Patients with advanced solitary fibrous tumors (SFTs) have a poor prognosis; treatment options for recurrent disease are particularly limited. Several novel targeted agents have recently shown promise against advanced SFTs, but the relative efficacy of new agents is difficult to assess because data on the efficacy of conventional chemotherapy for SFTs are limited. We thus sought to estimate the efficacy of conventional chemotherapy for SFTs by reviewing data on tumor response to therapy and progression-free survival from SFT patients who received this therapy.We retrospectively analyzed the clinical outcomes of 21 patients with grossly measurable, advanced SFTs  who received conventional chemotherapy and follow-up at The University of Texas MD Anderson Cancer Center between January 1994 and June 2007. Best tumor response to therapy was assessed using the Response Evaluation Criteria In Solid Tumors 1.1. The Kaplan-Meier method was used to estimate median progression-free survival (PFS) duration.Of 21 patients, 4 received more than 1 regimen of chemotherapy, for a total of 25 treatments. Doxorubicin-based chemotherapy was given in 15 cases (60%), gemcitabine-based therapy in 5 cases (20%), and paclitaxel in 5 cases (20%). First-line chemotherapy was delivered in 18 cases (72%). No patients had a complete or partial response, 16 (89%) had stable disease, and 2 (11%) had disease progression. Five patients (28%) maintained stable disease for at least 6 months after first-line treatment. The median PFS duration was 4.6 months. The median overall survival from diagnosis was 10.3 years.Conventional chemotherapy is effective in controlling or stabilizing locally advanced and metastatic SFTs. Our findings can serve as a reference for tumor response and clinical outcomes in the assessment of novel treatments for SFTs. Solitary fibrous tumors (SFTs) are rare sarcomas considered to be of fibroblastic origin ,2. HemanRecently, several novel targeted agents have shown promising results in the treatment of advanced SFTs. Temozolomide-bevacizumab combination therapy  as well To estimate the efficacy of conventional cytotoxic chemotherapy for advanced SFTs, we evaluated tumor response and progression-free survival (PFS) duration in advanced SFT patients who received this therapy at a single institution during a 13-year period.This study was approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center, and informed consent was waived for the proposed patient record review.We queried the soft-tissue tumor pathology database at MD Anderson Cancer Center to identify all patients whose tumor specimens had been evaluated by a sarcoma pathologist at our institution from January 1994 to June 2007 and who were diagnosed with SFTs. The clinical records of the patients identified by our database search were then reviewed using MD Anderson’s institutional electronic medical records database. These records included those of all clinical encounters at MD Anderson and all available outside records that had been scanned into the system. To be included in our retrospective analysis, patients had to have advanced SFTs with grossly measurable disease treated with systemic chemotherapy under the direction of an MD Anderson sarcoma medical oncologist, and patients’ available clinical records and radiologic scans were reviewed. Patients were considered to have advanced SFTs if they had unresectable metastatic disease or if they had primary tumors for which chemotherapy could improve local resectability. Excluded from our analysis were patients who had received adjuvant chemotherapy and who had no grossly measurable disease, patients who had been seen only at their initial visit without subsequent follow-up visits, and patients younger than 18 years of age at diagnosis.We collected data on patient characteristics, including age at diagnosis, sex, ethnicity, and vital status as of April 1, 2010, and on primary tumor characteristics, including histologic diagnosis (SFT), site, size, and presence of metastases. Tumor site and size were determined from patients’ pathology reports, operative reports, and radiologic scans or reports.We recorded the chemotherapy schedule and the regimen used for each patient. For patients with unresectable disease who had received chemotherapy only, we recorded the chemotherapy regimen, the number of cycles, the best tumor response reached, and the date of disease progression. For patients for whom radiologic scans were available, information on the best tumor response achieved and disease progression was ascertained from direct measurement of the image on the radiologic scan; otherwise, the information was collected from radiologic or clinical reports.The medical records showed that all patients who received chemotherapy were evaluated with radiologic scans at baseline, during treatment, and after treatment. We assessed radiologic response to treatment on available images following the Response Evaluation Criteria In Solid Tumors (RECIST) v.1.1 . For ourPatient characteristics were summarized using medians and ranges for continuous variables and frequencies and percentages for categorical variables. We used the Kaplan-Meier method and SPSS software to estimate median PFS duration and median OS duration. PFS duration was defined as the time interval from the beginning of chemotherapy until radiologic evidence of disease progression as defined by RECIST or death from any cause. OS duration was defined as the time interval from initial diagnosis or initiation of chemotherapy until death from any cause. Survival data were updated on April 1, 2010, and data for patients who were still alive were censored from survival analysis at that point.Our initial database search identified 128 patients diagnosed with SFTs, treated, and followed at MD Anderson from January 1994 to June 2007. Of these patients, 21 met our study inclusion criteria (had advanced SFTs with grossly measurable disease and were treated with systemic chemotherapy) and were included in our analysis.The clinical characteristics of the patients (12 men and 9 women) are summarized in Table The chemotherapy regimens used for patients in our retrospective analysis are listed in Table 2 of doxorubicin and 1 patient receiving 60 mg/m2 of doxorubicin. Doxorubicin was most often administered with 10 g/m2 of ifosfamide (in 12 of 15 cases). In all 15 patients, doxorubicin-based chemotherapy was the first-line treatment. Those patients not receiving doxorubicin-based therapy were prescribed gemcitabine-based chemotherapy (5 cases) and paclitaxel (5 cases).Fifteen patients received doxorubicin-based therapy, with 14 patients receiving 75 mg/mThe tumor responses to each type of regimen and to each line of chemotherapy are summarized in Table Of the 6 patients who were treated with second-line chemotherapy, 4 (67%) had stable disease, with 1 maintaining stable disease for longer than 6 months, and 2 (33%) exhibited disease progression. The patient treated with third-line chemotherapy exhibited disease progression after 2 cycles of chemotherapy.Kaplan-Meier curves for PFS duration and OS duration are shown in Figures 2) and cisplatin (120 mg/m2) for the metastatic tumor. Restaging studies after chemotherapy showed stable disease, and the patient underwent surgical resection of the tumor, which showed 50% necrosis. She received no further therapy and remained alive with no further evidence of recurrent disease after 11 years.Most patients in the group died from progression of their disease. Two patients, however, were still alive at the time of this analysis on April 1, 2010. The first of these was a 28-year-old woman with an SFT in the right gluteal region who presented with a solitary metastasis in her right ilium. She underwent 4 cycles of neoadjuvant chemotherapy with intra-arterial doxorubicin (75 mg/m2) and ifosfamide (10 g/m2). He demonstrated stable disease after cycle 2 of chemotherapy, but the disease progressed after cycle 4 and the tumor was surgically resected. No significant necrosis was seen. The patient received no further therapy and remained alive with no evidence of recurrence after 6.3 years.The second surviving patient was a 50-year-old man with a locally advanced SFT of the pleura. He received 4 cycles of neoadjuvant chemotherapy with doxorubicin  of 13 patients with SFTs treated with first-line doxorubicin- or ifosfamide-based chemotherapy demonstrated a partial response and that the median PFS duration was 4 months . In our Stacchiotti et al. recently published the results of response to chemotherapy in 31 patients with solitary fibrous tumor . In thisThe findings of our retrospective analysis were limited by our small sample size and thus by a lack of statistical power. The heterogeneity of our patients’ disease characteristics was another potential limitation. We also were only able to use RECIST, not Choi criteria, to assess tumor response to conventional chemotherapy ,20,21. ONevertheless, to our knowledge this retrospective analysis is one of the largest published series of patients with SFTs treated with conventional chemotherapy agents. The lack of objective tumor responses to chemotherapy observed in our patients suggests that treating patients who have advanced SFTs with first-line conventional doxorubicin- or gemcitabine-based chemotherapy might not be very effective to achieve tumor shrinkage. Thus, it may be useful to consider other therapies.Recently, several novel agents have shown promise in the treatment of advanced SFTs. We previously reported our experience using the combination of temozolomide plus bevacizumab, a monoclonal antibody directed against the activity of vascular endothelial growth factor (VEGF) . FourteeSeveral tyrosine kinase inhibitors have also shown promise in treating SFTs. Sunitinib, a multitargeted tyrosine kinase inhibitor whose targets include VEGF receptors 1–3 and platelet-derived growth factor receptors A and B, has shown therapeutic promise for SFTs. Stacchiotti et al. found that 6 (60%) of 10 patients with advanced, chemotherapy-refractory SFTs treated with sunitinib achieved a Choi partial response, and 5 maintained their response for more than 6 months. The tumor response rate to sunitinib when assessed using RECIST was 0% . SorafenOur findings indicate that conventional chemotherapy is effective in controlling or stabilizing locally advanced and metastatic SFTs. Our data on the clinical outcomes with traditional chemotherapy can serve as a reference point for comparing the efficacy of novel agents against SFTs. Despite the small number of patients treated with the temozolomide-bevacizumab combination and the sunitinib regimen to date, these agents appear to have greater efficacy than conventional chemotherapy for SFTs ,12. SFTsThe authors declare that they have no competing interests.MP wrote the manuscript, VP performed the statistical analysis, AC helped write the manuscript, SP, RS, provided some of the patient cases, JC, DL, gave scientific advice about the project, AL, WLW confirmed the pathologic diagnosis and DA helped write and edit the manuscript. All authors read and approved the final manuscript.


Hepatitis B virus (HBV) infection ranks among the most devastating health problems in the world.The most probable transmission routes of HBV are blood contact, sexual, and horizontal transfer. Other sources of HBV transmission are razor sharing, beauty treatments, tattooing, piercing, and manicures and other chiropody treatments.Many infections have been reported in South-East Asia, where barbers commonly share and reuse razors. Detection of HBV DNA in contaminated devices such as razor blades is important in the demonstration of transmission routes and indirect estimation of HBV prevalence in specific subpopulations such as barbershop clientele. Therefore, we aimed to detect the presence of HBV contamination on razor blades by nucleic acid testing.Used razor blades (n = 151) were purchased from different barber’s shops. Used razor blades purchased from chronic HBV patients (n = 8) were included as a positive control. The amplification and detection of HBV DNA was carried out by a semi-nested PCR method in a thermal cycler.The presence of HBV DNA was found in 10 (6.6%) used razor-blade samples by the detection of a specific positive band with agarose gel electrophoresis.In conclusion, used razor blades may be contaminated with HBV, and the practice of sharing used razor  blades may pose a risk of transmission. Nucleic acid detection methods involving PCR can be used to detect HBV contamination of razor blades. HBV control and prevention programs should educate barbers about the importance of contagious diseases, proper sterilization techniques, and avoiding reuse and sharing of contaminated equipment and supplies such as razor blades. As an infection control measure, prohibition of razor reuse can reduce the spread of HBV infection in rural areas, where the practice is often common at barbershops. Hepatitis B virus (HBV) infection ranks among the most devastating health problems in the world . The preWorldwide, the route of HBV transmission is mainly parenteral, although perinatal, sexual,household, and occupational transmission can occur. In Turkey, the most common transmission route is blood contact (41.1% of cases), whereas the routes for the remaining cases are unknown (49.4% of the cases) in Turkey . Other pDespite the fact that needles and razor blades can transmit HBV, a feasible detection method  for HBV does not exist to date 7]. Alth. Alth7].Used razor blades (n = 151) were purchased from different barbershops in Samsun, Turkey. Used razor blades purchased from chronic HBV patients (n = 8) were used as a positive control. Identifying information for each blade was recorded, and each blade was put in a single DNAse-free and RNAsefree tube (15 mL). Each used-razor-blade sample was stored separately at -20°C until the blades were studied. We prospectively collected each subject’s name, surname, sex, and ages.In the first step of DNA isolation, 200 μL of distilled water, 40 μL of proteinase K, and 200 μL of binding buffer were added to each tube, in turn. Tubes were vortex mixed vigorously. After that, HBV DNA was isolated with a high pure PCR template preparation kit  according to the instructions of the manufacturer. To elute the HBV DNA, a 50μL elution buffer was used.The amplification and detection of HBV DNA was carried out by a semi-nested PCR method in a thermal cycler. For amplification, 5 μL of HBV DNA were added to a 45-μL reaction mixture. HBV primers were used for the first round of PCR. The primers 377 (5’- GGATGTGTCTGCGGCGTTT -3’) and 840 were used in the second round of PCR . The PCR reaction mixture contained 200 mM of each nucleotide triphosphate, 0.5 μM of each primer, 1.5 mM of MgCl2, and 2 units of Hot Start Taq DNA polymerase (MBI Fermentas) in a 50-μL total reaction volume. Samples were thermocycled  for 35 cycles . Then, 2 μL of the PCR products were added to the second round PCR mixture. Second-round PCR products were separated in a 1.5% agarose gel, then stained with ethidium bromide and viewed under UV light. A result was considered positive when a band of the appropriate size was visible in the gel . Standard procedures for reducing contamination were strictly followed.All 151 subjects were male, and the mean age ± standard deviation (SD) was 34.6 ± 10.0 years. HBV DNA was detected in 10 (6.6%) razor-blade samples by the detection of a positive band with the agarose gel electrophoresis . All used razor blades (n = 8) purchased from chronic HBV patients were also positive.The whole virion, or Dane particle, is a 42-nm sphere that contains a core, or nucleocapsid, enclosing the DNA . Other HWe considered that used razor blades could have been contaminated with HBV through the normal bodily fluids of infected patients. Unfortunately, as of this writing, there are no feasible infectivity assays available to validate the route of HBV infection, either in cell cultures or in small animals, although several authors have reported different cell culture systems such as HepG2 9]10]. [10]. 9][. [10]. 9In this study, HBV DNA was detected in 10 (6.6%) used razor-blade samples. This positivity rate is plausible because Turkey is a country with an intermediate endemicity for HBV. The rate indicates that sharing razor blades can be risky in endemic areas. Particularly in hyperendemic areas, sharing razor blades should be considered a very important risk factor because of the higher numbers of HBV carriers. Our method can be used for the detection of HBV presence in razor blades and other similar devices. With this method, the extent of risk can be shown in detail, and appropriate infection control measures can be implemented.The main transmission routes of HBV are parenteral and perinatal. Sexual, household, and occupational transmissions are also important . HBV infDespite this knowledge, these two practices continue to be common many parts of the world.  For instance, a survey of barbers in India and Pakistan 20] reve reve20] The unawareness among barbers and healthy people at risk of contracting HBV through sharing razor blades may weaken the control of hepatitis B transmission, and end up with an increase in prevalence even in low-endemic areas. Candan et al.  found that barbers in Turkey may often be exposed accidentally to the blood and bodily fluids of their customers; indeed, the authors found that the prevalence rates of HBV and hepatitis C virus were higher in barbers  than in a comparison group  . HBV conIn the literature, there are few articles about hepatitis transmission and beauty treatment 21]22].[22].21][.[22].21]Because all used razor blades collected from chronic HBV patients were positive in our study, special efforts should be made to educate patients with chronic hepatitis B infection about the risk of spreading HBV via razor blades. They should particularly be warned of the risks of sharing personal articles like razor blades.In conclusion, nucleic acid detection methods can be used for the detection of HBV in devices such as razor blades. Reuse of razor blades is an important route for transmission of HBV infection, and general reduction of these exposures and assuring sterile practices are logical goals for intervention and prevention of hepatitis B infection. Future studies should focus on how to detect biologically active viruses quantitatively in risky devices.


Sustained ocular drug delivery is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Biodegradable subconjunctival implants with controlled drug release may circumvent these two problems. In our study, two microfilms  were surgically implanted with one type of microfilm in each eye. Serial anterior-segment optical coherence tomography (AS-OCT) scans together with serial slit-lamp microscopy allowed us to measure thickness and cross-sectional area of the microfilms. In vitro studies revealed bulk degradation kinetics for both microfilms, while in vivo studies demonstrated surface erosion kinetics. Serial slit-lamp microscopy revealed no significant inflammation or vascularization in both types of implants  over a period of 6 months. Histology, immunohistochemistry and immuno-fluorescence also revealed no significant inflammatory reaction from either of the microfilms, which confirmed that both microfilms are biocompatible. The duration of the drug delivery can be tailored by selecting the materials, which have different degradation kinetics, to suit the desired clinical therapeutic application. The eye is the vital organ for sight and unique because it is both anatomically and immunologically privileged. While the eye is protected physiologically, it is also resistant to penetration by drugs. Topical application is the mainstay of most ocular therapy, but ocular bioavailability is poor due to the efficient protective barriers Biodegradable polymers are proven vehicles for sustained drug delivery in vitro studies using commonly used ophthalmic drugs, latanoprost and 5-fluorouracil PLGA has been used extensively in studies to deliver a wide variety of drugs using various forms such as microparticles, emulsions, implants and hydrogels in vitro and in vivo, surgical feasibility and biocompatibility of both types of microfilms using three complementary techniques: slit-lamp microscopy, anterior segment optical coherence tomography (AS-OCT) and histology with various staining methods.In this study, we aim to develop and compare two different biodegradable microfilms for their potential application as vehicles for intraocular drug delivery, specifically on surgical insertion into the subconjunctival space. We evaluated the degradation behavior of both microfilms Polymers used in this study are poly , PLGA50/50  and poly  PLC70/30 , which were purchased from Purac Far East Pte. Ltd., Singapore. High-performance liquid chromatography (HPLC)-grade dichloromethane and chloroform were from Tedia Company. Phosphate buffer saline (PBS) tablets were obtained from CalBioChem, England.Samples of PLGA50/50 and PLC70/30 were weighed before dissolving the appropriate amount in dichloromethane. Following dissolution, the samples were dried in petri-dishes under a fume hood for a day, followed by drying in a vacuum oven at 37°C until the solvent level was less than 1% of the total weight, as measured using a thermo-gravimetric analyzer . After drying, all samples were cut manually into standard sized microfilms (6.0×3.0×0.5 mm) by using a sharp knife.Samples were immersed in a closed vial containing 5 ml Phosphate Buffered Saline . PBS was prepared by dissolving PBS tablets into 1 liter deionized water. All vials were incubated at 37°C throughout the study. The buffer was refreshed every week, and at every predetermined time point, samples were taken out, rinsed with deionized water and dried in 37°C vacuum oven for 7 days, before testing. Degradation of PLGA50/50 and PLC 70/30 was monitored by film thickness (measured by Elcometer 456), water absorption, weight loss and weight average molecular mass (Mw) and poly dispersity index(PDI). Dried samples were dissolved in chloroform (1–5 mg/ml) and filtered through 0.22 µm regenerated cellulose syringe driven filters before test. Weight average molar mass and poly dispersity of the sample were determined by gel permeation chromatography  at 35°C, using Agilent PLgel 5 µm mixed-C column, under a flow rate of 1 ml chloroform per minute, using a Refractive Index Detector (RID).All the samples were sterilized by ethylene oxide (ETO) at 37°C  in Tan Tock Seng Hospital (Singapore) prior insertion into animals.We obtained approval from the SingHealth Institute Animal Care and Use Committee  and all procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Nine New Zealand white rabbits (18 eyes) were used aged 4–6 months old with a weight range of 2–2.5 kg each. Each rabbit was anesthetized with intraperitoneal injection of ketamine hydrochloride (35–50 mg/kg) and Xylazil (5–10 mg/kg). After the animal had been adequately anaesthetized, the eye was cleaned with povidone–iodine (10%) and draped with sterile cloth. A subconjunctival pocket was created via blunt dissection just at the limbus with a 5–6 mm incision in the superior-temporal aspect of the rabbit's eye. Microfilms were sterilized in ethyl alcohol and chlorhexidine before soaking in sterile normal saline. The microfilm was then inserted into the subconjunctival pocket 1 mm from the limbus using a conjunctival forceps. Closure with 10-0 nylon sutures was done to ensure secure implantation of each microfilm. In each rabbit, PLC70/30 (n = 9) microfilms were inserted into the right eye, whilst PLGA50/50 (n = 9) microfilms were inserted into the left eye. Topical Tobradex (Tobramycin & Dexamethasone) was administered each eye 4 times a day for 5 days.Visual inspection of the operated eyes was conducted daily following surgery. The animals' eyes were also inspected for changes at the insertion site, gross appearance of the implant and for any evidence of infection. Slit-lamp examination of the exterior and anterior chamber of the eyes was done prior to surgery and weekly thereafter. All clinical and ocular observations were recorded on a chart. The test animals were also monitored for any gross changes such as eye discharge, squinting and, ocular discomfort. A modified Hackett McDonald ocular score was used to record the presence of conjunctival injection, swelling, discharge and corneal clarity www.meditec.zeiss.com).Anterior segment photographs and anterior segment optical coherence tomography  of the implanted eyes was performed at monthly intervals. The Visante OCT is a high-resolution biomicroscopic device for anterior segment imaging , based the principle of low coherence interferometry using a 1310 nm light emitting diode Three rabbits were randomly selected for euthanasia at 1, 3 and 6 months post-implantation. Euthanasia was carried out with intraperitoneal pentobarbitone (60–150 mg/kg) followed by enucleation of both eyes. The eyes were immediately immersed in a mixture of 4% glutaraldehyde and 2.5% neutral buffered formalin for 24 hours. The globes were dehydrated and embedded in paraffin, then sent to the histopathological laboratory for sectioning with a microtome, appropriate staining (Haematoxylin and Eosin and Masson Trichrome stain) and reading – for signs of inflammation, tissue damage, scarring and fibrosis. Immunohistochemistry stains were used to quantify the amount of encapsulation that developed in each eye.2O2/PBS solution, washed, and blocked with 20% Aquablock  in PBS/0.2% Tween-20 for 30 minutes. Sections were incubated with a rabbit anti-mouse CD45 monoclonal antibody (mAb) at 10 µg/ml . Detection of the primary antibodies was performed with biotinylated rabbit anti-rabbit IgG secondary antibodies  followed by incubation with 3,3′-diaminobenzidine  and counterstained with Hematoxylin QS (Vector Laboratories).The sectioned, paraffin-embedded slides were heated to 60°C for 1 hour, deparaffinized, and rehydrated. Endogenous peroxidase activity was quenched by a 30-minute incubation in 3% HThe sections were de-paraffinized with xylene and rehydrated. Antigen retrieval was performed before heating the sections to 100°C for 25 minutes. The sections were cooled and washed with PBS and 1%BSA for 30 minutes before incubating in a dark incubation chamber for 90 mins with 50 ul of primary antibody at room temperature. Images of the sections were then viewed and digitally captured with a fluorescent microscope.The amount of fibrosis and scarring was evaluated by measuring the thickness of the collagen capsule formed around each implant. The sections were stained with Masson Trichrome, and the average thickness of the collagen capsule was measured by integration of the collagen-stained area throughout the entire length of the implant. Next, the total inflammatory cell response was estimated by determining the percentage of the implant surface lined by inflammatory cells. We calculated the ratio L: C [L = total length of the implant surface and C = length of implant surface lined by inflammatory cells (CD45 stained) immediately adjacent to the implant surface]. In sections where the implant was not clearly visible, the implant site was sectioned and stained for inflammatory cells.Statistical analysis included descriptive statistics, where the mean and standard deviation (SD) was calculated for the continuous variables; while frequency distribution and percentages were used for categorical variables. The Student's t-test was used to analyze immunohistochemistry data and independent samples t-test was used to study the rate of degradation of the microfilms. P-values of less than 0.05 were defined as statistically significant. All data was expressed as mean ± SD unless otherwise stated. All analyses were performed using STATA version 11 .in vitro, such as water absorption, weight loss, change in thickness and change in molar mass, to analyze the degradation of both types of microfilms.We studied various factors wet−Wdry)/Wo %, where Wwet represents the weight of the wet sample after wiping by tissue, Wdry represent the final weight of the dried samples, and Wo represent the sample's initial weight. The amount of water absorbed in the microfilm increased with immersion time for both polymers /Wo %. In conjunction with water absorption, mass loss started earlier for PLGA 50/50. At 3 weeks, PLGA 50/50 started to lose significant mass, whereas PLC 70/30 did not demonstrate any notable mass loss until after day 56  in the first 21 days, but a sudden drop of 60% occurred on the day 28, and immeasurable on day 42,. From the observed changes on mass loss, PLGA50/50 lost 90% of its initial weight on day 42, and corresponded to the change in film thickness. In contrast, PLC70/30 maintained its shape throughout the entire duration of the study period, with minimal change in film thickness .P = 0.91, no significant inter-observer variability). The cornea, anterior chamber and lens remained clear with no evidence of inflammation or scarring. We did notice on external ocular examination that the PLGA50/50 microfilms appeared more pliable compared to the PLC70/30 microfilms, which retained its original shape. However, the animals did not display any signs of ocular discomfort, nor was there any extrusion of any of the implants from either material. At month 4 onwards, the PLGA50/50 microfilms had dissolved to such an extent that they were not grossly visible even with slit-lamp examination. In contrast, PLC70/30 microfilms persisted and visible up to 6 months.The gross appearance and examination of the implanted microfilms revealed minimal localized inflammation and vascularity using serial vascularity grading scales 2 = 0.8878 for PLC70/30), as measured by AS-OCT  in the subconjunctival space . No migry AS-OCT .P<0.001. Histological examination did not show an obvious foreign body encapsulation of the implanted films. This is important as excessive scarring and encapsulation often affects ocular function or lead to surgical failure in surgeries such as glaucoma filtration surgery Histopathological examination of all the enucleated eyes (n = 18) revealed minimal inflammation in the initial 1–2 weeks post-operatively. This inflammation was seen to resolve by 1 month and there was minimal or no fibrosis or scarring detected. By 4 months post-implantation, the PLGA50/50 microfilms were not significantly visible on histology sections within the subconjunctival space. There was no significant fibrosis or collagen capsule formation seen around the implant site . HoweverSections stained with immunohistochemistry for CD45 T cells were analyzed under polarized microscopy at 3 and 6 months post-operatively. The PLC70/30 microfilms had significant reduction in amount of inflammation surrounding the capsule from 3 months post-implantation to the end of 6 months . Importantly, there was minimal infiltration of T cells detected by immunohistochemistry surrounding the implant site of the PLGA50/50 microfilms and the inflammatory L/C ratio could not be calculated .Sections stained with immunofluorescence for CD45 T cells were analyzed for both PLC70/30 and PLGA50/50 at 3 and 6 months. There was minimal inflammatory reaction, with scattered T cells surrounding both implants. We also noted a reduction in T cells surrounding the implant for both PLGA50/50 and PLC70/30 when comparing implants at 3 and 6 months post-operatively .The current mainstay of ocular therapy is via topical administration. While it is easy to administer , there are many drawbacks - which include poor bioavailability and penetration of the drugs, frequent instillation leading to poor compliance and blurring of vision from viscous vehicles We have used biodegradable polymers to create implants that may be capable of sustained ocular drug delivery, to overcome the disadvantages of topical medications and the issues with compliance – a common problem faced by ophthalmologists in dealing with diseases such as glaucoma, the second leading cause of irreversible blindness in the world. This biopolymer microfilm placed in the subconjunctival space may significantly improve drug availability and reduce local ocular side effects, while overcoming poor patient compliance.in vivo over a prolonged period of 6 months. We used several techniques to demonstrate the biocompatibility, as well as behavior of these microfilms when inserted into the subconjunctival space of the eye. Slit-lamp microscopy allowed us to examine the location of the microfilms, while monitoring inflammation and scarring of the overlying conjunctiva. We did not find any significant increase in inflammation or vascularity using serial vascularity grading scales Our study demonstrates that both PLGA50/50 and PLC70/30 microfilms are biocompatible and safe to be inserted subconjunctivally in the rabbit eye. We also report for the first time, the use of a novel AS-OCT imaging technique to serially measure the degradation of the microfilms and describe the internal structures of the eye for encapsulation It is generally accepted that, in this class of polymers used in our study (poly α-hydroxy esters), there may be two different modes of degradation. In the first mechanism, which is often referred to as homogeneous or bulk degradation, the polymers degrade slowly with no appreciable mass or volume loss until the degradation products become water-soluble and leach out of the matrix, when mass loss is then detectable. In the second mechanism, the polymer degrades first at the surface, and the surface molecules decrease in molecular weight to the point where the surface molecules leach out, without affecting the interior of the material. In this mode of degradation, which is sometimes referred to as heterogeneous degradation or surface erosion, there is continuous decrease in mass and in the material dimensions.in vitro and in vivo. Although not as evident  becomes higher than the water ingress rate into the polymer (Rw). We hypothesize that in the in vivo situation, Rh is being increased relative to Rw, most likely due to the influence of enzymes (esterases) or proteins present in the eye. A surface erosion mode is the preferred mode in such applications, as bulk degradation may lead to “catastrophic” breakdown into small fragments causing localized irritation. Surface erosion also results in a constant release of incorporated drug. PLGA and PLC are anionic polymers that undergo bulk degradation in vitro. Embedded drugs are released from the matrix via diffusion initially, followed by degradation of the polymer matrix itself From the results of the study, the PLGA 50/50 films clearly exhibited bulk degradation decrease  without decrease , but thidecrease  decreaseThe subconjunctival space is a potential area in the eye that is useful for delivering ocular drugs in a sustained manner. Currently, peribulbar or subtenon injections are used to deliver short to intermediate duration of drugs to the eye in vitro, whereas PLC70/30 exhibited surface erosion in vivo. The observation of surface erosion in the sub-conjunctival space is significant for controlling the release of drugs locally, and opens the door for more efficient and sustained therapy.In conclusion, we report that biodegradable microfilms prepared from PLGA50/50 and PLC70/30, are non-toxic and well tolerated when implanted in the subconjunctival space and therefore has the potential use as an ocular drug delivery platform. PLGA50/50 always degraded at a faster rate than PLC70/30. Both PLGA50/50 and PLC70/30 demonstrated bulk degradation


Bacillus thuringiensis is considered a good alternative as a biocontrol agent for suppressing plant pathogens such as Fusarium. In this study, we used microscopy, flow cytometry, indirect immunofluorescence, and high performance liquid chromatography to determine the interaction between B. thuringiensis subsp. kurstaki LFB-FIOCRUZ (CCGB) 257 and F. verticillioides MRC 826, an important plant pathogen frequently associated with maize. B. thuringiensis showed a strong in vitro suppressive effect on F. verticillioides growth and inhibited fumonisin production. Flow cytometry analysis was found to be adequate for characterizing the fungal cell oscillations and death during these interactions. Further studies of the antagonistic effect of this isolate against other fungi and in vivo testing are necessary to determine the efficacy of B. thuringiensis subsp. kurstaki in controlling plant pathogens. This is the first report on the use of flow cytometry for quantifying living and apoptotic F. verticillioides cells and the B. thuringiensis Cry 1Ab toxin.Bacterial antagonists used as biocontrol agents represent part of an integrated management program to reduce pesticides in the environment.  Fusarium verticillioides (Saccardo) Nirenberg  is an important plant pathogen commonly associated with maize, which is capable of causing seedling diseases and stalk and cob rots F. verticillioides (Fv) contamination and the risks of fumonisin ingestion, many efforts have been made to control this fungus Fv contamination; nevertheless, Fusarium strains exhibit a remarkable capacity to adapt and to become resistant to these strategies Due to economic losses regarding BacillusPseudomonas, and StreptomycesPesticides have increased crop resistance for over four decades; nonetheless, the emerging, re-emerging, and endemic plant pathogens are still challenging crop safety worldwide Bacillus thuringiensis (Bt) is a ubiquitous gram-positive spore-forming bacterium that produces parasporal crystals containing insecticidal proteins called crystal (Cry) or cytolytic (Cyt) toxins. This bacterium is found in a wide variety of habitats such as soils, dead and living insects, the plant phylloplane, and as an endophyte. The high abundance of Bt strains is attributed to its long-lasting spore viability Bt strains are classified according to the serological response differences in their flagellar antigens, and there are at least 71 serotypes and 84 subspecies B. thuringiensis subsp. kurstaki (Btk), which produces lepidopteran-insecticidal Cry 1A proteins, including Cry 1Ab, is the most commonly employed strain used as a bioinsecticide against lepidopteran pest larvae B. thuringiensis insecticidal proteins, the bacterium may also act as a potential BCA against a variety of plant pathogens due to its production of antimicrobial molecules, including zwittermicin A, chitinases, chitin-binding proteins, and quorum sensing-quenching enzymes Bt spore germination and vegetative cell growth were associated with the development of fungal hyphae in the soil. Furthermore, vegetative cells have been observed growing with the fungal mycelia, likely due to the ability of Bacillus species to cause the death of the fungus and thus survive the hyphal lysis products Although many studies have focused on the activity of in vitro interaction of F. verticillioides and B. thuringiensis serovar kurstaki based on a multi-method approach. To our knowledge, this is the first report of the use of flow cytometry to quantify living and necrotic fungal cells and the Cry 1Ab toxin during the time-course of interaction between Btk and Fv.The aim of this study was to characterize the F. verticillioides MRC 826  was obtained from a culture growing on Spezieller Nährstoffarmer agar by monosporic isolation and was used in all experiments. This isolate is capable of producing high levels of fumonisins Bacillus thuringiensis subsp. kurstaki LFB-FIOCRUZ (CCGB) 257, which produces the Cry 1A toxin group, was provided by Dr. Leon Rabinovitch from the Culture Collection of Bacillus and Related Genera . This Btk strain was isolated from moist soil from the Morretes Village, Paranaguá, Paraná State, Brazil prior to 1995. It was identified and characterized by Dr. Leon Rabinovitch and Dr. Tania V. Guaycurus at the Bacterial Physiology Laboratory in the Oswaldo Cruz Institute in collaboration with the Pasteur Institute, Paris, France. The LFB-FIOCRUZ (CCGB) 257 strain exhibits proteolytic and amylolytic properties, is mesophilic, and produces a bi-pyramidal crystal. The serovar kurstaki , which produces flagellar antigens of serotype “H3”, was characterized by the Pasteur Institute.Btk LFB-FIOCRUZ (CCGB) 257 into Erlenmeyer flasks containing 250 mL of Nutrient Broth  and incubating at 30°C on a rotary shaker (100 rpm) for 72 h. The cultures were centrifuged at 12,000 rpm for 30 min at 4°C, and the cell pellets were stored at −20°C Btk spore and crystal biomass was made by diluting to 10 µg/mL, which was the minimum inhibitory concentration for Fv growth under our laboratory conditions (data not shown).The cultures were stored lyophilized; bacterial spores and crystal biomass were obtained by inoculating Fv (1×106 spores/mL) was centrally inoculated onto each PDA Petri plate. PDA medium was chosen since it can provide for Fusarium species' growth, in addition to bacterial growth. The microscopy, immunofluorescence, and cytometry experiments were conducted with five replicates after 3, 5, and 7 days of incubation under continuous white-light illumination at 25°C. The radius measurements of the pure and mixed (Fv+Btk) cultures were recorded for 20 days for five replicates each, after which the presence of the fumonisins B1 and B2 was determined.About 50 µL of Btk biomass (10 µg/mL) was centrally inoculated onto potato dextrose agar (PDA) using a micropipette. After inoculum adsorption, 50 µL of Five replicates were analyzed after 3, 5, and 7 days of incubation. The cultures were removed from the agar by scraping, transferred into a 50-mL tube containing sterile distilled water and Tween 80 (2 drops/100 mL water), and centrifuged at 7000 rpm for 20 min. The cells were rinsed with the Tween 80/water solution twice and resuspended in 20 mL phosphate buffered saline (PBS), after which a 4 mL aliquot was filtered through a 70-µm mesh-sized cell strainer  to obtain a uniform cell suspension. The cell solution was stained with 10 µL of 1 µg/mL Calcofluor White  at a final concentration of 0.05 µg/mL CFW and stained with 5 µL of 7-aminoactinomycin (7-AAD), utilizing the PE Annexin V Apoptosis Detection Kit  per the manufacturer instructions. The cells were incubated with CFW and 7-AAD in the dark for 15 min and stored on ice until the analysis.ex, 365 nm; λem, 430 nm), using violet laser excitation (405 nm) with detection in the Pacific Blue channel (405–450/50 nm), and 7-AAD , using blue laser excitation (488 nm) with detection in the PerCP (peridinin chlorophyll A protein) channel (650 nm). A minimum of 30,000 events per sample were acquired; the data were collected using a linear representation for the side scatter (SSC) and forward scatter (FSC) and a logarithmic representation for the fluorescent signals. The analyses were performed using FlowJo software . The necrotic fungal cell population was gated to separate it from the dead bacterial cell population. For verifying Fv autofluorescence and cross reaction between the Btk and CFW, bacterial cells stained with CFW and fungal cells not labeled with the fluorochrome were used as negative controls. For the 7-AAD negative controls, pure cultures of living bacterial and fungal cells were stained with 7-AAD. The negative controls were utilized to determine the background of each fluorescent marker. For the 7-AAD positive controls, the bacterial and fungal cells were incubated at 100°C for 30 min in a water bath The living and necrotic fungal cells were quantified by flow cytometry performed using a FACS Canto II system  equipped for CFW  glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 2 h at room temperature and then overnight at 4°C. The samples were rinsed with cacodylate buffer and fixed in 1% (w/v) osmium tetroxide before dehydration in a graded series of ethanol concentrations and embedment in Epon 812 resin. Ultrathin sections were collected on 200-mesh nickel grids coated with Formvar and stained with uranyl acetate and lead citrate. The grids were examined with a JEOL 1010 transmission electron microscope . For each replicate, three ultrathin sections were examined After the incubation periods on PDA, 1-cmThe pure cultures and treatments were placed in a 40% glutaraldehyde solution for 24 h, dried at 42°C for at least 48 h, and fixed to appropriate aluminum bases. The material was then sputtered with gold and examined with a Leo-440i-SEM scanning electron microscope  Fv and Btk were analyzed during the incubation period. The cells were removed by scraping, washed 3 times with PBS, and incubated for 30 min in PBS/BSA (1%) at room temperature before adding rabbit polyclonal antibody against Bt Cry 1Ab toxin ; the incubation was for 30 min in the dark under gentle rotation (60 rpm). The cells were washed three times with PBS/BSA and incubated with a goat polyclonal secondary antibody to rabbit IgG coupled to Alexa Fluor 488  under the same conditions. The cells were washed three times with PBS/BSA and 10 µL of CFW (1 µg/mL) was added. The cells were resuspended in 40 µL of a solution composed of glycerol (500 µL), PBS (400 µL) and n-propyl gallate (0.022 g) . Slides were prepared and analyzed with an EVOS FL Cell Imaging System  using the DAPI (diamidino-2-phenylindole) channel for CFW and the GFP (green fluorescence protein) channel for Alexa Fluor 488 . The background of each fluorescent marker was determined with negative controls of the Btk, Fv, and Btk+Fv treatments incubated with the secondary antibody, but not with the primary antibody. For the CFW controls, the bacterial cells were stained with the fluorochrome, while the fungal cells were not Pure and treated cultures of ex, 495 nm; λem, 519 nm) using blue laser excitation (488 nm) with detection in the FITC (fluorescein isothiocyanate) channel (530/30 nm). The collection and analysis were as previously described for the flow cytometry analysis of the fungal cells. For negative controls, the cells from all treatments were incubated only with the secondary antibody The cells were treated as described above, but omitting the CFW staining. Quantification of the Cry 1Ab toxin was performed with the FACS Canto II  equipped for Alexa Fluor 488 (λFv (1×106 spores/mL) and Fv plus Btk biomass (10 µg/mL) were inoculated onto PDA (five replicates/treatment) and incubated at 25°C for 20 days ex, 335 nm; λem, 335 nm). An acetonitrile/water/acetic acid  solution was used for the mobile phase. Chromatography was conducted with column temperature of 30°C, maintained in an oven, and a flow rate of 1.0 mL/min,at room temperature (22–23°C). The retention times of the FB1 and FB2 under these conditions were 9 and 20 min, respectively. Calibration curves were utilized for quantifying the fumonisin with correlation coefficients of 0.9927 (FB1) and 0.9950 (FB2). The quantification limit was 0.015 µg/g for FB1 and FB2, and the mean recoveries from five replicates for each were 92.38%  and 85.39%  for FB1, and FB2, respectively.p-values were <0.05. The statistical tests were conducted using Assistat software Statistical comparisons were performed using ANOVA and Tukey tests, as the data were normally distributed. The differences were considered significant when Btk LFB-FIOCRUZ (CCGB) 257 could be a potential BCA against Fv, flow cytometry tests were used to identify either apoptotic or living fungal cells. During the culture growth for the analyses, it was observed that Fv increased its biomass faster than did Btk on PDA medium, as demonstrated by the average of three radius measurements of the colonies alone versus those of interactions between Fv and Btk (five replicates) during the period of analysis  257 strain was found to show significant antagonistic activity against Fv from the second to the twentieth day, based on the radius measurements of the five replicates (Fusarium cells (data not shown). On the fifth and seventh days, the Fusarium cells appeared damaged, as evidenced by cell-wall irregularities and by disorganization of the cytoplasm. In contrast, the Fv pure cultures showed intact hyphae and abundant microconidia formation when the colonies were analyzed by TEM , although the percentage of cells remained virtually the same from the fifth to the seventh day (3.48%). Interestingly, fungal cell death was observed on the seventh day of incubation (5.23%), in agreement with the TEM and SEM observations  to the fifth day  257 strain, demonstrating its relevance as a BCA. The morphological alterations observed by TEM, SEM, and the immunofluorescence assays subsequently confirmed the flow cytometry results. No such changes were noted in the control cultures.By the end of the experiment, the treated cultures were primarily colonized by the Btk LFB-FIOCRUZ (CCGB) 257 strain was analyzed after 3, 5, and 7 days of incubation by utilizing specific antibodies followed by analysis with flow cytometry. This experiment was conducted based on the observations of the indirect immunofluorescence assay, which was possible to visualize high quantity of the toxin and Btk spores all over the treated culture  257 strain in comparison to the control group (p<0.05). We observed 15.54%, 20.86%, and 41.61% positivity rates on the third, fifth, and seventh days of incubation, respectively (The production of the Cry 1Ab toxin by the  culture . In Btk ectively . A signiectively .Fv strain produced lower levels of the fumonisins FB1 and FB2 during interactions with Btk LFB-FIOCRUZ (CCGB) 257 for 20 days. In the control group, the average production of FB1 and FB2 was 18.97 µg/g  and 0.86 µg/g , respectively. In the treated group, the average production of FB1 was 13.81 µg/g , while FB2 was not detected in any replicate. The five replicates of the control and treated groups were significantly different from one another (p<0.05).The Fv and Btk based on the combined use of microscopy, flow cytometry, and HPLC techniques. We also presented a practical and effective approach to identify these interactions using flow cytometry, which could be extended to other plant pathogens and antagonistic bacteria, thereby contributing to the ability to determine the potential of microorganisms as possible BCAs.The aim of this study was to characterize the interaction between Fv in terms of living and dead cell percentages and identify the Cry 1Ab protein produced by Btk during the experimental period.Flow cytometry has emerged as a high-resolution technology that supports the characterization of individual cell types within mixed populations. Cellular patterns can be identified by assessing the protein expression using fluorescent probes and antibodies coupled with fluorochromes B. thuringiensis forms a parasporal crystal during the stationary phase of its growth cycle; nevertheless, the co-culture can change the kinetics of both microorganisms, imposing the need to mark each population to monitor their outcome during the interaction.Interaction studies of two or more microorganisms are challenging because the populations are always changing over time. In general, fungal and bacterial cells can grow and divide, leading to an increase in their biomass. The fungal cells were labeled with the fluorochrome CFW that binds to chitin, a cell-wall polysaccharide that maintains the fungal cell integrity and confers structural rigidity during growth and morphogenesis Btk cells were treated with rabbit polyclonal antibody to the Bt Cry 1Ab toxin, and a goat polyclonal secondary antibody to rabbit IgG coupled to Alexa Fluor 488, to quantify the Cry 1Ab toxin and determine the bacterial sporulation during the analysis period. Due to the parasporal characteristic of the crystals, which are produced during the stationary phase of Bt growth Btk and Btk+Fv groups. For both groups, background fluorescence was absent when control cells were treated only with the secondary antibody. This technique enabled the quantification of the Cry 1Ab toxin in terms of percentage. To our knowledge, this is the first report of flow cytometry use for characterizing Cry 1Ab production.The in vitro antagonistic effect of the Btk LFB-FIOCRUZ (CCGB) 257 strain on Fv MRC 826 was shown. Previous studies have reported Bacillus spp. as potential biological controls of plant pathogenic fungi such as F. oxysporum, F. sambucinum, and F. graminearumBacillus spp. reduced Fv colonization and the accumulation of fumonisin in corn A strong Fv culture. Sectors of intumescent hyphae were observed in response to the bacterial effects, with a paucity of fungal growth and numerous Bt cells seen in the SEM and indirect immunofluorescence assays. In fact, swollen hyphae can be associated with a decrease in fungal growth and higher levels of non-viable cells due to hyphal transformations and death resulting from Fusarium growth kinetics Bacillus spp. are able to overcome such barriers and cause severe fungal cell injuries Fv MRC 826 and Btk LFB-FIOCRUZ (CCGB) 257 together for 7 days demonstrated that Bt strains can be very aggressive towards fungi and are able to circumvent this fungal defense strategy.The microscopic examination by SEM and TEM revealed no marked changes on the third day of incubation; however, the antagonistic effect was evident on the fifth and seventh days, showing fungal cell wall and cytoplasm damage in comparison to the control Fusarium can reside in the internal tissues of living plants without causing any immediate or negative effect, but may turn pathogenic during host senescence. The interactions between host plants and endophytes in natural populations are poorly understood; however, they can actively compete against other microorganisms for niche or infection sites Fv is an endophyte and one of the most commonly reported soil-borne fungal pathogens infecting maize worldwide in vitro results corroborate this theory, as Fv MRC 826 growth was restricted by Btk LFB-FIOCRUZ (CCGB) 257 for over 20 days. A reduction in the cell numbers in the treated cultures was also observed on the third (10.93%), fifth (4.19%), and seventh (3.48%) days through the flow cytometry analysis.Fungal endophytes such as Btk on Fv can also be correlated with secreted inhibitory molecules. A number of antifungal compounds, including polypeptides that interact with the fungal membrane, are produced by Bacillus species Bt can secrete chitinases and chitin-binding proteins; together these proteins control fungi by binding to cell wall chitin and disrupting the cell polarity, leading to fungal growth inhibition, and thus conferring an advantage as a competitor in its niche. Degraded chitin can also serve as a nutrient and further contribute to Bt growth and proliferation The suppressive effect of Btk was interacting with Fv. The accumulation of Cry 1ab was enhanced during the interaction, particularly after 7 days of incubation. This response probably occurred as a consequence of the stressful conditions during the Fv and Btk co-cultivation, which may promote more aggressiveness in this Btk isolate. Given that Bt crystals are produced concurrently with the spores, there was also an increase in spore production during the experimental period. Despite the controversial hypotheses about Bt spore longevity, some reports have demonstrated the environmental persistence of Bt spores, which confer resistance and a competitive advantage against other microorganisms Our results showed increased levels of the Cry 1Ab toxin when Fv and Btk interactions, less accumulation of fumonisin was observed. Reduced FB1 levels have been reported previously, when Fv was treated with Bacillus spp. in maize During the Btk LFB-FIOCRUZ (CCGB) 257 exhibited strong biocontrol potential for suppressing Fv growth and inhibiting fumonisin production in vitro. Moreover, flow cytometry was demonstrated to be sensitive enough to characterize fungal cell oscillations and death during its interaction with Btk. Future studies regarding the antagonistic effect of this bacterium against other fungi and in vivo tests are necessary to determine the efficacy of Btk in controlling plant pathogens.In conclusion, our findings are important for agricultural applications since


Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by both a chronic inflammation and tissue remodelling; as indicated by extracellular matrix protein deposition, basement membrane thickening, goblet cell hyperplasia and subepithelial edema, with reduced vessels and glands. Although remodelling is generally considered to be consequence of persistent inflammation, the chronological order and relationship between inflammation and remodelling in polyp development is still not clear. The aim of our study was therefore to investigate the pathological features prevalent in the development of nasal polyps and to elucidate the chronological order and relationship between inflammation and remodelling, by comparing specific markers of inflammation and remodelling in early stage nasal polyps confined to the middle turbinate (refer to as middle turbinate CRSwNP) obtained from 5 CRSwNP patients with bilateral polyposis, mature ethmoidal polyps from 6 CRSwNP patients, and normal nasal mucosal tissue from 6 control subjects. Middle turbinate CRSwNP demonstrated significantly more severe epithelial loss compared to mature ethmoidal polyps and normal nasal mucosa. The epithelial cell junction molecules E-cadherin, ZO-1 and occludin were also expressed in significantly lower amounts in mature ethmoidal polyps compared to healthy mucosa. Middle turbinate CRSwNP were further characterized by significantly increased numbers of subepithelial eosinophils and M2 type macrophages, with a distinct lack of collagen and deposition of fibronectin in polyp part. In contrast, the turbinate area of the middle turbinate CRSwNP was characterized by an increase in TGF-β activated myofibroblasts expressing α-SMA and vimentin, an increase in the number of pSmad2 positive cells, as well as increased deposition of collagen. These findings suggest a complex network of processes in the formation of CRSwNP; including gross epithelial damage and repair reactions, eosinophil and macrophage cell infiltration, and tissue remodelling. Furthermore, remodelling appears to occur in parallel, rather than subsequent to inflammation.  Nasal polyposis is a common inflammatory disease of the nasal and paranasal mucosa, affecting 1-4% population ,3. FurthRemodelling has long been considered to be a secondary phenomenon and developing late in the disease process as a consequence of persistent inflammation. However, findings from our studies suggest that there may not be a direct link between inflammation and remodelling in upper airway disease. A comparison of the inflammatory patterns in the upper airways of Caucasian and Chinese CRSwNP patients demonstrated that there were clear differences in the inflammatory patterns in these ethnicity groups, although the remodelling patterns appeared to be similar . In partA total of 17 subjects  were recruited into the study at the department of Oto-Rhino-Laryngology of the University Hospital of Ghent, Belgium. The diagnosis of chronic rhinosinusitis with polyps (CRSwNP) was based on the history, clinical examination, nasal endoscopy and computed tomography according to the current EP3OS guidelines . Atopy wAs controls, samples were obtained from the inferior turbinate during septal surgery. For the middle turbinate CRSwNP specimens, samples were taken from patients with bilateral CRSwNP and small nasal polyp formation on the medial aspect of the middle turbinate. These polyps were considered to occur at a rather late stage in CRSwNP, and thus of younger age (early stage) than the mature ethmoidal specimens from the selected CRSwNP patients. Special attention was paid to collecting the whole polyp with the stalk and parts of the surrounding mucosa . The study was approved by the ethical committee of the Ghent University Hospital and performed at Ghent University Hospital, Ghent, Belgium. All patients gave written informed consent before participation.Tissue was fixed in formalin  and embedded in paraffin, prior to being cut at a thickness of 4mm and being air-dried for 24 hours at 37°C on microscope slides. The dried tissue sections were deparaffinised by heating in citrate buffer (pH= 6), and endogenous peroxidase activity was blocked by incubation with hydrogen peroxidase (Dako Envision + kit) for 5 minutes at room temperature. The sections were then washed with TBS for 10 minutes and incubated overnight at 4°C in the presence of one of the primary mouse or rabbit antihuman monoclonal antibodies for detection of a marker of interest as shown in Collagen was assessed by picrosirius red staining as previously described  . After Analysis of epithelial loss was performed in Haematoxylin-Eosin (HE) stained samples, viewed at x100 magnification microscope. Images of the entire epithelium of a specimen were recorded on a digital camera  without overlapping zones and analyzed using ImageJ software .The length of missing epithelium and the total length of the basement membrane were measured, and epithelial loss was expressed as the percentage of the missing epithelium length over total basement membrane length. The epithelium was considered incomplete when the basement membrane was completely denuded or when it was covered by only a single layer of basal cells with no intact ciliated cells or goblet cells .10 random images(x400 magnification) from each sample were recorded on a digital camera  without overlapping zones, and then assessed by two independent observers blinded to the tissue staining protocol . As most all of the epithelium was missing in the polyp area of the middle turbinate samples, it was not possible to include these samples in the assessment, and a semi-quantitative analysis was therefore performed on the intact areas of epithelium in other samples, using an arbitrary 5-point visual staining scale as follows: 0 absent; 1 weak; 2 moderate; 3 intense; and 4 very intense staining .Eosinophils were counted in HE stained sections. For α-SMA and vimentin positive cells, only scattered positive cells detected in the lamina propria were counted, while immune-labelled cells arranged in groups around vessels or glands were not taken into account . 10 random images(x400 magnification) from each sample were recorded on a digital camera  without overlapping zones, and then assessed by two independent observers blinded to the tissue staining protocol. The results were expressed as the number of cells per field at x400 final magnification.10 random images(x400 magnification) from each sample were recorded on a digital camera  without overlapping zones, and then assessed by two independent observers blinded to the tissue staining protocol, using ImageJ software as we have described previously [ 10 random images(x400 magnification) from each sample were recorded on a digital camera  without overlapping zones, and then assessed by two independent observers blinded to the tissue staining protocol. Fibronectin staining in ECM was assessed semi-quantitatively on a 4-point staining intensity score as follows: 0, negative; 1, mildly positive; 2, intermediately positive; 3, strongly positive .The sections were analysed with a microscope equipped with filters to provide circularly polarized illumination. The lower filter was placed above the microscope’s field iris diaphragm ring, while the upper filter was placed below the linear polarizer aligned such that its transmission axis was at 45. Collagen content was quantified under polarized light microscopy. 10 random images from each sample were recorded under bright-field at a final magnification of 400x without overlapping then analysed using ImageJ software. After subtraction of the background, original images were converted to an 8-bit grayscale. The total collagen amount was calculated for each section expressed as percentage of the total area [2 tissue culture flask  for 4 days. At the end of this incubation period, the non-adherent cells were removed by change of the culture medium and the attached cells cultured further for 2 weeks, by which time the cells had established as a confluent monolayer of fibroblast-like cells. The cells were harvested and cultured further for 3-5 passages, before being used in further experiments. During culture and in between the passages, the medium was changed every 3 days.Nasal tissues harvested from control subjects and CRSwNP patients with mature ethmoidal polyps were rinsed and suspended in sterile RPMI 1640 tissue culture medium ; supplemented with 2mM L-glutamine , 50IU/ml penicillin and 50mg/ml streptomycin  and 0.1% bovine serum albumin . The tissues were cut into small pieces and then incubated (at 37°C in a 5% CO2 in air atmosphere) in PBS containing 0.25% trypsinase for 45 min. Following this enzyme treatment, the tissues were gently passed through a cell mesh filter and washed twice by centrifugation at 10g and suspension in PBS. The final washed cells were suspended in RPMI 1640 supplemented with 2mM L-glutamine, 50IU/ml penicillin and 50mg/ml streptomycin, 10% foetal calf serum, and plated in a 25mmAt the end of culture, the cells were harvested, seeded into 6-well plates at a concentration of 1×105Cells /well, and stimulated by incubation in the presence of 5ng/ml TGF-β1 , for 48h at 37°C in a 5% CO2 in air atmosphere. For each sample in each experiment, a negative control was also employed using cells incubated with only culture medium. At the end of culture the cell cultures were centrifuged at 4°C and the supernatants collected and stored at -20°C until use. One proportion of the stimulated cells was tested for the expression of α-SMA by flow cytometry and another proportion was collected for total RNA extraction and stored at -80°C.Concentrations of fibronectin in cell culture supernatants were measured by Elisa  according to the manufacturer’s instructions.6/ml and 100μL of supernatant was collected. Cells were fixed and permeabilized using permeabilizing solution 2 and then stained with 10μL of anti-human α-SMA -FITC (Abcam) for 20 min at room temperature. Corresponding isotypes were used as control. The cells were washed twice and analysed by flow cytometry .The expression of α-SMA was detected by flow cytometry. The cells were adjusted to a concentration of 10GACCAATGCCAGGATTCAGAGAC, homo-fibronectin1-R GACAGGACCACTTGAGCTTGGATAG). The PCR protocol consisted of 1 cycle at 95°C for 2 min followed by 40 cycles each at 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. β-actin, hydroxymethyl-bilane synthase, and elongation factor 1 were used as internal control genes for normalization. The relative number of molecules for each gene was expressed in relative expression units quantified per 20 ng of cDNA sample and determined by the △△ct value method.Total RNA was isolated using RNAisoplus , and cDNA was synthesized from 5μg of RNA using the SuperScript® III First Strand Synthesis Kit (Invitrogen) in accordance with the manufacturer’s instructions. Amplification reactions were performed with Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) using specific primer sets . Data are expressed as box-and-whisker plots presenting medians, ranges, and interquartile ranges. Baseline variables were analyzed using a one-way ANOVA test and the Fisher’s exact text. The Kruskal-Wallis test was used to assess the significance of intergroup variability using paired comparisons and the Mann-Whitney U 2-tailed test was used for between-group comparisons. P-values <0.05 were considered statistically significant. The demographic and clinical characteristics of the subjects in the three study groups are shown in p=0.037 and p=0.006, respectively). Furthermore, with the epithelial loss was significantly greater in the middle turbinate polyp compared to the mature ethmoidal polyp  and middle turbinate CRSwNP groups  demonstrated a significantly higher epithelial loss compared to the control group , gap junctions, adherence junctions (AJs) and desmosomes ,17. TJs Earlier studies have indicated that events leading to polyp formation may include damage of the mucosal epithelium by mechanical forces or inflammatory mediators ,21. The  A markedly increased number of alternatively activated MMR-positive (M2) macrophages in the polyp area of the middle turbinate CRSwNP compared to control mucosa and mature ethmoidal CRSwNP also support the Th2 signature. Moreover, elevated expressions of CD68 ,28 and M4β1 integrin expressed on human eosinophils [Here we also report an increased expression of fibronectin in mature ethmoidal polyps in CRSwNP compared to normal mucosa, and an even higher expression of fibronectin in the polyp part of middle turbinate CRSwNP. This is in accordance with the findings of other studies, which have also shown high expression of fibronectin in mature polyps in CRSwNP patients and suggested that it may contribute to edema formation and growth of nasal polyps by interaction with eosinophils ,40. Fibrinophils , prolonginophils , and stiinophils ; all actinophils .Although, we did not find any marked increase in inflammation, e.g. accumulation of eosinophils and macrophages, in the turbinate part similar to that noted in the polyp part of the middle turbinate CRSwNP, we did find signs of a pronounced remodelling, as indicated by accumulation of α-SMA positive and vimentin positive myofibroblasts, as well as an increase in the number of TGF-β triggered pSmad2 positive fibroblasts. Moreover, large amount of fibres arrayed in parallel and formed thick fibre bundles in the turbinate to form a defensive wall against the polyp part.It has been suggested that proliferation of preexisting stromal fibroblasts is the most important origin of myofibroblasts.When eng Chronic airway remodelling has been suggested to be the result of an aberrant repair cycle which occurs in response to chronic airway inflammation. However, evidence from studies over the last decade suggests that inflammation and remodelling occur in concert, rather than remodelling being a consequence of long-standing airway inflammation . Airway This study is somewhat limited due to the comparatively small numbers of samples with polyp formation on the medial aspect of the middle turbinate in patients with bilateral CRSwNPs, as this condition is not frequent to find. These polyps are developing late in the disease process, but were formed only recently before the surgery; they are of younger age (early stage) than the mature ethmoidal specimens from CRSwNP patients. Furthermore, biopsies of the middle turbinate CRSwNP were limited in size, which restricted other possible investigations. However, despite these limitations, we were able to demonstrate significant differences in the inflammatory cells and ECM deposition between early and late stage nasal polyp disease, suggesting that this merits confirmation in further investigations involving larger patient cohorts recruited from several study centres across the country. In summary, our study has indicated a number of pathophysiologic differences between the early stage polyps and mature polyps that have not been described before in CRSwNP patients. The epithelial loss was more prominent in the early stage polyps in the middle turbinate in CRSwNP patients, coupled with increased numbers of especially M2 type macrophages and markedly high expression of Fibronectin. Taken together, these findings suggest that aggravated epithelial damage and Fibronectin expression play a crucial role in the adhesion and penetration of the basement membrane by bacteria in the initial stages of polyp formation. Similarly, the increased numbers of macrophages in the polyp area of the middle turbinate CRSwNP suggests a defective host defense mechanism in the early stage of the disease. In contrast, a fibrotic response build up a defence mechanism involving increased deposition of dense collagen fibre bundles in the underlying mucosa to prevent spread and generalization of edema and inflammation. Overall, these findings suggest a complex network of processes in the formation of the CRSwNP; including gross epithelial damage and repair reactions, eosinophil and macrophage cell infiltration, and tissue remodelling. Furthermore, remodelling appears to occur in parallel, rather than subsequent to inflammation, as has been shown in CRS patients without nasal polyps .


To date, the role of male factor contributing in evaluation of spontaneous recurrent pregnancy loss (RPL) has been less investigated and there is discrepancy in the role of Y chromosome microdeltions in RPL. Therefore, the current study was designed to examine whether Y chromosome microdeletions were associated with RPL in an Iranian population.One hundred men from couples, experiencing three or more RPLs, and one hundred normal men from couples with at least one child and no history of miscarriages as control group were included. Genomic DNA was extracted from peripheral blood and tested for Y chromosome microdeletions in AZFa, AZFb and AZFc regions using two multiplex PCR.None of the men in the case and control groups had any microdeletions in the AZFa, AZFb and AZFc regions.It seems that Y chromosome microdeletion is not associated with recurrent pregnancy loss, therefore performing this test in Iranian couples with RPL is not recommended. Recurrent pregnancy loss (RPL) is usually defined as three or more consecutive pregnancy losses, which affects 0.5-3% of all couples.2] RPL i RPL i2] To date, the clinical investigation of couples with RPL has mainly focused on the female partner. The maleApproximately 1-4% of couples with recurrent miscarriage have cytogenetic abnormalities. MutationAccording to the recent studies, there is a potential association between Y chromosome microdeletions and RPL.12] The  The 12] This is a case-control genetics association study. One hundred men from couples with history of three or more consecutive miscarriages, who referred to Avicenna Infertility Clinic, Tehran, Iran, during 2009-2010, were included. One hundred healthy men from couples with at least one live birth and no miscarriages were considered as control group. Our study was approved by Avicenna Research Institute's Ethics and Human Rights Committee and an informed consent was obtained from each man in the case and control group. In the case group, all men and their spouses were analyzed for the karyotype and those with cytogenetic abnormalities were excluded. All clinical information of couples including history of all previous miscarriage, age and clinical work-up to determine cause of miscarriage was obtained from Avicenna Infertility Clinic. All women with RPL examined with hysterosalpingography and ultrasonography for detection of anatomical abnormalities of the genital tract. Hematological disorders such as, anti-thrombin III activity, proteins C and S activity, activated protein C resistance, factor V Leiden mutation, protrombin mutation, immunological risk factors such as natural killer cells activity (NK Cells), lupus anticoagulant, anticardiolipin antibodies, antinuclear antibodies (ANAs) and for endocrinological disorders, hormone analysis including, follicle-stimulating hormone (FSH), luteinizing hormone (LH), free testosterone, prolactin, thyroid stimulating hormone (TSH) were checked in all women with at least 3 miscarriages.Genomic DNA was extracted from 3 ml peripheral blood samples using salting-out method. The scre2, 1U Taq DNA polymerase , 10 nmol each NTPs, and 10 pmol of each primers. Amplification conditions were started with an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 62°C for 45 seconds, and extension at 72°C for 90 seconds, ended by a last extension at 72°C for 10 minutes and cooling to 4°C. The PCR products were separated on 3% Agarose gel, stained with ethidium bromide and visualized under UV light. Genomic DNA from fertile men and water used as positive and negative control, respectively  was used for statistical analysis and p values <0.05 were considered statistically significant.The clinical characteristics of cases and controls were shown in Recurrent pregnancy loss, is estimated to affect 0.5-3.0% of all couples.2] The c The c2] Three AZF regions on the long arm of Y chromosome are essential for normal spermatogenesis. AZF deleRecent studies showed that Y chromosome microdeletion in AZF region may be a possible etiologic factor of RPL.12] Acco Acco12] They used 4 sequence tagged sites: sY67, sY129, sY150 and sY152. The STS The aim of the present study was to determine the association between Y chromosome microdeletions and RPL in Iranian population. All 100 patients and 100 control men were checked for AZF microdeletion using 6 STS recommended by EAA/EMQN guideline (2004) and also the two markers sY150 and sY152 which used by Dewan et al., Y chromoDespite of the previous study, recently published article by Kaare et al. (2008) with 46 Finish couples and analyzing 32 STS, including EAA/EMQN recommendations and STS used by Dewan et al. (2006) showed absence of Y chromosome microdeletion in male partners. In anothThere are important differences between the prevalence of Y chromosome microdeletion and RPL in previous studies. For understanding causes of discrepancies, we must consider that for AZF microdeletions screening, there is validated guideline endorsed by the EAA/EMQN which could detect up to 95% of all reported AZF microdeletions. However,Finally, it seems that Y chromosome microdeletions are not associate with RPL and more research is needed, therefore performing this test in Iranian couples with RPL is not recommended.


Crocodylus niloticus persist in Chad, Egypt and Mauritania. Although crocodiles were widespread throughout the Sahara until the early 20th century, increased aridity combined with human persecution led to local extinction. Knowledge on distribution, occupied habitats, population size and prey availability is scarce in most populations. This study evaluates the status of Saharan crocodiles and provides new data for Mauritania to assist conservation planning.Relict populations of gueltas and seasonal tâmoûrts were the most common occupied habitats. Crocodile encounters ranged from one to more than 20 individuals, but in most localities less than five crocodiles were observed. Larger numbers were observed after the rainy season and during night sampling. Crocodiles were found dead in between water points along dry river-beds suggesting the occurrence of dispersal.A series of surveys in Mauritania detected crocodile presence in 78 localities dispersed across 10 river basins and most tended to be isolated within river basins. Permanent Gueltas are crucial for the persistence of mountain populations. Oscillations in water availability throughout the year and the small dimensions of gueltas affect biological traits, including activity and body size. Studies are needed to understand adaptation traits of desert populations. Molecular analyses are needed to quantify genetic variability, population sub-structuring and effective population size, and detect the occurrence of gene flow. Monitoring is needed to detect demographical and genetical trends in completely isolated populations. Crocodiles are apparently vulnerable during dispersal events. Awareness campaigns focusing on the vulnerability and relict value of crocodiles should be implemented. Classification of Mauritanian mountains as protected areas should be prioritised.Research priorities in Chad and Egypt should focus on quantifying population size and pressures exerted on habitats. The present study increased in by 35% the number of known crocodile localities in Mauritania.  The Sahara is the largest desert in the world and it is characterised by the occurrence of vast dune fields and featureless plains subjected to low precipitation levels and high temperature ranges Palaeogeological events and climatic shifts constitute driving factors of current species distribution and diversity patterns in the Sahara. The progressive coolness that followed the arid LGM allowed “humid” species of Mediterranean and sub-Saharan affinity to spread over the shrunken Sahara and the mild climatic conditions of the mid-Holocene made available suitable aquatic environments for many nowadays-extinct fauna, such elephants, giraffes or hippopotamus Olea laperrini) and the false smooth snake (Macroprotodon cucullatus), and of sub-Saharan affinity, like the savannah toad (Amietophrynus xeros) and the Guinea baboon (Papio papio) Remarkably, relict populations have persisted in mountains where suitable climatic conditions endured. Saharan mountains constitute refugia for species of Mediterranean affinity, such as the olive tree (Crocodylus niloticus) is one of the species occurring in the Sahara that experienced historical range contractions and is currently vulnerable to population isolation. Crocodiles were widespread throughout the Sahara at least from the mid-Holocene up to Roman times , the Hoggar and Tassili ‘n’Ajjer mountains (Algeria) and the lower Drâa river (Morocco) , when torrential waterfalls fill up the pools. The size of gueltas varies according to the geomorphology of mountain slopes but generally are small, ranging between 0.001 ha and 1.0 ha; and 2) floodplains, locally known as tâmoûrt, located on the foothills of the mountains, which are larger in size and reach frequently more than 1,000 ha. Nevertheless, water is usually shallow and tâmoûrts are mostly arid during the dry season (October to June), thus crocodiles are forced to find shelter in nearby rock outcrops during this period oued), lakes and dams In Mauritania, crocodiles were reported in the Tagant Varanus niloticus) has been suggested to be a predator of crocodile eggs and a prey for adult crocodiles, indicating a possible predator-prey relationship Crocodile populations in Mauritania are fragmented and many are constituted by a small number of individuals This study aims to evaluate the status of crocodiles in the Sahara and for Mauritania, in particular, it is aimed to update the distribution, characterise occupied habitats, provide local counts of crocodiles, and assess possible dispersal events. The results of this study are intended to increase the knowledge about distribution and occupied habitats by crocodiles in the Sahara, and to assist conservation planning in Mauritania, particularly to provide additional data for the recent listing of the “Gabbou basin” in the Ramsar Convention Ethics statement: Fieldwork in Mauritania developed with permission from the Ministère Délégué auprès du Premier Ministre, Chargé de l’Environment. Parc National du Banc d’Arguin, Nouakchott (Permit: 460/MDE/PNBA). There are no animal husbandry, experimentation and care/welfare concerns.The study area encompasses the mountains of Tagant, Assaba and Affolé, in southern Mauritania . AltitudAcacia ehrenbergiana, Acacia tortilis, Balanites aegyptiaca and associated species Most of the study area is covered by open and sparse grasslands 49.3%;  with vegTwo field missions were developed in 2008 (10 October to 18 November) and 2009 (31 March to 5 May), following previous short-timed visits in 2003 (21 to 25 November) and 2007 (9 to 16 December), totalling 109 days of fieldwork. A total of 102 localities (water points) were visited, of which 19 were visited in two or more occasions . Each viCrocodiles found dead were measured, photographed and the probable cause of death was estimated. In some cases locals informed us that the crocodile had been deliberately killed and clear beating marks were usually found in the head. Inquires were also used to complement information about annual water availability, permanent or seasonal, and the month when locality dries. To identify dispersal events, vehicle-based surveys were conducted whenever possible along the river-beds between localities with known presence of crocodiles. Riverbeds were in many occasions the main overland route between localities, which facilitated sampling and inquiries to locals. Occurrence of dispersal was considered when dead crocodiles were found along the dry river-beds in between water localities. The coordinates of localities with crocodiles and crocodiles found dead outside water points were gathered from a Global Positioning System (GPS).Localities with presence of crocodiles were collected from bibliographic references, including for the Saharan Holocene Status of populations in Mauritania was ranked in four categories: 1) present, when crocodiles were observed during field missions, when faeces or footprints were found, or when recent bibliographic references (after year 2000) reported presence but field missions did not sampled these localities; 2) possible, when locals reported presence of crocodiles in apparently suitable habitats but individuals or their signs were not observed; 3) not confirmed, when localities referenced recently in bibliography (after year 2000) with presence were sampled during field missions but individuals or their signs were not observed; 4) extinct, when inquiries to locals and bibliographic references reported the extinction of crocodiles and field missions also did not found evidences for their presence.Populations of the Nile crocodile in the Sahara are currently known from three countries, Chad, Egypt and Mauritania . An apprPopulations of crocodiles in Chad are best known from Guelta Archei in the eastern Ennedi mountains, where several individuals have been reported since the 1930s  [18], [2th century they became largely restricted to the Nile south of Aswan and probably went extinct during the 1960s , but presence was detected also in oueds (9.9%), sources 8.5%), lakes (8.5%) and dams (5.6%)  and 5. C%, lakes The total number of crocodiles observed was 178, including yearlings to adults . The numFour localities along the Krâa Naga oued  were sampled in the period 2000–2002 A larger number of crocodiles were observed after the rainy season in comparison with the dry season. In localities with crocodiles sampled in late 2008 (N = 20), an average of 5.9 individuals were observed, whereas in localities sampled in early 2009 (N = 13), an average of 3.5 crocodiles were observed. Also, in localities sampled during daylight and at night (N = 20), more crocodiles were observed on average at night (6.1±5.3) than during daylight (2.7±4.6).A total of 10 crocodiles were found dead, of which five had been killed by locals, two died from apparently natural reasons and three by unknown reasons (only bone remains were found after digging). Seven of these crocodiles were found in between water points along the dry river-bed and were considered to be dispersing . All disThe 78 localities are dispersed across 10 river basins and most tended to be isolated within river basins . SummaryNile crocodiles occur in the Sahara desert in fragmented populations throughout several mountains. Although the mechanisms explaining the presence of crocodiles in the Sahara are well understood e.g., in realCrocodylus cataphractus), which was reported along the Senegal river The present study increased in by 35% the number of known crocodile localities in Mauritania. Presence was confirmed in 60 localities and another 11 were identified as of possible presence. The increase in known localities is probably related to previous lack of sampling and cryptic behaviour of crocodiles. The remote character of southern Mauritanian mountains, associated to with logistical fieldwork constraints, has prevented detailed sampling. Also, crocodiles were found spending large portions of time hidden inside caves or burrows Crocodiles were mostly found in gueltas and tâmoûrts, which is probably related to their higher abundance in comparison to other water habitats. Gueltas are apparently crucial for the persistence of crocodiles in mountains, as already emphasised for other vertebrates with isolated populations Hoplobatrachus occipitalis), while in December, no amphibians were detected and the tâmoûrt was dry, suggesting that crocodiles should feed only during the active period of amphibians. The relatively short feeding period and available prey of small size, mostly fishes and amphibians, probably affects growth rates and reduces maximum body size Oscillations in water availability throughout the year and the relatively small dimensions of gueltas have dramatic consequences in the activity and ecology of populations. During the dry season, individuals are forced to aestivate Dead crocodiles were found between water points along dry river-beds, suggesting the occurrence of dispersal . MovemenThe present study identified localities completely isolated without any possibility of rescuing-effects The isolation and vulnerability of mountain populations apparently induces behavioural shifts in aggressiveness patterns of crocodiles. Individuals are extremely shy and plunge into water at the first sign of human disturbance. Interestingly, this behaviour was also reported for the extinct Algerian populations Climate change scenarios for the region predict significant warming and rainfall decrease Text S1Detailed data on localities, crocodile observations, population status, and conservation issues affecting habitats in Mauritania.(0.07 MB DOC)Click here for additional data file.Table S1Distribution, status and date of last observation of Nile crocodile populations in the Sahara excluding extant localities for Mauritania.(0.10 MB DOC)Click here for additional data file.Table S2Distribution, status and date of last observation of Nile crocodile populations in Mauritania.(0.16 MB DOC)Click here for additional data file.Figure S1Distribution of sampled localities in the three main mountains of southern Mauritania. Dots represent sampled localities (water points). Only the river basins (names) with crocodile populations are presented.(1.77 MB TIF)Click here for additional data file.Figure S2Presence signs of crocodiles in Mauritania. Tracks and footprints (A) at tâmoûrt Kour, faeces (B) at guelta Garaouel and burrows (C) at tâmoûrt Bougâri were taken as presence signs of crocodiles.(6.55 MB TIF)Click here for additional data file.Figure S3Photos of extremely small localities with presence of Nile crocodiles in Mauritania. A. Five to six crocodiles occur at source Aouînet Nanâga; B. two juveniles were observed at source Oumm Icheglâne; C. one adult and two sub-adults were observed inside a spring-fed water trough at Chegg el Mâleh.(6.54 MB TIF)Click here for additional data file.