In our study, we have examined the age-associated changes in the NK cell population and their subsets at different age groups of males and females (at a range from 41 to 80 years). We found that the total lymphocyte count significantly dropped upon aging in both genders. Although, the level of total immune cells also dropped on aging, and surprisingly the total NK cell population was remarkably increased with the majority of NK cells being CD56dim. Subsequently, we evaluated the proliferation potential of NK cells and our results showed that the NK cell proliferation ability declines with age. Overall, our findings prove that there is an increase in the circulating NK cell population upon aging. However, the proliferation rate upon aging declines when compared to the young age group (<41 yrs).Age-associated changes in natural killer (NK) cell population, phenotype, and functions are directly attributed to the risk of several diseases and infections. It is predicted to be the major cause of the increase in mortality. Based on the surface density of CD56, NK cells are subdivided into two types, such as CD56 However, this NK cell subset produces an array of cytokines and chemokines which influence immunomodulation and thus these cells are commonly referred to as “cytokine producers.” In contrast, CD56dim NK cells are predominant (90%) in the circulatory system and are potent mediators of natural and antibody-dependent cytotoxicity [bright : NKdim ratio is impaired upon aging which could be a reason why elderly people become more prone to several diseases, infections, and cancers.Natural killer (NK) cells are considered the primary defense lymphocyte against virally infected and virally transformed cells. The coverage of their defense system has been extended to include antimicrobial response , 2, elimeristics . Brieflydominant 0% in the minimal % populatdim cell through the granule exocytosis pathway [dim cells, which directly influences the NK cell-mediated cytolysis on the senescent cell. Unlike the NKdim cell, the total population, phenotype, and functions of NKbright cells decline due to aging, which is attributed to poor immunomodulation, poor resolution of inflammation, and poor induction of adaptive immunity. One study examined the effect of aging on the cytokine production of NKbright cells and reported that the production of cytokines  is significantly lower in older NKbright cells than in younger NKbright cells [Physiological aging is an evolutionarily conserved process which is associated with a defective or impaired function of immune cells, including NK cells. The impaired function of NK cells is known as NK cell immunosenescence. Age-associated NK cell immunosenescence contributes to the higher incidence of viral infection and cancer induction. Also, NK cell-mediated elimination of senescent cells declines on aging and results in the accumulation of aged cells in tissue or organs which impairs tissue homeostasis and their function. NK cell-mediated elimination of senescent cells is a direct elimination  process which is accomplished by the NK pathway . Howeverht cells .dim population and a decrease of the CD56bright population [Several studies signify that the NK cell number and subpopulations vary upon aging with an increase of the CD56pulation –15. In epulation , liver fpulation , infectipulation . Thus, apulation , 20. In pulation . NK cellPeripheral blood was obtained from 40 donors from different age groups (ranging from 41 to 80 years old), with no significant medical illnesses or a history of cancer. All enrolled donors signed informed consent forms approved by the medical research ethics committee, University Malaysia Medical Centre, Malaysia. NK cell-containing peripheral blood mononuclear cells (PBMCs) were isolated by lymphoprep  density gradient medium. Isolated PBMCs were washed twice with magnesium and calcium free phosphate buffered saline (PBS). The PBMCs were counted in an automated cell counter .6/ml as per the protocol described by us earlier [The isolated PBMCs were finally resuspended in NK cell expansion media containing 10% autologous plasma and 700 IU/ml of IL-2. Suspended PBMCs were seeded at a density of 1 × 10 earlier .Flow cytometry analysis was performed to qualify and quantify the NK cell population in peripheral blood and cultured cells using NK specific antibodies especially CD56-PE and CD3-PC5 . Staining was performed as per the manufacturer's instruction. Stained cells were washed and resuspended in PBS and analyzed by flow cytometry . The acquired data were analyzed using CXP software provided by the manufacturer.t-test. A value of #,@,∗P < 0.05 was considered to be statistically significant.Data from each group were expressed a mean and standard error (SE) of at least three separate experiments performed. Statistical comparison between groups was analyzed using Student's n = 20 male and n = 20 female) who were segregated into four groups with each group containing 5 males and 5 females; Group 1 (41–50 years old), Group 2 (51–60 years old), Group 3 (61–70 years old), and Group 4 (71–80 years old). The absolute lymphocyte count was performed at different age groups using an autoanalyzer and the results showed that the average lymphocyte count significantly decreased upon aging , 9.6 ± 1.8% (51–60 years old), 10.8 ± 2.3% (61–70 years old), and 14.7 ± 3.0% (71–80 years old)  among aging populations may be a result either of an accumulation of long-lived NK cells or an impaired routine process of elimination of increased-senescence cells.The percentage of NK cells was analyzed in the peripheral blood of each age group. The median percentage of CD56ars old) . After te gender . Next, we groups . This in+NKbright cells at all age groups, while a very minimal number of CD56+NKdim cells were observed in all age groups (+NKbright cells are equally expressed in all age groups even though NK cell fold changes between different age groups significantly decreases upon aging in both males and females.An average of more or less a similar number of PBMCs from different age groups were seeded on day 0 and then cells were allowed to grow for 14 days with NK cell-specific culture medium. After 14 days, the count was performed using the trypan blue exclusion method. NK cells were grown successfully in all age groups with more than 90% purity. With regard to NK cell fold change between different age groups, after 14 days of culture, the NK cell fold changes were significantly decreased upon aging in both male and female genders . Flow cye groups . These rManipulations are more predominant in the case of aging with newer approaches and strategies. The most evident modification is the deregulation of the immune function which contributes to the increased vulnerability to infection of the aged people . The stubright NK cells declines with age, which may have a huge proposition for NK cell function in the elderly individuals [bright NK cells were lower in all four groups compared with CD56dim. The total NK cell population was higher with advancing age; however, the age-related increase may be an accumulation of long-standing NK cells in older adults [The results reveal that the number of CD56ividuals . Our datr adults . The lytr adults .bright population which is responsible for innate immune response and cytokine production [bright cells could delay the fold changes in an elderly cohort [In this study, we used the optimized protocol to produce large numbers of activated NK cells from healthy donors of the different age cohorts. The use of clinical gate IL-2 plays essential roles in NK cell development, activation, and expansion , 31. In oduction , 32. Howy cohort .bright NK cells decreased with age while the number of CD56dim NK cells increased. However, after in vitro activation with IL-2, the expressions of CD56bright cells were higher and the fold changes increased. Overall, our optimized protocol managed to expand the number of CD56bright NK cells. However, a more elaborate approach needs to be carried out to evaluate the cytotoxic activity of these variant NK cell populations.In our study, we have shown that the number of lymphocytes and CD56


A novel omics-like method referred to as “particle morphomics” has been proposed in the present study. The dynamic images of >2,000,000 particles per sample in sediments, soils and dusts were collected by a Sympatec GmbH QICPIC particle size and shape analyzer, and the morphological descriptors of each particle including equivalent diameter, sphericity, aspect ratio and convexity were extracted as the “particle morphome”. Various multivariate analyses were adopted to process the high-throughput data of particle morphome including analyses of alpha and beta diversities, similarity, correlation, network, redundancy, discretion and principal coordinate. The outcome of particle morphomics could estimate the morphological diversity and sketch the profile of morphological structure, which aided to develop a morphological fingerprint for specific particle samples. The distribution and properties of particle assemblages of specific morphology could also be evaluated by selecting particles with respect to filter criteria. More importantly, the particle morphomics may be extended to investigate and explain the biogeochemical and environmental processes involved with particle morphology if linked with external variables. For instance, the size and density of fine sediment particles govern the settling velocity according to the classic Stokes’ law2. In terms of aerodynamics, shape has a direct impact on the drag forces acting on ash particles of floating in the air3. Thus, quantifying particle morphology is a fundamental issue in particle research.The morphology, that is size and shape, is an important characteristic of solid particles which determines their many properties4. Due to outstanding features such as fast measurement and simple operation, laser diffraction over the past 40 years has gradually become the most frequently applied method for particle size analysis5. However, laser diffraction technique lacks of reliable and quantitative particle shape characterization. In the last 10–20 years, image analysis is probably the mainstream approach to simultaneously characterize the size and shape of particles, which uses a two-dimensional image of a three-dimensional particle and calculates various size and shape parameters from this image1. Especially, the dynamic image analysis technique can capture the three-dimensional morphology of particles by rotating particles with random orientation which allows the measurement of the true characteristics of particles6.Sieving is probably the most used method to determine the particle size distribution. This traditional method is time consuming and expensive, and has deviations when particle shape is involvedCurrently, particle morphological analyses focus on size/shape distribution or average information such as volume mean diameter (VMD), while the morphological diversity and structure extracted from information of millions particles have not been addressed. The heterogeneous particles or specific particle assemblages in a particle sample are also difficult to reveal using conventional morphological analyses. Moreover, the morphological information of particles is often not associated with external factors , which veils the scientific value of particle morphology.7, which aims at the collective characterization and quantification of pools of various information. In this paper, attempts have been made to establish an omics-like method referred to as “particle morphomics” to comprehensively analyze the large dataset of particle morphological descriptors (“particle morphome”). The particle morphomics would open up a research approach for particle morphological analyses and reacquaint the importance of particle morphology.The development of high-throughput “omics” approaches over the last few years has considerably accelerated the pace of discovery in multidisciplinary sciences8, and the morphological descriptors of each particle including equivalent diameter, sphericity, aspect ratio and convexity were extracted by the WINDOX 5 software9. The measurement principle of QICPIC system is described in SI Text To this end, a set of particle samples including 7 sediments, 4 soils and 3 dusts were collected (Supporting Information (SI) Table 10 and instrumental property, the measure parameters of dynamic image analysis were optimized to achieve a minimum number of calculated particles per measurement of above 2,000,000 and an optical concentration of within 0.5–1% during each measurement, assuring the accuracy and reliability of measured data. Table According to ISO 14488 standardq2)11:d and VMDs are the VMD calculated by dynamic image analysis and sieving method, respectively, and VMDmean is the mean value of VMDs. The obtained q2 is 0.565, indicating the sieving method is relatively reliable for acquiring the mean size information of particle samples. However, besides time consuming, the sieving method cannot provide an accurate profile of particle size distribution due to the limited size class. For instance, some small peaks in the distribution curve cannot be identified by sieving method  and 2.23–3.04 , respectively.Table 16, and represents both the irregularity of particle form and roughness of particle surface. The calculated mean particle sphericity of the measured samples was between 0.72 to 0.84  and 3.25–3.95 , respectively. The aspect ratio is defined as the ratio of the Feret’s minimum length to the Feret’s maximum length16, and has been used historically to classify the general form of particles . The shape of average aspect ratio distribution was similar with that of sphericity distribution that is close to the normal distribution and left-skewed  were observed in the measured samples . Size, sphericity and convexity factors showed strong associations with each other, while aspect ratio exhibited a relatively independent character Fig. . Most moDiscrete analysis of morphology distribution at each size or shape class may provide the information on which fraction of particles showed the largest variability among the measured samples. SI Fig. 13, and here we used it to describe the particle diversity between a set of particle samples. Bray-Curtis similarity analysis was employed to estimate the similarity or disparity between particle samples in terms of morphology distribution. Figure Beta diversity originally referred to the ratio between regional and local species diversity in ecologyPrincipal coordinate analysis (PCoA) diagram was further used to visually illustrate the beta diversity of particle morphology among samples Fig. . The sca17. Discriminating these specific particle assemblages will be of great help for investigations on biogeochemical processes 18, ecosystem structure 19 and dispersion of pollutants 20 in marine environments. Since different particle assemblages have specific morphological characteristics, morphome may provide useful information for characterization of particle assemblages.It is of importance to classify and characterize the specific particle assemblages in a heterogeneous sample. For instance, the suspended particles in marine environments comprise of various assemblages such as phytoplankton-dominated particles, organic non-algal particles, and mineral-dominated particlesThe WINDOX 5 software provides a function of particle selection with respect to filter criteria of particle size and shape. For exhibition, the impurity fiber particles in the measured samples were picked up under the filter condition of “aspect ratio ≤0.25 and sphericity ≤0.25 and convexity ≤0.9”. The results showed the fiber particles were very few in the samples, and only 407 fibers were identified with a mean length of fiber (LEFI) of 1.02 mm (0.36–3.08 mm), mean diameter of fiber (DIFI) of 25.47 μm (12.99–73.01 μm), and mean elongation (EL = DIFI/LEFI) of 0.028 (0.007–0.082) Fig.  indicate21, discriminating the geographical origin of food produce by using nuclear magnetic resonance (NMR)22, and identifying the source contribution of sediment by using compound specific stable isotopes (CSSI)23.The fingerprint technique is to collect and analyze the profiles of chemical compositions or physiochemical properties of investigated samples by various analytical techniques to identify and compare the disparity between samples. The fingerprint technique has wide applications such as controlling the quality of herbal medicine by using high-performance liquid chromatography (HPLC)16, X50, X84, expectation, kurtosis and skewness) and Shannon diversity index. Figure To visually and quantitatively compare the difference of particle samples in particle size and shape, a morphological fingerprint was constructed by adopting the morphology distribution parameters  as tracers26, few have investigated the effects of particle morphology on sediment source signatures or employed particle morphology as a tracer. In fact, all the processes of soil erosion and detachment, sediment transportation, deposition and abrasion are particle morphology dependent, and the selectivity of particle morphology acts over the entire landscape27. Not only are sediment transport processes particle morphology selective, the properties used to trace sediments may have different affinities  to particle morphology28. Particle morphomics can be a powerful tool to analyze particle morphology, and provided information can aid in understanding of particle morphology-biogeochemical tracer interactions and improving the predictability and accuracy of sediment source tracing.Volcanic ash particles research. Volcanic ash particles carry a wealth of information on various processes occurring during volcanic eruptions, and their morphology has been recognized to reflect numerous eruptive parameters such as magma viscosity, volatile content, degree of interaction with external water, particle transport and sedimentation29. Many investigations of ash morphology have been carried out with multi-targets at unravelling the origin of ash and the related magma fragmentation dynamics30, determining the physical properties of magma and its degree of interaction with external water31, modeling the processes of ash dispersal and settling32, forecasting the continuation or ending of an eruptive episode29. The particle morphomics methodology outlined in this study can promote volcanic ash particles research to dredge up the intrinsic nature of morphology, e.g., diversity, backtrack the generation process and mechanism by linking the eruptive parameters, and predict the behavior and fate by linking the environmental conditions.Marine particle monitoring. Marine particles are a ubiquitous and important component in the marine environment, and comprise bio-derived  and nonbio-derived  particles. Current researches adopted various optical sensors to study the spatial and temporal variability of concentration and size distribution of suspended marine particles34. However, classification and characterization of specific marine particle assemblages  is always a challenge. The dynamic image analysis and particle morphomics described here may have the potential ability to discriminate different marine particle assemblages based on their distinctive morphological nature. Particle morphomics can also benefit the study on the ultimate supply, transport, and fate of marine particles.Activated sludge characterization. The activated sludge is the critical component in a biological process of wastewater treatment plant, and its morphology is correlated with its physical characteristics , biological characteristics , pollutant removal efficiency38, and dewaterability39. The previous studies focused more on sludge size impact and ignored the shape influence. The particle morphomics based on image analysis can greatly improve sludge morphological characterization by combining size and shape descriptors and better explain the underlying mechanism of the morphology-dependent sludge properties. Moreover, the discrimination of different particle assemblages  by morphomics analysis can be conducive to evaluation of sludge performance.This paper has presented the independent application of particle morphomics. Besides, particle morphomics may play a greater role in geoscience and environmental studies when combing with the external factors Fig. . When coThis paper has proposed the concept of particle morphomics, and demonstrated its potential in exploring the morphological diversity and the intrinsic relations with morphological descriptors, evaluating the sample similarity and clustering the samples based on their morphological characteristics. Like other omics, multivariate statistical analyses such as PCoA, RDA and clustering analysis are the useful tools for morphome data mining. Moreover, the particle morphomics may play a greater role in biogeochemical and environmental studies when combing with external factors. Considering the wide use of dynamic image analysis (SI Sheet 2), particle morphomics has wide prospects, and can expand and deepen the current researches related to particle morphology.The particle sampling took place in March-May, 2017. Samples 1–7 are sediments, of which 1–5 were collected along the Yangtze River in Shanghai City, and 6 and 7 were collected in a small river in Xinghua City. Samples 8 and 9 are agricultural soils and were collected in the edge of the Yangtze River; samples 10 and 11 are urban soils and were collected at a construction site. Each sediment/soil sample of approximately 1 kg was collected by either a stainless steel grab or an Ekman dredge. Samples 12–14 are dusts, of which 12 was collected with a brush at the windowsill of a student dormitory at Fudan University, and 13 and 14 were collected at the ground near a building materials factory. The detailed sampling information and characteristics of particle samples are described in SI Table For traditional sieving, an air-dried sample of approximately 500 g were successively passed through a series of sieves, i.e., 10 mesh (2 mm), 18 mesh (1 mm), 35 mesh (0.5 mm), 65 mesh (0.25 mm), 150 mesh (0.1 mm), and 300 mesh (0.05 mm). The volume of each sieve fraction was measured and used for determination of particle size distribution.QICPIC analysis was conducted at a wet dispersing systems with deionized (DI) water as the carrier liquid. Approximately 0.1–1 g of a particle sample was dispersed in 500 mL DI water to make the injecting liquor, which passed through a flow cuvette (10 mm × 30 mm) with a rate of 3 mL/s during the measurement. A high-speed camera with the detection range of 11–22528 μm was employed to obtained the dynamic images of particles with a frame rate of 40 Hz in 90–120 s. For achieving high-resolution digital images of individual particles, the optical concentration of particle flow was kept within 0.5–1% during each measurement.The morphological data were processed by the WINDOX 5 software. Each particle image was stored, and four morphological descriptors, i.e., equivalent diameter, sphericity, aspect ratio and convexity, were calculated. For fiber-like particles , the fiber length, diameter, and elongation were also determined. The detailed definitions of these morphological descriptors are described in SI Text p value of < 0.01 indicated a significant correlation. Before Pearson correlation analysis, the data normality was checked by Kolmogorov-Smirnov test. Network analysis was carried out using the software Cytoscape (http://www.cytoscape.org) with input Pearson correlation coefficient to explore the associations between morphology distribution parameters and diversity indices. Redundancy analysis (RDA) was executed using Canoco 4.5 for Windows41 to investigate effects of morphology distribution parameters on the diversity indices. Forward selection based on the Monte Carlo permutation test (n = 499) was conducted to rank the most important distribution parameters that explained the particle morphological diversity. The heatmap constructed by R software (www.R-project.org) with the pheatmap package was used to illustrate the particle morphology distribution in each sample. Clustering of samples was conducted by the PRIMER 5.0 software  using the mode of group average based on Bray-Curtis similarity. PCoA analysis based on the particle morphology distribution was performed to compare the differences of samples using the software PRIMER 5.0. Discrete analysis was conducted to reveal the dispersion of particle morphology at each morphological class among the measured samples with OriginPro 8 software .The morphological diversity was estimated by Simpson and Shannon indices, which were calculated by the following equations D = 1 − ∑p*p and H = −∑p*Ln(p), respectively. p (%) is the relative volume abundance of each morphological descriptor class. The bivariate correlations among morphology distribution parameters and diversity indices were estimated by Pearson correlation analysis using SPSS 13.0 for Windows . The two-tailed test was used to compute the statistical significance of the correlation coefficient. The Supporting Information


Objectives: The objective of this study was to evaluate new bone formation derived from freshly crushed extracted teeth, grafted immediately in post-extraction sites in an animal model, compared with sites without graft filling, evaluated at 30 and 90 days. Material and Methods: The bilateral premolars P2, P3, P4 and the first mandibular molar were extracted atraumatically from six Beagle dogs. The clean, dry teeth were ground immediately using the Smart Dentin Grinder. The tooth particles obtained were subsequently sieved through a special sorting filter into two compartments; the upper container isolating particles over 1200 μm, the lower container isolated particles over 300 μm. The crushed teeth were grafted into the post-extraction sockets at P3, P4 and M1 (test group) , while P2 sites were left unfilled and acted as a control group. Tissue healing and bone formation were evaluated by histological and histomorphometric analysis after 30 and 90 days. Results: At 30 days, test site bone formation was greater in the test group than the control group (p < 0.05); less immature bone was observed in the test group (25.71%) than the control group (55.98%). At 90 days, significant differences in bone formation were found with more in the test group than the control group. No significant differences were found in new bone formation when comparing the small and large alveoli post-extraction sites. Conclusions: Tooth particles extracted from dog’s teeth, grafted immediately after extractions can be considered a suitable biomaterial for socket preservation. There is compelling evidence that immediate grafting of extraction sockets using various ridge preservation techniques—including the placement of graft materials and/or the use of occlusive membranes—will prevent alveolar bone loss during the repair phase of wound healing. However, the technique lacks the capacity to promote osteogenesis and osteoinduction, and so its usefulness is limited in terms of the formation of viable bone .Human demineralized dentin matrix created from extracted human teeth was first developed in 2008 and its use in implant dentistry has been evaluated on the basis of its osteoinductive, osteoconductive, and remodeling capacity. By weight, dentin and bone are composed of 30% collagen, 60% hydroxyapatite, and 10% body fluid ,5; by voKim and his team have been investigating the development of biomaterials using human teeth since 1993, and have recently reported the promising results of their research ,15,16,17TM device has been designed to crush and sort extracted teeth into tooth particles of specific sizes. A chemical cleanser and a buffer are applied for 15–20 mins to eliminate bacteria. This novel procedure can be indicated for most tooth extractions, although endodontically treated teeth are counter-indicated due to contamination by foreign materials.The “Smart Dentin Grinder”The objective of this study was to evaluate immature bone and new bone formation after filling fresh extraction sockets in an animal model with tooth particle graft compared with empty sockets, after a 90-day follow-up.Six Beagle dogs about one year old, weighing 14–15 kg each were used in the study. The University of Murcia Ethics Committee for Animal Research approved the study protocol A1320140404 (12-02-2015), which followed guidelines established by the Directive of the Council of the European Union of February 1, 2013/53/CEE, guidelines for animal studies.The animals were quarantined for the application of rabies and vitamin vaccines. The animals were kept in cages before and after the operation and received appropriate veterinary attention during the study period. All the animals presented intact dental arches, without viral or fungal mouth lesions.The animals were pre-anesthetized with 10% zolazepam at 0.10 mL/kg and acepromazine maleate  0.12–0.25 mg/kg and medetomidine 35 mg/kg . The mixture was injected intramuscularly into the quadriceps femoris. The animals were taken to the operating theatre where, at the first opportunity, an intravenous catheter (diameter 22 G or 20 G) was inserted into the cephalic vein, and propofol infusion was administered at a rate of 0.4 mg/kg/min at a constant infusion rate.Anesthetic maintenance was performed with volatile anesthetics and the animals underwent tracheal intubation with a Magill probe for the adaptation of the anesthetic device and for the administration of volatile isoflurane diluted in oxygen (2 V%). In addition, local anesthesia  was administered at the surgical sites. These procedures were carried out under the supervision of a veterinary surgeon.Mandibular premolars and first molars  were extracted bilaterally  under geTeeth with multiple roots were hemisectioned in bucco-lingual direction, protecting the remaining bony walls. Immediately after extraction, the clinician eliminated the organic coating of the root surfaces that includes periodontal ligament tissue and bacterial biofilm, which was removed using a tungsten carbide bur, or hand-piece drill and water.The teeth were cleaned, dried with an airflow syringe, and crushed immediately after extraction isolating the crown and root using the “Smart Dentin Grinder” , specially designed for this procedure. The tooth particles obtained were sized at 300–1200 μm, which were sieved through a special sorting system into two compartments: the upper container has a grid to isolate larger particles of over 1200 μm, while the lower container is more rigid, allowing the accumulation of smaller particles of over 300 μm .More than 95% of the tooth particulate accumulates in a sterile “drawer chamber” that isolates particles of 300–1200 μm , isolating smaller and larger particles in separate trays. The particulate volume is about 2–3 times greater than the original volume of the tooth. The particle size criteria is based on the fact that small particles facilitate quick bone resorption (300 μm) and bone maintenance, while larger particles (over 1200 μm) protect against and reduce bone resorption. Both type of tooth particles were obtained in two different compartments and the proportion of both particles used were 80% of big particles and 20% of small particles to avoid inflammation.The particulate teeth were immersed in a basic alcohol cleanser in a sterile container for 15 min to dissolve all organic waste and bacteria. The particles were then washed with sterile saline for 5 min. Then the particles were dried, and the small and large tooth particles were mixed. The particulate was then grafted into post-extraction sites of premolars P3, P4 and first molar M1 (test group), they were filled with freshly extracted teeth , while tEthanol 70% denatures and precipitates proteins and so is bactericidal. The present experiment used a 30% solution of 70% ethanol, which, in addition to its main bactericidal action, strongly enhances the action of NaOH, which eliminates organic compounds, while ethanol does not. So, the combination of ethanol and NaOH completely dissolves every organic substance and acts as a very effective disinfectant. Ethanol only affects organic compounds on the outside surface of the mineral . It does not penetrate the mineral and so does not affect collagen trapped in the mineral itself .Continuous non-absorbable sutures  were used to protect and maintain the grafted areas . The stiMandibular teeth from canine to canine were maintained and no surgeries were performed in the upper maxilla. All surgical techniques were performed under the supervision of the University veterinarian, assigned to the University of Murcia’s Animal Research Unit.3) to aid post-surgical recovery. Following the guidelines established by the animal research ethics committees, anti-inflammatory and antibiotic drugs were administered after surgery and every other day for seven days to prevent postoperative infection and inflammation: the anti-inflammatory, Metacam® , 1–2 mL intramuscularly; and the antibiotic Alsir® (enrofloxacin), 2 mL intramuscularly.During the surgical procedure, the animals were hydrated with glucose-saline .After 30 and 90 days healing, a local anesthetic was applied to the buccal and lingual gums and crestal incisions were made in the area regenerated from the canine to the second molar . A full The bone cores obtained were individually preserved in 10% formaldehyde for 20 days. The samples were then decalcified for 30 days using TBD-2 . After dehydration and inclusion in paraffin, sections of 20 μm thickness were prepared; samples were stained with picrosirius-hematoxylin and hematoxylin to distinguish between immature and mature bone.For histomorphometric analysis, images were enlarged 20×, and eight fields per sample were evaluated digitally . Microimage 4.0 software  was used for image analysis.All analyses were carried out by the same technician, who was blinded to each sample’s group assignation (test or control).The total area of newly formed bone and connective tissue were evaluated and the percentage of immature bone was measured.p < 0.05). The Mann–Whitney U test, a non-parametric test, was applied and the ANOVA test was used to analyze the differences between variables.Statistical analysis was performed using SPSS 21.0 software . Descriptive statistics were calculated (mean and standard deviation) for both groups. For the comparison of means, a non-parametric Friedman test was applied with a significance level of 95%  and 6 mm by 10 mm length  a total of 36 small and 12 big alveolus filled for both test and control specimens.Control samples showed large amounts of newly formed immature bone covering the bone defect, and highly disorganized tissues with high rates of cellularity and large medullary cavities.The tooth particulate group showed newly formed bone with irregular disposition and high levels of capillarity, but to a lesser extent than the control group a,b.p < 0.05) , while in the untreated defects (control group) new bone formation was 55.87 ± 0.32%, with statistically significant difference between the groups ( < 0.05) .p < 0.05)*. , compared with the control group (58.92 ± 0.32%) with statistically significant difference ( 0.05)*. .The control group showed greater bone organization compared with the 30-day evaluation. All bone defects were completely covered by newly formed osteons, while other areas were occupied by non-organized immature bone c. The teAt 30 days, histomorphometric analysis found immature bone (25.71 ± 0.25%) in defects treated with tooth particulate with significant difference between the test and control groups (55.98 ± 0.16%). At 90 days, histomorphometric analysis found total immature bone of 14.2 ± 0.66% in the test group with significant differences in comparison with the control group (35.17 ± 0.74%) .Our last published work described how fresh extraction sockets can be grafted with tooth particles made by crushing the extracted teeth; bone formation was evaluated after 30 days and 90 days  finding more immature and mature bone at test sites than control sites at both study times 22). Although in the present study, the statistical method was different, the present results were similar to the earlier study, observing more immature bone at 30 days and more mature bone at 90 days, with less connective tissue. These data show how the tooth particles resorb and are replaced by new bone at 90 days.Teeth that suffer trauma have ankylosed roots, which continuously resorb over time and are replaced by bone, eventually resorbing the entire root, while the alveolar bone is conserved during this period. Malmgren et al. described the decoronation method, offering possible explanations for the favorable outcome of this technique and proposing guidelines for deciding the correct time for intervention. After decoronation, the alveolar ridge was maintained in buccal/palatal direction, and the bone level increased, so maintaining normal alveolar conditions . Pang etThe present results reveal a close and direct interaction between mineralized dentin, bone matrix, and the grafted tooth particle material. When the extraction of a tooth is necessary, the Smart Dentin Grinder makes it possible to prepare the tooth particles from the freshly extracted autologous tooth, ready for use in as little as 15 min during a single surgical session. With no antigenicity, it improves bone remodeling capabilities and can be considered a viable graft option given its autogenous origin and favorable clinical and histological results. Moreover, despite its inductive properties, mineralized dentin integrates into newly formed bone, creating a stable site for dental implant insertion. In fact, several clinical studies have shown that implant insertion and loading can be performed in both upper and lower jaws as early as 2–4 months after grafting with crushed and demineralized tooth and bone ,26.Since mineralized dentin is remodeled very slowly, as compared with cortical bone, the aesthetic and structural integrity of the alveolar ridge is maintained for many years ,28. The Autogenous demineralized dentin matrix from extracted tooth grafted to extraction sockets for the augmentation of vertical dimension was as effective as augmentation using anorganic bovine bone. Both groups showed favorable wound healing, similar amount of implant stability, and histologically confirmed new bone formation. Thus, the results of this study suggest that autogenous tooth graft material is a viable option for alveolar bone augmentation following dental extraction .It also overcomes patients’ rejection of materials derived from animal or synthetic sources, as well as providing excellent biocompatibility without provoking an immune response, foreign material reaction, or infection. Unless it is contaminated by an infectious lesion, a tooth does not cause problems, even when the root tip is left in the alveolar bone. The use of the patient’s own tooth is entirely legal, providing the patient agrees. In the present study, the crushed tooth particulate produced more bone regeneration at 90 days than control samples. As the dentin graft maintains the collagen structure, it preserves both the height and width of bone crests.Within the limitations of the present animal study, it would appear that tooth particle graft (both enamel and dentin) can be viewed as a useful autogenous biomaterial for socket preservation due to its similar characteristics to autologous bone. Currently, this graft material can be used in combination with autologous bone debris as an excellent graft material without compromising the capacity for bone regeneration.


Disrespectful and abusive behavior during childbirth and maternity care is violation of fundamental right of women and unborn child. There is scarce of data on disrespectful and abusive behavior during childbirth and maternity care in Ethiopia. The aim of this study was to determine disrespectful and abusive behavior during childbirth and maternity care in Ethiopia.Seven studies were included in this meta-analysis of disrespectful and abusive behavior during childbirth and maternity care. The pooled prevalence of disrespect and abuse care during childbirth and maternity care was 49.4% (95% CI 30.9–68.1). Whereas physical abuse was 13.6% (95% CI 5.2–31.2), non-confidential care was 14.1% (95% CI 7.3–25.4), abandonment care was 16.4% (95% CI 14.7–18.2), and detention was 3.2% (95% CI 0.9–11.5). This study showed that disrespectful and abusive behavior during child birth and maternity care is high. Whereas, abandonment care is high. This study indicates that health care providers shall not leave women during childbirth and maternity care and listen women, federal minister of health and regional health bureau also identifying root of cause disrespect and abuse and to alleviate mistreatment during childbirth and maternity care.The online version of this article (10.1186/s13104-019-4118-2) contains supplementary material, which is available to authorized users. Disrespectful and abusive behavior during child birth is a public health problem, which violates fundamental rights of women and unborn child. Disrespect and abuse treatment during childbirth in health facilities are a burning issue all over the world . It is aPregnancy and childbirth are historic events that sustain offspring of families in every community across the globe. During pregnancy and childbirth woman’s positive and negative experience stay with her throughout her lifetime , 8. MoreIn Ethiopia has huge maternal and neonatal morbidity and mortality, concurrently low utilization of maternity care. The main to deterring pregnancy related complication and associated adverse pregnancy outcomes is ensuring regular and holistic care for all women throughout pregnancy and childbirth. And to increase maternity care utilization woman’s relationship with maternity care providers and the health facility system during pregnancy and childbirth is fundamental. Maternity care providers isn’t only the apparatus to utilize maternity care and potentially lifesaving maternity care services, while the feeling with maternity providers at this time have the impact to women’s memories of their childbearing experiences stay with them for a longtime and are often shared with other women and influences their decision to seek care from health facility , 11–13.Every woman has the right to get quality of health care and with respect to her right during child birth and maternity care , 15. TheThis systematic review and meta-analysis was carried out based on published studies. Articles were searched through an electronics on data bases: including, PubMed, Medline and HINARI, Google Scholar, Google and Cochrane Library were used to search articles. Articles were accessed by two reviewers (ZY and SH) using the following key terms, “attitude of health personnel” AND “delivery obstetrics*/nursing”, “maternity care” AND “disrespect”, “disrespect” OR “abuse”, “parturition” AND “prevalence”, “Professional Misconduct” AND “Professional-Patient Relations”, “disrespect “AND “Ethiopia”. For those studies having similar outcome of interest with the current objectives, their abstracts and the full-text were reviewed accordingly. The quality of each article was appraised by two independent reviewers (ZY and SH) using the Joana Brigg’s Institute (JBI) critical appraisal checklist for simple prevalence  using niData entry and analysis were done using comprehensive meta-analysis (version 3.1). The summary of pooled prevalence of disrespect and abuse during childbirth with 95% CI was done using the random effects model, due to the possibility of heterogeneity among the studies.2 statistics and the Egger’s test respectively [2 test statistics. I2 test statistics result of 25%, 50%, and 75% was declared as low, moderate and high heterogeneity respectively. Statistical significant publication bias was declared at p-value less than 0.05 [Heterogeneity and publication bias were assessed using Iectively . The hethan 0.05 .2 = 98.49%, p-value is 0.000 and publication bias .A total of 31 articles were identified through electronic data base search PubMed, Medline and HINARI, Google Scholar, Google and Cochrane Library. Articles were screened using their titles, abstracts and through full article review. Whereas 10 articles excluded due to duplication and 21 articles were reviewing full articles and 14 articles were excluded after full article reviewing due unreported of prevalence  . The Cohran’s Q and IDisrespectful and abusive behavior during childbirth and maternity care are violation of fundamental rights of woman and unborn child. It is a common problem in health facilities. In fact, promoting respectful maternity care during childbirth a vehicle to utilize maternal health care and preventing maternal and child morbidity and mortality by augmenting woman center care.Disrespectful and abusive behavior during childbirth and maternity care have been documented in both high and low income countries, making this a truly a global agenda. Its operational definition is not straightforward and having a difference an intentional community. Bowsers and Hill’s in 2010 revealed that the evidence of disrespectful and abusive behavior during child birth and maternity care at facilities. They categorized into seven; physical abuse, non-confidential care, non-consented care, non-dignified care, abandonment of care, discrimination and detention in the facilities , 27.The aim of this systemic review and meta-analysis was to assess disrespectful and abusive behavior during childbirth and maternity care in Ethiopia. Seven studies were included in this systemic review and meta-analysis. The pooled prevalence of disrespectful and abusive behavior during child birth and maternity care was 49.4% in Ethiopia. This finding is higher than the study was done in India 28% , in MexiThis finding is lower than study done in Pakistan 97.4% , in PakiIn this meta-analysis the highest prevalence was abandonment care during childbirth and maternity care 16.4%, whereas the lowest prevalence was detention during childbirth and maternity care 3.2%.Implication of this study; produce pooled prevalence of disrespectful and abusive behavior during child birth and maternity care in Ethiopia. Decision makers, policy planners and clinician on maternal and child health to achieve sustainable development goal three to ensure health lives and promote wellbeing for all ages and the women has the right to get maximum standard of care during maternity care. Therefore, this meta-analysis is an input for policy planners for evidence-based strategy to alleviate disrespect and abuse during childbirth and maternity care at facilities.This study showed that disrespectful and abusive behavior during child birth and maternity care is high. Whereas abandonment care is high. This study indicates that health care providers shall not leave women during childbirth and maternity care and listen women, federal minister of health and regional health bureau also identifying root of cause disrespect and abuse and to alleviate mistreatment during childbirth and maternity care.Potential limitations of this study, due to the nature of disrespectful and abusive behavior during childbirth and maternity care. Another important limitation is that different definition of disrespect and abusive behavior during childbirth and maternity care. The current estimates are limited to childbirth and maternity care. Disrespectful and abusive behavior can be occurred at family planning and another medical care. An important limitation is the use of childbirth and maternity care as search term.Additional file 1: Figure S1. The forest plot Prevalence of physical abuse during childbirth and maternity care in Ethiopia. Figure S2. The forest plot Prevalence of non-confidential care during childbirth and maternity care in Ethiopia. Figure S3. The forest plot Prevalence of detention during childbirth and maternity care in Ethiopia.Additional file 2: Table S1. PRISMA 2009 Checklist for disrespectful and abusive behaviors during childbirth and maternity care in Ethiopia.


The 20 μM melatonin treatment reduced ABA content in seeds by 42.13–51.68%, while the 20 μM melatonin treatment increased GA3 content in seeds to about 1.7–2.5 times that of seeds germinated without melatonin. This study provides new evidence suggesting that low concentrations of melatonin can promote cotton seed germination by increasing the activity of antioxidant enzymes, thereby reducing the accumulation of MDA and regulating plant hormones. This has clear applications for improving the germination rate of cotton seeds using melatonin.Seed germination is considered the beginning of the spermatophyte lifecycle, and it is a crucial stage in determining subsequent plant growth and development. Although many previous studies have found that melatonin can promote seed germination, the role of melatonin in cotton germination remains unexamined. The main objective of this study is the characterization of potential promotional effects of melatonin  on cotton seed germination. This experiment demonstrated that low concentrations of melatonin can promote germination, while high concentrations failed to promote germination and even inhibited germination. Together, these results indicate that a 20 μM melatonin treatment optimally promotes cotton seed germination. Compared with the control, germination potential (GP), germination rate (GR), and final fresh weight (FW) increased by 16.67%, 12.30%, and 4.81%, respectively. Although low concentrations of melatonin showed some improvement in vigor index (VI), germination index (GI), and mean germination time (MGT), these effects were not statistically significant. Antioxidant enzyme activity during seed germination was most prominent under the 20 μM melatonin treatment. Superoxide dismutase (SOD) and peroxidase (POD) activities were significantly increased by 10.37–59.73% and 17.79–47.68%, respectively, compared to the melatonin-free control. Malondialdehyde (MDA) content was reduced by 16.73–40.33%. Two important plant hormones in seed germination, abscisic acid (ABA) and gibberellins (GAs), were also studied. As melatonin concentration increased, ABA content in seeds decreased first and then increased, and GA Gossypium hirsutum L.) is a globally important cash crop, fiber crop, and oil crop, and it plays an important role in China’s economic development [Seed germination is the process by which the radicle breaks through the seed coat; it is an early and crucial stage in the spermatophyte life cycle, referring to the physiological process starting from the uptake of water by the dry seed and ending with radicle protrusion . Seed geelopment . The quaN-acetyl-5-methoxytryptamine), a highly conserved and efficient molecule, exists widely in plants and animals [Lycopersicon esculentum) and cucumbers (Cucumis sativus) in 1995 [Melatonin  during seed aerobic metabolism leads to lipid peroxidation, which is an important cause of seed deterioration and inhibition of seed germination. Melatonin is the most potent endogenous compound known to scavenge free radicals, at rates twice and four times as high as those of vitamin E and glutathione, respectively , 22. In Gibberella fujikuroi in 1938 by Japanese scholars [1 and GA3, demonstrating activity. Most related compounds are GA synthesis precursors or products inactivated by metabolism. The active components of plant gibberellins mainly consist of GA1, GA3, GA4, and GA7. GAs can break seed dormancy, promote seed germination, trigger seed germination in suitable environments and time periods, and prepare for subsequent seedling growth [In 2004, Hernández-Ruiz et al. first proposed that melatonin may be a plant growth hormone . It is wscholars . GAs regg growth .ABA is also an important hormone in the regulation of plant growth and development and accordingly plays a critical role in regulating various physiological processes in plants. It is derived from carotenoid precursors, which can delay seed germination and promote seed dormancy. ABA levels increase when plants are subjected to certain abiotic stresses, such as water deficits, and during seed development.The positive effect of melatonin on seed germination observed in several plant species motivated this research. The objectives of the study were to investigate (1) whether melatonin can improve germination of cotton seeds, (2) whether melatonin can improve the physiological activity of seeds, and (3) whether melatonin affects the hormones that regulate seed germination. This study aims to provide insight into the roles of melatonin in the germination of cotton seeds.N-acetyl-5-methoxytryptamine) was obtained from Sigma-Aldrich . All other reagents were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd. . All chemicals used in all experiments were of analytical grade.Melatonin , Hebei Province, China. All cotton seeds used in this study were of the ‘NDM601’ cotton cultivar. ‘NDM601’ is a cotton cultivar that is widely grown in the Yellow River Valley region of China and was developed by Hebei Agricultural University.Selected cotton seeds were surface sterilized with 1% NaClO for 30 min and rinsed in distilled water five times. Fifty seeds were then placed in Petri dishes (15 × 15 cm) containing double filter paper  wet with 10 mL of the different treatment solutions and incubated at 25°C in a darkened growth chamber. The following treatments were used: M0 , M10 (10 μM melatonin), M20 (20 μM melatonin), M50 (50 μM melatonin), M100 (100 μM melatonin), and M200 (200 μM melatonin). Germination tests were performed with six replicates.Seedlings were harvested at 7 days after sowing (DAS) for the determination of seedling fresh and dry weights and of seed germination rates. Germination assay samples were harvested at days 2, 4, and 6, and were stored at -80°C until the analyses of malondialdehyde (MDA) contents, antioxidant enzymes activities, and hormones were conducted.Seeds were considered to be germinated when the total radicle and hypocotyl length exceeded half the length of the seeds. Germination of seeds was recorded daily.Gi / Ti), where Gi is the number of germinated seeds per day corresponding to Ti, and Ti is the day of the germination test. Vigor index (VI) was calculated as VI = GI × fresh weight (FW) of germinated seeds on day 7. Mean germination time (MGT) was calculated according to the method by Ellis and Roberts [NiTi) / ∑ Ni, where N is the number of seeds germinated at time i, and Ti is day of the germination test.The numbers of germinated seeds on the day 3 and 7 after initiation were germination potential (GP) and germination rate (GR), respectively ,31. Germ Roberts , i.e., MSuperoxide dismutase  activity was estimated based on the method by Giannopolitis and Ries  using th3 and ABA concentrations were determined using an indirect ELISA technique [GAechnique ; the antP-values less than 0.05 in a one-way ANOVA.Analysis of variance was performed using IBM SPSS Statistics 22.0 . All data are reported as mean ± standard deviation (SD) values. We performed six independent replicates for each treatment. The statistical significance was considered for We conducted an extensive set of germination assays using cotton seeds to determine how different concentrations of melatonin (0–200 μM) affect seed germination. The seeds started to germinate on the second day of incubation at 25°C. By focusing solely on the GP of the seeds, it is clear that the M20 treatment seeds germinated well. Under the M20 treatment, the GP of the seeds was 16.7% higher than that of the M0 group and reached a significant level. During seed germination days 4–7, the GR of the M20 treatment group continued to increase but not significantly. Therefore, this data indicates that the promotion of seed germination by melatonin is likely to occur at the early stage of seed germination. P < 0.05) while MGT was reduced in the M100 and M200 treatments. On day 7 of seed germination, we obtained the fresh weight of the seeds. The M10–M200 treatments increased the FW of the seeds, with the M20 seeds reaching a significantly higher weight. The FW of cotton seed in the M20 treatment increased by 4.81% in comparison with M0  with melatonin promoted hypocotyl growth. Wen et al.[Current research indicates that exogenous melatonin has a variety of effects on plants and can be used to regulate plant growth and development. For example, melatonin can promote the growth of lateral roots  and slowen et al. investigOur experiments assessed GR, GP, GI, VI, MGT, and FW in order to accurately evaluate the germination vigor and quality of cotton seeds. GP is used to assess germination speed and uniformity. The higher the value, the stronger the germination potential is, which is one of the key indicators of seed quality. After the cotton seeds in the experiment were treated with different concentrations of melatonin, the GP of melatonin-treated seeds at concentrations of 10–50 μM was improved compared with the control group, with the 20 μM melatonin treatment performing significantly better. In contrast, treatment with 100–200 μM melatonin significantly inhibited GP. Our study revealed that high melatonin concentrations  significantly inhibited the GR of cotton seeds .Stevia seed germination compared with the 100 and 500 μM treatments. Previous studies have found that melatonin application significantly increased osmopriming effects, such that 50 μM melatonin achieved maximal results [GI indicates the germination speed, and the larger the value, the faster the germination speed . The ger results . Similar results .Arabidopsis compared with high-concentration (200–400 μM) applications. Our results showed that melatonin (10–200 μM) can increase the FW of the seeds, with the 20 μM melatonin treatment having the strongest effect and also reaching a significant level , and free radical forms, such as O2•−. The antioxidant system formed during plant evolution contributes to the balance of active oxygen metabolism. The antioxidant defense system includes the enzymatic system and non-enzymatic system. SOD and POD are essential antioxidant enzymes that can effectively eliminate superfluous ROS, such as H2O2, hydroxyl, and superoxide anions, from plant cells [2 and H2O2, while H2O2 is further decomposed by other antioxidant enzymes such as catalase.As a byproduct of aerobic metabolism , 54, reant cells , 56. SOD2O2 scavengers. Melatonin can scavenge reactive oxygen species by donating electrons and can also affect some oxidative and antioxidant enzymes in cells and tissues through its receptors, for example, by enhancing SOD and POD levels, which act to scavenge free radicals. Among antioxidants, SOD is a particularly active substance in the living body, where it eliminates harmful substances generated by plant metabolism. It is important to determine whether melatonin can also reduce the inhibition of seed germination by ROS [Melatonin and its metabolites are endogenous free radical scavengers, broad-spectrum antioxidants, and Hn by ROS , 58. Ourn by ROS  and Shi n by ROS  found non by ROS . This waPlant damage is closely related to membrane lipid peroxidation induced by reactive oxygen species accumulation. The accumulation of ROS and free radicals in organisms often leads to membrane lipid peroxidation and causes metabolic disorders. The integrity of cell structure is the basis of seed vigor, so once the cell membrane system is destroyed, leakage increases throughout the progression of germination. MDA is a product of lipid peroxidation, so it represents the level of lipid peroxidation and the degree of membrane damage in cells. The MDA content under the 20 μM melatonin treatment was the lowest, but not significantly, while the high-concentration (100–200 μM) treatments significantly increased the MDA content . This inSeed germination, as the beginning of the agricultural cycle from the perspective of the plant, is the first stage of crop growth. Hormones inside the seed control the germination of seeds . For exa3 and GA4, thus promoting subsequent germination processes under salt stress [3 content may slightly differ among the days of seed germination, both 10 μM and 20 μM melatonin treatments, especially the latter, can significantly increase the GA content of seeds. Compared with the control treatment, melatonin had no effect on GA3 content and even inhibited it at concentrations of 50–200 μM. At present, there are few studies on the effects of melatonin treatment on gibberellin content  leaves, exogenous melatonin treatment reduced ABA content and delayed its aging process [ABA is the most important plant hormone regulating seed dormancy and germination . It indu process . In our 3 content and reduced ABA content. Correspondingly, high concentrations of melatonin generally showed no effect or even inhibited germination somewhat.The present study demonstrates the positive effects of melatonin on seed germination in cotton. Low concentrations of melatonin (10–50 μM) had a positive effect on cotton seed germination compared with high concentrations (100–200 μM), of which 20 μM melatonin was the optimal treatment. This concentration also resulted in low levels of MDA concentration as well as high SOD and POD activity. Additionally, melatonin may promote seed germination by regulating the endogenous synthesis of phytohormones, as the hormone content of seeds treated with low concentrations of melatonin, which promoted seed germination, also showed a corresponding pattern, in which the 20 μM melatonin treatment achieved the best germination and significantly increased GAS1 File(XLSX)Click here for additional data file.


Pteridium arachnoideum) highly contaminated areas, investigating the associations between these parameters; and 2) to investigate the presence of papillomavirus DNA by polymerase chain reaction (PCR) in the UDT papillomas (n = 47) from 30 cattle that also had UDT SCCs. There were statistically significant associations between clinical signs and tumor localization in the UDT; between histological grade of differentiation and tumor localization; and a trend towards significant association between histological grade of differentiation and presence of metastases. The average age of cattle with oropharyngeal SCCs was 7.39 years, with statistically significant difference comparing to cattle with esophageal SCCs (8.6 years). No statistical association was observed among other clinical-pathological parameters (growth pattern and primary site of the tumor) analyzed. No BPV DNA was detected in papillomas by PCR. Therefore, these results suggest the possibility that papillomas of the UDT are not necessarily associated with BPV infection.Upper digestive tract (UDT) cancer is rare in cattle, however in Southern Brazil, the UDT squamous cell carcinomas (SCCs) are relatively common and have been associated with bracken fern consumption and the presence of papillomas. Although a theory of pathogenesis considers bovine papillomavirus type 4 (BPV-4) as a cofactor in the development of these SCCs, some aspects of the etiopathogenesis of this disease need to be more investigated. In fact, detection of BPV-4 in UDT papillomas is scarce in other regions of the world and has not been performed in Brazil. Therefore, this study had two aims: 1) to analyze the epidemiological, clinical and pathological aspects of 100 natural cases of SCCs in the UDT of cattle grazing on bracken fern ( Pteridium spp.) poisoning and presence of UDT papillomas [Squamous cell carcinomas (SCCs) are the most common oral neoplasms of humans, cats, horses, and livestock species and are the second most common of dogs, behind only the melanocytic tumors –4. Oral pillomas , 5, 7–9.Some studies show that UDT papillomas are caused by a productive bovine papillomavirus type 4 (BPV-4) infection , 11. AltIn Brazil, the relationship between these neoplasms and chronic ingestion of bracken fern is well established , 9, 14, Pteridium arachnoideum) highly contaminated areas in Southern Brazil, investigating the associations between these parameters; and 2) to investigate the presence of papillomavirus DNA in the UDT papillomas by PCR.Therefore, this study had two different aims: 1) to analyze the epidemiological, clinical and pathological aspects of 100 natural cases of SCCs in the UDT of cattle grazing for years on bracken fern , esophageal  or ruminal (rumen entrance). When cattle had more than one SCC in the UDT, the largest tumor in centimeters was considered for this study . The groPart of the papillomas (n = 47) from 30 cattle was collected and kept fresh or frozen for PCR. Another part of the papillomas and SCCs (n = 100) from 100 cattle were fixed in 10% buffered formalin, routinely processed for histopathology and stained with hematoxylin and eosin (HE). Microscopically, each SCC was graded as well, moderately, or poorly differentiated. Briefly, well differentiated SCCs were characterized mainly by islands of neoplastic keratinocytes with abundant eosinophilic cytoplasm, intercellular bridges, and concentric laminated deposits of keratin (keratin pearls). Nuclear pleomorphism and mitotic activity was minimal. Moderately differentiated SCCs were characterized by ribbons, cords or cellular aggregates of tumor cells, with less eosinophilic cytoplasm, nuclei showing greater pleomorphism and hyperchromatism and more numerous mitotic figures. Keratin pearls and intercellular bridges were less frequent. Poorly differentiated SCCs show little squamous differentiation. The cytoplasm appears amphophilic and the nuclei were pleomorphic, hyperchromatic and with marked mitotic activity. Neoplastic cells were deeply invasive, often appearing as single cells or small groups of cells surrounded by abundant desmoplastic reaction , 16.In this study we did not perform any animal experiments. All the data were obtained from the archives of necropsy reports. All the samples (paraffinized and frozen) used in this work came from the archives of the diagnostic routine of the veterinary pathology laboratory.Frozen (n = 42) and fresh (n = 5) fragments (50–100mg) of papillomas collected from 30 bovine oral or esophageal mucosa that also had UDT SCCs were submitted to total DNA extraction using the phenol and chloroform method. The samples were lysed and digested using 1x sodium dodecyl sulfate solution and 1mg/ml of proteinase K. The samples were incubated during 1–3h at 56°C. The organic and DNA phases were separated using phenol and chloroform solution and centrifugation . DNA precipitation was performed using ethanol and total DNA was eluted in 100μl of Tris-EDTA solution.6 DNA copies. PCR reactions were performed in 25μl volume, using 2μl of template DNA, 12.5μM of each primer, 2.5mM of MgCl2, 10mM of dNTPs, 1x reaction buffer and 1 unit of Taq DNA polymerase (ThermoFischer Scientific). PCR conditions were: initial denaturation (95°C for 10 min), followed by 30 cycles of 95°C–60s; 45°C–60s for primer annealing and 72°C–60s for primer extension; and a final extension of 7 min at 72°C. Products were visualized in a 1.5% agarose gel, stained with Gel Red  and visualized under UV light.Total DNA was submitted to a PCR to amplify a 165 bp segment of the BPV-4 L1 gene. Initially, the sensitivity of the PCR was analyzed using the primers described by Borzacchiello et al. . The posPanpapillomavirus PCR was also performed, using primers FAP59/64 and conditions described by Forslund et al. , and DNA5’-3’: TGTTCCAGTATGATTCCAC) and reverse (3’-5’: TCCACCACCCTGTTGCTGTA).DNA of all extracted samples was initially submitted to endogenous control PCR to verify acid nucleic integrity. For this, partial sequence of housekeeping GAPDH gene (841bp) was amplified using primers forward  or the Mann–Whitney U test when not normally distributed. A value of P <0.05 was considered statically significant.Statistical analysis and graphics were performed using R Core Team (2016) statistical software . Chi-square or Fisher’s exact tests were used to examine the associations between clinicopathological parameters . Age and tumor localization were displayed as bar graphs. Average age by tumor localization was displayed as mean ± standard deviation. Age of bovines according to tumor localization was compared using the Student’s The mean age of animals with UDT SCCs was 7.8 (±2.3) years, ranging from 3 to 13 years. Ninety-six were females and 4 were males. Eighty-nine were crossbreed (mixture of breeds), 6 Holstein, 4 Jersey and 1 Charolais.The most common reported clinical signs were weight loss (n = 95), ruminal atony (n = 77), dysphagia (n = 62), coughing (n = 59), diarrhea (n = 53), regurgitation of ruminal contents through the mouth or nostrils (n = 36), bloating (n = 35), and salivation (n = 30). Less commonly signs were halitosis (n = 27), anorexia (n = 14), dyspnea (n = 8), neck extension (n = 6), weakness (n = 5) and kyphosis (n = 2). Sometimes, the presence of large amounts of undigested food in the feces was reported.The SCCs were more commonly observed in the oropharyngeal n = 41; , followeEighty-one SCCs were of endophytic growth pattern and 19 were exophytic. The endophytic tumors were deeply infiltrating lesions, with relatively few surface manifestations  or extensive ulcers . The exoIn addition to the SCC large tumors, smaller tumors (<5cm in diameter) were observed, sometimes multifocally, along the UDT in 57 cattle. These small tumors had the same gross features of large tumors and were more frequent in the rumen entrance (n = 31) and esophagus (n = 27), followed by epiglottis (n = 17), base of tongue (n = 16), and pharynx (n = 12).The SCCs were graded as well n = 62; , moderatThroughout the UDT, from the soft palate to the rumen entrance, all 100 cattle had a few or multiple papillomas , rangingGrossly, adjacent to the SCCs, the mucosa was often irregular, with focal patches of increased prickle layer or nodules with markedly irregular surfaces, small erosions or ulcers. Histologically, these squamous intraepithelial lesions were characterized by squamous hyperplasia, mild, moderate or severe dysplasia and carcinomas in situ. Moderate to severe dysplasia and carcinoma in situ were frequently accompanied by moderate lymphoplasmacytic inflammatory infiltrate.6 DNA copies. The same was observed in panpapillomavirus PCR (FAP59/64) using bovine papillomavirus type 1 as positive control. Furthermore, the total DNA integrity of all analyzed samples was preserved, and it was confirmed by a PCR using the GAPDH housekeeping gene as target.PCR failed to amplify a segment of the BPV genome from the (n = 47 papillomas of 30 cattle) extracted samples. The BPV-4 PCR was able to detect at least 1.56 x 10A significant (p<0.001) association between clinical signs and tumor localization was observed . CoughinThe association between histological differentiation grade and tumor localization was statistically significant  was not significantly associated with the presence of metastasis (p = 0.284).Considering the primary site, epiglottis tumors metastasized in 80% of cases; n = 4/5, followed by those located in the distal esophagus , pharynx , tongue , rumen entrance  and cranial and middle esophagus . However, no significant association between tumor primary site and presence of metastasis (p = 0.486) was observed.t test) than cattle with esophageal SCCs (8.6 years). No significant difference was found among the mean age of cattle with esophageal and ruminal tumors (7.79 years) or oropharyngeal and ruminal tumors  analyzed.One hundred cases of SCCs of the UDT of cattle grazing on bracken fern in Southern Brazil were studied. Studies analyzing other aspects of the disease, with 30  and 40 cCattle with oropharyngeal SCCs were significantly younger than cattle with esophageal tumors; however, the reason for this was not determined. Cranial neoplasms may progress more rapidly than tumors of other sites in the UDT, or the clinical signs are more rapidly detectable in cranial SCCs .Although cows are overrepresented in the present study, this does not represent a predisposition for females to develop these tumors. It can be explained by the fact that this category represents the largest bovine population on farms of that geographic area and also because cows have a longer life than the other categories of cattle, being more likely to develop the lesions of chronic poisoning by bracken fern . AccordiCachexia, ruminal atony and dysphagia were the most common clinical signs in cattle grazing on bracken fern areas, similar to that already observed by other authors , 9. TherHistological differentiation grade was the main factor predicting the prognosis of SCCs in the UDT of cattle. The poorly differentiated tumors were more associated with the presence of metastasis. Although the prognostic value of histological classification of oral SCCs remains a controversial topic in human medicine and there have been a few studies to determine whether the histological grade also is prognostic in other domestic animals, this should be further evaluated because it is possible that the subtype of the SCCs and histological grade may be useful to predict tumor behavior, especially in cases in which complete clinical staging is not possible , 21. TheThe site of the primary tumor is predictive of nodal metastasis and prognosis in human SCCs . Human SSCCs of oral cavity occurred more frequently in the pharynx 63.41%; 26/41), followed by the base of the tongue  and epiglottis . In humans, SCCs occur mainly in the oral cavity (67%) followed by pharyngeal region (30%) %; 26/41,. It is iMetastases were present at a much higher frequency (52% of cases) in our study when compared to oral SCCs in dogs and cats  , 20. RegEndophytic SCCs were much more frequent (81% of cases) than exophytic tumors, in accordance with the local invasive nature of this tumor, but no statistical association was observed between the growth pattern and clinical or pathological factors. Of all endophytic tumors, 13 had a specific gross presentation as annular stenotic esophageal SCCs, which were detailed elsewhere . In huma5 genomes per cell, indicating the presence of high viral load in infected tissues.The presence of BPV DNA was investigated in papillomas by PCR with primers specific for BPV-4 and with panpapillomavirus degenerate primer. Despite the relatively low sensitivity of the PCR used in this study, high copy numbers of BPV-4 DNA sequences have been regularly detected in both naturally occurring or experimentally induced papillomas, as observed by Campo et al.  who deteThe absence of DNA detection of BPV-4 by PCR and the absence of intranuclear inclusion bodies in papillomas suggest that papillomas of the UDT in cattle may not be necessarily associated with papillomavirus infection in Southern Brazil.Additionally, the presence of squamous intraepithelial lesions found throughout the UDT epithelium that do not appear to pass through a papillomatous stage may demonstrate direct action of bracken fern carcinogens in the epithelium without the need for viral involvement . In BrazPteridium spp. toxicity. In a study of spontaneous cases of bracken fern-related SCCs of the UDT in Southern Brazil, lymphopenia was observed only in three out of 40 cases [Oral papillomatosis in cattle is rare and it is observed in specific cases of immunosuppression . Accordi40 cases .The absence of papillomavirus DNA in the papillomas, the absence of lymphopenia in most cows and the presence of squamous intraepithelial lesions in the UDT, keep open the possibility that papillomas of the UDT may not be necessarily associated with papillomavirus in cattle from our region and that papillomas and SCCs could be both independently induced by bracken fern. This possibility was also considered in a broad bovine and human comparative papillomavirus review . ConsequS1 File(PDF)Click here for additional data file.S1 FigNuclear immunoreactivity within the superficial keratinocytes in a bovine cutaneous papilloma used as positive control. Inset shows an immunolabelled keratinocyte (A). Absence of immunoreactivity in a bovine upper digestive tract papilloma. Inset shows absence of immunostaining in epithelial cells (B).(TIF)Click here for additional data file.


Lasius niger ant and the evaluation of their potential application in dye bioremediation. Strains were identified and classified as judged by rRNA 16S. The most abundant isolates were found to belong to Actinobacteria (49%) and Firmicutes (47.2%). We analyzed the content in laccase, azoreductase and peroxidase activities and their ability to degrade three known dyes  with different chemical structures. Strain Ln26 (identified as Brevibacterium permense) strongly decolorized the three dyes tested at different conditions. Strain Ln78 (Streptomyces ambofaciens) exhibited a high level of activity in the presence of Toluidine Blue (TB). It was determined that 8.5 was the optimal pH for these two strains, the optimal temperature conditions ranged between 22 and 37 °C, and acidic pHs and temperatures around 50 °C caused enzyme inactivation. Finally, the genome of the most promising candidate  was sequenced. Genes coding for two DyP-type peroxidases, one laccase and one azoreductase were identified and account for the ability of this strain to effectively oxidize a variety of dyes with different chemical structures.Industrial synthetic dyes cause health and environmental problems. This work describes the isolation of 84 bacterial strains from the midgut of the  Consequently, the wastewater generated by these processes often contains a variety of chemicals that can seriously pollute the environment2. Industrial effluents can often contain quantities of reactive synthetic dyes ranging from 2% to 50% (w/v)3. However, copper-enzymes can catalyze the oxidation of different natural and xenobiotic compounds, such as phenols, polyphenols, amines, substituted polycyclic aromatic hydrocarbons, pesticides and synthetic dyes, by using atmospheric oxygen. Among these dyes, thiazine, azo and anthraquinone are the organic groups most commonly found, and all are recalcitrant and toxic to both the environment and humans in varying degrees4. Therefore, eliminating these compounds is essential in order to protect living beings and the natural world. Although different chemical methods have been proposed to achieve this goal, many are expensive and generate the problem of disposing the resulting sludge5. In recent years, there has been a trend toward developing eco-friendly solutions for degrading dyes based on bioremediation strategies. One of the best options for bioremediation is the use of microorganisms that contain oxidoreductases capable of degrading these types of dyes. Moreover, it is known that laccases, azoreductases and peroxidases are three types of enzymes frequently involved in these kinds of oxidative processes.Synthetic dyes are widely used in different types of production including the pharmaceutical, cosmetic, hair coloring, food coloring supplements, carpet and leather manufacturing and printing industries7. As a result, laccases have received much attention, and in particular fungal laccases8, owing to their potential application in biotechnological processes. Furthermore, ever since laccase-like activity was initially detected in the bacteria Azospirillum lipoferum, the use of prokaryotic cells expressing these enzymes has increased due to their potential applications12.Among the first and most frequently used enzymes are laccases (E.C. 1.10.3.2), since they have the well-known ability to degrade resistant organic materials such as lignin13. Azoreductases are flavoenzymes that require a redox cofactor for catalyzing the degradative reactions, typically NAD(P)H14. These enzymes are also used to degrade azo dyes15, because they are able to cleave azo bonds producing simple amines and other decolourized aromatic compounds. In recent years, several bacterial azoreductases have been discovered, such as Kocuria rosea, Bacillus subtilis, Xenophilus azovorans, Enterococcus faecalis and Shewanella S1220, and used for similar types of purposes.Azoreductases (E.C 1.7.1.6) are enzymes that exist in a number of fungi and bacteria that allow these microorganisms to use azo dyes as an alternative carbon source22. Furthermore, the use of peroxides to remove azo dyes and other xenobiotic compounds from textile effluents is well established23. These enzymes are presented in both eukaryotic and prokaryotic organisms15 and new microbial peroxidases have also been recently described24.Peroxidases (E.C. 1.11.1.x) form the third group of enzymes that can be potentially used for dye degradation. These enzymes catalyze the oxidation of a number of compounds using hydrogen peroxide as the substrate25. In addition, dye decolorizing bacteria have been isolated from different niches including soil, water, the animal gut, and even the human intestine27. Some reports indicate that there are other easy-to-handle and promising ecological niches, such as insects, that are practically unexplored. Preliminary studies have indicated that bacteria from the termite gut contain putative laccases involved in the oxidation of polyphenols and other related compounds present in plant biomass28. Bacterial strains isolated from the ant midgut have also been described in diverse species of the genera Camponotus and Cephalotes, suggesting oxidative degrading enzymes contribute to the digestion of xenobiotics ingested by these insects30. Furthermore, a beneficial symbiosis between the actinomycetes inhabiting the surface of black garden ants (Lasius niger) and their host has also been reported31. However, there are no studies related to the detailed nature of the living microbiota in ants and their potential application in dye degradation and bioremediation processes.Although fungi are the most commonly used microorganisms in the treatment of azo dyes, the use of bacteria has been increased in recent years as a way to improve the end result of the oxidative processes. Prokaryotic cells are able to carry out dye degradation faster and with wider specificityLasius niger and the evaluation of their potential application in bioremediation. We explored the nature of the oxidative enzymes contained in the strains identified that were found to effectively degrade three common dyes with different chemical structures. The correlations among oxidoreductases and the sort of dye preferentially degraded are discussed, and those oxidoreductases have been partially characterized. Data on the optimal pH and temperature for the two most active strains Ln26 and Ln78 have been presented and a complete genome sequence analysis of the strain Ln26 genome has been carried out to establish possible correlations between the genes identified and enzyme activities. The advantage and novelty of the present case is the use of ant microbiota as source of enzymes for bioremediation. We were looking for strains with wide specificity containing several complementary oxidoreductases acting on dyes of different chemical structure.The present work describes the isolation and identification of a number of bacterial strains from Lasius niger) were collected aseptically from soil in Calvarrasa de Arriba (N40°54′24.19″ O5°35′31.16″), Salamanca, Spain and subjected to surface-sterilization and crushing treatments. After a week of incubation on agar plates, a total of 93 microorganisms were isolated, of which 84 were bacterial strains and 9 were eukaryotes (4 filamentous fungi and 5 yeasts). Only the bacterial isolates were selected for this study and identified through sequencing the 16S rRNA gene. Fifty-five of these strains showed an overall similarity higher than 99% between their rRNA sequences and those of strains registered in the databases, and the strains were correctly identified and classified (Table Streptomyces (11), Brevibacterium (6), Micrococcus (6), Rhodococcus (2), Micromonspora (1) and Dermacoccus (1). There were also 26 isolates from the phylum Firmicutes, which was represented by the genera Bacillus (20), Paenibacillus (4), Staphylococcus (2). Two isolates from the phylum Alphaproteobacteria were isolated, represented by the genera Roseomonas (1) and Sphingomonas (1). The largest group, in terms of the number of species recovered, was the genus Streptomyces (5 species), followed by Bacillus, Brevibacterium and Staphylococcus (2), and Roseomonas, Micromonospora, Dermacoccus, Paenibacillus, Sphingomonas and Micrococcus (1).A total of 15 black garden ants (2O2 as enzymatic substrate). The bacterial strains exhibited varying abilities to generate decolored halos on plates containing CR, TB and RBB. Forty-one strains were able to oxidize at least one of the three dyes. The decoloration activity was expressed in qualitative terms and classified as weak (W), positive (+) and strong positive (++), according to the size and time of appearance of a decolored halo (Table Biodegradation of the synthetic dyes Congo Red (CR), Toluidine Blue (TB) and Remazol Brilliant Blue (RBB) by the bacterial strains under aerobic conditions was studied using a decoloration assay in agar plates. The assay conditions were laccase-like (no extra additions), azoreductase-like (addition of NADH as enzyme cofactor) and peroxidase-like  by 16 strains.Interestingly, the degradation of each dye showed a certain correlation with some of the three conditions assayed. CR was decolorized by 19 strains under laccase-like conditions, but only 8 strains were able to act on this azo dye in the presence of NADH or H2O2 enhanced the decolorizing potential of some strains in comparison to laccase-like conditions but decreased the action of the others. Although these specific features are interesting regarding the nature of the enzymes contained by the different strains, the application of these differences in bioremediation processes is not clear yet.Some strains showed specific activity, where Ln16, Ln30, Ln49 and Ln93 only showed activity in the presence of CR under the three different conditions. In turn, Ln49 only degraded RBB under laccase-like conditions and strains Ln39 and Ln41 were capable of degrading TB and RBB, but only in the presence of the NADH cofactor. On the other hand, the addition of NADH or HOn the other hand, Table Brevibacterium permense , showed strong oxidizing activity for TB degradation, irrespective of the absence or presence of cofactors  were also found. Other features of the bacterial chromosome are summarized in Table Brevibacterium were searched at the NCBI protein database (https://www.ncbi.nlm.nih.gov/protein/). All sequences found were compared with the Ln26 Brevibacterium permense genome using Blastp32. Some genes were identified encoding possible laccase and azoreductase enzymes that had not been previously annotated. In parallel, Cu-oxidase, Dyp-type and FMN-dependent protein domains were found using Motif scan tool (https://myhits.isb-sib.ch/cgi-bin/motif_scan) and NCBI’s Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210587/). All of these studies and searches are included in Supplementary Material  have previously been described as symbionts isolated from the midgut of fire ants34. The two other isolates (Streptomyces and Dermacoccus) have been described as being associated with leaf-cutting-ants35. Moreover, it has been reported that the genus Streptomyces can act as an ectosymbiont on the surface and nests of Lasius niger31, suggesting that Streptomyces is a common symbiont of ants and could play a role in their lifecycle. The remaining 6 genera  have never been previously described as being associated with any type of ants, including Lasius niger.In this study, we isolated and characterized a number of bacterial strains obtained from the ant midgut. In fact, ant microbiota is an easy and essentially unexplored source. The classification and identification of the strains was performed using 16S rRNA sequences. All bacterial isolates were classified into 11 genera. Three of these (36. Azoreductases are oxidoreductases that act on nitrogenous organic compounds using NAD(P)H as cofactors14. According to their name, most azoreductases are able to cleave azo bond in dyes containing this chemical group, although sulfonated dyes are resistant to degradation37. Peroxidases are powerful oxidoreductases, as they use hydrogen peroxide as co-substrate. We have chosen three standard dyes, with different chemical structures, widely used in textile and biotechnological processes. In total, three types of dyes were used: Red Congo ; Blue Toluidine ; and Remazol Brilliant Blue R .In turn, according to our results, some isolates may contain oxidoreductases. Briefly, laccases are multicopper oxidoreductases that show relatively wide specificity using atmospheric oxygen as the oxidant substrate38, we found that a significant number of strains (19) were able to degrade CR  (19 vs 17). In sum, the addition of NADH generally does not improve the efficiency of this sulfonated azo dye, suggesting that the action of active bacterial strains is mostly due to laccases rather than to azoreductases.17 and the results presented here, azoreductases are capable of decolorizing TB. Most of our strains effectively degrade TB in the presence of NADH (21), but some strains are also active in the absence of this cofactor. This point out that both laccases and azoreductases are able to act on basic thiazine dyes.According to previous reports40. Our results indicate that RBB is mostly degraded in the presence of H2O2 (peroxidase-like conditions). Sixteen strains are able to decolorize this dye under these conditions, but only 8 are capable of catalyzing under laccase-like conditions, and only 5 under azoreductase-like conditions. However, most of the strains active in the presence of H2O2 presented weak activity (10 out 16), indicating that this dye is not easily decolorized even by bacterial strains presumably containing peroxidase-like enzymes.As far as we know, the effect of laccases and azoreductases on RBB and related sulfonated anthraquinones is not described in the literatureIn addition to the classification of these types of enzymes, the use of efficient strains with wide specificity in industrial processes, together with simple and cheap conditions, is particularly interesting. Laccases seem to be the most efficient and inexpensive enzymes for dye decoloration, as they do not require the addition of NADH or hydrogen peroxide. The addition of these molecules to environments inoculated with bacteria for the purpose of bioremediation would enhance the contribution of azoreductases or peroxidases, but this would however be more difficult to carry out and more costly.42, azoreductases43 and peroxidases24. Ln 26 was able to partially decolorize the three dyes in liquid medium after around 3,5 days, although RBB was the most resistant one  and a highly promising strain with wide specificity and the ability to decolor all type of dyes in different conditions (Ln26). Thus, the strain Ln26 seems to be the best candidate for using in bioremediation processes and, consequently, its genome was sequenced and genome mining was performed in order to examine the presence of genes encoding oxidoreductases. Genome sequencing is an effective tool for searching for correlations among the enzymatic activities measured and the occurrence of genes encoding different forms of oxidoreductases. Using both RAST and BlastKoala tools, we detected genes encoding the 3 different types of oxidoreductases previously described, confirming the potential use in bioremediation processes for degrading dyes. These three enzyme types appear to be commonly found in Lasius niger midgut. Initially, the trapped ants were disinfected using a 2.5% Cl2Hg solution for 10 min then washed five times with sterilized H2O. The disinfection was performed to ensure that ants did not contain any external microorganisms on their bodies. Afterwards, the ants were crushed and plated under aseptic conditions on Yeast Mannitol Agar (YMA)48 and Yeast Extract Dextrose medium (YEDP) . All plates were incubated at 28 °C for 7 days. Colonies were selected based on colour, shape, and size and the individual colonies were re-streaked on fresh YMA or YEDP plates until a pure culture was obtained.All bacterial strains were isolated from the TM Plant PCR Kit (Sigma) following the manufacturer’s instructions. First, amplification of the 16S rRNA was performed using bacterial DNA as the template, The primers 27F (5′AGAGTTTGATCCTGGCTCAG) and 1522R (5′AAGGAGGTGATCCANCCRCA) were used for the PCR reaction under the following conditions: preheating at 95 °C for 9 min, 35 cycles of denaturation at 95 °C for 1 min, annealing at 54 °C for 1 min and extension at 72 °C during 1 min. The final extension in the last cycle was performed at 72 °C for 7 min. The amplicons (approximately 1200 bp) were checked on a 1% (w/v) agarose gel and the GeneRuler 1 kilobase DNA Ladder (Thermo Scientific TM) was used as the size marker. For purification of DNA fragments, GeneJet Gel Extraction and DNA Cleanup Micro Kit (Thermo Scientific TM) was used according to the manufacturer’s instructions. The sequences obtained were assembled using BioEdit software49 to obtain contig sequences for each strain. The isolates were identified using NCBI’s Blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch).DNA for amplifying and sequencing 16S rRNA was extracted from pure colonies using the REDExtract-n-Amp2O2 favouring azoreductase and peroxidase activities were also evaluated. All dyes were purchased from Sigma and were added to the media after autoclaving as sterile-filtered water solutions to avoid any chemical decomposition due to high temperature. Dye decoloration was carried out in agar plate assays using squared Petri dishes containing 50 ml of YE medium  supplemented with 0.05% (w/v) of the each dye49. In addition to the control series, lacking any additive that may have facilitated laccase-like activity, another series of plates were supplemented with either 0.1 mM NADH or 1 mM H2O2 as cofactors, in order to enhance the oxidative ability of bacterial strains containing azoreductase-like or peroxidase-like activities, respectively. Another set of plates, in addition to the plates containing the standard dyes, were supplemented with 1 g/L 2,6-dimethoxyphenol (DMP)50 as an auxiliary indicator of the oxidative ability of bacterial strains (see below). All agar plates were inoculated by adding 5 µl aliquots of bacterial solutions (around 107 cells mL−1) and incubated at 28 °C for 7 days. The plates were monitored daily by visual inspection. Oxidizing activity was considered positive when a diffuse (weak activity) or clear halo (+) was observed around the colonies at the end of the 7th day, although in the most active strains, halos were already evident during the 1st-3rd days of incubation (marked as strong positive (++) at Table Congo Red (CR), Toluidine Blue (TB) and Remazol Brilliant Blue R (RBB) were used as the test dyes for the detection of bacterial strains able to decolorize recalcitrant compounds. These dyes are widely used in industrial processes and were selected based on their different chemical structures: azo (CR), thiazine (TB) and anthraquinone (RBB) Fig. . The abi7 cells mL−1). Then, flasks were incubated at 28 °C and aerated by gentle shaking at 180 rpm during 5 days. We used three replicates for each condition. At the beginning of the experiment (time zero) and subsequently at several periods of time, samples of 1 mL were pipetted out from the flasks and centrifuged at 10.000 rpm to settle down the bacterial pellet. The clear supernatants containing the residual decolorized dye were monitored by measuring the absorbances of the samples at wavelengths 468, 498, 590 and 590 nm for DMP, CR, TB, and RBB respectively. Some supernatants should be diluted 10-times before absorbance determination to get appropriate linear range between absorbance and concentration. According this approach, the monitoring was not possible longer than 4 days due to artifacts in the color of residual dyes related to pH changes and late phase of the bacterial growth.Quantitative estimation of dyes decoloration by Ln26 was also carried out in liquid media assay using laboratory flasks containing 30 mL of YE medium supplemented with 0,05%(w/v) of each dye. Other laboratory flask was supplemented with 1 g/L DMP used control of the oxidizing potential of the bacterial strain. All flasks were inoculated by adding 1 mL of a fresh bacterial sample  or 100 mM sodium phosphate buffer . Aliquots of 20 µl from each tested bacterial strain extract (protein concentration were 2.2 and 3.1 mg/ml for Ln26 and Ln78 respectively) were added to 180 µl of DMP solution in the appropriate buffer and temperature . Initial A468 were measured, and the plates were incubated overnight and A468 were again determined. Negative controls reactions were carried out under identical condition, but 20 µl of the appropriate buffer was added instead of the bacterial extracts. Commercial laccase from Trametes versicolor was purchased from Sigma and used as positive control. A calibration curve was plotted using amounts of the laccase ranging from 0.08 to 87 units to correlate A468 and units of laccase activity. According to the instructions provided by the manufacturer for laccase originating from Trametes versicolor one unit corresponds to the amount of enzyme that converts 1 µmole of catechol per minute at pH 5.0 and 25 °C. All experiments were performed in triplicate.Oxidative enzymatic activity under different of pH and temperatures conditions was determined using DMP as the chromogenic substrate52. Gene calling and annotation was performed using RAST 2.0 (Rapid Annotation using Subsystem Technology)53 and The SEED viewer framework54 and BlastKOALA tools55 were used to search for genes related to dye decoloration.The genomic DNA for genome sequencing was obtained from bacterial cells of Ln26 strain grown on TSA plates and collected after 24 h at 28 °C, using the ZR Fungal/Bacterial DNA MiniPrep (Zymo Research). The draft genome sequence of the isolated was obtained by shotgun sequencing on an Illumina MiSeq platform via a paired-end run (2 × 251 bp). Sequence data was assembled using Velvet 1.2.10All the bacterial strains used in this study were isolated before the signed of the Nagoya Protocol.Figure 1SFigure 2S