Autologous fat grafting procedures in plastic surgery have been extensively used to reinforce soft tissue in congenital or acquired tissue impairments. With this background, the aim of this study is firstly to examine the impact of a selective centrifugation on existing adipose stem cells (ASCs) in terms of stemness profile maintenance and, secondly, to investigate the effect of restoring volume in reconstruction on patients affected by soft tissue damage. After centrifugation, the fat graft products were separated into two layers and subsequently examined in vitro for the expression of CD34, CD90, CD117, CD105, CD29, CD31, CD44, CD73, CD133, CD14 and CD45 markers by flow cytometry and gene expression analyses were performed for Sox2, WNT3A, END, CD44, FUT4, COLL1, CTNNB1, hbEGF, KRTLG, MMP2 and VIM genes. The results showed that in the middle-high density (MHD) layer there was a peak concentration of ASCs, compared to another layer obtained after centrifugation. Research carried out on patients under treatment for soft tissue regeneration using cells obtained from MHD layer selection will be fundamental in comparative analysis. These studies will lead to an adequate standardization of outcomes, provided that treatment is performed through cell selection. Therefore, a unique procedure in tissue reconstruction and regeneration through fat grafting is presented here. Fat grafting procedures are frequently used to restore volume and to regenerate tissues following the existence of stem cells in human adipose tissue, as revealed by Zuk et al. in 2002 . StudiesAdipose tissue contains adipocytes with a surrounding and supporting heterogeneous cell population known as stromal vascular fraction (SVF), which accommodates the adipose stem cells (ASCs) ,12. ThesAutologous fat grafting, also known as lipofilling, is based on various techniques differing in harvesting centrifugation or infiltration methods. Fat grafting often provides favorable, albeit unpredictable long term outcomes. Some authors have attempted to minimize fat graft loss and to obtain a high rate of adipose stem cells that are easily administered into soft tissue, accessible for tissue regeneration. The purpose of this study is therefore to assess the stemness potential of cells within aspirated and centrifuged fat tissue, with particular attention paid to examining the molecular and cellular features of fat distributed cells after centrifugation . MoreoveThe fat of healthy patients was aspirated and centrifuged according to a technique, which separated the various components of the aspirated fat into three levels. The top level was the least dense and consisted mainly of oil. The central part was composed primarily of adipose tissue. The lower level was composed of blood, water, and other watery elements. The top and bottom layers were discarded. The middle layer (ML) was divided into two distinct part, as previously described by some authors ,23: a loA total of 25 female patients were operated on consecutively. The age of the patients ranged from 41 to 67 years (mean = 52.8 years). The mean number of sessions performed on each reconstructed breast was 3.4 ± 1.9 (range = 1–6 sessions) with an average volume of 245.5 ± 18 cc of autologous fat injected in each session (range = 200–300 cc). The average interval between two sessions was 3.9 ± 1.3 months (range = 2–6 months). The clinical outcome following fat grafting in mammary reconstruction included both volume increase and enhancement of regenerative potential. In all cases, the skin of the reconstructed or irradiated breast was supple, and the depression expansible, ideally with no evidence of post-radiation lesions such as telangiectasia or severe post-irradiation fibrosis. In addition, settlement of superficial irregularities and major pliability were observed. The aforementioned clinical benefits allow the rehabilitation of skin dystrophy, and are the results of regeneration on injection of ASCs layer . OvercorPostoperative 2D/3D sonography  and MRI A total of 15 patients were operated on consecutively. The age of the patients ranged from 42 to 67 years (mean = 54.8 years). All patients had previously undergone various unsuccessful treatments. The mean number of sessions performed on each pathological scar was 1.4 ± 0.9 (range = 1–3 sessions) with an average volume of 24.5 ± 10.8 cc of autologous fat injected in each session (range = 9–41 cc). The average interval between two sessions was 3.9 ± 1.3 months (range = 2–6 months). In the pathological scar group, the clinical outcome included, most importantly, a significant improvement in skin scar colour, pliability and texture. We observed, in all cases, a general enhancement in the quality of the scars, leading to improvements in the scar assessment scores assigned by both the observers and patients A. We obsAutologous fat grafting has several beneficial characteristics, such as lack of immunogenicity, simple surgical procedure, low cost, and easy accessibility, and has therefore become a popular technique for numerous reconstructive procedures ,4,7,8,10The aim of this study is to assess the different densities of ASCs within the injectable layers following centrifugation. Previous works have shown that the lower layer of the injectable fat yields the greatest density of viable cells ,23. MoreIn these scenarios, this technique has also shown its clinical effectiveness in the treatment of atrophic retraent scars. In this clinical series, patients reported an important articular functional recovery in terms of range of motion, and a significant improvement of the skin, resulting in better pliability and less tension. Research has identified that cell inflammation is characteristic of fibrotic disease and may stimulate fibroblast development and expansion via cytokine promulgation . ASCs arThe methods and techniques used for grafting autologous adipose tissue remain varied with no evidence in literature of a unique procedure to process harvested fat. The fat grafting technique has improved over the years and has demonstrated that centrifugation at 1300 rpm for 5 min preserves the cell integrity and the characteristics of stemness, yielding minimal resorption rates . Moreove2HPO4 10 mmol/L, KH2PO4 1.8 mmol/L), and centrifuged at 1300 rpm (250× g) for 5 min.Liposuction was performed in the Plastic and Reconstructive Surgery Clinic and adipose tissue was collected. The aspirate from the liposuction was set in NaCl 0.9%, and washed twice with phosphate-buffered saline , adipose (middle) and fluid (bottom). The adipose portion was divided equally into two layers: the lower two-thirds and the upper third . The twoAfter digestion, at least 500,000 fresh cells were incubated with primary antibody for 30 min at 4 °C, washed twice in PBS and incubated with a secondary antibody. Antibodies used in this study were as follows: anti-CD117 PE (c-kit) , anti-CD34 FITC and PE , anti-CD90 FITC , anti-CD105 FITC , anti-CD29 Cy , anti-CD31 FITC , anti-CD133 PE , anti-CD73 PE , anti-CD44 FITC , anti-CD45 Cy and PE , and anti-CD14 PE . A FACS Vantage (Becton Dickinson) was used. CellQuest software was used for analyses.After digestion, the RNeasy Lipid Tissue kit  was used to obtain total RNA, as well as DNase digestion via RNase-Free DNase Set (Qiagen). Two different layers were obtained from the centrifuged lipoaspirate of the adipose tissue and from the lipoaspirate specimens that were not centrifuged. A RT2 First Strand kit (Qiagen) was utilized to reverse transcribe 800 ng of RNA. PCR in real time was carried out conforming to the instruction manual for the RT2 Profiler Human Mesenchymal Stem Cells PCR array , and with SYBR Green ROX FAST Master Mix (Qiagen). A Rotor Gene Q 100 (Qiagen) was used to perform thermal cycling and to detect fluorescence. The data were examined with Excel-based PCR Array Data Analysis Templates (SABiosciences) and the results were recorded in ratios in relation to the mRNA expression of uncentrifuged fat.Between March 2009 and March 2013, injections of autologous fat were administered to 40 patients. The inclusion criteria for the procedure were disease-free patients with close clinical follow-up, a minimum of eight months after completion of radiotherapy and the patient’s refusal to undergo autologous tissue reconstruction. Based on the inclusion criteria, a total of 25 patients were enrolled. In seven cases, lipoinjection was performed following quadrantectomy/lumpectomy, and radiation therapy. The remaining 18 patients underwent mastectomies as follows: ten patients received modified radical mastectomy with subsequent radiation treatment in two cases. Eight patients underwent nipple-sparing mastectomy with radiation treatment in one case. Of the 25 patients who underwent autologous fat grafting to improve the cosmetic result of reconstructive breast surgery, 10 patients had undergone parietal radiotherapy prior to breast reconstruction with implant. In these cases, fat injections were administered multidirectionally, from superficial to deep layers, spreading the fat throughout the irradiated area. In the modified radical mastectomy case, fat was transplanted in two different overlay planes in multiple tunnels. Following injection sessions, the final prosthesis was implanted under the pectoralis major muscle.In 15 cases, the technique was used for treatment of post-traumatic scars. The injection phase was completed with small fat parcels implanted in multiple tunnels allowing full access of the fat graft to the surrounding blood supply.We recorded the procedure date, the amount of injected fat, follow-up duration, and events of any complications that occurred. The pre-operative and post-operative images were analyzed by two masked surgeons who rated them according to the following rating scale: good/very good, moderate, or inadequate.g) for 5 min. Following the centrifugation procedure, three layers were obtained from the specimens: an oily yellow top layer acquired from the destroyed fatty tissue fragments, a central layer of adipose tissue graft, and a bottom layer composed of blood. After discarding the oil (superiorly) and the blood (inferiorly), we collected the middle layer and in particular, due to the short oil derived residual properties, we selected the lower two-thirds of purified lipoaspirate deposited in the lower part of the syringe near the needle after the removal of red cells.The patients were informed of standard preoperative instructions, including the use of antibiotics and recommendations against the use of aspirin and derivatives at least one week before surgery. For all the patients, the same technique of autologous fat grafting was performed. Tumescent injection was used . The fat donor sites were the hips, the abdomen, the trochanteric area, and the inner sides of the thigh and knees. The fat was collected using a two-holed blunt harvesting cannula (3-mm inner diameter) attached to a 10-mL Luer-Lok syringe , progressively applying a gentle negative pressure and pulling gradually back on the plunger. The capped 10-mL Luer-Lok syringes were placed in a centrifuge and spun at 1300 rpm , Vancouver Scale and photo documentation, as well as the measurement of microcirculation using the Doppler ultrasound.t-test. The threshold for statistical significance was established at p-values < 0.05. Repeatability was considered as a standard deviation to calculate differences between measurements using SPSS 16.0 software  for testing analysis.All data were statistically analyzed using One-way ANOVA test. The POSAS and Vancouver score results were compared using an unpaired The study respects all ethical requirements in its objectives and methodologies. We strictly comply with widely recognized international codes of practice such as the Nuremberg code, the Helsinki agreement, the conventions of the Council of Europe on human rights and biomedicine, with particularly attention to EU legislation: 2001/83/EC, 86/609/EEC and FP7 Decision nr 1982/2006EC. Human biological samples are necessary because we need to test human cells, which have unique biological characteristics, distinct from those of animals. The overall intention in the project is to reduce the number of animal experiments. Only adult patients who are able to give consent will be included. All the patients that are the subjects of our study donated their consensus to scientific treatment and publication of their clinic situation and images. We have obtained written informed consent from all patients. This study was approved by our Internal Ethical Committee (RegStem01–03/2009) without any registration in public registry because this study is not a clinical trial.In conclusion, there are several ways to optimize a fat grafting procedure and our study has provided evidence that stem cells selected from the middle-high density layer of human adipose tissue, after centrifugation, constitute a large, ideal source of cells for tissue regeneration, and tissue-based clinical therapies. However, other techniques may be concurrently used, including PRP  or Rigen


Previous studies have shown that the bone marrow micro-environment supports the myeloproliferative neoplasms (MPN) phenotype including via the production of cytokines that can induce resistance to frontline MPN therapies. However, the mechanisms by which this occurs are poorly understood. Moreover, the ability to rapidly identify drug agents that can act as adjuvants to existing MPN frontline therapies is virtually non-existent. Here, using a novel predictive simulation approach, we sought to determine the effect of various drug agents on MPN cell lines, both with and without the micro-environment derived inflammatory cytokines. We first created individual simulation models for two representative MPN cell lines; HEL and SET-2, based on their genomic mutation and copy number variation (CNV) data. Running computational simulations on these virtual cell line models, we identified a synergistic effect of two drug agents on cell proliferation and viability; namely, the Jak2 kinase inhibitor, G6, and the Bcl-2 inhibitor, ABT737. IL-6 did not show any impact on the cells due to the predicted lack of IL-6 signaling within these cells. Interestingly, TNFα increased the sensitivity of the single drug agents and their use in combination while IFNγ decreased the sensitivity. In summary, this study predictively identified two drug agents that reduce MPN cell viability via independent mechanisms that was prospectively validated. Moreover, their efficacy is either potentiated or inhibited, by some of the micro-environment derived cytokines. Lastly, this study has validated the use of this simulation based technology to prospectively determine such responses. The tumor micro-environment is a complex system of host cells that work, inadvertently, to promote the growth of neoplastic cells. Host cells within a typical tumor micro-environment include fibroblasts, endothelial cells, and immune cells , 2. WithThe Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) encompass polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). There are approximately 22 cases of PV, 24 cases of ET, and 1.46 cases of myelofibrosis for every 100,000 people, which amount to approximately 68,000 patients with PV, 74,000 with ET, and 4,500 with MF in the United States . A very ex vivo. This predictive simulation approach differs from other biological modeling methodologies in that it incorporates integrated cancer physiology networks that cover all disease phenotypes in the simulation and provides semi-quantitative outputs and trends [Predictive simulation modeling is an emerging technology in the realm of personalized medicine. A computational simulation avatar is created via genomic profiling information that is derived for a given cell line or patient biopsy. A digital library of molecularly targeted drugs is tested on this individual avatar (N=1), where drug agents are combined at different doses via simulation-based computation, resulting in analyses of the effects of a large number of drug combinations on various tumor phenotypes, including cell proliferation, apoptosis, and viability. A critical feature of this approach is that the drug library can be composed of drugs that have been approved for other indications and/or new investigative drug agents. The assessment of drug impact can be done under variations of the bone marrow micro-environment from the paracrine perspective, and along with the genomics driven autocrine loops, can provide insight into factors that are interfering with drug response. Shortlisted drugs and drug combinations are then experimentally validated on immortalized cell lines that represent the disease of interest or patient cells d trends –13. The Given the importance of the tumor micro-environment on both MPN disease pathogenesis and drug resistance, we hypothesized herein that predictive simulation modeling could be employed in order to better understand the role of inflammatory cytokines on drug efficacy and resistance, and to develop a methodology to tease out the causal factors for enhancing or reducing the response of the prospectively validated shortlisted drug combinations. To test this, we selected two representative MPN cell lines, Human erythroleukemia 92.1.7 (HEL) and SET2, and obtained their genomic profile. The HEL cell is largely defined by the Jak2-V617F mutation, along with deletion of CDKN2A, RB1 and TP53 –16; whilThe simulation model used herein includes representation of important signaling pathways implicated in cancer as detailed in the methods below Figure . The SETet al. [et al. [et al. [In order to determine the confidence of the virtual MPN HEL and SET2 cell lines, we evaluated the correlation of predicted outcomes with that various known experimental datasets in the literature including Rubert et al. , Will et [et al. , and Par [et al. . Figures [et al. .et al. also presented results on the enhancement of efficacy of JAK inhibitor-1 on apoptosis in SET2 cells, by the inclusion of the BCL2 inhibitor, ABT737 [Figure , ABT737 . This co, ABT737 , therebyet al. similarly tested the ability of the Jak2 kinase inhibitor, TG101209, to inhibit HEL cell proliferation [Pardanani feration . Our preferation , therebyThus, when taken together, the data in Figure The predictive simulation modeling and the subsequent therapeutic drug simulations indicated that the Bcl2 inhibitor, ABT737, and the pre-clinical Jak2 kinase inhibitor, G6, would be efficacious against Jak2 driven pathogenesis, when used alone and synergistically when used in combination. ABT737 is a BH3 mimetic and inhibits several proliferative genes including Bcl-2, Bcl-xL, and Bcl-w . G6 is a50 concentrations of the two drugs. Combinations of the drugs at the varied doses indicates that a combination of Jak2 inhibitor, along with BCL2 inhibitor at half the IC50 G6 dose, along with 1/3rd of the ABT737 dose, indicated by the black cross on the left, could achieve the same 50% inhibition as the individual IC50 doses. Similarly 1/3rd of G6 dose with half of the ABT737 dose, as indicated by the black cross on the right, also gave an IC50 inhibition, indicating that these two drugs are synergistic in reducing HEL cell viability.Figure Collectively, predictive cellular modeling and subsequent drug simulations indicated that ABT737 and G6 would significantly decrease HEL cell viability when used individually, and synergistically, when used in combination. The subsequent prospective validation on cultured HEL cells validated those predictions. As such, these studies validate the virtual HEL cell line and how two investigative drug agents affect it.per se, can render MPN cells resistant to Jak kinase inhibitors including Ruxolitinib and atiprimod [Cytokine storm is a common syndrome within MPN patients. It is characterized by increased levels of inflammatory cytokines, both in the bone marrow and the peripheral blood –25. Withtiprimod , 26. IntOverall, the results in Figures Dysregulated Jak2 kinase signaling has been implicated in a number of human pathologies including hypertension, diabetes, and cancer . Within In PV, >90% of the patients harbor the Jak2-V617F mutation and in most instances, this is both the key and only aberration. In such profiles, there is a Jak2 driven up-regulation of anti-apoptotic genes BCL2, MCL1, BCL2L1 , 28. TheAcross the various cytokines measured in the plasma of MPN patients, eight chief cytokines have been described; MIP-1β, TNF-α, IL-6, GM-CSF, IFN-γ, G-CSF, IFN-α, and Rantes , 25, 34.in silico in various profiles and disease models across a large number of phenotypes and biomarkers. This can provide an in-depth insight into efficacy and mechanistic details without putting in extensive resource, time, and efforts into running experiments. The data provided herein have been used as the foundation for a personalized medicine approach for several hematological disorders. Specifically, peripheral blood and/or bone marrow samples are being collected from myelofibrosis, multiple myeloma, acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome patients under an IRB approved protocol (NCT02435550). After collection, the samples are being subjected to next-generation sequence (NGS) analysis, protein mutation analysis, and metabolomic analysis. The profiling data are then being fed into the predictive simulation modeling, where a short list of novel drug combinations is subsequently generated for each patient. The drug combinations are then being validated on ex vivo cultures using the same patient's primary cells. The most efficacious therapy is then being tested in the patient. Consequently, the studies described in this current work have served as the basis for a hematological-based, precision medicine campaign that can readily be leveraged into other cancers as well.The predictive simulation modeling that was employed in this work integrates signal transduction, epigenetic regulation, protein homeostasis, metabolic pathways, and other phenotypes representing not just MPN, but all cancers Figure . The plaa priori in context of their genomics and microenvironment, the in silico simulation model provides an innovative approach to precision medicine for not only MPN, but other neoplastic disorders as well.In summary, this work identified two investigative drug agents that act significantly when used alone and synergistically when used in combination, to reduce the viability of two MPN model cell lines. We found that their efficacy is potentiated by some inflammatory cytokines that are typically elevated in MPN. Thus, by accurately predicting responses of cells to targeted agents The simulation experiments and analyses were performed using the predictive tumor model, a comprehensive and dynamic network of signaling, and metabolic pathways in the context of cancer physiology as described . The simControl stage: The model is simulated for 50,000 seconds simulation time during which the different species  attain a steady state concentration. This concentration is dependent upon the balance between the rate of reaction nodes producing the species and the reaction nodes utilizing/degrading the species. This is the un-triggered control baseline.Disease stage: At simulation time of 50,000 seconds, the genomic aberrations for a profile (cell line or patient biopsy) is overlaid on the control network through representing the aberrations as increased or decreased expression/activity of genes or proteins to represent copy number variations and mutations respectively. The system is then simulated further to 125,000 seconds simulation time. During this time, the system attains a new steady state which aligns to the network dynamics of the tumor cell line or patient profile depending on the genomic input file.Disease + Drug Stage: Once the disease baseline is created, the drug is introduced in the system at 125,000 seconds by perturbing the target reaction nodes to represent the drug mechanism of action. The system is simulated further until 200,000 seconds simulation time. The percentage change seen in values of the biomarkers and phenotypes such as viability and proliferation index from the disease baseline give the read out for the therapeutic potential of the drug.Disease + Microenvironment + Drug: To the disease baseline we first add in the changed cytokine and chemokine paracrine inputs, either cumulatively or individually, and simulate to create the disease baseline variant with the microenvironment. The drug can then be simulated on this modified disease baseline in presence of the microenvironment factors to assess response, and compare against the response without the microenvironment factors.The simulation protocol for the testing of the drug combinations in the virtual MPN cell line models for HEL and SET2 in context of the different microenvironment based cytokine variation consisted of the following steps:To create the MPN cell line models SET2 and HEL, we used the simulation protocol described above. Genetic profiles (mutation and CNV) of these cell lines were derived from sequencing and cytogenetic data in the literature. Data obtained from the cBioportal and Sanger databases were used as input files to trigger the system to a neoplastic disease state , at the site of action and the dose response of the drug entails varying the percentage target inhibition of the primary drug target that could equate to different concentrations of the drug in the in vivo setting.To simulate the effect of a drug in the 2 humidified atmosphere and passaged at least two times per week. The Jak2 inhibitor, G6, was obtained from the NIH/NCI drug repository (NSC33994). The Bcl2 inhibitor, ABT737, was obtained from Selleck Chemicals. The IL-6, TNFα, and IFNγ were purchased from R&D Systems .HEL cells were purchased from ATCC . SET-2 cells were obtained from DSMZ . The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% COLog phase cells were seeded in 96-well plates at 30,000 cells per well in 100 μl of media containing either 0.25% DMSO or varying concentrations of drug. Cell viability was determined 68-72 hours later via the CellTiter 96AqueousOne Solution Reagent from Promega  according to the manufacturer's protocol. Specifically, 20 μl of reagent was added to each well and plates were incubated for 2-3 hours at 37°C and absorbance was then read at 490nm using a 96-well plate reader. Data are expressed as a percentage of vehicle-treated cells (0.25% DMSO), which was arbitrarily defined as 100% viable.t-test as well as a repeated measures analysis of variance followed by Bonferroni and Student-Newman-Keuls post hoc tests for multiple comparisons. P values of <0.05 were considered statistically significant. There is no statistical variation seen in the predictive results since it is based on a kinetic model that will achieve the same results with repeated perturbations. The variation can be seen by varying the perturbations/inputs.All results were expressed as mean ± SD. Statistical comparisons were done using the Student's


The objective of this research was to study and to prove the effectiveness of hyperbaric oxygen therapy (HBOT) starting time on liver morphofunctional changes after ischemia-reperfusion in the hind limb of rabbits.This research used a complete randomized design with 4 groups and 6 repetitions on each. After 6 h artery femoral is ligation, reperfusion was performed for 100 min (G1), HBOT for 90 min after 10 min reperfusion (G2), 10 min reperfusion (G3), and HBOT 90 min after 60 min reperfusion (G4). Then, all of the rabbits were sacrificed. The liver and blood were taken for histopathological changes examination as well as for measuring the level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The statistical test using Kruskal–Wallis and Mann–Whitney showed that the score of degeneration, necrosis, and portal inflammation in groups without HBOT (G1 and G3) were not significantly different, as well as in group with HBOT (G2 and G4) (p>0.05). However, the scores of histopathological changes in G1 and G3 were significantly different from those in G2 and G4 (p<0.05). The levels of AST and ALT in the groups without hyperbaric therapy (G1 and G3) were not significantly different from those in the groups treated with hyperbaric therapy (G2 and G4) (p>0.05).Hind limb ischemia injury reperfusion can trigger damage for liver morphology, but not lead to liver dysfunction. Reperfusion can trigger increased activity of neutrophils, while neutrophil infiltration in the organ will lead to dysfunction. HBOT can inhibit the activity of neutrophils and the dysfunction of organs caused by ischemic reperfusion.HBOT for 90 min, both 10 and 60 min after the reperfusion, can protect hepatocytes from damage. Ischemic condition can occur in a wide variety of disease status. Nevertheless, an ischemic condition often occurs in a variety of surgical procedures such as in a surgical process of vascular or organ transplantation. In such circumstances, ischemic injury can occur when blood flow to a tissue is reduced or stopped in a long time, resulting changes in the metabolism of muscles, from aerobic state into anaerobic one. Consequently, adenosine triphosphate (ATP) depletion and calcium (Ca) extracellular transfer into muscle cells occur, leading to abnormalities in metabolism and thrombosis, as well as tissue inflammation .Blood flow improvement (reperfusion) is the main effort to overcome tissue ischemia (ischemia-reperfusion [IR]). However, reperfusion has negative side effects because it can cause damage and impaired organ function when it is not given appropriately. Inappropriate reperfusion then can trigger activation of proinflammatory mediators as well as formation of reactive oxygen species (ROS), resulting injury to the tissue . In seveMany researchers actually have already been conducted on the effects of various drugs in reducing the negative impacts of reperfusion. Those previous researchers focused on the impacts of reperfusion used hyperbaric oxygen therapy (HBOT), a method of therapy using 100% oxygen with a high atmospheric pressure in a special room. HBOT has been proved to be very helpful to reduce the negative impacts of reperfusion in various tissues by reducing the activity of mediators of inflammation and oxidative stress induced by the reperfusion process .Oryctolagus cuniculus) as animal models after hind limb reperfusion.Unfortunately, there have not been a lot of researchers aimed to know the appropriate time for the administration of HBOT in patients with various organ function disorders after reperfusion, especially its impact on liver organ. In patients with stroke, HBOT is known to be effective if given exactly 3 h after reperfusion since if given more than 3 h, it will not give any effect . This reThis research was conducted under the ethical use of laboratory animals and also was approved by the Ethical Committee of Faculty of Veterinary Medicine, Universitas Airlangga.This research was conducted at the Faculty of Veterinary Medicine, Universitas Airlangga, and also at the Department of Underwater and Hyperbaric Medicine, Hospital Dr. Ramelan, Surabaya. Preparations for histopathological examination and blood tests were made in the Department of Pathology and Veterinary Hospital, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya.Experimental animals used in this research were 24 male New Zealand rabbits aged 6 months old and weighed 2-2.5 kg. Most studies use male animal models because of their stable hormonal status during treatment, and these animals are not at risk of being affected by hormonal (estrogen/progesterone) fluctuation related to the reproductive cycle that may confound the results of the study. Reperfusion injury using the rabbit as animal model showed significantly less infarct in female hearts as compared to males .Materials used in this research were mineral water and food for rabbits, 70% alcohol, 10% ketamine, 2% xylazine hydrochloride, atropine sulfate, sterile thump, 10% buffered formalin, materials for staining hematoxylin-eosin (HE), materials for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) examinations, and materials for the euthanasia of rabbits at the end of the research.Tools used in this research were rabbit hutches, minor surgery equipment, masks, gloves, 10 cc syringe, syringes 3 cc, silastic band, hyperbaric chamber, a place to store organs, light microscope, object, cover glass, as well as blood container tube without anticoagulants.ad libitum. Next, femoral artery ligation was conducted on all the experimental animals. They were then divided into four groups, namely, G1, G2, G3, and G4.Those rabbits were adapted for 1 week before the research. They were fed and given to drink Afterward, before anesthesia was conducted for surgery, all those rabbits were fasted for 12 h. Next, anesthesia was performed by weighing each rabbit and then injecting them with 0.02 mg/kg of atropine sulfate intramuscularly. After 10 min, anesthesia was carried out by injecting a combination of ketamine-xylazine with doses of 35 mg/kg and 5 mg/kg intramuscularly .Although 30-50% of rabbits produce endogenous atropinase enzymes, because using atropine sulfate 0.1-0.2 mg/kg bw/sc, im. in some studies often helpful to decrease salivary production, bronchial secretion and preventing the occurrence of vagal which can cause bradycardia .After all of those rabbits were anesthetized, an incision was made in the groin area of their sinister. Next, dissection was performed to separate the femoral artery from the nerve and the vein. Ischemia was induced by ligating the femoral artery with silastic band for 6 h, then followed with reperfusion by opening or removing the ligation to restore normal blood circulation in accordance with the treatment group.Group 1 (G1): Femoral artery ligation for 6 h, followed with reperfusion by releasing ligation for 100 min (IR 100 min).Group 2 (G2): Ligation for 6 h, followed with reperfusion by releasing ligation for 100 min. HBOT was given 10 min after the reperfusion for 90 min (IR 100 min with HBOT 90 min).Group 3 (G3): Ligation for 6 h, followed with reperfusion by releasing ligation for 150 min (IR 150 min).Group 4 (G4): Ligation for 6 h, followed with reperfusion by releasing ligation for 150 min. Hyperbaric therapy was given 60 min after the reperfusion for 90 min (IR 150 min with HBOT 90 min).At the end of the research, all the rabbits were sacrificed. Their liver and blood were taken for examining histopathological changes in their liver using HE staining as well as for measuring the levels of AST and ALT. Histopathological changes were evaluated to assess the degree of liver damage as a result of the treatment given. Scoring method used in this research was a modification of Knodell method . The typScoring of histopathological changes in the livers was focused on necrotic lesions, degeneration, portal inflammation, and congestion. The results showed that within groups without hyperbaric therapy (G1 and G3), the scores of necrosis, degeneration, and portal inflammation were not significantly different (p>0.05). Similarly, within groups with hyperbaric therapy, namely, G2 and G4, the scores of necrosis, degeneration, and portal inflammation were not significantly different (p>0.05). However, the scores of histopathological changes in G1 and G3 were significantly different from those in G2 and G4 (p<0.05).Moreover, scoring results of congestion degree had a different pattern, in which there was no congestion in G1, G2, and G4. Unlike the other three groups, congestion only appeared in G3 (p<0.05) . The degDifferences in the scoring results of histopathological changes between the groups without hyperbaric therapy, furthermore, did not seem affected by the length of reperfusion duration. For instance, there was no difference in histopathological scoring results and AST/ALT levels between Group G2 and Group G4 although both had different reperfusion duration, namely, 10 and 60 min before the administration of hyperbaric therapy.In addition, results of the analysis of AST and ALT levels in each treatment group also showed a different pattern of results derived from the scoring results of histopathological changes in the livers. The results showed that the levels of AST and ALT in the groups without hyperbaric therapy (G1 and G3) were not significantly different from those in the groups treated with hyperbaric therapy (G2 and G4) (p>0.05) . The levHepatic ischemia-reperfusion injury is a multifactorial pathophysiological process. Numerous studies have shown that this process includes excessive intracellular Ca, increased free oxygen radical, and increased of apoptosis or necrotic liver parenchymal cells ,11. The In this research, necrotic, degenerative, and inflammatory lesions on those livers were not accompanied with changes in the function of those livers by examining the levels of AST/ALT. This may be caused by the degree of hepatocyte damage classified into mild to moderate one. In addition, it can also be caused due to compensatory mechanism owned by those livers, where healthy hepatocytes would take over the function of hepatocytes suffering from the injury.Similarly, a previous research also showed that limb ischemia suffered by mice for 2 h, then followed by reperfusion can cause injury to their lungs, but not in their liver and kidneys . Unlike Studies conducted directly with hepatic ischemia-reperfusion injury (HIRI) showed that this treatment cause hepatocytes apoptosis accompanied by elevate levels of AST and ALT, reflect severe hepatocyte damage ,11. ThesVital organs, including liver, are very sensitive to ischemia that occurs on injury due to IR. Ischemia-reperfusion is usually accompanied by oxidative stress and then continued with stimulation of inflammatory response, eventually leading to injury to the liver . OxidatiVarious factors, such as oxygen free radicals, migration and activation, abnormalities in the microcirculation, damage to endothelial cells sinusoid, coagulation cascade activation, and Kupffer cell activities to release inflammatory mediators and proteolytic enzymes, plays a role in causing injury to the liver due to IR ,19. LesiIn this research, furthermore, HBOT could improve morphological function of liver, namely, in Group G2 and Group G4. On the other hand, according to Kihara , HBOT caet al. [Da Silveira et al.  stated tBiochemistry profile parameters, including AST/ALT, in rabbits, were influenced by various factors such as breed, age, sex, feed, liver disease, environmental conditions, stress, pregnancy, and heart rhythm -27. NeveHind limb ischemia injury reperfusion can trigger a damage for liver morphology, but not lead to liver dysfunction. HBOT for 90 min, 10-60 min after the reperfusion, can protect hepatocytes from damage.BSL and YM carried out the main research works, WMY performed the statistical analysis and analyzed the main data in the experiments and BSL, WMY and YM approved the final manuscript. All authors read and approved the final manuscript.


Tactile sensory feedback would make a significant contribution to the state-of-the-art prosthetic hands for achieving dexterous manipulation over objects. Phantom finger sensation, also called referred sensation of lost fingers, can be noninvasively evoked by transcutaneous electrical nerve stimulation (TENS) of the phantom finger territories (PFTs) near the stump for upper-limb amputees. As such, intuitive sensations pertaining to lost fingers could be non-invasively generated. However, the encoding of stimulation parameters into tactile sensations that can be intuitively interpreted by the users remains a significant challenge. Further, how discriminative such artificial tactile sensation with TENS of the PFTs is still unknown. In this study, we systematically characterized the tactile discrimination across different phantom fingers on the stump skin by TENS among six subjects. Charge-balanced and biphasic stimulating current pulses were adopted. The pulse amplitude (PA), the pulse frequency (PF) and the pulse width (PW) were modulated to evaluate the detection threshold, perceived touch intensity, and the just-noticeable difference (JND) of the phantom finger sensation. Particularly, the recognition of phantom fingers under simultaneous stimulation was assessed. The psychophysical experiments revealed that subjects could discern fine variations of stimuli with comfortable sensation of phantom fingers including D1 (phantom thumb), D2 (phantom index finger), D3 (Phantom middle finger), and D5 (Phantom pinky finger). With respect to PA, PF, and PW modulations, the detection thresholds across the four phantom fingers were achieved by the method of constant stimuli based on a two-alternative forced-choice (2AFC) paradigm. For each modulation, the perceived intensity, which was indexed by skin indentations on the contralateral intact finger pulp, reinforced gradually with enhancing stimuli within lower-intensity range. Particularly, the curve of the indentation depth vs. PF almost reached a plateau with PF more than 200 Hz. Moreover, the performance of phantom finger recognition deteriorated with the increasing number of phantom fingers under simultaneous TENS. For one, two and four stimulating channels, the corresponding recognition rate of an individual PFT were respective 85.83, 67.67, and 46.44%. The results of the present work would provide direct guidelines regarding the optimization of stimulating strategies to deliver artificial tactile sensation by TENS for clinical applications. Amputation inevitably brings huge damage to both physical and mental health for upper-limb amputees Kejlaa, . ProstheThe sense of touch originated from normal hands carries complicated and comprehensive information like shape, temperature, size and texture of objects. Manipulation over objects by prosthetic hands can be slow, stiff and non-intuitive without tactile feedback , pulse frequency (PF), and pulse width (PW). To determine the effective parameter range, we measured the detection thresholds and upper limits which would elicit uncomfortable sensations. And then, within available parameter ranges, we further assessed the perceived intensities indexed by the indentation depth on the contralateral intact finger pulps. The just-noticeable difference (JND), also called the difference threshold, and Weber fractions were evaluated to estimate the subjects' capability to distinguish among different stimuli. Finally, the phantom finger recognition was characterized under simultaneous stimulation.Ten volunteers were randomly recruited. Prior to the psychophysical experiment, an interview was first conducted to find out each volunteer's medical history, phantom limb sensations and whether they experienced phantom limb pain now or in history. In our psychophysical experiments, the participants had a unilateral forearm amputation, and remained psychologically healthy with PFTs near the stump. And then six adult forearm amputees  were recruited. The other four volunteers were excluded without phantom finger sensation. One of them was with congenital forearm deficiency (Subject 7), one as a forearm amputee (Subject 8), and the other two with shoulder-level amputation (Subjects 9 and 10). All the ten volunteers were right handed before amputation, and the general information were presented in Table For all the six subjects, phantom finger sensations were evoked when certain skin regions near the residual stump were touched by a stylus pen with 4 mm in diameter. These regions were confirmed as PFTs. Subjects 1–3 and 6 possessed five independent PFTs, which was designated in the experiments as phantom digits D1–D5. Subject 5 had four independent PFTs without phantom sensation of the ring finger (D4). Subject 4 also had five PFTs, but the territories D2 and D3 could not be clearly discriminated. In order to be consistent for comparing the tactile discrimination among the six subjects, four PFTs labeled as D1, D2, D3, and D5 were investigated under TENS to produce phantom finger sensations corresponding to lost thumb, index, middle and pinky fingers, respectively.The detailed procedures for locating PFTs were described as follows: (1) The subject sat in a wooden chair comfortably with his/her amputated stump naturally placed on the table, and then the stump skin was cleaned with alcohol wipes. The subject's eyes were covered with an eyeshade. (2) A stylus was used to touch the volar side of the residual stump skin, and the subject was required to quickly report if specific phantom finger sensations were produced or not. Then the stylus was moved to the next point until the whole volar side was covered. In the end, the sites corresponding to the same phantom finger were connected to form a PFT outline. The most sensitive point (MSP) referred to a finger pulp in each PFT was also clearly identified and marked. This whole process was repeated twice for each subject to validate the PFTs. Each process took approximately 35 min, and a break of 1–2 min was randomly given to allow the subjects to have a relax.Sometimes in the procedure of identifying the PFTs, a gentle touch on the stump skin by the stylus pen only produced a local sensation of the stump skin, and the phantom finger sensations were evoked with much stronger press. The regions originated from the phantom finger pulp, back, sides, and root were all covered inside a PFT. Although skin sites referred to the phantom palm and opisthenar were also reported, these were not involved within the PFTs. Two typical PFTs were shown in Figure The current stimulator  can generate four-channel independent stimulating current pulses, which are cathodic-first, biphasic and charge-balanced Figure . The PA To quantitatively characterize the perceived intensity of phantom finger sensation under TENS, a compact punching machine Figure  was desiTo impose electrical stimuli on the MSP in a PFT, the flexible electrode array  was utilized. All the electrodes were coated with a thin layer of conductive hydrogel adhesive. Two adjacent circular electrodes were defined as the stimulating and reference electrodes, respectively. Each electrode was 7 mm in diameter, and the center-to-center distance was 12 mm. The psychophysical experiments were carried out in the laboratory at 26°C.To characterize the phantom finger sensation through TENS, a set of four experiments Figure  were carThe detection thresholds under TENS in each PFT were tested in terms of PA, PW and PF modulations. The procedures were double-blinded for both the experimenter and subjects. The PA, PF, and PW were set as the predetermined typical values of 1.5 mA, 50 Hz, and 200 μs, respectively. Obvious and comfortable phantom finger sensations were elicited for all the six subjects with these typical stimulating parameters. Prior to finding out the detection thresholds, the upper stimulus limits leading to an uncomfortable sensation were obtained by the method of minimal change.Urban  pointed In stage 1, the rough thresholds of PA, PW and PF were measured using the method of minimal change, which provided a solid basis for the selection of testing values in the fine determination of detection thresholds in stage 2. During stage 1, the stimulating pulse trains lasted 3 s. With PF at 50 Hz and PW of 200 μs, PA increased from a lower value of 0.4 mA by a step of 0.1 mA until the subject reported that the stimuli were perceived. Similarly, for rough determination of PW, PA and PF were respectively set as 1.5 mA and 50 Hz, and PW started from 20 μs with an increasing of 20 μs at each step. Also, for rough determination of PF, PF increased from 1 Hz by 1 Hz with PA and PW set as 1.5 mA and 200 μs, respectively. For four PFTs among six subjects, the rough thresholds of PA ranged from 0.6 to 1.5 mA, those of PW from 60 to 120 μs, and those of PF from 1 to 17 Hz.n.On the basis of the rough threshold ranges and the output precision of the stimulator, as listed in Figure In stage 2, detection thresholds were finely determined based on the method of constant stimuli by adopting two-alternative forced-choice 2AFC) paradigm Figure , where tFC paradiFour stimulating blocks were presented in terms of four PFTs. Within each block, each trial was repeated 7 times for PA, PW and PF modulations, respectively, and the stimulus order within these two intervals in one trial was pseudo-randomized. Then the responses to every trial in each block were fitted by a sigmoid function. Within each trial during stage 2, the expected probability of correct judgment was 50% if the subject did not detect the stimuli at all, or otherwise the probability would rise to 100% if the subject readily detected the stimuli. Therefore, the detection thresholds were defined as the values of PA, PW, and PF that each subject could correctly identify 75% of the stimuli Figure . The samThe PA, PW, and PF are the three common stimulus parameters which can be independently manipulated to introduce sensory feedback. In the previous work , so the nearest frequencies to achieve these reference percentages were used. For example, for the 50 Hz reference, the test values were 25, 40, 45.5, 55.6, 62.5, and 76.9 Hz Figure .During the electrical stimulus discrimination, each trial was repeated 7 times within one block. Both the order of a reference/test stimulus pair and the stimulus order within the pair were pseudo-randomized and double-blinded for both the experimenter and the subjects in each block.The experiment of phantom finger recognition was carried out in three levels with typical stimuli, i.e., PA of 1.5 mA, PW of 200 μs and PF of 50 Hz. The participant was required to point out which phantom finger or fingers were perceived. Figure P > 0.05).The rough upper limits to induce uncomfortable sensation were about 3 mA, 400 Hz, and 600 μs for PA, PW, and PF, and the detection thresholds were much lower than these upper limits. Figure During TENS of the PFTs, the subjects experienced a wide range of perceived intensities indexed by the indentation depth in the contralateral intact finger pulps. Figure In Figure In terms of the operational definition about detection thresholds, the subjects still had a probability of less than 75% to perceive the subthreshold stimulation. Different from this definition, the subjects definitely knew that there would be a stimulus applied to the PFTs during the experiments of perceived intensity quantification. Consequently, the subjects could perceive the electrical stimulation under small stimulus intensities. This could be the main reason why there was some difference between the lowest values in Figure The plateau in the plots in Figure DLu and DLl in Equation (1).During this experiment, the subjects were required to judge whether the test or reference stimulus was stronger within every trial. Responses by participants were converted into a probability value based on their accuracy of identifying the correct interval with the stronger stimulus. A sigmoid was fitted and upper and lower limits on this probability function were defined as 25% and 75% probability of correctly identifying the stronger stimulus. For a given reference stimulus, the JND was yielded by averaging DLu and DLl respectively denoted the differences between the reference stimulus with the upper limit (Lu) and lower limit (Ll) of the discriminated test stimuli. Figure As shown in Figure In this experiment, the average Weber fractions ranged from 0.11 to 0.18 for the PA modulation, 0.14–0.32 for PF, and 0.1–0.265 for PW. The relationships of Weber fraction with different stimuli were plotted in Figure By ignoring low intensities of 1 mA and 100 μs, and high intensity of 400 Hz, the proposed “optimal range” of the stimuli, which elicited a clearly discriminative sensation without uncomfortable feeling such as pain, were 1.2–2.8 mA in PA, 10–350 Hz in PF and 150–600 μs in PW. And then the corresponding Weber fractions were defined as 0.1 in PA, 0.2 in PF and 0.1 in PW.The recognition performance of different PFTs was assessed in terms of three levels with typical values of PA, PF, and PW. The more the possible number of PFTs under simultaneous stimulation, the poorer the recognition performance of the individual PFT. For Levels 1–3, the correct recognition ratios about individual PFTs were 85.83% (103/120) (chance level: 25%), 67.67% (203/300) (chance level 10%), and 46.44% (209/450) (chance level 6.7%), respectively. For Level 1 Figure , the leaOur normal hand is so dexterous, with 27 degrees of freedom. Hand muscles are innervated by thousands of afferent nerve fibers which convey different (sometimes overlapping) information about objects under manipulation . All the subjects or participants were informed of the whole experimental procedure, and signed the informed consent form before experiments.XS contributed to the design of the overall psychophysical experiments and data analyses. ML conducted the psychophysical experiments and data analyses. ML wrote the first draft and XS also contributed to the whole manuscript revision. DZ, LH, and YC contributed to subject recruitment and the experiment. XC, YC, and JG were involved in the establishment of the experimental setup. All authors were active in the editing and revising processes of the manuscript. All authors read and approved the final manuscript.LH was employed by company Shanghai Health 51 Net Technology Co., Ltd. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Actinobacteria is one of the most ubiquitously present bacterial lineages on Earth. In the present study, we try to explore the diversity of cultivable rare Actinobacteria in Sigangli Cave, Yunnan, China by utilizing a combination of different sample pretreatments and under different culture conditions. Pretreating the samples under different conditions of heat, setting the isolation condition at different pHs, and supplementation of media with different calcium salts were found to be effective for isolation of diverse rare Actinobacteria. During our study, a total of 204 isolates affiliated to 30 genera of phylum Actinobacteria were cultured. Besides the dominant Streptomyces, rare Actinobacteria of the genera Actinocorallia, Actinomadura, Agromyces, Alloactinosynnema, Amycolatopsis, Beutenbergia, Cellulosimicrobium, Gordonia, Isoptericola, Jiangella, Knoellia, Kocuria, Krasilnikoviella, Kribbella, Microbacterium, Micromonospora, Mumia, Mycobacterium, Nocardia, Nocardioides, Nocardiopsis, Nonomuraea, Oerskovia, Pseudokineococcus, Pseudonocardia, Rhodococcus, Saccharothrix, Streptosporangium, and Tsukamurella were isolated from these cave samples.The phylum  Caves provide a quasi-extreme environment for living organisms owing to relatively low organic nutrient input and lack of light . Some ‘sProteobacteria, Acidobacteria, and Actinobacteria to be among the dominant taxa on cave environments .The Sigangli Caves, located in Cangyuan County, Yunnan Province, China, are part of a series of karst caves of the Yunnan–Guizhou Plateau formed from the dissolution of limestone and other calcareous rocks. The plateau, covering an area of over 1.3 × 10Actinobacteria were collected from different part of the caves . The samples (Table 1) include the hard rock forms  and the weathered rock forms . The samples were collected using sterile scalpels or spades and were transferred immediately in falcon tubes or Ziploc bags. These samples were then stored under a low temperature environment until processing for isolation.Samples for isolation of Actinobacteria is listed in Table 2. Samples (2 g) were suspended in 20 ml sterile distilled water and kept in a rotary shaker  for 1 h. The suspensions were serially diluted and aliquots of 100 μl of the diluted suspension was plated on freshly prepared agar media (in triplicates). The following isolation media were used: Humic acid-vitamin medium (HV) (Streptomyces Project (ISP) 5 medium (-1) and nalidixic acid (20 mg L-1) to prevent the growth of fungi and fastidious bacteria. Following incubation for 2 weeks at 28°C, all the colonies developed on the isolation media were counted. Depending on the isolation conditions and the media used, total colony forming units (CFUs) from each treatment were determined. Apart from the spore-forming strains, viabilility of the other vegetative cells were determined by subculturing on YIM 38 medium (A set of pretreatments and inoculation procedures adopted for the isolation of culturable ium (HV) ; Interna5 medium ; Cellulo5 medium ; Trehalo5 medium ; Starch-5 medium ; B-4 med5 medium , and Wat8 medium . HeatmapTM Genomic DNA purification kit according to manufacturer’s instructions. The DNA preparations were used as template for PCR amplification using the universal primers 27F: 5′-CAGAGTTTGATCCTGGCT-3′ and 1492F: 5′-AGGAGGTGATCCAGCCGCA-3′. PCR reactions were conducted using iCycler Thermal Cycler  by applying the following conditions: initial denaturation at 94°C for 4 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. Amplified PCR products were verified on 0.8% agarose gel with 2 Kb DNA ladder (Fermentas) as a molecular size reference and sent for sequencing .Genomic DNA was extracted using TIANGENActinobacteria, were not reflected in the current study. Relative abundance of the Actinobacteria based on the number of strains from each genera were plotted into a scatter-plot using Microsoft Excel 2013.16S rRNA gene sequences of the isolates were compared with the published 16S rRNA gene sequence database in EzBioCloud server  on the bKX274728–KX274786, MF431270–MF431414 , more CFUs was determined in samples incubated at 40°C for 2 days prior to isolation (Treatment c) than in samples incubated at higher temperatures . It is, however, interesting to note that samples kept at room temperature (Treatment b) yield lower CFUs than the one incubated at 40°C. Among the five media during this process, HV agar seems to be a better isolation medium . When pH was used as the isolation criteria, it was determined that more CFUs were obtained in media adjusted to neutral pH, fewer in alkaline pH and least in circumneutral pH . If the sample pH is taken into consideration, there is a gradual decrease in the number of CFUs with increase in sample pH, irrespective of the sample types. Lastly, considering the use of calcium salts in isolation, it was determined that CaCO3 yielded more Actinobacteria than (CH3COO)2Ca and CaCl2 . However, it was not just the salt that is important, the concentration of each salt in the selection media also played an important role. In our case, it was determined that higher CFUs was determined when salt concentration was proportionately at lower concentration  than in its absence or at high concentration.Actinobacteria, while the strains isolated from Type 2 samples were distributed to 21 genera and 16 families. The relative abundance of the strains is represented in Figure 3, and the 16S rRNA gene sequence profile listed in Supplementary Table Streptomyces, the rare actinobacterial genera Nocardia and Rhodococcus were relatively abundant in both the sample types . While the genus Micromonospora was relatively more abundant in Type 2 samples , the genus Mycobacterium was more in hard rock (7) than in the weathered rock samples (3). Other rare genera that were common to both the sample types include Jiangella, Kribbella, Nocardioides, Nocardiopsis, Nonomuraea, and Streptosporangium. Apart from these common genera, few rare actinobacterial genera were restricted to only one particular sample type. The genera Amycolatopsis, Beutenbergia, Cellulosimicrobium, Gordonia, Isoptericola, Microbacterium, Mumia, Oerskovia, and Pseudokineococcus were isolated only from Type 1 samples while the rare genera Actinocorallia, Actinomadura, Agromyces, Alloactinosynnema, Knoellia,Kocuria, Krasilnikoviella, Pseudonocardia, Saccharothrix, and Tsukamurella were isolated from Type 2 samples.Of these total colonies observed, morphologically distinct colonies were further selected, subcultured, and preserved. These include 87 isolates from Type 1 samples and 117 from the Type 2 samples. Sequence analysis of 16S rRNA gene indicated that the strains from the Type 1 samples were distributed to 20 genera in 14 families of the phylum 6 cells/g of rock , relatively high humidity (90–100%) and total darkness or low level of light, and are often mystical and inaccessible for study. Above that, caves usually constitute a oligotrophic ecosystem with total organic carbon of less than 2 mg/L . Despite of rock . Despitepressive . In anotbacteria .Actinobacteria, because of its different versatile morphology, were detected and isolated from even the most extreme of environments  or exogenously  in response to environmental stress . An advantages of air-drying is that many Actinobacteria produces spores, and that dry spores have low respiration rate and can survive for longer period of time. During this period of low level of endogenous respiration, the spores did not germinate, but can be germinated readily when a defined medium with organic energy source is provided . These highest OTUs were represented in samples treated at low ambient temperatures. The levels of CFUs was found to decrease with increased pretreatment’s conditions. Least CFUs were obtained in the samples pretreated at 110°C for 1 h. However, if the pretreatment at 110°C for 1 h is accompanied by incubation at 40°C for 2 days, the numbers of sporulating cells that survived the heat treatment increased as indicated in the Figure 2A. This finding could be related to the study of Actinobacteria obtained and the temperature used for pretreatment. While no strict thermophilic Actinobacteria such as members of the genera Thermomonospora, Dactylosporangium, etc. were isolated during our study, a fair number of rare Actinobacteria with aleurispores (Micromonospora), arthrospores (Actinomadura), sporangiospores (Streptosporangium), or other spore-bearing structures (Amycolatopsis etc.) were obtained. However, in addition to these Actinobacteria, endospore-forming non-Actinobacteria such as Bacillus strains were also obtained during the isolation process. The co-occurrence of these bacteria are, however, found to be very less as confirmed by our preliminary sequencing analysis of the 16S rRNA gene (data not shown).Among the physical enrichments method for actinobacterial isolation, air drying, dry heating, moist incubation, and desiccation have been found to be an effective for selection of spore-forming rare bacteria . Air-dryomycetes . Similarnomadura . Air-drybacteria . Genus Mr 30 min . An effe for 2 h . Preferebacteria . In the provided . During Actinobacteria are capable of growing under selective conditions of pH or salinity . During the current study, more actinobacterial CFUs were detected in neutral pH . This may be because of the easy maintanence of cell’s cytoplasmic pH at close to neutrality , Actinobacteria-coated spots on the cave walls was found to uptake carbon dioxide gas which is available in abundance in cave . This finding is also in congruence with the finding of Figure 2C). Among the two types of samples, more CFUs were observed in Type 2 than in Type 1 samples. The reason could be implicated on the lower cell concentration on the surface of hard rock . While SC and ISP media were introduced for the isolation of mycelial-producing Actinobacteria particularly genus Streptomyces . The reason behind the larger CFUs in HP could not be ascertained from the current study, however, it is possible that degradation of cellulose required complex enzyme-system and that many Actinobacteria were not able to use cellulose as their energy sources. On the other hand, water agar, which is found to stimulate growth of spore-forming microorganisms, was not effective during our study for the growth of rare Actinobacteria . Inhibition of certain rare actinobacterial strains by the preferential treatments, however, cannot be completely ruled out.In all the above cases, isolation media play the key role for providing the favorable condition for isolation and growth of rare bacteria , lest Acorangium . It may ptomyces , B-4 medcrystals . The lar sources . Among tActinobacteria  and non-spore formers .Despite the isolation of varied actinobacterial groups after applying a set of pretreatments and modification of isolation media, our study suffers from few limitations. Firstly, the physicochemical parameters of the sampling site and the co-existence of different minerals/metals were not measured during the study. Lack of these data prevent us from indirect establishment of the interrelationship between the occurrence of different actinobacterial groups and their physiological roles in cave. Secondly, the study was limited to isolation of culturable W-JL, B-ZF, and JC designed research and project outline. JC, M-XH, MX, and NS performed isolation, deposition, and identification. B-ZF, J-YJ, and D-QW contructed the heatmap and other related bioinformatic plots. B-ZF, NS, MX, and W-JL drafted the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Head injuries are the leading cause of death among cyclists, 85 % of which can be prevented by wearing a bicycle helmet. This study aims to estimate the incidence of pediatric bicycle-related injuries in Olmsted County and assess differences in injuries between those wearing helmets vs. not.Olmsted County, Minnesota residents 5 to 18 years of age with a diagnostic code consistent with an injury associated with the use of a bicycle between January 1, 2002, and December 31, 2011, were identified. Incidence rates were calculated and standardized to the age and sex distribution of the 2000 US white population. Type of injuries, the percentage requiring head CT or X-ray, and hospitalization were compared using a chi-square test. Pediatric intensive care unit (PICU) admission, permanent neurologic injury, seizure, need for mechanical ventilation, and mortality were compared using Fisher’s exact test.p < 0.001) and to experience a brain injury .A total of 1189 bicycle injuries were identified. The overall age-adjusted incidence rate of all injuries was 278  per 100,000 person-years for females and 589  for males. The corresponding rates for head injuries were 104  for females and 255  for males. Of patients with head injuries, 17.4 % were documented to have been wearing a helmet, 44.8 % were documented as not wearing a helmet, and 37.8 % had no helmet use documentation. Patients with a head injury who were documented as not wearing a helmet were significantly more likely to undergo imaging of the head (32.1 percent vs. 11.5 %; Children and adolescents continue to ride bicycles without wearing helmets, resulting in severe head and facial injuries and mortality. Unintentional injuries remain the leading cause of death among children and adolescents in the United States of America (USA) Rezendes . BicycleThe Cochrane Collaborative conducted an analysis of controlled studies evaluating the effect of bicycle helmets on injury prevention. The review found that helmets provide up to a 63 to 88 % reduction in the risk of head, brain, and severe brain injury for cyclists of all ages as well as a 65 % reduction of injuries to the upper- and mid-face . This study was designed to investigate the incidence of bicycle-related injuries in Olmsted County among children ages 5 to 18 years between January 1, 2002, and December 31, 2011, enumerate the location and types of injuries, and analyze the differences in injuries between those children wearing and not wearing bicycle helmets.Olmsted County, located in southeast Minnesota, is relatively isolated from other urban centers, rendering it amenable to epidemiologic research. The county consists of urban, suburban, and rural communities and has a population of 144,248 persons, of whom 86.5 % are white, 51.1 % are female, 24.9 % are under the age of 18 years, 94 % have graduated from high school or pursued a higher degree, and 8.5 % fall below the federal poverty level; the median household income is 66,667 dollars annually.Local medical providers, consisting primarily of Mayo Clinic and its two affiliated hospitals, Olmsted Medical Center (OMC) and its affiliated hospital and the Rochester Family Medicine Clinic, deliver nearly all health care to the county population. All three institutions participate in the Rochester Epidemiology Project (REP), an infrastructure that permits the sharing of patient medical records to support population-based research . The REP database comprises the records of all persons living in Olmsted County, MN. REP medical records include the details of every inpatient hospitalization at the three affiliated area hospitals; every outpatient visit to an office, clinic, urgent care, or emergency department; as well as every laboratory result and correspondence concerning each patient, and all individual facility records are linked to a single REP identification number for each Olmsted County resident. These records are accessible and easily retrievable because the REP has created extensive indices based on clinical and histological diagnoses and surgical procedures dating to the early 1900s.Utilizing the REP electronic indices, all residents of Olmsted County between the ages of 5 and 18 years who had a discharge, primary, or secondary diagnosis of crash involving a bicycle between January 1, 2002, and December 31, 2011, were identified. Individual records were then retrospectively reviewed. Any subject using an alternative form of transport  or riding as bicycle passenger was subsequently excluded from the study. Similarly, those children and adolescents who had been seen for another primary indication, but had been noted incidentally in the chart to have had a bicycle crash without further evaluation, were also excluded. Mayo Clinic Institutional Review Board (IRB), Olmsted Medical Center IRB, and REP approval, as well as a University of Minnesota IRB Authorization Agreement were obtained.Once identified, patients’ records were reviewed and data were collected including date of birth, sex, street address, and zip code; date of injury; circumstance of injury and potential associated risk factors ); helmet use; date and time of acute care visit; mode of transport to medical facility; location and type of injury; Glasgow Coma Score (GCS); modality of diagnostic imaging performed and results; occurrence of trauma-related seizure; presence of residual permanent neurologic injury; need for inpatient hospital admission, pediatric intensive care unit (PICU) admission, and lengths of stay; need for mechanical ventilation, operative procedure, or discharge to a long-term rehabilitation care unit; and mortality. We defined a brain injury as a radiologically apparent sub-calvarial injury impacting brain parenchyma including epidural hematomas, subdural hematomas, subarachnoid hemorrhages, intraparenchymal bleeding/contusions, penetrating trauma, diffuse white matter injury, cerebral edema, or herniation; head injuries included the more superficial injuries of fractures, contusions, abrasions, and/or lacerations of the bony or soft tissues of the head. All data were tabulated into the REDCap database .p values are reported with values ≤0.05 considered statistically significant.Crash characteristics are summarized using mean ± standard deviation (SD) for continuous variables and frequency percentages for categorical variables. In order to calculate incidence rates, the identified cases were allocated to calendar period, age- and sex-specific categories for the numerator with the corresponding Olmsted County population counts used as the denominator  were male and 367 (30.9 %) female. Patient characteristics and circumstances of the bicycle-related crashes are presented in Table For both male and female youth, the age-adjusted incidence of all bicycle-related injuries remained relatively consistent over the 10-year calendar period  per 100,000 person-years for females and 589  for males. The corresponding rates for head injuries were 104  per 100,000 person-years for females and 255  for males. The corresponding rates for brain injuries were 20  per 100,000 person-years for females and 50  for males. Figure p < 0.001) more likely to have documentation available regarding helmet use, with significantly (p < 0.001) more documented as not wearing a helmet. Of the 500 patients with head injuries, 17.4 % were wearing a helmet, 44.8 % were not wearing a helmet, and 37.8 % had no documentation regarding helmet use. Among the 689 patients without head injuries, the corresponding percentages were 14.1, 23.1, and 62.8, respectively.Of the 1189 bicycle-related injuries, 184 (15.5 %) were documented as wearing a helmet, 383 (32.2 %) were documented as not wearing a helmet, and 622 (52.3 %) had no documentation regarding helmet use. Compared to those without head injuries, patients with head injuries were significantly (Table p < 0.001) and significantly more likely to experience a brain injury .Compared to those wearing a helmet, those who were not wearing a helmet were significantly more likely to undergo an X-ray or computed tomography (CT) scan of the head . The 17-year-old female, also struck by a motor vehicle, arrived in the emergency department with an absent pulse, a GCS of 3, and fixed and dilated pupils. Neither of these adolescents was wearing a helmet.Of the remaining nine subjects with head and/or facial injuries, eight were not wearing a helmet. Helmet use was not documented for one patient. All nine suffered brain injuries , six suffered skull fractures , five were admitted to the PICU, three had seizures, and two suffered permanent neurologic damage. None required mechanical ventilation or discharge to a long-term rehabilitation care unit.Four patients admitted to the PICU with bicycle-related injuries were admitted for injuries that did not involve the head and/or face, including two patients with liver lacerations, one patient with a laceration of the spleen who was also observed for risk of a cervical spine injury, and one patient with a femur fracture who required an operative procedure.Our study aimed to estimate the incidence of bicycle-related injuries in children, describe the types and location of injuries, and analyze the differences in injuries between those children wearing and not wearing helmets in a county with no helmet ordinance. The incidence of all injuries and head injuries has remained stable over the 10-year period studied. Bicycle-related injuries were more common among males, in the summer months, and were predominantly single-bicycle events. Children and adolescents presenting with head injuries were more likely to have documentation of helmet use/non-use in the medical record and were more likely to have not been wearing a helmet. Among patients with head injuries, those not wearing a helmet were more likely to undergo X-ray or CT scan of the head and more likely to have suffered a brain injury.The overall age-adjusted incidence rate of all bicycle-related injuries in our study ranged from 211 per 100,000 to 560 per 100,000 person-years; this rate is only slightly lower than the range of 392 per 100,000 to 663 per 100,000 children 18 years and younger found by Mehan et al. in a recent retrospective analysis of the National Electronic Injury Surveillance System . Similarly, a study of 108 cyclists, none of whom wore a helmet, performed by Munivenkatappa et al. , while helmet use in higher income areas has been found to be as high as 73 % even prior to passage of mandatory helmet legislation (Parkin et al. The American Academy of Pediatrics recommends that all pediatricians discuss bicycle helmet use as a part of anticipatory guidance (American Academy of Pediatrics & Committee on Injury and Poison Prevention Twenty-one states and the District of Columbia mandate bicycle helmet use for children, and many cities have local ordinances that require bicycle helmets for some or all riders (Insurance Institute for Highway Safety & Highway Loss Data Institute There are several limitations to this study. Helmet use was documented in only 47.7 % of medical records, making conclusions regarding the protective effect of helmets difficult; in addition, data regarding appropriate helmet size and use are not available. Importantly, any bicycle-related injury occurring during this time period that did not present to an acute care setting will not be captured. This would affect the calculated incidence and may bias our study toward capturing more severe injuries, and the proportion of severe to minor injuries, such as concussions, is not known. Furthermore, the REP does not represent hospital catchment area, and some patients may have presented to a facility outside of Olmsted County, particularly those patients with relatively trivial injuries at county borders.As described, the Olmsted County population is largely white, educated, and of middle class income with a low poverty rate; hence, our results may only be generalizable to communities of similar populations. Notably, however, evidence suggests that children of families with middle income are more likely to wear helmets than those of families of low income. Furthermore, legislation may increase helmet use among these children to match, or potentially supercede, that among children of families with higher income, which may predict the effect of mandatory legislation in this county (Parkin et al. Children and adolescents continue to ride bicycles without wearing helmets, resulting in severe head and facial injuries and mortality. Educational campaigns, provider counseling, and mandatory helmet legislation may encourage helmet use and reduce bicycle-related head injuries.


The objective of this study was to evaluate the alterations in sleep and circadianparameters during the aging process. The study sample comprises volunteers older than18 up to 90 years of age that answered the Pittsburgh Sleep Quality Index (PSQI) andthe Horne and Östberg circadian preference questionnaire. We observed that the shiftto morningness with increasing age is associated with a significant worsening insleep quality. We discuss that this sleep profile characterized by morningness andworse sleep quality observed in elderly, when compared to younger people, reflectsnot necessarily a pathological state, but an expected profile for this age group. Individual differences in the sleep-wake behavior, encompassing aspects such as habitualbedtime, wake time, sleep duration and structure have been observed in several studies. MoreoveIt has been proposed that sleep is regulated by two processes: circadian andhomeostatic. These two processes are generated independently, but their interactionregulates sleep-wake cycles and from it emerges the sleep phenotypes and sleep-wakedisorders . Easily Self-reported diurnal preference can be measured by the Horne and Östberg (HO)questionnaire , which hSleep patterns change ontogenetically and are very different in older people. Reports oforal temperature measurements have shown evidence of a weakened 24-h periodicity in theelderly, even in those individuals with generally regular lifestyles . CompareIn spite of its biological and medical relevance, few studies have been specificallydesigned to correlate sleep quality and diurnal preference in an ontogeneticperspective. Thus, the aim of the present study was to evaluate concomitantly thealterations in sleep and circadian parameters during the aging process. Specifically,this report evaluated the association between changes in sleep quality and chronotypethroughout the aging process.We contacted the volunteers in schools, universities, and companies, such as clinicallaboratories and offices, and explained the purpose of this study. After a verbalexplanation, their written consent was obtained. One thousand and thirty-fivevolunteers living in São Paulo, SP, Brazil, were initially recruited to answer to thePSQI and HO questionnaires.Participants older than 18 years of age, of both genders and who had answered all thequestions in both questionnaires, were drawn from the initial recruitment, resultingin a final sample of 812 volunteers. All participants belonged to the Brazilianpopulation, which is very ethnically interbred, and consists mainly of European(Portuguese) and Brazilian aboriginal backgrounds that were later mixed with avariety of African groups and with a variety of European ethnicities  and Asiatic ethnicities (mainly Japanese) at the beginning of the 20thcentury .The study was approved by the Ethics Committee of Universidade Federal de São Pauloand informed consent was obtained from the participants (#CEP 1471/07).The PSQI is a self-rated questionnaire that assesses the sleep quality anddisturbances of an individual over the month prior to completing the questionnaire.The nineteen questions are combined into seven clinically derived component scores,each weighed equally from 0–3. The sum of scores for these seven components yieldsone global score ranging from 0–21, with higher scores indicating worse sleep quality. To asseThe Horne-Östberg (HO-MEQ) questionnaire provides a quantitative measure of diurnalpreference , which hTo analyze answers from HO and PSQI questionnaires, the volunteers were classifiedaccording to their self-reported age in 10-year categories, except for the flanks ofthe distribution. They were categorized as the following age ranges, respectingadolescence and elderly age concepts: 18 to 24 (group 1), 25 to 34 (group 2), 35 to44 (group 3), 45 to 54 (group 4), 55 to 64 (group 5), and 65 years old and older(group 6).Post hoc analyses were performed using the Fisher test.Spearman's test was used to analyze the correlations between PSQI index and HO score,and the correlations between each of them with age. Multiple correlation was used toanalyze the PSQI index with age and HO as predictors. The level of significance wasset at P<0.05.We used ANOVA and ANCOVA to analyze differences among age groups with HO scores as acovariate. The sample was composed by 812 individuals, average age of 39.7 years (18–94 years), and64.6% were women. The male/female proportion, the number of subjects, the mean valuesfor HO scores and PSQI by age group, are shown in -6). Ingeneral, considering the HO score distribution by age, late chronotypes (evening types)presented worse sleep quality (r= -0.11 and P<0.05).Chronotype and sleep quality correlated with age , however multiple correlation considering age and HO scores as predictors ofsleep quality reveals a stronger correlation  and group 2 (25 to34 years old) were compared. When we analyzed the distribution of HO scores according to the age categories, weobserved a clear and progressive shift to morningness with increasing age =63,156, P<0.001). Fisher -6, HO covariate mean: 53.9, Additionally, considering HO score as a covariant, we observed that after 65 years ofage (group 6), there was a significant worsening in sleep quality =11,864, are related to worse quality of sleep in all ages.Moreover, two extreme sleep profiles emerged as representative of the ontogeny ofhomeostatic and circadian sleep regulation; morningness and worse quality of sleep arecharacteristic of the elderly while eveningness and better quality of sleep arecharacteristic of young people.Considering other reports in the literature ,23, it i


Fire as a dominant disturbance has profound implications on the terrestrial carbon cycle. We present the first ever multi-decadal, spatially-explicit, 30 meter assessment of fire regimes across the forested ecoregions of Canada at an annual time-step. From 1985 to 2015, 51 Mha burned, impacting over 6.5% of forested ecosystems. Mean annual area burned was 1,651,818 ha and varied markedly , with 25% of the total area burned occurring in three years: 1989, 1995, and 2015. Boreal forest types contained 98% of the total area burned, with the conifer-dominated Boreal Shield containing one-third of all burned area. While results confirm no significant national trend in burned area for the period of 1985 to 2015, a significant national increasing trend (α = 0.05) of 11% per year was evident for the past decade (2006 to 2015). Regionally, a significant increasing trend in total burned area from 1985 to 2015 was observed in the Montane Cordillera (2.4% increase per year), while the Taiga Plains and Taiga Shield West displayed significant increasing trends from 2006 to 2015 . The Atlantic Maritime, which had the lowest burned area of all ecozones (0.01% burned per year), was the only ecozone to display a significant negative trend (2.4% decrease per year) from 1985 to 2015. Given the century-long fire return intervals in many of these ecozones, and large annual variability in burned area, short-term trends need to be interpreted with caution. Additional interpretive cautions are related to year used for trend initiation and the nature and extents of spatial regionalizations used for summarizing findings. The results of our analysis provide a baseline for monitoring future national and regional trends in burned area and offer spatially and temporally detailed insights to inform science, policy, and management. Globallmbustion ; howevermbustion . Across mbustion ; howevermbustion , due to mbustion –8 as welmbustion  and a membustion .Under a changing climate, patterns in a number of wildfire characteristics such as area burned, size, occurrence, and seasonality are expected to change as the weather becomes more fire conducive , althoughttp://www.nrcan.gc.ca/forests/fire-insects-disturbances/fire/13159) [Information on fire event characteristics over Canada’s forested ecosystems has conventionally been derived in recent times from a combination of data sources, primarily, GPS (i.e. fire boundaries recorded from aircraft or helicopters), air photo interpretation, and satellite imagery. Given that the mandate for forest management in Canada is the purview of provincial and territorial agencies, data are compiled by each responsible jurisdiction and shared through the Canadian Wildland Fire Information System. As Canadian forests are composed of both managed and unmanaged land crossing provincial and territorial borders, multiple jurisdictions are involved in stewardship; leading to issues in both temporal and spatial compilations ,9. The ie/13159) .The opening of the Landsat archive and the associated processing and analysis opportunities  enables As a result, fire events can now be comprehensively detected and characterised across the entire forested land-base of Canada, availing upon the 30 m spatial resolution data in the Landsat archive. This unprecedented capacity to systematically detect burned areas allows for detailed temporal and spatial analysis of wildfire: providing a marked improvement in our understanding of where fires have occurred, both spatially and temporally, the characteristics of these wildfires, and changes in these characteristics over time across the forested ecosystems in Canada. In this paper we demonstrate how Landsat time-series information can be used to systematically analyze, across the entire forested landbase of Canada, the year and spatial extent of fires from 1985–2015. To do so, we compare burned area estimates across Canadian ecozones and predefined homogenous fire regimes, to examine if any temporal trends in size or frequency are evident over the time period, and demonstrate how these refined estimates of the burned area inform upon national fire regimes for Canada.http://nfi.nfis.org), along with wetlands and lakes, among other cover types. As a contrasting scale of analysis we also examine the pattern of fire events using a more recently developed regionalization proposed by Boulanger et al. [Canada’s forests represent approximately 10% of global forests and 30% of global boreal forests. In 2014, Canada’s forest industry contributed 20.1 billion dollars to Canada’s GDP and accounted for 30.7 billion dollars’ worth of exports . Naturalr et al.  of homogst ± 30 days (corresponding to the average growing season) from 1984 to 2015. Approximately 100,000 images were ultimately used to produce annual best-available-pixel (BAP) composites. As summarized in White et al. [Canada is covered by over 1200 Landsat scenes (path / rows) of the Landsat Worldwide Referencing System (WRS-2) . Candidae et al. , all elie et al. . Clouds,e et al. .Annual BAP image composites were then created using the pixel-scoring functions described in White et al. . FollowiChange attribution accuracy was assessed using an independent evaluation sample set, where reference classes were manually interpreted using the Landsat annual image composites and high spatial resolution imagery from Google Earth. Ancillary data, including the CNFDB, were used to support the visual interpretation of change attribution. Representative reference samples were allocated using a restricted random selection , which iTo examine and report on burned patches and regimes we utilised the two stratifications of Canada  and a 50Fire regimes were assessed for each ecozone or fire regime area using a set of key criteria ,14. BurnAll statistical analysis was completed in R . First, Over the entire analysis period, from 1985 to 2015, a total of 51,206,361 ha in Canada’s forested ecozones burned, representing 6.5% of Canada’s forested ecosystems. The total area burned annually varied markedly. On average, the annual total area burned was 1,651,818 ha ; however in some years, the total area burned greatly exceeded this average. For example, fires in 1989, 1995, and 2015 accounted for 25% of the total area burned from 1985 to 2015 . The total area burned in 1989, 1995, and 2015 was greater than the area burned in 14 other years combined .-1) followed by the Boreal Shield West (0.67% yr-1), Taiga Plains (0.52% yr-1), Boreal Plains (0.30% yr-1), Taiga Shield East (0.24% yr-1), and Boreal Cordillera (0.23% yr-1). Compared to the boreal ecozones, the coastal ecozones in the west (Pacific Maritime) and east (Atlantic Maritime) had markedly lower PAAB (0.01% yr-1). In 1989, the area burned was predominantly in the Boreal Shield West and Taiga Shield East, whereas in 1995, the largest areas burned were concentrated in the Boreal Shield West and Taiga Plains  and a slight increase (2.4% yr-1), respectively. As the annual burn rate was lowest in the Atlantic Maritime (0.01% yr-1), a decrease of 2.4% yr-1 only represents a decrease of 20.6 ha yr-1, while the increase of 2.4% yr-1 in the Montane Cordillera represents an increase of 709.3 ha yr-1. For the 2006–2015 period, there were significant increasing trends in burned area in both the Taiga Plains (26.1% yr-1) and the Taiga Shield West (12.7% yr-1), as well as an increase of 11% yr-1 nationally . By homogeneous fire regime regionalization 3 of the 16 units  showed significant trends in burned area from 1985 to 2015, all increasing by 2.8–2.9% yr-1, and all occurring within western Canada, essentially overlapping Taiga Shield West and Montane Cordillera ecozones. Only the Montane Cordillera ecozone had a significant increase in the number of large (> 200 ha) burned patches from 1985 to 2015 (3.5% yr-1). Alternatively, four of the 16 fire regimes had a significant increase in large burned patches from 1985 to 2015 , while two fire regimes had a significant decrease in the number of large patches (3.1% yr-1 for ET and 4% yr-1 for WJB).The percent area burned annually by ecozone and fire regime area is presented in Figs 2) were most numerous; however, these small patches comprised only a small proportion of the total burned area in most ecozones. In contrast, only 3% of burned patches nationally were greater than 200 ha in size; however, these burned patches accounted for 63% of the total burned area. The Atlantic and Pacific Maritime ecozones showed evidence of fire regimes dominated by small burned patches. In contrast, the Boreal Shield East and West ecozones exhibit much larger variability in patch size than the other ecozones, with the greatest proportion of burned area occurring in patches that were 200–500 km2 in size. The distribution of burned area in the Taiga Plains is skewed towards larger burned patches, with the largest proportion of burned area (~30%) occurring in burned patches that were greater than 1000 km2 in size.For each ecozone, we computed the mean fire return interval using exponential fitting See , Table 1Through a systematic and automated approach, we have analysed the spatio-temporal variability in burned area across Canada’s forested ecosystems for the past 30-years. Differences in fire activity between ecozones is a function of many competing factors, including fuel availability, fuel flammability, the probability of lightning strikes, climate, drought, and human influence ,30. ClimIn addition to longitudinal trends in fire activity, latitudinal trends also exist as a function of human influence and climate. Public safety and resource protection lead to an emphasis on fire suppression in the southern regions of Canada resulting in low area burned along the southern fringes of the forested ecozones, while unmanaged forests in northern Canada are left to burn naturally , explainThe area burned across Canada can vary markedly from year to year due to variations in fire weather conditions . Above average burn years typically occur when conditions are hotter and drier than normal . In 19892 scenario [While large changes in fire weather are expected across the Canadian boreal ,53, we oscenario . Expectescenario .Varying fire return intervals across Canada influence the age and structure of forest stands. Kneeshaw and Gauthier  demonstrLandsat-based assessments of burned area across Canada can augment historical fire information, such as CNFDB, which is a multi-source compilation of fire polygon data collected by fire management agencies across Canada. While compiling the CNFDB required years of data collection and coordination across management agencies, Landsat time-series analysis allows researchers to reconstruct disturbance history over the 30 m Landsat record for the entire landmass of Canada. Kasischke and Turetsky  noted a The CNFDB is an extremely valuable and irreplaceable data source for fire monitoring in Canada. The Landsat time series approach presented herein allows the precise area of burns to be determined; however, local knowledge and proactive monitoring by fire management agencies can provide information on ignition source, start date, and suppression efforts. This specific information is valuable for assessing fire cause and action taken and is missing from the Landsat time series approach applied herein. Further, while burned patches are derived in the Landsat time series approach, it is difficult to quantify the number of fires, as single fire patches can be detected as a series of many disconnected burned patches.Practices implemented by provincial and territorial resource management agencies result in burned patches being grouped into fire events representing the life cycle of a given fire. These events often include unburned islands and water bodies, which increases the burned area associated with each fire event. Capture and tracking of fire events enables a detailed assessment of changes in the number of fires and fire sizes across Canada in a manner that is particularly informative for fire management and suppression efficacy point of view. Therefore, while the Landsat time series approach allows for a more detailed assessment of burned area, the CNFDB provides additional attributes for fire events, such as fire start and end date, causal agent, and suppression efforts. Besides differences in data sources, dissimilarities in methodological approaches explain the divergence in reported results (via consideration of patches) and those of previous studies (where events are considered). For example, Stocks et al.  found thThe results of this study provide a spatially and temporally consistent representation of burned area from a single remote sensing data source, across Canada’s forested ecosystems, and a regionalisation of fire regimes, that can be used to inform both national monitoring programs and scientific applications. With recent significant temporal trends in burned area found during our 30-year study period, these results provide a critical baseline upon which future area burned can be compared. In addition, fusion of these data with more conventional fire event databases will allow a comprehensive examination of fire regimes across Canada. Given to the systematic nature of satellite based analysis, additional years of data can be incorporated as these data become available, allowing trends to be observed over a longer time period, in a near-real time enabled by data availability and computing. Additionally, with the launch of Sentinel 2 in 2014, which has similar imaging characteristics to the Landsat series , enhance