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Introduction The development of factor VIII (FVIII) inhibitor is the main complication of replacement therapy in patients with haemophilia A ( HA). A ratio of 5-7% of individuals HA develops antibodies (inhibitors) against the FVIII infused during the treatment, thereby reducing their pro-coagulant activity. The immunomodulatory cytokine genes have been related to the risk of development of alloantibodies in several studies, mainly in HA with severe form. Aim We investigated the polymorphisms in regulatory regions of cytokine genes ( IL1A, IL1B, IL1R, IL1RA, IL4RA, IL12, INFG, TGFB1, TNF, IL2, IL4, IL6, IL10) that could influence the risk of developing inhibitors in patients with severe HA. Methods The genotyping of cytokine genes of 117 patients with HA was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP) using the protocol recommended by the manufacturer (Invitrogen kit Cytokines®, Canoga Park, USA) Results From the cohort of 117 patients with severe HA, 35 developed inhibitors. There was a higher frequency of +874 T allele in INFG and of +869 TT and TG/TG in TGFB1 genes on patients with inhibitors. Conclusion This suggests that polymorphisms in INFG and in TGFB1 genes are related to risk of developing inhibitor, and could contribute to a genetic profile of the individual HA for the risk of inhibitors development to FVIII. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to analyze the ability of G-CSF and TGF-β1 to mobilize periodontal ligament stem cells to obtain populations with better potential for proliferation and osteogenic differentiation., Design: Primary cultures were established from the periodontal ligament of Wistar rats. After a cell migration assay, four experimental groups were obtained: PDLSC, composed of the primary culture, non-mobilized cells; MPDLSC, the spontaneously migrated cells; MPDLSC-GCSF, the cells mobilized with G-CSF; and MPDLSC-TGF-β1, the cells mobilized with TGF-β1. The expression of mesenchymal stem cell markers was assessed by flow cytometry. Clonogenicity, viability, proliferative potential, and osteogenic differentiation capacity were also analyzed., Results: All the study groups expressed well-known mesenchymal stem cell markers and exhibited clonogenic capacity. The higher proliferation potential was seen in the PDLSC and MPDLSC groups, while the MPDLSC and MPDLSC-TGFβ1 groups showed a higher number of mineralized deposits in vitro and higher ALP activity after osteogenic differentiation induction. Cells of all the groups also expressed mRNA of genes associated with osteogenic differentiation without previous induction., Conclusions: Both agents were able to mobilize stem cells from the periodontal ligament, but G-CSF did not show an advantage, whereas TGF-β1 appears to direct the cells towards a state of increased osteogenic differentiation. Furthermore, spontaneous cell migration through a membrane was sufficient to enrich the cell population., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The aim of this study was to analyze the effect of chronic ethanol ingestion on dendritic cell repopulation during the repair process of rat oral mucosa and in the rat spleen by analyzing the immunohistochemical expression of dendritic cell markers. Wistar rats ingested 20% ethanol solution for 28 days; a surgical wound was performed on the rat tongue after this period. The repair process and the number of CD1a+, CD11c+, and CD207+ cells in the regions adjacent to the wound were determined at day 1, 3, and 7 following the wound as well as in the rat spleen. The wound-only group (no ethanol exposure) had complete reepithelization after 7 days, but this did not occur in the ethanol + wound group at this time point. The inflammatory infiltrate was significantly reduced in animals exposed to ethanol, which also showed significantly lower counts of CD1a+, CD11c+, and CD207+ cells than the wound-only group at all experimental time points. In addition, ethanol exposure also resulted in lower densities of CD11c+ and CD207+ cells in the rat spleen. In conclusion, chronic ethanol intake had a negative impact on dendritic cell numbers, a fact that may contribute to delay in oral mucosa repair., (© 2022 Scandinavian Division of the International Association for Dental Research. Published by John Wiley & Sons Ltd.)

Mesenchymal and epithelial stem cells were identified in dental tissues; however, knowledge about the odontogenic stem cells is limited, and there are some questions regarding their temporo-spatial dynamics in tooth development., Objective: Our study aimed to analyze the expression of the stem cell markers CD146 and p75NTR during the different stages of odontogenesis., Methodology: The groups consisted of 13.5, 15.5, 17.5 days old embryos, and 14 days postnatal BALB/c mice. The expression of CD146 and p75NTR was evaluated by immunohistochemistry., Results: Our results showed that positive cells for both markers were present in all stages of tooth development, and the number of positive cells increased with the progression of this process. Cells of epithelial and ectomesenchymal origin were positive for CD146, and the expression of p75NTR was mainly detected in the dental papilla and dental follicle. In the postnatal group, dental pulp cells were positive for CD146, and the reduced enamel epithelium and the oral mucosa epithelium showed immunostaining for p75NTR., Conclusions: These results suggest that the staining pattern of CD146 and p75NTR underwent temporal and spatial changes during odontogenesis and both markers were expressed by epithelial and mesenchymal cell types, which is relevant due to the significance of the epithelial-ectomesenchymal interactions in tooth development.

In order to know the behaviour and performance of a vertical flow constructed wetland (VF-CW) operating with earthworm, this study evaluated the earthworm presence linked to hydraulic loading rates applied for domestic wastewater treatment. Two VF-CW units (710 cm², 0.75 m deep, with sand as filter media and planted with Heliconia rostrata ) operated with a variable hydraulic and organic loadings rates (200 mm d -1 /123 g COD m -2  d -1 ; 280 mm d -1 /186 g COD m -2  d -1 ; 160 mm d -1 /94 g COD m -2 d -1 ). Although the efficiency of COD load removal was similar (around 70%) for both CWs, the efficiency of total nitrogen load removal was around 95% throughout the evaluated period. The nitrification-denitrification process was identified with and without the presence of earthworm. VF-CW with earthworms showed higher hydraulic conductivity values (from 0.11 to 0.14 m h -1 ) compared with the VF-CW wetland without earthworms (0.07-0.09 m h -1 ). This study showed that the use of earthworms in CWs can be associated mainly with a preventive measure of clogging, which requires measures to maintain earthworms inside the bed media. Additionally, the earthworm presence has an ingestion mechanism of organic and inorganic solid particles in wastewater which excretes them as finer particles.

Inflammatory periodontal disease known as periodontitis is one of the most common conditions that affect human teeth and often leads to tooth loss. Due to the complexity of the periodontium, which is composed of several tissues, its regeneration and subsequent return to a homeostatic state is challenging with the therapies currently available. Cellular therapy is increasingly becoming an alternative in regenerative medicine/dentistry, especially therapies using mesenchymal stem cells, as they can be isolated from a myriad of tissues. Periodontal ligament stem cells (PDLSCs) are probably the most adequate to be used as a cell source with the aim of regenerating the periodontium. Biological insights have also highlighted PDLSCs as promising immunomodulator agents. In this review, we explore the state of knowledge regarding the properties of PDLSCs, as well as their therapeutic potential, describing current and future clinical applications based on tissue engineering techniques., Competing Interests: Conflict-of-interest statement: There is no conflict of interest associated with any author and/or coauthor that contributed to this manuscript., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

A vertical subsuperficial tidal flow constructed wetland (TFCW) operated under flooded time (FT) variation, was evaluated in the removal of carbonaceous, nitrogenous, and phosphorous matter from urban stream water. The TFCW downflow (117 L) was filled with bricks (44% porosity) and vegetated with Althernanthera philoxeroides (32 plants m -2 ). The TFCW was operated under different flooded times - Stage A (48 h), B (36 h), C (24 h), and D (12 h), organic loading rates of 19.58-43.83 gCOD m -2 d -1 , 3.68-6.94 gTN m -2 d -1 and 0.93-2.00 gTP m -2 d -1 and volumetric load rates of 46.8, 58.5, 78.0 and 11.7 L d -1 . No significant differences were observed in the removal efficiencies to Chemical Oxygen Demand (COD 66 to 94%), Total Ammonia Nitrogen (TAN 58 to 87%), and Total Nitrogen (TN 53 to 78%) among the stages, and nitrate concentrations lower than 6 mg L -1 in the effluent. High Total Phosphorus removal was obtained in FT of 48 h (TP 79%). Total phosphorus loading rate was a limiting factor in TP removal, which reduced along with the reduction of FT. The nitrifying community was present over time since ammonia-oxidizing bacteria (Nitrosospira) and nitrite-oxidizing bacteria (Nitrobacter and Nitrospira) were identified in operational stages with variation in relative abundance, but TAN removal efficiency did not show significant differences. There was no change in the denitrifying community structure, indicating that FT did not influence the TN removal. A. philoxeroides was responsible for phytoextraction of 2.1% of TN and 2.7% of TP from the total removed by TFCW. TN removal (65%) was attributed to adsorption in the filtering material and microbial metabolism during the rest time. The findings of this study suggest FT of 12 h to remove COD and TN, and equal to or higher than 48 h to remove TP., Competing Interests: Declaration of competing interest This statement is to certify that all Authors have seen and approved the manuscript being submitted. We have no conflict of interest to declare. We warrant that the article is the Author's original work. We warrant that the article has not received prior publication and is not under consideration for publication elsewhere. On behalf of all Co-Authors, the corresponding Author shall bear full responsibility for the submission. We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This research has not been submitted for publication nor has it been published in whole or in part elsewhere. We attest to the fact that all Authors listed on the title page have contributed significantly to the work, have read the manuscript and attest to the validity and legitimacy of the data and its interpretation, and agree to its submission to the Science of the Total Environment. All authors agree that the author list is correct in its content and order that no modification to the author list can be made without the formal approval of the Editor-in-Chief, and all authors accept that the Editor-in-Chief's decisions over acceptance or rejection or in the event of any breach in the Ethics in publishing and Policies and Ethics in the Science of the Total Environment. No additional authors will be added post submission, unless editors receive agreement from all authors and detailed information is supplied as to why the author list should be amended., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study aimed to evaluate the influence of two different feeding modes on wastewater treatment performance and oxygen consumption rate (OCR) of the unsaturated (UVF wetland) and partially saturated (SVF wetland) vertical flow constructed wetlands operated in parallel under subtropical climate for four years. Each wetland had a superficial area of 7.5 m 2 and was planted with Typha domingensis. Both units have a filter media depth of 0.75 m, composed by sand (effective diameter of 0.29 mm and uniformity of 4). UVF wetland operated typically unsaturated, while SVF wetland had the bottom part saturated (57% of total depth). Two feeding modes were evaluated for both wetlands. The feeding mode was operated within the limits recommended by the German standard, with a hydraulic loading rate (HLR) of 75 mm d -1 and specific pulse volume (SPV) of 19 L m -2 for both wetlands and a specific hydraulic loading rate (SHLR) of 8 and 9 L m -2  min -1 for UVF and SVF wetlands, respectively. Meanwhile, the second feeding mode was applied for both wetlands, being an HLR of 103 mm d -1 , 26 L m -2 of SPV, and 4 L m -2  min -1 of SHLR. The load removal efficiency of SVF wetland was higher than the UVF wetland for all parameters and feeding modes. No statistical difference was identified for OCR values between wetlands and feeding mode. The results showed that operating UVF and SVF wetlands with a SHLR around 4 L m -2  min -1 and SPV equal of 26 L m -2 is preferable. This fact could represent a significant reduction in inlet pumping power requirements and also less superficial area requirements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Aiming at more effective and safer cell therapies, the objective of this study was to evaluate the biological properties of human apical papilla cells cultured in the absence of serum supplementation in comparison to cells cultured with fetal bovine serum (FBS)., Design: Two apical papilla cell populations were isolated from third molars with incomplete rhizogenesis, and cultured in four different media: minimum essential Eagle medium - alpha modification (alpha-MEM); alpha-MEM supplemented with FBS (alpha-MEM + FBS); Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12); and DMEM/F12 supplemented with FBS (DMEM/F12 + FBS). We evaluated their proliferation, clonogenicity, and in vitro osteogenic and chondrogenic differentiation potential., Results: Apical papilla cells cultured in DMEM/F12 + FBS and alpha-MEM + FBS were more proliferative than those grown in serum-free media, and also exhibited greater efficiency in colony cell formation. Despite this, all study groups showed immunostaining for the marker of mitosis anti-PHH3. Also, alpha-MEM + FBS, alpha-MEM, and DMEM/F12 + FBS exhibited higher amount of mineralized deposits in vitro than DMEM/F12, while only cells cultured with FBS were able to form spheres in chondrogenic differentiation assay., Conclusions: Our results showed that, although the cultivation of apical papilla cells in a serum-free medium has reduced the properties of cell proliferation and differentiation, these cells are still capable of maintaining their desirable characteristics., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Data on the occurrence and clinicopathological characteristics of actinic cheilitis (AC) and lip squamous cell carcinoma (LSCC) are well studied; however, they are based on studies limited to a single centre. Herein, we described the frequency of AC and LSCC submitted to microscopic examination from representative geographic regions of Brazil. A retrospective multicentre study was performed on biopsies obtained from 1953 to 2018 at 10 Brazilian oral and maxillofacial pathology centres. A total of 198,709 biopsy specimens were surveyed. Sociodemographic data and clinicopathologic characteristics were analysed. A total of 2017 cases of ACs (1.0%) and 850 cases of LSCCs (0.4%) were recorded. A strong fair-skinned (> 87%) male (> 70%) predilection was observed in both conditions. The mean age was 54.8 ± 18.7 for individuals with AC and 57.8 ± 19.0 for individuals with LSCC. The most commonly affected site was the lower lip (> 90%). This is a large multicentre study of AC and LSCC from Brazil. The frequency and clinicopathological features of AC and LSCC were similar to those described worldwide. This study provides robust and representative epidemiological data of these conditions for the scientific community.

Granular activated carbon (GAC) fixed bed adsorption technology was applied to remove carbamates carbaryl, methomyl and carbofuran from public water supply. In order to minimize the effect of clogging and to evaluate adsorbent saturation for carbamates, the microfiltration (MF) was previously used to adsorb and the backwash procedure of the GAC bed was carried out. The determination and quantification of the carbamates were performed by analytical technique in high performance liquid chromatography with post-column derivatization and fluorescence detector. The MF of the water previously adsorbed in the GAC fixed bed allowed the greater removal of 100% of the carbamates pesticides with an initial concentration of 25 μg L -1 during the first 48 h of operation. The saturation of the GAC fixed bed occurred in 240 h, due to the partial removal of the natural organic matter by the MF, consequently the competition for adsorptive sites of the GAC was smaller. The backwashing procedure contributed to the partial recovery of the performance of the hydraulic filtration and allowed to identify the saturation of the adsorption column, since the sealing phenomenon preceded the saturation. Finally, the use of public water supply was considered a relevant and positive aspect, since it allowed the identification of the performance of this technology in the removal of carbamates pesticides considering the presence of substances inherent to the public water supply.

The reduction of greenhouse gases (GHG) emissions is important challenge in the wastewater treatment plants. In this way, the present study aimed to evaluate the GHG emissions and carbon dioxide fixation by duckweed ponds (DWP) applied to treat municipal wastewater in a polishing stage. Two pilot DWP (3000 L) were operated in a series with real wastewater receiving a flow rate of 200 L d -1 and organic load rate of 39 g COD ha -1 d -1 . Beyond the standard physicochemical parameters for wastewater monitoring, the gases emissions from pond surface were measures by using a static flux chamber with infrared probes installed inside to detect CO 2 and CH 4 concentration. Operating the DWP with a load of 18.1 kg TN ha -1 d -1 and 2 kg TP ha -1 d -1 , across 425 days of monitoring, higher COD and nutrient removal efficiency was identified (79%, 93% and 84% for COD, TN and TP, respectively). The CO 2 emission rate ranged from 3048 to 6017 mg CO 2 m -2 d -1 and the fixation rate ranged from 19,592 to 42,052 mg CO 2 m -2 d -1 . Methane emission was not detected (less than 0.1%). Moreover, low abundance of archaeal community was identified in both DWP. The results showed that in presented conditions, under low COD loading rate DWP could fix at least three times more CO 2 than it emits, highlighting the sustainability of this natural technology., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The oxygen transfer rate (OTR) has a significant impact on the design and operation of vertical flow constructed wetlands (VFCWs) intended for organic matter removal and nitrification. Despite its key role, the information on real oxygen input in VFCWs is limited, being usually estimated by mass balance (stoichiometry), through which is calculated only the oxygen consumption rate (OCR). In this study, for the first time, the gas tracer method was applied to evaluate the oxygen transfer capacity of a real-scale VFCW (24.5 m 2 ) applied to the treatment of domestic wastewater. Propane was used as tracer. The OCR and the OTR were evaluated in VFCW under hydraulic loading rates (HLR) of 60, 90, and 120 mm d -1 corresponding to recirculation rations of 0%, 50%, and 100%. The OTR in standard conditions (20 °C) ranged from 120 to 176 g O 2  m -2  d -1 . The highest OTR was found for the lowest HLR. For the operating conditions tested, the OTR obtained with gas tracer were higher than the OCR calculated by stoichiometry in VFCW, which ranged from 20.6 to 27.8 g O 2  m -2  d -1 . Besides, the OTR were sufficient to satisfy the VFCW oxygen demand for organic matter removal and nitrification. These results show that the gas tracer method for OTR determination may allow advances on the understanding of treatment processes and on the design of new VFCWs since its treatment performance requires aerobic conditions., (Copyright © 2019. Published by Elsevier B.V.)

Expression of proteins related to cell surveillance has been described in tumors presenting resistance to photodynamic therapy (PDT). The aim of this study was to verify whether there was upregulation of proteins related to resistance in oral squamous cell carcinoma (OSCC) after PDT. OSCC was chemically induced in rats and treated after one cycle of PDT mediated by 5-aminolevulinic acid (5-ALA-PDT). Immunolabeling of p-NFκB, Bcl-2, survivin, iNOS, p-Akt, p-mTOR and cyclin D1 was performed after the treatment. There was increased expression of Bcl-2 (P = 0.008), iNOS (P = 0.020), p-Akt (P = 0.020) and p-mTOR (P = 0.010) by surviving neoplastic cells after PDT when compared to the control. In conclusion, after one cycle of 5-ALA-mediated PDT, Bcl-2, p-Akt, p-mTOR and iNOS were upregulated in neoplastic cells of OSCC, suggesting an activation of antiapoptosis and cell proliferation pathways. This fact must be considered in the establishment of PDT protocols for OSCC treatment, mainly those in which PDT will be combined with chemotherapy drugs targeted at the studied proteins., (© 2018 The American Society of Photobiology.)

Introduction: Dental pulp is a complex tissue with highly differentiated cells, which makes its reconstruction a challenging task. The apical papilla is an undifferentiated tissue considered as the remnant of the dental papilla that forms the dentin-pulp complex. Aiming to analyze morphologic features of the tissue formed in an in vivo pulp model, we used human apical papilla as a cell source without the use of exogenous growth factors., Methods: A construct was built using newborn mice molar crowns treated with TrypLE (Fisher Scientific, Loughborough, UK) and EDTA. The crowns were filled with PuraMatrix (Corning Inc, Corning, NY) and a pool population of human apical papilla cells. As a control, we used crowns filled only with PuraMatrix and empty crowns. The constructs were transplanted under severe combined immunodeficient mice kidney capsules. Immunohistochemistry for lamin A, dentin sialophosphoprotein, and dentin matrix protein 1 was performed., Results: Morphologic analysis of all transplanted crowns showed the formation of a loose connective tissue of variable cellularity with the presence of well-formed functional vessels. In the study group, lamin A-positive cells represented the majority of cells within the pulp chamber and a few cells in the vessel lining. We also found positivity for dentin sialophosphoprotein and dentin matrix protein 1, an indicator of odontoblast differentiation., Conclusions: In our study model, human transplanted apical papilla cells mixed with the host cells and formed a vascularized viable tissue, and these cells were able to differentiate into odontoblastlike cells without the use of exogenous growth factors., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Nitrogen dynamics and its association to metabolically active microbial populations were assessed in two vertical subsurface vertical flow (VF) wetlands treating urban wastewater. These VF wetlands were operated in parallel with unsaturated (UVF) and partially saturated (SVF) configurations. The SVF wetland exhibited almost 2-fold higher total nitrogen removal rate (5 g TN m -2 d -1 ) in relation to the UVF wetland (3 g TN m -2 d -1 ), as well as a low NO x -N accumulation (1 mg L -1 vs. 26 mg L -1 in SVF and UVF wetland effluents, respectively). After 6 months of operation, ammonia oxidizing prokaryotes (AOP) and nitrite oxidizing bacteria (NOB) displayed an important role in both wetlands. Oxygen availability and ammonia limiting conditions promoted shifts on the metabolically active nitrifying community within 'nitrification aggregates' of wetland biofilms. Ammonia oxidizing archaea (AOA) and Nitrospira spp. overcame ammonia oxidizing bacteria (AOB) in the oxic layers of both wetlands. Microbial quantitative and diversity assessments revealed a positive correlation between Nitrobacter and AOA, whereas Nitrospira resulted negatively correlated with Nitrobacter and AOB populations. The denitrifying gene expression was enhanced mainly in the bottom layer of the SVF wetland, in concomitance with the depletion of NO x -N from wastewater. Functional gene expression of nitrifying and denitrifying populations combined with the active microbiome diversity brought new insights on the microbial nitrogen-cycling occurring within VF wetland biofilms under different operational conditions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: The aim of this work was to evaluate the expression of the cancer stem cell (CSC) markers CD44, ALDH1 and p75NTR in the ultraviolet-induced lesions actinic cheilitis (AC) and lip squamous cell carcinoma (LSCC), and to correlate it with p53 expression., Methods: Immunohistochemistry was performed in 4 cases of normal lip (NL), 43 of AC and 20 of LSCC., Results: All cases were positive for CD44, showing a membranous staining without differences between the groups. ALDH1 showed cytoplasmic staining and it was invariable amongst the grades of epithelial dysplasia and between AC and LSCC. p75NTR presented membranous/cytoplasmic staining in the basal and parabasal layer of NL and AC, while LSCC presented cytoplasmic staining in the peripheral layers of the tumor islands. p75NTR showed different expression amongst the dysplasia grades (p < 0.001) but no differences between AC and LSCC. p53 expression was similar amongst the dysplasia grades and between AC and LSCC. CD44, ALDH1 and p75NTR were unrelated amongst themselves and to p53 expression., Conclusions: CSC markers are expressed in potentially malignant and malignant lesions of the lip. Their expressions were invariable between AC and LSCC and unrelated to p53. p75NTR expression increased with the worsening of epithelial dysplasia grade.

Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic ® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic ® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering., (© 2017 Wiley Periodicals, Inc.)

Photodynamic therapy (PDT) has been indicated for oral squamous cell carcinoma (OSCC) at early stages. Chemo and radio-resistance are frequently observed in OSCC, but it is unknown whether this tumor can develop resistance to PDT. It was investigated the process of acquiring resistance to multiple cycles of PDT by using OSCC cells. We also analyzed the expression of anti-apoptotic proteins and those related to Akt/mTOR pathway. Sub-lethal doses of PDT were applied, consisting of a constant concentration of 5-aminolevulinic acid (5-ALA) (1 mM, 4-h incubation) and increasing irradiation dose with LED (from 5.86 to 10.54 J/cm 2 ). Cell viability, migration capacity, intracellular expression of protoporphyrin IX (PpIX), mitochondrial density, and pro-survival proteins were investigated in PDT-resistant cells. Six OSCC cell generations resistant to PDT were isolated. The resistant cells exhibited a survival phenotype characterized by a reduction in the expression of endogenous PpIX, increase in mitochondrial density, increase in migration capacity, and up-regulation of p-NFκB, p-survivin, iNOS, p-Akt Ser 473 , cyclin D1, p-mTOR Ser 2481 , and p-mTOR Ser 2448 . OSCC cells are able to survive doses of 5-ALA-PDT by reducing PpIX synthesis and activating signaling pathways related to the inhibition of apoptosis and improvement of cell proliferation. Further studies are necessary to confirm whether this PDT-resistance phenotype can be clinically present, mainly in OSCC showing clinical recurrences after exposure to different PDT protocols., (© 2017 Wiley Periodicals, Inc.)

The effect of effluent recirculation on the removal of total nitrogen (TN) and eight pharmaceuticals and personal care products (PPCPs) was evaluated during 9months in an experimental hybrid constructed wetland (CW) system applied in the treatment of urban wastewater. An Imhoff tank was followed by three stages of CWs (two 1.5-m 2 vertical subsurface flow (VF) beds alternating feed-rest cycles, a 2-m 2 horizontal (HF) and a 2-m 2 free water surface (FWS) wetland in series). A fraction of the final effluent was recycled back to the Imhoff tank with a recirculation rate of 50% (hydraulic loading rate=0.37md -1 ). The system's performance varied throughout the study. In Period I (summer) consistently high load removal efficiencies of TN (89±5%) and a removal rate of 6.6±1.4gTNm -2 d -1 were exhibited. In Period II (fall), the poor performance of the FWS during the senescence of macrophytes caused a large increase in organic matter, solids and nutrient concentrations, drastically deteriorating water quality. The determination of PPCPs was conducted during this period. Recalcitrant compounds, namely sulfamethoxazole, carbamazapine, TCEP and sucralose were negligibly removed in all CWs. However, noteworthy was the ≈30% removal of sucralose in the VF wetland. Caffeine (80%) and fluoxetine (27%) showed similar elimination rates in both VF and HF units, whereas trimethoprim and DEET were significantly better removed in the VF than in the HF. The concentration of the four latter compounds showed a severe increase in the FWS, indicating possible desorption from the sediment/biomass during adverse conditions. Harvesting of the aboveground biomass in this unit returned the system's performance back to normality (Period III), achieving 77±7% TN removal despite the winter season, proving effluent recirculation as an effective strategy for TN removal in hybrid CW systems when stringent restrictions are in place., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The dynamics of the active microbial populations involved in nitrogen transformation in a vertical subsurface flow constructed wetland (VF) treating urban wastewater was assessed. The wetland (1.5m 2 ) operated under average loads of 130gCODm -2 d -1 and 17gTNm -2 d -1 in Period I, and 80gCODm -2 d -1 and 19gTNm -2 d -1 in Period II. The hydraulic loading rate (HLR) was 375mmd -1 and C/N ratio was 2 in both periods. Samples for microbial characterization were collected from the filter medium (top and bottom layers) of the wetland, water influent and effluent at the end of Periods I (Jun-Oct) and II (Nov-Jan). The combination of qPCR and high-throughput sequencing (NGS, MiSeq) assessment at DNA and RNA level of 16S rRNA genes and nitrogen-based functional genes (amoA and nosZ-clade I) revealed that nitrification was associated both with ammonia-oxidizing bacteria (AOB) (Nitrosospira) and ammonia-oxidizing archaea (AOA) (Nitrososphaeraceae), and nitrite-oxidizing bacteria (NOB) such as Nitrobacter. Considering the active abundance (based in amoA transcripts), the AOA population revealed to be more stable than AOB in both periods and depths of the wetland, being less affected by the organic loading rate (OLR). Although denitrifying bacteria (nosZ copies and transcripts) were actively detected in all depths, the denitrification process was low (removal of 2gTNm -2 d -1 for both periods) concomitant with NO x -N accumulation in the effluent. Overall, AOA, AOB and denitrifying bacteria (nosZ) were observed to be more active in bottom than in top layer at lower OLR (Period II). A proper design of OLR and HLR seems to be crucial to control the activity of microbial biofilms in VF wetlands on the basis of oxygen, organic-carbon and NO x -N forms, to improve their capacity for total nitrogen removal., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of this study was to characterize the nitrogen transforming bacterial communities within a partially saturated vertical subsurface flow constructed wetland (VF) treating urban wastewater in southern Brazil. The VF had a surface area of 3144m 2 , and was divided into four wetland cells, out of which two were operated while the other two rested, alternating cycles of 30days. The nitrifying and denitrifying bacterial communities were characterized in wetland cell 3 (764m 2 surface area) over a period of 12months by using the FISH technique. Samples were collected monthly (from Feb 2014 to Feb 2015) from different layers within the vertical profile, during operation and rest periods, comprising a total of 6 sampling campaigns while the cell was in operation and another 6 when the cell was at rest. This wetland cell operated with an average organic loading rate (OLR) of 4gCODm -2 d -1 and a hydraulic loading rate of 24.5mmd -1 . The rest periods of the wetland cell presented influences on the abundance of ammonia-oxidizing bacteria (AOB) (8% and 3% for feed and rest periods, respectively), and nitrite-oxidizing bacteria (NOB) (5% and 2% for feed and rest periods, respectively). However, there was no influence of the rest periods on the denitrifying bacteria. AOB were only identified in the top layer (AOB β-proteobacteria) in both operational and rest periods. On the other hand, the NOB (Nistrospirae and Nitrospina gracilis) were identified in feed periods just in the top layer and during rest periods just in the intermediate layer. The denitrifying bacteria (Pseudomonas spp. and Thiobacillus denitrificans) were identified from the intermediate layer downwards, and remained stable in both periods. Based on the identified bacterial dynamics, the partially saturated VF wetland operated under low OLR enabled favorable conditions for simultaneous nitrification and denitrification., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Context: The preservative solution ADSOL (adenine, dextrose, sorbitol, sodium chloride and mannitol) maintains red cell viability for blood trans-fusion for 6 weeks. It would be useful to know about its preservation qualities over longer periods., Objective: To determine some red cell biochemical parameters for peri-ods of up to 14 weeks in order to determine whether the red cell metabo-lism integrity would justify further studies aiming at increasing red cell preservation and viability., Design: Biochemical evaluation designed to study red cell preservation., Setting: São Paulo University erythrocyte metabolism referral center., Sample: Six normal blood donors from the University Hospital of the Universidade Federal do Paraná, Curitiba, Brazil., Main Measurements: Weekly assay of erythrocyte adenosine-5;-triphosphate (ATP), 2, 3-diphosphoglycerate (2,3DPG), hexokinase (HX), phosphofructokinase (PFK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconic dehydrogenase (6-PGD), glyceraldehyde-3-phosphate dehydrogenase (GAPD), glutathione reduc-tase (GR), glutathione peroxidase (GSHPx), plasma sodium and potas-sium, blood pH, and membrane proteins of red cells preserved in ADSOL were studied during storage for 14 weeks storage., Results: During ADSOL preservation, erythrocyte ATP concentration decreased 60% after 5 weeks, and 90% after 10 weeks; the pH fell from 6.8 to 6. 4 by the 14th week. 2,3-DPG concentration was stable during the first week, but fell 90% after 3 weeks and was exhausted after 5 weeks. By the end of the 5th week, an activity decrease of 16-30% for Hx, GAPD, GR, G-6-PD and 6-PGD, 35% for PFK and GSHPx, and 45% for PK were observed. Thereafter, a uniform 10% decay was observed for all enzymes up to the 14th week. The red blood cell membrane pro-teins did not show significant alterations in polyacrylamide gel electro-phoresis (SDS-PAGE) during the 14 weeks., Conclusion: Although the blood viability was shown to be poor from the 6th week up to the 14th week of storage due to ATP and 2,3-DPG depletion, the other biochemical parameters remained in fairly good condition for longer storage. As there is a gradual and uniform decay in activity throughout these 14 weeks, it seems that ADSOL-preserved red cells may be used as red cell enzyme standards and membrane proteins as well.

Red blood cells (RBC) are viable if kept in an adequate preservative solution, although gradual changes in morphology and metabolism may occur. There is a gradual decrease in adenosine-5'-triphosphate (ATP) concentration, pH, glucose consumption, and enzyme activity during preservation. The normal discocyte shapes are initially replaced by echinocytes and stomatocytes and, at final stages, by spherocytes, the last step before splenic sequestration. Post-transfusional survival has been correlated with the ATP concentration. RBC preserved in ADSOL, a solution containing adenine, dextrose, sodium chloride, and mannitol, are viable for transfusion for up to 6 weeks. Erythrocytes from 10 blood units taken from healthy adult donors were preserved for 12 weeks in ADSOL at 4 degrees C. We now report a significant correlation (r2 = 0.98) between the percentage of discocytes (89 to 7%) and ATP (100 to 10%) concentration in ADSOL-preserved RBC. The results suggest that the percent of discocyte shapes used as an indicator of ATP concentration may be a useful indicator for quality control of RBC viability in centers which have limited assay facilities.