250 ppm NAg than control. All NAg groups and SBMP presented similar cytotoxicity in each period. Adhesives with NAg 200 and 250 ppm and SBMP (control) presented the highest µTBS values, similar to that of SBMP control, in both instances (24 h and 6 months) (p > 0.05). CONCLUSIONS: The commercial primer containing NAg 250 ppm showed both antibacterial effect and reliable bond strength with no cytotoxicity increase. The addition of NAg to primers seems promising for the improvement of conventional dental adhesives efficacy. CLINICAL SIGNIFICANCE: The addition of low concentrations of NAg (250 ppm) to primers were effective to improve antibacterial effect preserving the bond strength and the biocompatibility of the commercial product. NAg/primer association could protect the tooth-adhesive interface increasing dental restoration longevity.

Expression of tumor suppressors miR-195 and let-7a as potential biomarkers of invasive breast cancer.

OBJECTIVE: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the posttranscriptional level. Some miRNAs, including let-7a and miR-195, have been described as tumor suppressors. However, the roles of these microRNAs in breast cancer progression remain controversial. The aim of this study is to evaluate miR-195 and let-7a expression as potential biomarkers of invasive breast cancer. METHODS: In the present study, 200 individuals were separated into three groups: (i) 72 women constituting the control group who were selected according to rigorous and well-established criteria; (ii) 56 patients with benign breast tumors; and (iii) 72 patients with malignant breast cancers of different clinical stages. The miR-195 and let-7a expression levels in serum were evaluated by real-time PCR. The results were assessed alone and in combination, and the analysis included an estimation of sensitivity and specificity in ROC curves. RESULTS: Compared with the benign and control groups, both microRNAs were downregulated in the malignant breast cancer patient group. Compared with the malignant group, the combination of both biomarkers in the control and benign groups showed good sensitivity and specificity in the serum with AUCs of 0.75 and 0.72, respectively. The biomarker combination for the control group versus the malignant group exhibited a better sensitivity and specificity than for the benign group versus the malignant group. CONCLUSION: These findings support the evidence that the analysis of miR-195 and let-7a can be used as a non-invasive biomarker for breast cancer detection.

Photobiomodulation therapy and vitamin C on longevity of cell sheets of human dental pulp stem cells.

Photobiomodulation therapy (PBMT) can improve processes relevant to tissue regeneration, such as survival, proliferation, migration, and differentiation of cells, including stem cells. Thus, PBMT could be applied as auxiliary therapy for tissue regeneration. Cell sheets (CSs) induced by vitamin C (VC) can generate large amount of cells, which would also be useful for tissue regeneration. VC and PBMT cause opposite effects on cell metabolism (e.g., VC is antioxidative, and PBMT generates reactive oxygen species); however, hDPSC CSs were formed when VC and PBMT+VC were applied. Thus, this study showed that PBMT does not interfere with the formation of cell sheets induced by VC. Additionally, PBMT improved the functional differentiation of the cells isolated from the CSs. Thus, due to the clinical benefits of PBMT, the association of this therapy with cell sheets seems promising for future applications in tissue regeneration.

MicroRNA-27a-5p regulation by promoter methylation and MYC signaling in prostate carcinogenesis.

Upregulation of MYC and miRNAs deregulation are common in prostate cancer (PCa). Overactive MYC may cause miRNAs' expression deregulation through transcriptional and post-transcriptional mechanisms and epigenetic alterations are also involved in miRNAs dysregulation. Herein, we aimed to elucidate the role of regulatory network between MYC and miRNAs in prostate carcinogenesis. MYC expression was found upregulated in PCa cases and matched precursor lesions. MicroRNA's microarray analysis of PCa samples with opposed MYC levels identified miRNAs significantly overexpressed in high-MYC PCa. However, validation of miR-27a-5p in primary prostate tissues disclosed downregulation in PCa, instead, correlating with aberrant promoter methylation. In a series of castration-resistant PCa (CRPC) cases, miR-27a-5p was upregulated, along with promoter hypomethylation. MYC and miR-27a-5p expression levels in LNCaP and PC3 cells mirrored those observed in hormone-naíve PCa and CRPC, respectively. ChIP analysis showed that miR-27a-5p expression is only regulated by c-Myc in the absence of aberrant promoter methylation. MiR-27a-5p knockdown in PC3 cells promoted cell growth, whereas miRNA forced expression in LNCaP and stable MYC-knockdown PC3 cells attenuated the malignant phenotype, suggesting a tumor suppressive role for miR-27a-5p. Furthermore, miR-27a-5p upregulation decreased EGFR/Akt1/mTOR signaling. We concluded that miR-27a-5p is positively regulated by MYC, and its silencing due to aberrant promoter methylation occurs early in prostate carcinogenesis, concomitantly with loss of MYC regulatory activity. Our results further suggest that along PCa progression, miR-27a-5p promoter becomes hypomethylated, allowing for MYC to resume its regulatory activity. However, the altered cellular context averts miR-27a-5p from successfully accomplishing its tumor suppressive function at this stage of disease.

Dynamic Influence of pH on Metalloproteinase Activity in Human Coronal and Radicular Dentin.

The aim of this study was to evaluate the effect of pH on the activation of matrix metalloproteinases (MMPs) of human coronal (CD) and radicular dentin (RD). CD and RD were pulverized to powder, and proteins were extracted with 1% phosphoric acid. The extracted proteins and the demineralized powder were separately incubated in the following solutions: 4-aminophenylmercuric acetate (control) or a buffer solution at different pHs (2.5, 4.5, 5.0, 6.0, and 7.0). After incubation, proteins were separated by electrophoresis to measure MMP activities by zymography. To assess the solubilized dentin collagen, the demineralized dentin powder was sustained in incubation buffer, and the amount of hydroxyproline (HYP) released was measured. Zymography revealed MMP-2 gelatinolytic activities for CD and RD in all experimental groups. For both substrates, the lowest pH solutions (2.5, 4.5, and 5.0) yielded higher gelatinolytic activity than those obtained by the highest pH solutions (6.0 and 7.0). For HYP analysis, no detectable absorbance values were observed for pHs of 2.5 and 4.5. The amount of HYP was higher for pH 7.0 than those of all other groups (p < 0.05), except for pH 6.0. No statistical differences were found between pHs 6.0 and 5.0 and control (p > 0.05). The MMP-2 enzyme from human CD and RD is dynamically influenced by pH: at low pH, the extracted enzyme activates this latent form, whereas collagen degradation by the matrix-bound enzyme is only observed when pHs are close to neutral.