The resting metabolic rate (RMR) decrease, observed after an obesity reduction therapy is a determinant of a short-time weight regain. Thus, the objective of this study was to evaluate changes in RMR, and the associated hormonal alterations in obese patients with a very low-calorie ketogenic (VLCK)-diet induced severe body weight (BW) loss.From 20 obese patients who lost 20.2 kg of BW after a 4-months VLCK-diet, blood samples and body composition analysis, determined by DXA and MF-Bioimpedance, and RMR by indirect calorimetry, were obtained on four subsequent visits: visit C-1, basal, initial fat mass (FM) and free fat mass (FFM); visit C-2, − 7.2 kg in FM, − 4.3 kg in FFM, maximal ketosis; visit C-3, − 14.4 kg FM, − 4.5 kg FFM, low ketosis; visit C-4, − 16.5 kg FM, − 3.8 kg FFM, no ketosis. Each subject acted as his own control.2 = 0.36; p < 0.001).Despite the large BW reduction, measured RMR varied from basal visit C-1 to visit C-2, − 1.0%; visit C-3, − 2.4% and visit C-4, − 8.0%, without statistical significance. No metabolic adaptation was observed. The absent reduction in RMR was not due to increased sympathetic tone, as thyroid hormones, catecholamines, and leptin were reduced at any visit from baseline. Under regression analysis FFM, adjusted by levels of ketonic bodies, was the only predictor of the RMR changes  contains supplementary material, which is available to authorized users. It is widely accepted that during periods of energy deficit or restriction , the human body tends to diminish energy expenditure by increasing the efficiency in its use and by decreasing the resting metabolic rate (RMR) . This phPublished research has shown that a very low calorie ketogenic (VLCK)-diet was able to induce a significant weight loss and maintained their efficacy along 2 years , 11. BecThe main target of this study was to observe the changes in RMR induced by a VLCK-diet in obese subjects, as well as the hormonal and metabolic alterations associated with that change.This is a follow up study on a published clinical trial . It was The patients attending the Obesity Unit at the Complejo Hospitalario Universitario of Santiago de Compostela, Spain to receive treatment for obesity were consecutively invited to participate in this study.2, stable body weight in the previous 3 months, desire to lose weight, and a history of failed dietary efforts. The main exclusion criteria were, diabetes mellitus, obesity induced by other endocrine disorders or by drugs, and participation in any active weight loss program in the previous 3 months. In addition, those patients with previous bariatric surgery, known or suspected abuse of narcotics or alcohol, severe depression or any other psychiatric disease, severe hepatic insufficiency, any type of renal insufficiency or gouts episodes, nephrolithiasis, neoplasia, previous events of cardiovascular or cerebrovascular disease, uncontrolled hypertension, orthostatic hypotension, and hydroelectrolytic or electrocardiographic alterations, were excluded. Females who were pregnant, breast-feeding, or intending to become pregnant, and those with child-bearing potential and not using adequate contraceptive methods, were also excluded. Apart from obesity and metabolic syndrome, participants were generally healthy individuals.The inclusion criteria were, age 18 to 65 years, body mass index (BMI) ≥30 kg/mThe study protocol was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee for Clinical Research of Galicia, Santiago de Compostela, Spain (registry 2010/119). Participants gave informed consent before any intervention related to the study. Participants received no monetary incentive.All the patients followed a VLCK diet according to a commercial weight loss program (PNK method®), which includes lifestyle and behavioral modification support. The intervention included an evaluation by the specialist physician conducting the study, an assessment by an expert dietician, and exercise recommendations. This method is based on a high-biological-value protein preparations obtained from cow milk, soya, avian eggs, green peas and cereals. Each protein preparation contained 15 g protein, 4 g carbohydrates, 3 g fat, and 50 mg docohexaenoic acid, and provided 90–100 kcal.The weight loss program has five steps . At this point, the patients underwent a progressive incorporation of different food groups and participated in a program of alimentary re-education to guarantee the long-term maintenance of the weight loss. The maintenance diet, consisted of an eating plan balanced in carbohydrates, protein, and fat. Depending on the individual the calories consumed ranged between 1500 and 2000 kcal/day, and the target was to maintain the weight lost and promote healthy life styles.During this study, the patients followed the different steps of the method until they reach the target weight or up to a maximum of 4 months of follow-up, although patients remained under medical supervision for the following months.Throughout the study, the patients completed a maximum of 10 visits with the research team (every 15 ± 2 days), of which four were for a complete (C) physical, anthropometric and biochemical assessment, and the remaining visits were to control adherence and evaluation of potential side effects. The four complete visits were made according to the evolution of each patient through the steps of ketosis as follows: visit C-1 (baseline), normal level of ketone bodies; visit C-2, maximum ketosis (approximately 1–2 months of treatment); visit C-3, reduction of ketosis because of partial reintroduction of normal nutrition (2–3 months); visit C-4 at 4 months, no ketosis .All anthropometric measurements were undertaken after an overnight fast (8 to 10 h), under resting conditions, in duplicate, and performed by well-trained health workers. Participant’s body weights were measured to the nearest 0.1 kg on the same calibrated electronic device , in underwear and without shoes. BMI was calculated by dividing body weight in kilograms by the square of height in meters (BMI = weight (kg)/heightThe RMR was measured by indirect calorimetry using a portable desktop metabolic system  and under overnight fasting conditions. Participants were instructed to arrive at the hospital by car, to minimize vigorous physical activity during the 24 h prior to the measurement, and to avoid drinking caffeinated beverages for at least 12 h before testing. All participants rested supine for at least 20 min. During this resting time, the body composition  was determined, and then rested in sitting position in a quiet and darkened room for a further 15 min before the test.Test-re-test validation was performed and after resting, oxygen consumption was measured continuously for 15 min under thermo-neutral conditions, and the final 10 min of data were used to calculate RMR. The FitMate uses a turbine flow meter for measuring ventilation and a galvanic fuel cell oxygen sensor for determining the fraction of oxygen in expired gases. Moreover, it has sensors for the measurement of temperature, humidity, and barometric pressure for use in internal calculations. The FitMate uses standard metabolic formulas to estimate oxygen consumption, and RMR is calculated using a predetermined respiratory quotient (RQ) of 0.85. During the measurement period, participants remained sitting, breathed normally, and were instructed to remain awake, and to avoid talking, fidgeting and hyperventilating. The reliability of measuring RMR with Cosmed’s FitMate metabolic system have been determined in several previous studies , 15, 21,For the purposes of this study measured RMR or the crude values provided by the method were obtained and expected-RMR was defined as the variation in energy expenditure that could be explained by the observed changes in fat-free mass (FFM), because FFM is the main determinant of RMR . FirstlyThis process is summarized by the following equation:On the other hand, metabolic adaptation has been described as the change in RMR not explained by changes in FFM , 19, andBody composition was first measured by dual-energy X-ray absorptiometry .Daily quality control scans were acquired during the study period. No hardware or software changes were made during the course of the trial. Subjects were scanned using standard imaging and positioning protocols, while wearing only light clothing. For this study, the values of bone mineral density, lean body mass and FM that were directly measured by the GE Lunar Body Composition Software option. Some derivative values, such as bone mineral content, regional lean mass, FFM, and fat mass percentage (FM%), were also calculated.Multifrequency bioelectrical impedance (MF–BiA) was also used for determining body composition. FM, FM%, FFM, total body water, intra- and extracellular water, and skeletal muscle mass, were calculated with In Body 720 . This technology is non-invasive and uses eight contact electrodes, which are positioned on the palm and thumb of each hand and on the front part of the feet and on the heels.Multifrequency bioelectrical impedance uses the body’s electrical properties and the opposition to the flow of an electric current by different body tissues. The analyzer measures resistance at specific frequencies  and reactance at specific frequencies . The participants were examined lightly dressed, and the examination took less than 2 min and required only a standing position. The validity of this technology has been documented in previous studies .Ketosis was determined by measuring ketone bodies, specifically β-hydroxy-butyrate (β-OHB), in capillary blood by using a portable meter . As with anthropometric assessments, all the determinations of capillary ketonemia were made after an overnight fast of 8 to 10 h. These measurements were performed daily by each patient during the entire VLCK diet, and the corresponding values were reviewed on the machine memory by the research team in order to control adherence. Additionally, β-OHB levels were determined at each visit by the physician in charge of the patient. The measurements reported as “low value” (< 0.2 mmol/l) by the meter were assumed as to be zero for the purposes of statistical analyses.During the study all the patients were strictly monitored with a wide range of biochemical analyses. However, for the purposes of this work only certain values are reported. Serum tests for total proteins, albumin, prealbumin, retinol-binding protein, red cell and white cells counts, uric acid, urea, creatinine and urine urea were performed using an automated chemistry analyzer .Thyroid-stimulating hormone (TSH), free thyroxine (FT4), and free triiodothyronine (FT3) were measured by chemiluminescence using ADVIA Centaur . All the biochemical parameters were measured at the 4 complete visits.The overnight fasting plasma levels of leptin were measured using commercially available ELISA kits . The fasting plasma levels of fractionated catecholamines  were tested by high pressure liquid chromatography .The data are presented as means (standard deviation). Each subject acted as his own control (baseline visit). The sample size of the current trial was calculated taking the weight loss after treatment (main variable) into account. It was calculated for an effect size ≥15 kg, and a α = 0.05, and a power (1-β) of 90%. Thus, the sample size was established at a minimum of 19 volunteers who finished the nutritional treatment. The sample size provided sufficient power to test for effects on a number of other metabolic variables of interest.p < 0.05 was considered statistically significant. Changes in the different variables of interest from the baseline and throughout the study visits were analyzed following a repeated measures design. A repeated measures analysis of variance (ANOVA) test was used to evaluate differences between different measurement times, followed by post hoc analysis with Tukey’s adjustment for multiple comparisons. In addition, multivariate linear regression models were fitted to assess the potential predictive factors of RMR at each complete visit. The regression models included fat-free mass, FT3, catecholamines , leptin and β-OHB as plausible determinants of RMR.All statistical analyses were carried out using Stata statistical software, release 12.0 . A Twenty obese patients, 12 females, age from 18 to 58 years (47.2 ± 10.2 yr) completed the study. Participants at baseline have a BMI of 35.5 ± 4.4 and body weight (BW) of 95.9 ± 16.3 kg, 45.6 ± 5.4% of which was fat. Other baseline characteristics and their corresponding changes during the study are presented in Tables Although the patients underwent a total of 10 visits, the RMR and body composition analyses were synchronized with the ketone levels in four visits Fig. . Visit CMost of the initial BW loss was in the form of fat mass (FM) with a minor reduction in fat free mass (FFA). The reduction in kg for FM and FFM respectively from baseline were; visit C-2 7.2 kg and 4.3 kg; visit C-3 14.4 kg and 4.5 kg; visit C-4 16.5 kg and 3.8 kg. Table .The measured RMR was not significantly different from the baseline at any time during the study, although a downward trend in these values was observed Fig. . CompareTo investigate how much of the mild and non-significant decrease in RMR could be accounted for by FFM change, we used the baseline RMR data to generate an equation for calculating the expected-RMR in accordance with variations in FFM Table . The difThe observation that the VLCK-diet preserved the RMR in accordance to variations in FFM, thus avoiding the metabolic adaptation was reinforced by the maintenance of the RMR/FFM quotient during the study Table . When, mThe concern regarding the possible preservation of the RMR as a consequence of the presence of stressing factors induced by the VLCK-diet and the rapid weight loss was focused by a strict analysis of the protein metabolism. Although there were some differences in protein status, renal function and nitrogen balance-related parameters, none of them was considered as clinically relevant Table . It is nBesides the FFM, that is considered the major contributing factor, several variables have been described as positive determinants of the RMR, including thyroid hormones, catecholamines, leptin and ketone bodies. In this study, the level of influence of these mentioned factors on the measured RMR was determined during the study. As Fig. p < 0.05, Table Linear regression models reveals that when adjusted by β-OHB, FFM was the best predictor of RMR  the rapid and sustained weight reduction induced by the VLCK-diet did not induce the expected drop in RMR, 2) this observation was not due to a sympathetic tone counteraction through the increase of either catecholamines, leptin or thyroid hormones, 3) the most plausible cause of the null reduction of RMR is the preservation of lean mass (muscle mass) observed with this type of diet.The greatest challenge in obesity treatment is to avoid weight recovery sometime after the previous reduction. In fact, after one or few years the most obese patients recover or even increase their weight, previously reduced by either, dietetic, pharmacological or behavioral treatments , bariatrWe have observed that the obesity-reduction by a VLCK-diet (Pnk method ®) was maintained 1 and 2 years after its completion , 11. AltAs the mechanisms supporting the metabolic adaptation phenomenon are not known, unraveling the reasons behind the present findings is challenging enough in itself. Changes in any circulating hormone that participate in thermogenesis could be the explanation for the absence of a reduction in RMR, for example a concomitant increase in the sympathetic system activity, either directly or indirectly. An increase in thyroid hormones generated by the VLCK-diet was discarded because free T3 experienced the well described reduction after losing weight , 24 withThen, an expected increase in thyroid hormones, catecholamines, or leptin levels was discarded as explanation for the observed minor or absent reduction in RMR. This was also endorsed by the undertook multiple regression analysis Table . In thisThe strength of this study is its longitudinal design, which allows the evaluation of the time-course of changes of RMR during a VLCK diet, by comparing each subject to himself, as his own control. The scarce number of subjects and the short duration of this study might be a limitation, since one cannot make claims regarding the RMR status long-term after the completion of the VLCK diet. However, no significant variations in body weight had been observed after 4 months in previous studies , 11. In In summary, this study shows that the treatment of obese patients with a VLCK-diet favors the maintenance of RMR within the expected range for FFM changes and avoids the metabolic adaptation phenomenon. This finding might explain the long-term positive effects of VLCK-diets on weight loss. Although, the mechanisms by which this effect could be justified are unclear, classical determinants of the energy expenditure, as thyroid hormones, catecholamines as well as leptin were discarded. The relative good preservation of FFM (muscle mass) observed with this dietetic approach could be the cause for the absence of metabolic adaptation.Additional file 1:Figure S1. Nutritional intervention program and schedule of visits. Visit C-4 was performed at the end of the study according to each case, once the patient achieved the target weight or maximum at 4 months of follow-up. (PDF 390 kb)Additional file 2:Figure S2. Bland Altman plots of Resting Metabolic Rate (RMR) for Cosmed’s Fitmate device compared to the Deltatrac. (PDF 11 kb)Additional file 3:Table S1. Independent effects of fat-free mass, free triiodothyronine, catecholamines, leptin and β-hydroxy-butyrate on resting metabolic rate at each visit. (DOCX 32 kb)


To close a refractory full thickness macular hole (FTMH) by adjacent subretinal fluid application to release the elastic retina from the retinal pigment epithelium (RPE).A 83 years old patient presented an old FTMH with a diameter of 1444 μm. After confirming intraoperatively the complete release of the epiretinal membrane around the FTMH, we installed 3 small subretinal blebs around the hole, to release the adjacent retina from the RPE. The mobilized retina was gently moved towards the macular center. A silicone oil tamponade was installed to secure a proper healing and observation of the FTMH. The closure of the 1444 μm FTMH was seen on indirect ophthalmoscopy and confirmed by OCT 5 days after surgery by restoring the retinal architecture. A late reopening was not apparent at the postoperative observations. Visual acuity improved from hand motion to 20/200 at 4 weeks postoperative.Although FTMH develop by epiretinal tangential traction, large FTMH may persist even after complete release of its epiretinal traction. Subretinal fluid application may release the flexible retina from the RPE to achieve a relocation at the central fovea facilitating an anatomical closure of the macular hole. Full thickness macular hole (FTMH) is a defect of all neuroretinal layers at the foveal center. Treatment of FTMH by pars plana vitrectomy (PPV) and consecutive gas tamponade was first described by Kelly and Wendel and has been refined since then, to improve the anatomical functional outcome . The curAttempts to treat refractory macular holes may include installation of silicone oil, application of autologous platelet concentrates  and moreHere we present a refractory large macular hole after repeated attempts with ILM peeling and secondary silicone oil installation, which remained open. In this case we used a novel technique by the application of subretinal fluid to shift the released retina towards the center to achieve an anatomical closure of the macular hole.A 83 years old patient presented a large sized FTMH in his left eye. Uneventful ppV with consecutive chromovitrectomy, application of brilliant blue dye (BBD) and consecutive 12% C2F6 gas application remained unsuccessful. Although the postoperative optical coherence tomography (OCT) defined no residual epiretinal membrane on the retina, the macular hole remained unchanged . During reoperation we confirmed this observation by restaining the retinal surface by BBD and determined a negative staining up to the upper and lower arcade. Due to the extensive ILM removal during the first surgery, additional ILM patch transplantation was not feasible in this situation anymore and a silicone oil tamponade was installed for 6 week. However, the macular hole remained also unchanged on postoperative OCT and after oil removal . The next step was the removal of the decaline by shifting the bleb over the optic disc and removing it safely. Then we applied BSS fluid at one retinotomy until the subretinal blebs connected to a small perifoveal serous neurosensory detachment. The released paracentral retina was gently massaged towards the foveal center using the Tano diamond dusted scraper.After fluid air exchange, we installed silicon oil to secure the healing of the sealed macular hole, and to observe the retina during the early postoperative period. Five days after surgery we observed by OCT a closure of the giant macular hole.Moreover, there was a restoration of the anatomical architecture Fig.  with sigNumerous secondary surgical attempts have been described to achieve a surgical closure of refractory FTMH after successful chromovitrectomy with ILM removal: (a) while restaining the retinal surface may confirm the complete release of the ILM, (b) repeated gas or silicone oil application may enhance the duration of the tamponade and time to close especially large holes. (c) More recent ILM patches may release growth factors or guide as sheath to the adjacent retina to heal the macular hole . (d) TheWe hypothesize, that in giant FTMH not necessarily the release of the tangential traction, but the firm adhesion of the perpendicular photoreceptors to the RPE may prevent a successful closure of the macular hole. In limited macular translocation we learned that the retina may shift by more than 1700 μm  by creating a serous detachment and scleral imbrication.Massaging the neurosensory retina with a scrapper is also potentially a traumatic maneuver. Controversly, we discussed if the mechanical brushing of the parafoveal retinal surface with a Tano scraper is essential and required in all cases. An iatrogenic trauma may be induced by the extend of the epiretinal massage. Thus, we proposed in future cases with primary failed larger FTMH closure, to apply solely subretinal fluid with a gas endotamponade in order to close even larger macular holes without secondary mechanical maneuvers.A primary release of the centripetal force by the ILM removal, followed by a release of the RPE-photoreceptor adherence may mobilize the elastic retina from epiretinal and subretinal adhesions completely. Secondary, stretching of the retina by application of subretinal fluid and tactile massage may enlarge the retinal surface covering potentially large macular holes. The here presented novel subretinal approach may serve as a first prove of principle to close residual FTMHs after subretinal fluid injection successfully.


The dimerization and sulfhydration of PDE 5A and soluble guanylatecyclase (sGC) were measured. PDE 5A and protein kinase G (PKG) activities were tested. Intracellular cGMP content was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that NaHS relaxed isolated rat vessel rings at an EC50 of (1.79 ± 0.31)×10−5mol/L, associated with significantly increased PKG activity and cGMP content in vascular tissues. Sulfhydration of sGC β1 was increased, while the levels of sGC αβ1 dimers were apparently decreased after incubation with NaHS in vascular tissues. Moreover, PDE 5A homodimers were markedly decreased, and accordingly the PDE 5A activity demonstrated by the content of 5′-GMP was significantly decreased after incubation with NaHS or GYY4137. Mechanistically, both NaHS and GYY4137 significantly enhanced the PDE 5A sulfhydration in vascular tissues. DTT partially abolished the effects of NaHS on PDE 5A activity, cGMP content and vasorelaxation. Therefore, the present study for the first time suggested that H2S exerted vasorelaxant effect probably via sulfhydration-associated PDE 5A dimerization.The study was designed to examine if the vasorelaxant effect of hydrogen sulfide was mediated by sulfhydration-associated phosphodiesterase (PDE) 5A dimerization. The thoracic aorta of rat was separated and the vasorelaxant effects were examined with  It was revealed that endogenous H2S could reduce blood pressure, relax blood vessels, and inhibit the proliferation of smooth muscle cells, demonstrating important roles in cardiovascular regulation [2S by targeting potassium channel and L-calcium channel pathway in vascular smooth muscle cell membrane [2S could also target other pathways to relax blood vessels [Hgulation –17. Prevmembrane –24. Mean vessels –27. The 2S could regulate protein function via modifying the thiol group of protein (-SSH) [2S could affect the formation of disulfide bond on androgen receptor (AR) by its S-sulfhydration [2S increased intracellular cGMP levels [2S signaling and biological activity [2S exerted its vasorelaxant effects through inhibiting the disulfide bond-dependent heterodimer formation of sGC and/or PDE is yet to be explored. The present study was, therefore, designed to examine if H2S exerts vasorelaxant effect by PDE dimerization in association with its sulfhydration.Recently, Gao, et al. discovered another mechanism for sGC activation, namely sulfhydryl-dependent dimerization formation. In this mechanism, heterodimers are formed by disulfide bonds of sGC protein α and β subunits, activating sGC and its downstream pathway, and thus resulting in vasodilatory effects. Similar to sGC, the other key regulatory molecules in sGC/PDE/cGMP/PKG pathway, such as PDE are also constituted of two subunits, regulating its activity by the disulfide bond-dependent heterodimer formation . Previoun (-SSH) , 30. Zhaydration . A previP levels . They shactivity . Sofia-Iactivity . However−5mol/L. The maximum diastolic effect of NaHS was (60.49 ± 1.48)% at the concentration of 500 μmol/L. Whereas, the vasorelaxant effect of NaHS was (55.71 ± 1.65)% at a concentration of 1000 μmol/L ×10P < 0.05, n = 8, Figure PKG is a key molecule in the sGC/cGMP signaling pathway. We determined the effects of NaHS on PKG activity in the vascular tissues by detecting the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) using Western blotting. The results demonstrated that compared with the basal line, after incubation with NaHS at the concentration of 50 and 300 μmol/L for 15 min, the p-VASP/VASP ratio was significantly increased by 112.9% and 78.8%, respectively  and a β subunit (β1). The dimerization of α and β1 subunits of sGC is required for its catalytic activity. Therefore, we detected the level of sGC β1 monomer and sGC αβ1 dimer with antibody of sGC β1 by non-denaturing Western blotting method. We found that compared with the basal value, after incubation with NaHS at 3, 50 and 300 μmol/L for 15 min, the levels of sGC dimers in vascular tissues were significantly decreased by 37.9%, 42.4% and 53.3%, respectively . Infection of ASMCs with CSE lentivirus markedly increased CSE protein expression compared with vehicle lentivirus infected ASMCs /cAMP pathway, we wondered if AC/cAMP pathway was involved in the mechanisms by which H2S induced vasorelaxation. The data showed that SQ22536, an AC inhibitor, at 100 μmol/L could significantly reduce the vasorelaxant effect of NaHS at a concentration of 50 μmol/L .In this study, we showed that the vasorelaxant effect of H2S, in the light of the concentration range of the vasorelaxant effect of H2S and endogenous H2S level in vivo (55.01 ± 11.07 μmol/L), we mainly studied two concentrations of NaHS, 50 and 300 μmol/L, in the regulation of PDE/cGMP/PKG pathway in vascular tissues. Firstly, we examined cGMP content and PKG activity in vascular tissues. The results showed that compared with the baseline, both 50 and 300 μmol/L NaHS incubations enhanced cGMP content and PKG activity, indicating that H2S activated the cGMP/PKG pathway.To explore whether the PDE/cGMP/PKG pathway was involved in the vasorelaxant effect of H2S increased cGMP content. Intracellular cGMP level was mainly maintained by the balance of its synthesis and degradation. The synthesis of cGMP from GTP is catalyzed by sGC. sGC is a heterodimer of an α subunit (α1 or α2) and a β subunit (β1). The dimerization of sGC αβ1 is essential for its catalytic activity. We found that NaHS and GYY4137 decreased sGC αβ1 dimerization in vascular tissues. The sGC αβ1 dimers were also reduced by endogenous H2S by CSE overexpression in ASMCs. These results indicated that H2S could inhibit sGC activity and cGMP synthesis. The observed rise in cGMP content in vascular tissues after incubation with NaHS dose not result from increased cGMP synthesis through sGC.Next, we examined the possible mechanisms by which H2S on the degradation of cGMP. It is known that PDE 5A is a key enzyme for cGMP degradation. Therefore, the activity of PDE 5A was detected in the study by measuring the rate of cGMP degradation to 5′ GMP in vascular tissues. The results showed that both NaHS and GYY4137 inhibited the rate of cGMP degradation to 5′ GMP, suggesting that H2S inhibited the activity of PDE 5A for cGMP degradation.Intracellular cGMP concentration is modulated by the balance between cGMP synthesis and degradation. Thus, we explored the regulatory effect of H2S inhibited the activity of PDE 5A have been unclear. Recently, Gao, et al. found an interesting mechanism for the regulation of sGC and PDE 5A activity, namely sulfhydryl-dependent dimerization formation. Heterodimers are formed by disulfide bonds of sGC protein α and β subunits, which activates sGC and its downstream pathway, causing the relaxation of blood vessels [2S could increase protein function by modifying the thiol group of protein (-SSH) [2S could improve the formation of disulfide bonds on androgen receptor (AR) by its S-sulfhydration [2S could inhibit PDE 5A dimerization in association with an enhanced sulfhydration. We interestingly found that NaHS, GYY4137 or endogenous H2S inhibited the PDE 5A dimerization, implying that H2S could inhibit PDE 5A dimerization and activity. Meanwhile, the sulfhydration of PDE 5A in vascular tissues was enhanced by NaHS or by GYY4137. The sulfhydryl modification of PDE 5A in vascular tissues was increased significantly after the incubation with NaHS at concentration of 3 μmol/L, 50 μmol/L, 300 μmol/L or 200 μmol/L GYY4137 for 15 min. Moreover, endogenous H2S also enhanced the sulfhydration of PDE 5A in ASMCs. The sulfhydration inhibitor DTT partly abolished the effects of NaHS on purified PDE 5A activity and vasorelaxation. The above mentioned results implied that H2S could promote PDE sulfhydration, inhibit its dimerization, and further reduce PDE activity, increasing cGMP content in vascular tissues.Up to now, the mechanisms by which H vessels . We founn (-SSH) , 30. Meaydration . Therefo2S inhibited the PDE 5A activity to reduce the cGMP degradation and suppressed sGC β1 activity to decrease the cGMP synthesis, the net effect after balance likely resulted in an increased cGMP level in vascular tissues, exerting the vasorelaxant effect of H2S.Moreover, on the basis of the data showing that H2S played a role in the regulation of AC/cAMP pathway. We found that SQ 22536, an inhibitor of AC, could significantly reduce the vasorelaxant effect of NaHS, indicating that the vasorelaxant effect of H2S is mediated by both AC/cAMP and cGMP signaling pathways.In addition to cGMP, AC/cAMP is also a powerful vasodilatory signal. Previous studies reported that HThe present study also had limitations. The exact residue of PDE 5A responsible for this sulfhydration, and the possible residue of sulfhydration of a protein have not been clarified. Further studies were needed to clarify the exact residue of PDE.2S enhanced PDE 5A sulfhydration to inhibit its dimerization and reduced its activity, accordingly inhibiting cGMP degradation, thereby activating the cGMP/PKG pathway for vascular relaxation. The study elucidated that the mechanism by which H2S exerted the vasorelaxant effect. It also provided potential targets of drug treatment in cardiovascular diseases by H2S in the future.In summary, the present study suggested that HMale adult Wistar rats (240–260 g) were purchased from the Experimental Animal Center of Peking University Health Science Centre and housed under standard conditions , provided with adequate tap water and pelleted food. This study was approved by the Animal Research Ethics Committee of Peking University First Hospital (permit no.: J201437).NaHS and GYY4137 were purchased from Sigma, freshly prepared with physiological saline before use. Phenylephrine hydrochloride (PE), dithiothreitol (DTT), S-methylmethanathiosufonate (MMTS), and PDE5A antibody were all purchased from Sigma. sGCβ1 antibody was bought from Cayman Chemica Company. PDE activity assay kit was purchased from EnzoBiochem Company. Anti-VASP and anti-phosphorylated VASP antibodies were purchased from Cell Signaling Company, β-actin monoclonal antibody from Santa Cruz company, and cGMP content assay kit from New East Biosciences Company. Conventional chemical reagents were all analytically pure and were purchased from Beijing Chemical Reagent .2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM glucose) with 95% O2 and 5% CO2 as described previously [2–5% CO2, which was helpful to keep tissue alive. The real-time tension change data were collected with a Force Transducer connected to a Power Lab Data Acquisition System (AD Instruments). The aortic rings were initially stretched passively to a tension of 1.5 g and allowed to equilibrate for 1 h. Then PE (1 μmol/L) was used to precontract the aortic rings. The concentration-response curve of H2S on the vasorelaxant effect was evaluated by cumulative addition of different concentrations of H2S after administration with PE. To examine whether the vasorelaxant effect of H2S was associated with its sulfhydration, DTT, the sulfhydration inhibitor (2 mmol/L) was added 30 min before the PE challenge. To examine the involvement of cAMP signaling, the AC inhibitor SQ22536  was added 30 min before the PE challenge. The dilation effect was expressed as percentage PE contraction.Urethane  was used for intraperitoneal anesthesia. Thoracic aorta was then quickly removed by thoracotomy and placed in pre-cold Krebs buffer  for 15 min. The treated aortic tissues were frozen in liquid nitrogen, and non-reducing lysis buffer  was added. After lysis and homogenization, the mixture was centrifuged at 13,000 g for 15 min at 4°C. The protein concentrations of the supernatants were determined using a BCA protein assay kit with BSA standards . Protein samples (20 μg/lane) in non-denaturing buffer  were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. The membranes were then blocked with TBS-Tween  containing 5% skim milk, and incubated with rabbit anti-sGCβ1 antibody , rabbit anti-PDE5A , and mouse anti-β-actin  overnight at 4°C, respectively. Afterwards, the blot was washed four times, each time 10 min, followed by addition of horseradish peroxidase-linked secondary antibodies for 60 min , and washed four times (×10 min). The bands were visualized using Super Signal West Pico Chemiluminescent Substrate Kit (Thermo Fisher Scientific). Band densities were detected by Kodak Image Station 4000 MM Digital Imaging System and analyzed by Alpha EaseFC software . The expression of β-actin was used as an internal control to normalize the expressions of target proteins. The percentage of relative density was calculated as the ratio of target protein expression to β-actin expression.Rat aortic rings were immersed in 95% ORat aortic rings were incubated with different concentrations of NaHS  for 15 min, respectively. Thoracic aortic rings were homogenized and extracted in ice-cold protein lysis buffer, and cell lysates were incubated with EZ-linkTMPEO-iodoacetyl biotin  for 12 h at 4°C and then incubated with 20 μl of Ultra LinkTM Immobilized NeutravidinTM (Pierce) for 4 h on a roller system at 4°C. The beads were washed three times with 1 ml of ice-cold PBS. The mixtures were centrifuged at 5,000 g for 10 min at 4°C. The supernatants were removed, and 50 μl of lysis buffer added. The measurement of sulfhydryl groups in the sGC and PDE protein was analyzed by Western blotting and probed with rabbit anti-sGC and PDE antibody .Aortic rings were reserved in liquid nitrogen immediately after incubation with saline or different concentrations of NaHS  for 15 min, according to manufacture’ guidelines using cGMP kit . Lysis buffer with 0.1 M HCl was used to lyse the tissue, and after homogenization, centrifugation at 4°C  was used to collect the supernatant, which was then subject to detection of protein and cGMP concentration according to the manufacturer's instructions, using a commercial kit. The resultant absorbance was determined at 450 nm with a microplate reader . A standard curve was prepared by plotting the absorbance value for each standard vs. its concentration, from which the cGMP concentration of each sample was determined.Aortic rings were rapidly frozen in liquid nitrogen after incubation with NaHS for 15 min and IBMX (100 μmol/L) for 30 min. The tissue was lysed with lysis buffer, and after homogenization and centrifugation at 13,000 g , the supernatant was collected and used to detect PDE activity with a PDE activity assay kit (EnzoBiochem). Turpentine was used to remove phosphate anions from the sample prior to analysis. To detect the activity of purified PDE 5A, the PDE 5A from bovine brain was incubated with different concentrations of NaHS , DTT (2 mmol/L) plus NaHS (50 μmol/L), or GYY4137 (200 μmol/L) for 1 h at room temperature. Thereafter, the cGMP substrate, 5′-nucleotidase, tissue samples or purified PDE 5A, or 5′-GMP standards were added, according to the kit requirements, then incubated with shaking for 30 min at room temperature. Biomol Green reagent was added after 60 min, and the resultant absorbance was measured at 620 nm with a microplate reader after incubation for 30 min. A standard curve was prepared by plotting the absorbance value for each 5′-GMP standard vs. its concentration, from which the 5′-GMP concentration of each sample was determined.Detection of PKG was performed in accordance with the previous literature. Rat aortic rings were rapidly frozen in liquid nitrogen after incubation with saline or different concentrations of NaHS  for 15 min. Thoracic aortic rings were homogenized and extracted in ice-cold protein lysis buffer containing 50 mmol/L Tris-Cl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L ethylenediamine tetra-acetic acid, 1% NP-40, 0.25% sodium deoxycholate, 1 mmol/L phenyl methyl sulphonyl fluoride (PMSF), protease inhibitors, and phosphatase inhibitors. Western blotting analysis was used to detect the phosphorylation level of a PKG substrate , which reflected PKG activity. The experimental steps were the same as the non-denaturing western blotting. Western blotting analyses were performed according to a previous method, and the primary antibodies were phospho-VASP (Ser-239) antibody  and VASP antibody .2 and saturated humidity, and the 4th to 6th generations were used for the experiments. ASMCs were seeded in culture flasks of 25 cm2 for virus transfection. When cells were grown to 40% to 50% confluence, lentivirus containing the cDNA encoding CSE  was added to infect ASMCs for 24 h at a multiplicity of infection of 20. The cells were selected with puromycin (4 μg/mL) to acquire the stably transfected cells expressing CSE.Rat ASMCs were purchased from Wuhan PriCells Biomedical Technology Co., Ltd  and cultured in DMEM  supplemented with 10% FBS, 100 U/mL penicillin, 2 mmol/L glutamine, 100 μg/mL streptomycin, and primary vascular smooth muscle cell growth supplement . ASMCs were cultured in an incubator at 37°C with 5% COt test. Mean values of more than two groups were compared using one-way ANOVA test, with the Student-Newman-Keuls test for post hoc testing of multiple comparisons. P values less than 0.05 were considered statistically significant. Half-maximal effective concentration (EC50) for the H2S relaxation curve represented the corresponding concentration of H2S that achieved 50% of the maximum relaxation effect. SPSS20.0  and GraphPad  software were used for statistical analysis and graphing.The results were expressed as means ± SEM. The significant difference between two groups was analyzed by Student's


Cisplatin (cis-diamminedichloroplatinum) is widely used as a chemotherapeutic drug for genitourinary, breast, lung and head and neck cancers. Though effective in inducing apoptosis in cancer cells, cisplatin treatment causes severe hearing loss among patients. Steroids have been shown to mitigate cisplatin-induced hearing loss. However, steroids may interfere with the anti-cancer properties of cisplatin if administered systemically, or are rapidly cleared from the middle and inner ear and hence lack effectiveness when administered intra-tympanically. In this work, we deliver prednisolone-loaded nanoparticles magnetically to the cochlea of cisplatin-treated mice. This magnetic delivery method substantially reduced hearing loss in treated animals at high frequency compared to control animals or animals that received intra-tympanic methylprednisolone. The method also protected the outer hair cells from cisplatin-mediated ototoxicity. Cisplatin (cis-diamminedichloroplatinum) and other platinum based drugs are the antineoplastic drugs of choice for various genitourinary cancers, certain forms of breast cancers, and as radiosensitizers for most head and neck cancers. However, these platinum-based drugs are very toxic to the kidneys  of both sexes (23–27 gm body weight) from the Jackson Laboratory . All animal studies were conducted in accordance with the policies and recommendations of the National Institute of Health Guide for the Care and Use of Laboratory Animals, and under approval from the Institutional Animal Care and Use Committee of the University of Maryland.The mice were anesthetized via intraperitoneal injections of ketamine 100 mg/kg and xylazine 20 mg/kg supplemented as necessary and were placed on a warming pad  to maintain body temperature at 37°C.The overall study design is shown in Table N = 6 mice per group for group A, B and D and N = 4 mice for group C. For all groups, ear treatment was administered 1 day before the second and third cisplatin cycles respectively. Group A mice received 1.8 μL of intra-tympanic saline into their left ears. Group B mice received 1.8 μL of intra-tympanic methylprednisolone  at a concentration of 40 mg/ml  into their left ears. Group C mice received 1.8 μL of 300 nm diameter magnetic nanoparticles without prednisolone into their left ears. Group D mice received 1.8 μL of 300 nm diameter magnetic nanoparticles that were loaded with prednisolone sodium phosphate at a concentration of 82 μg/mL (the maximum drug loading these particles could carry), also into their left ears. Both unloaded and prednisolone loaded particles were labeled with Texas red fluorescent dye for easy visualization in tissue samples . A 0.5 Tesla magnet  was then placed contralateral near the right eye of each animal in group C and group D for 20 min to pull the nanoparticles from the middle ear into the inner ear. For all animals in all groups, the right ears remained as untreated same-animal controls.The animals were divided into four different groups, with The hearing thresholds of the animals in all groups were measured by performing auditory brain stem response (ABR) assays before and after the cisplatin treatment and recovery periods. The mice were anesthetized and placed inside a sound booth . Two recording electrodes  were inserted postero-ventral to the auricular area of the left and right ears. A reference electrode was placed at the apex of the head. A ground reference electrode was placed subcutaneously in the lumbar area. Using our ABR recording system , the animals were then presented in free field with 600 sweeps of 5 ms long bursts  at varying intensities beginning at a 94 dB sound pressure level (SPL) and proceeding in 5 dB decrements down to a 14 dB SPL. The electrophysiological signals were recorded for 10 ms. These cycles of sound intensities were repeated for different sound frequencies . Hearing threshold at each frequency was determined as the lowest intensity at which a definite cochlear response could be identified (waves I & II). Figure The cochleas from the different groups were dissected to study the pathophysiology of the cisplatin treatment on the organ of Corti, as well as any effect of the otoprotective treatments. The animals were euthanized using carbon dioxide and the cochleas rapidly isolated. The cochleas were continuously perfused with ice cold 4% paraformaldehyde into the round window membrane and out of a small hole pierced in the apex of the cochlea, and then placed in paraformaldehyde overnight at 4°C. This was followed by 3X wash with 1X PBS at pH 7.4 and decalcification in 0.5 M EDTA for 3–4 days. After washing the cochleas three times using 1X PBS, they were micro-dissected into three turns  using an ophthalmic knife . The tectorial membrane was removed. Cochlear outer and inner hair cell layers were stained using Alexa Fluor 488 Phalloidin  for 45 min. The turns were mounted on a glass slide using Fluoromount-G with DAPI (Electron Microscopy Sciences). Images of each cochlear turn were taken at 40X magnification using an LSM 710 confocal microscope (Zeiss) in z-stack mode. The outer hair cells in these images were counted for the presence of nuclei and cell membranes over a 200 μm distance of the different cochlear turns using Zen 2010 software (Zeiss) and the assessments were made blind to avoid biases.In our experiments, the hearing thresholds of the animals in the different groups were determined after the completion of the cisplatin treatment by using ABR assays. The hearing loss experienced by the animal at a particular frequency was determined by the ratio of change in threshold post-treatment to the initial hearing threshold at the same frequency. After systemic cisplatin treatment, this threshold increase is known to occur first at high frequencies, progressing to the lower frequencies as treatment continues, eventually reaching speech frequencies  ears, as compared to control group A, B, and C ears which exhibited almost complete hearing loss at high frequencies (**p < 0.05). Overall, the prednisolone loaded nanoparticles mitigated cisplatin induced ototoxicity at high frequencies, with 95% statistical significance. At 8 and 16 kHz, magnetic delivery also seemed to reduce the degree of hearing loss, but at these lower frequencies a statistical significance of 95% was not reached, in part because cisplatin caused less hearing loss in mice at these lower frequencies  ears the hearing loss was greatest at high frequency (at 32 kHz), as compared to at 16 and 8 kHz Figure . In treaCisplatin is known to induce apoptosis in the three rows of outer hair cells starting at the outer row and progressing to the inner row  vs. from ears of animals that received cisplatin plus one of the three types of ear treatments  is shown in Table The benefit of magnetic delivery is that it actively and directly transports drug into the cochlea, in contrast to intra-tympanic administration where drug diffuses passively into the cochlea from the middle ear. Although it was not possible to measure the amount of steroid delivered into mouse cochleas in this study , in initial adult human cadaver studies we have observed significantly greater delivery of steroid to the cochlea with magnetic delivery as compared to intra-tympanic administration without magnetic fields and particles. Improved delivery to the cochlea, and also to the vestibular system, is of interest not only for protection of hearing from chemotherapy, but also for other conditions of the inner ear such as sudden and noise-induced hearing loss, for presbycusis, for tinnitus, and for Ménière's disease. This study achieved efficacy by delivering prednisolone, a generic off-the-shelf steroid. However, magnetic nanoparticles can be loaded with many other therapies. Magnetic delivery of new and emerging therapies  is anticipated to yield even greater benefits than delivery of a generic drug. Conversely, even the best new drug or therapy will not be effective if it does not reach the cochlea in sufficient quantities to be therapeutic.The cisplatin regimens that were administered to the animals caused hearing loss primarily at high frequencies. In human patients also, hearing loss due to cisplatin is known to occur first at higher frequencies and then to progress to lower frequencies as treatment continues . The other authors do not have any financial relationship to the research to disclose. 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.


Mycoplasma hyopneumoniae has proven difficult to culture since it was first isolated in 1965. A reliable solid medium has been particularly challenging. Moreover, clinical and pathological samples often contain the fast-growing M. hyorhinis which contaminates and overgrows M. hyopneumoniae in primary culture. The aim of this study was to optimise the culture medium for recovery of M. hyopneumoniae and to devise a medium for selection of M. hyopneumoniae from clinical samples also containing M. hyorhinis. The solid medium devised by Niels Friis was improved by use of Purified agar and incorporation of DEAE-dextran. Addition of glucose or neutralization of acidity in liquid medium with NaOH did not improve the final yield of viable organisms or alter the timing of peak viability. Analysis of the relative susceptibility of M. hyopneumoniae and M. hyorhinis strains to four antimicrobials showed that M. hyopneumoniae is less susceptible than M. hyorhinis to kanamycin. This was consistent in all UK and Danish strains tested. A concentration of 2 μg/ml of kanamycin selectively inhibited the growth of all M. hyorhinis tested, while M. hyopneumoniae was able to grow. This forms the basis of an effective selective culture medium for M. hyopneumoniae.The fastidious porcine respiratory pathogen  The disease is of global significance both in reducing growth efficiency and in promoting susceptibility to concurrent bacterial and viral infections . Both liquid and solid media are available. These support the growth of M. hyopneumoniae but the constituents of this commercial medium are not publicly available. Solidification of Friis medium with Agar did not allow growth of colonies of M. hyopneumoniae, suggesting that the agar was inhibitory to growth or sequestered an essential nutrient. The use of a better defined solid medium would allow greater flexibility for research, such as transformation of M. hyopneumoniae .eumoniae  and the yorhinis . The ide2.24·7H2O, 2 g/l MgCl2.6H2O, 3.7 g/l CaCl2·2H2O), 12.5 ml sterile solution B , 50 ml pig serum (Invitrogen) heat-treated at 56 °C for 20 min, 50 ml heat-treated horse serum (Invitrogen), 1 ml phenol red solution (0.6% in 0.1 M NaOH), 25 μl of each of 100 mg/ml azlocillin and flucloxacillin and adjusted to pH 7.4 with 1.0 M NaOH.Friis medium was prepared largely as described by 2.3® solid medium was prepared according to the manufacturer’s instructions . Cultures were incubated in a humidified incubator at 37 °C in 5% CO2.Concentrated (2.8×) Friis medium was prepared by the addition of 88 ml water to 4.3 g BHI, 4.6 g PPLO sterilised by autoclaving, 51.4 ml yeast extract, 35.7 ml sterile solution A, 35.7 ml sterile solution B, 143 ml heat-treated horse serum, 143 ml heat-treated pig serum, 2.8 ml 0.6% phenol red solution, azlocillin and flucloxacillin , pH 7.4. Friis agar was prepared by mixing concentrated Friis medium (2.8×), mixed at a ratio of 35: 65 with either 1.8% agar No. 1 , Purified agar  or agarose (Invitrogen). Where indicated, DEAE-dextran  was added to agar at 0.1 mg/ml. Mycoplasma ExperienceCharcoal-treated agar was prepared by incubating a 1.3% solution of Bacteriological agar with 2% w/v of activated charcoal  for 10 min at 55° C, the charcoal was then removed using a sieve. Following sterilisation, each agar type was mixed with medium supplement  or concentrated Friis medium at a ratio of 35: 65 (supplement: agar) and plates poured.2.42 at 37 °C. Colonies were counted, from those dilutions showing the largest number of separate colonies, using a dissecting microscope; the triplicate counts were combined and the viability calculated. Growth was also estimated from the metabolic activity recorded as a colour shift of the phenol red pH indicator from pH 7.4 to 6.8 as described by Ten-fold serial dilutions in Hanks’ Balanced salts Solution  were spotted (10 μl in triplicate) on to solid medium. Cultures were incubated for 7 days in 5% CO2.5Solutions of kanamycin (Sigma), tetracycline (Sigma) and puromycin (Fisher) were prepared in de-ionised water. Chloramphenicol (Sigma) was dissolved in ethanol at 10 mg/ml final concentration. All were sterilised through a 0.2 μm filter . A final antibiotic concentration range was achieved by two-fold serial dilutions of each antibiotic in sterile water or ethanol as appropriate. Antibiotics were incorporated into solid medium over a range of concentrations 20–0.01 μg/ml (kanamycin), 4.0–0.03 μg/ml (tetracycline), 20–0.06 μg/ml (chloramphenicol and puromycin)M. hyopneumoniae and M. hyorhinis inocula were prepared in 1 x sterile phosphate buffered saline  as a suspension containing approximately 103 CFU/ml. A sample of 10 μl of each strain was inoculated onto each plate in triplicate. Plates were incubated at 37° C with 5% CO2 and observed for growth of colonies between day 2 and day 10. The MIC was determined as the highest dilution of antibiotic giving an absence of growth as determined by the formation of mycoplasma colonies.33.1M. hyopneumoniae to the commonly used colour shift in liquid medium was determined (8.9 at approximately 2.5 days incubation. This corresponded to a colour shift between 2 (pH 7.2) and 3 (pH 7.1). After this point there was an exponential decline reaching 106 CFU/ml after 8 days (beyond colour shift 4).The viability of termined . Culture3.2The concentration of glucose in Friis medium is approximately 2 mM: 1 mM is derived from the brain heart infusion broth and another 1 mM from the diluted serum. To investigate whether glucose is limiting the growth, further glucose was supplemented into the liquid medium to give 4 mM (×2) and 8 mM (×4) glucose, and viable counts performed through the growth cycle. The growth curve was unchanged by the increased glucose concentrations . Friis s3.3M. hyopneumoniae, different agar preparations were mixed with Friis base and or Mycoplasma Experience® (ME) supplement and investigated for the ability to support the growth of M. hyopneumoniae colonies. The results are shown in In order to use Friis medium as the base for a solid medium to culture M. hyopneumoniae 277/94 & 325/95. When Agar no.1, used widely in the solidification of bacteriological culture medium, was mixed with either Friis medium or ME supplement there was no growth of M. hyopneumoniae. Pre-treatment of the agar with activated charcoal, in an attempt to remove any growth-inhibiting components, did not alter the ability of medium made with either supplement to support M. hyopneumoniae. However, when either Purified agar or agarose were used as the solidifying agent, Friis medium, but not the ME supplement, grew colonies of M. hyopneumoniae  and appeared to be superior to the medium commercially available  of M. hyopneumoniae strain 277/94 and M. hyorhinis Mhr1/09 was used to assess the ability of kanamycin to selectively suppress the growth of M. hyorhinis on solid medium. After 8 days incubation in the absence of kanamycin, M. hyorhinis colonies outgrew M. hyopneumoniae as recognised by colony morphology .Initial experiments indicated that the purity of the agar used to solidify modified Friis medium greatly affected the growth of M. hyorhinis or M. hyopneumoniae. This suggested that an essential constituent for growth was absent from the liquid supplement and only present within the agar component. Solidifying Friis medium with Purified agar treated with DEAE-dextran confers a substantial improvement on the commercially available medium for growth of M. hyopneumoniae. Since DEAE is a positively charged group able to bind negatively charged ions, its action may be explained by chelation of the agaropectin present in agar which may be inhibitory to growth of M. hyopneumoniae. Agaropectin is the mixture of smaller molecules, comprising alternating residues of l- and d-galactose modified with acidic side-groups (particularly sulfate). This is consistent with the beneficial use of purified agarose, which is depleted of agaropectin, over agar no. 1 in this study. No equivalent improvement was seen from DEAE in culture of M. hyorhinis, suggesting that it is less sensitive to the acidic components of agaropectin.When the commercial ME supplement was solidified with agar other than the supplied agar component it was unable to support the growth of either M. hyorhinis. This requires rabbit antiserum which is expensive and not always available for regular culture. In testing the relative sensitivity of M. hyopneumoniae and M. hyorhinis to four antimicrobials, our results showed that the latter was consistently more sensitive to kanamycin. M. hyorhinis and M. hyopneumoniae were susceptible to kanamycin (0.1 and 0.5 μg/ml respectively). However, different isolates were used, and determination of the MIC by Williams was in liquid medium. Nevertheless, incorporation of 2 μg/ml of kanamycin into the agar medium appeared to prevent the growth of M. hyorhinis following 8 days incubation. When mixed in equal volumes with M. hyopneumoniae strain 325, the M. hyorhinis did not form colonies while controls, without the addition of kanamycin, yielded only M. hyorhinis colonies from the mixed culture. Screening of M. hyopneumoniae and M. hyorhinis strains showed that this difference in kanamycin susceptibility was present between all M. hyopneumoniae and M. hyorhinis strains tested.M. hyopneumoniae, effectively preventing the growth of M. hyorhinis while allowing colonies of M. hyopneumoniae to form. The relative kanamycin resistance of M. hyopneumoniae has greatly improved the primary isolation of M. hyopneumoniae from abattoir tissue samples originating from 15 different farms. False negatives, resulting from M. hyorhinis overgrowth have been overcome using this selective medium.The addition of kanamycin can be used in place of the hyperimmune rabbit serum and gives a clean selection for the culture of None.


The incidence of zoonotic cutaneous leishmaniasis (ZCL) makes it the most widespread parasitic disease in Tunisia and the Arab world. Yet, few studies have addressed its psychological and psychosocial effects. The purpose of this study was to examine the psychosocial impact of ZCL scars among Tunisian women.We conducted an exploratory study, we administered Revised Illness Perception Questionnaire (IPQ-R), World Health Organization Quality Of Life-26 (WHOQOL-26) and Psoriasis Life Stress Inventory (PLSI) to a group of girls and women with ZCL scar in the region of Sidi Bouzid. This group was randomly selected from volunteers who came to primary health care facilities to seek for treatment for any pathology.Descriptive statistics showed that the collected scores from the three scales exhibit heterogeneous distributions: IPQ-R , PSLI , WHOQOL-Physical , WHOQOL-Psychological , WHOQOL-Social , and WHOQOL-Environmental . The correlation analyses performed on Inter and intra-subscales showed that the emotional representations associated with ZCL were correlated with the loss of self-esteem and feelings of inferiority . In addition, high education level and the knowledge about ZCL are positively correlated with cognitive and emotional representation in the IPQ-R . "Rejection experiences" and the "anticipation and avoidance of stress" were respectively negatively correlated with age . Correlations between the scores on IPQ-R domains and PLSI factors were significant. The results showed that anticipation of rejection and avoidance of stress are strongly correlated with a negative perception of ZCL. Quality of life scores were not correlated with either age, education level, time of illness, or the number of facial or body scars. However, the correlations between quality of life scores and the multiple IPQ-R domains were all insignificant. Finally, there was a negative correlation between the scores on the perceived quality of social life and the knowledge about ZCL .This makes it vital to strengthen preventive health education. Conducting studies on ways to establish a holistic support system for managing ZCL, a system that covers the psychological challenges and the barriers it causes to women’s social and professional integration, is a vital first step. Zoonotic cutaneous leishmaniasis (ZCL) is the most common form of leishmaniasis in Tunisia. The disease is not severe and heal spontaneously with a definitive scar causing a social impact mainly when the lesion occurs in the face. Yet, few studies have addressed these psychological and psychosocial effects. To examine these issues among Tunisian women suffering from ZCL, we administered Revised Illness Perception Questionnaire (IPQ-R), World Health Organization Quality Of Life-26 (WHOQOL-26) and Psoriasis Life Stress Inventory (PLSI) to a group of girls and women with ZCL scars in the region of Sidi Bouzid. This study demonstrated the wide range of psychological effects  and psychosocial impacts  of ZCL permanent scars; mainly when it is located in the face; among women and girls. This study suggests the need of social and psychological support and professional integration for women and girls in rural remote areas first and second conducts studies on ways to establish control measures for ZCL, mainly in vulnerable exposed population. Cutaneous leishmaniasis is the most common form of leishmaniasis. It is a skin infection caused by a unicellular parasite transmitted by infected sandflies that feed on the blood of rodents bred in caves or burrows in degraded environments. It is known also as Oriental Sore, Biskra Button, Aleppo boil or Tabaa Sidi Bouzid, depending on the geographic region concerned.Cutaneous leishmaniasis is endemic in many parts of the world. There are about twenty different species of Leishmania capable of infecting humans. The distribution of cutaneous leishmaniasis is very closely related to the geographical characteristics and the ecological specificities of the endemic areas.Cutaneous leishmaniasis always heals spontaneously, but leaves permanent scars. New treatment options and new molecules are still in the process of getting validated. ZCL is the most widespread parasitic disease in Tunisia. It mostly affects people who live in the central and south-western parts of the country.ZCL is not a fatal disease, but it causes significant changes in victims that affect their psychosocial condition and quality of life. Work done in Pakistan and Afghanistan  suggestsThe theoretical framework for this study is the self-regulatory theory framework by which Leventhal, Meyer & Nerenz  explain identity: what the illness is called and the symptoms that affect the patient and are associated with the illness; the cause: the patient’s opinion on the causes of the illness. An illness with no identifiable cause is a devastating experience that causes impotence and difficult adjustment; the timeline: chronic, acute or cyclical; the consequences: for the patient and his entourage; and cure and control: this domain refers to personal perceptions of one’s potential power to control the illness and its spontaneous development or treatment.According to Leventhal, Nerenz and Steele , there aemotional representation, relating to all the negative feelings caused by the illness or the coherence of the illness, a metacognitive domain relating to the general understanding of the illness and to the degree of integration of its various dimensions, symptoms and causes.Apart from these five domains of illness perception, there are other domains such as Our study intended to evaluate the psychological and social impact of ZCL on women with facial scars in the region of Sidi Bouzid. Understanding perceptions of ZCL and quality of life among patients with ZCL and ZCL scars can help us develop psychosocial intervention strategies to fight against stigma and self-deprecation, and to develop strategies for preventing harmful behaviour patterns on ZCL.The study was conducted in Sidi Bouzid, a predominantly rural or semi-rural region with scattered groups of extended families attached to their farmland. Sidi Bouzid is a region that provides over 20% of the country's agricultural production. There is no psychological services available for this population. For the facility and the feasibility of this first study and the disponibility of the population, the sample was made up of 41 girls and women from El Hichria (n = 31) and Ouled Mhamed (n = 10) in the Sidi Bouzid Governorate . We condTo assess the perception of health and illness, we used the "Revised Illness Perception Questionnaire (IPQ-R )." This The IPQ-R contains 71 questions rated on a four-level Likert scale on level of agreement with the item . It explores the illness identity, timeline, consequences, cure, control, and coherence domains and the emotional representation domain.During scaling and scoring, we did reverse recoding of several items. The results for the domains are expressed in percentages.The assessment of the psychosocial impact of leishmaniasis was done using the Psoriasis Life Stress Inventory (PSLI) questionnaire . The queTo assess the quality of life of adolescents and women affected by ZCL, we chose to use the World Health Organization Quality Of Life-26 (WHOQOL-26) scale . This scFour major factors of quality of life were examined to enable us to have a sufficiently comprehensive perception of the perceived quality of life of the respondents according to four domains: 1. The physical domain, which covers items on physical pain feeling, the need for any medical treatment, energy for everyday life, sleep satisfaction, functional capacity, energy and work capacity; 2. The psychological domain, which contains items exploring aspects of perceived satisfaction about the meaning of life, the capacity of concentration, the bodily appearance, self-image, and negative feelings; 3. The domain of social relationships, which includes items related to satisfaction about personal relationships, sex life, and social support; 4. The environmental health domain, which comprises items related to perceived safety in daily life, financial resources, information availability, leisure activities, condition of living place, accessibility to health services, and transportation.The questionnaires were administered in the following order: the IPQ-R, the PLSI and WHOQOL Bref.Statistica 6 software after verifying that it was entered properly. We performed statistical analysis using parametric tests after checking for the normal distribution of variables. The correlation analysis were done using the Pearson correlation coefficient. For comparison, we used Student’s t-test or variance analysis. We didn’t perform multivariate analysis because of the small sample size.The information gathered was processed using The questionnaires were administered by a psychologist and/or a health educator on ZCL patients in their homes or at the local clinic. All persons who participated in this study did so willingly. The study was approved by the "Ethic committee of Pasteur Institute of Tunis (IPT)".All women provided informed consent, and parents of any child participant provided also informed consent on their behalf. The informed consent given by all subjects was oral since the majority of them were illiterate, the psychologist and the health educator explained the aim and the procedure of the study and asked them for consent. The "Ethic committee" approved the study since we didn't need any clinic or biological investigation from all subjects with the guarantee from the research team about confidentiality and the respect of individual information. The response on the questionnaire attest the oral consent of participants.For the respondents in the survey, this disease, "leishmaniasis", is linked with sand flies. All the respondents stated that they consider ZCL scars as marks of the disease, and that the number of scars varies from person to person. Seventy-eight per cent considered that ZCL is sex-specific. Seventy-eight per cent declared that it is seasonal and spares no age group. Seventy per cent believed the insect bites eventually become scars. The respondents had different views on whether this disease was found recently (56%) or a long time ago and on the possible disappearance of the scars over time (51%). Further, 68% stated that ZCL is dangerous.Concerning perceptions on the causes of ZCL, the respondents mentioned the human responsibility for the environmental changes. The majority considered that ZCL is not hereditary (80%). It was not related to the quality of food (92%), or stress in everyday life (78%). However, 73% linked the environment (stagnant water) and physical activity  with the incidence of ZCL. About 66% thought extreme temperatures contribute to the incidence of ZCL. Close to three quarters (73%) saw a correlation between stagnant water, sand flies and the incidence of ZCL. About fifty percent made a link between ZCL and abundant rainfall that frequently ends by forming stagnant water. This may be because these events rarely occur in Sidi Bouzid. The summer is usually very hot and associated easily with sand flies and increased risk of sand fly bites and illness.Almost 71% of the respondents mentioned the notion of vectors and reservoirs. Close to two thirds linked rat’s sand flies to ZCL transmission. Yet, barely 43% said that killing rats and sand flies vectors is relevant to preventing risk of exposure to ZCL.Eighty five per cent of the respondents thought that sand flies are responsible for the disease. Eighty seven per cent admitted there is a correlation between working with animals and exposure to ZCL. None of the subjects believed that the sandfly that are related to ZCL are small and almost invisible. Seventy per cent said that the sand fly that are linked to ZCL are the same size as other mosquitoes.We found that the respondents’ perceptions on the time it takes for scars to develop ranged from an uncertain period (some scars are permanent while others appear and then fade away) to chronicity, sometimes with an increase in the number and appearance of the scars. Almost half the number of respondents stated that the scars do not fade quickly, but they were carefully optimistic that the scars would fade gradually with time.The respondents said unanimously that ZCL has had no positive effects on their lives. The consequences have rather been negative. About 73% suffered social exclusion and stigmatization. Their relationships have been broken and they face more interpersonal conflicts in society, regardless of the context . The consequences were seen also in their chances of getting employment. ZCL affected women who stay at home more than those who study or are already working. It reduced marriage prospects for men (75%) and women (59%). The consequences were felt also on aesthetic features, for the scars alter women’s beauty (58%).With respect to individual ability to control ZCL, the most common perception was an absolute lack of power to control and cure the disease. The respondents thought there is nothing useful they can do to control the disease, its symptoms or its progress. Drugs were seen to have little or no effect, and the risk of getting the disease again is still likely.When it comes to understanding the problem, we noted that patients still cannot explain how they contracted ZCL. There is little understanding of the problem, and respondents keep asking themselves "why am I the one who is infected".We found that the emotional representations associated with ZCL were associated with the loss of self-esteem, feelings of inferiority and the idea that the disease is equal to an obvious social disadvantage. There was also a strong sense of shame.The inter-correlation matrix for multiple IPQ-R domains  showed tThis correlation analysis suggested also that patients who have a more coherent perception of ZCL do have stronger emotional reactions  and face more severe consequences . Finally, those who are more inclined to perceive ZCL as an illness with negative consequences were more likely to claim that they experience severe emotional difficulties .All these significant correlations are maintained even after testing them on the "age" variable and the "education level" variable, using partial correlations.The descriptive data focused more on anticipation of a negative reaction from others and the efforts made to display anticipatory avoidance behaviour meant to deny such reactions, than on convictions from the presence of negative experiences that patients have had through social interactions on the state of their skin ."Rejection experiences" and the "anticipation and avoidance of stress" were respectively, significantly and negatively correlated with age . The younger the subject, the higher the number of experiences tied to stigma; and the older the subject, the more such feelings are dismissed. Thus, the correlation analysis showed that stigma assessment indices were not correlated with level of education, "total knowledge" score, or time of illness. They were correlated only with age: the younger the subject, the more they tend to experience stigma.Furthermore, there are moderate correlations and just around the significant level between the total number of body scars and "rejection experiences" , and between the number of facial scars and the scores on "Anticipation and avoidance of stress" . It is important to note that this correlation between the number of scars and stigma indices was at the same threshold of significance when checked against age-related variations. Hence, the number of body scars had a strong link to experience with stigma, regardless of a patient’s age.We also noted correlations between the scores on multiple IPQ-R domains  and PLSI factors . Environmental quality of life is seen as significantly less fulfilling (p<0.001) than quality of social life. According to the respondents, it appeared to be far less fulfilling than (p<0.001) the physical quality of life. Quality of mental life did not differ much from environmental quality of life. However, it is said to be significantly more impaired than quality of social life (p<0.001) and the physical quality of life (p<0.001). The physical quality of life was not described as significantly different from quality of social life .Quality of life scores were not significantly correlated with either age, education level, time of illness, or the number of facial or body scars . In addiOn the other hand, quality of life scores had interesting correlations with PLSI scores on stigma Figs  and 4. TFinally, there was a significant and negative correlation between the score on quality of social life and that on "Total Knowledge". It seems that the higher a person’s knowledge of ZCL, the less likely they are to view their quality of social life in a negative manner . The quality of knowledge seems therefore to be tied closely to the quality of social life .It was important also to note that the correlation between scores on 'Rejection experience" and on mental quality of life index was on the verge of the threshold of significance . Mental quality of life tends to be perceived negatively, if the subject claims to have suffered many rejection experiences. The mental quality of life index tended to be associated with the total stress and stigma score .The objective of this study is to explore the perceptions of ZCL, the stigma associated with the disease, and the quality of life of people with ZCL in Sidi Bouzid.We studied the perceptions of CL based on the theoretical model by Leventhal et al. , which ith of February, 2016, we found that ScienceDirect contained 1 758 articles on "IPQ-R" and 21 831 articles on "leishmania", but no entry on "IPQ-R" and "leishmania".It is the first time this type of model, and the assessment tool associated with it (IPQ-R), is applied to study CL. For example, on the 18It is the first time also that the psychological and psychosocial aspects of CL are studied in the Arab world, which is one of the areas with the highest number of cases of CL in the world. All works dealt basically with the epidemiological and/or biological aspects of the issue. Despite the originality of this work, our results should be taken with caution and have to be confirmed because we only did an explanatory study on a small sample size.Based on the results from the IPQ-R, we see that patients tend to consider ZCL as a disease that affects entire generations and is more rampant in some regions more than others. The disease mostly affects women and spreads in cyclical trends, caused by "mosquito" bites associated with the presence of rats, and related to climate change and the favourable ecological context in Sidi Bouzid. The patients were incline to think ZCL has negative consequences on their personal, family, social and professional life. They generally believe that ZCL considerably alters their natural beauty and reduces their prospects of getting employment, getting married and being valued in society. They see ZCL as a mysterious, incurable and uncontrollable disease.After analysing the responses we had gathered under the “causes” domain in the survey questionnaire, we were able to identify patients’ beliefs on the underlying causes of ZCL. The contents relate basically to biological and/or ecological causes. The patients did not talk about irrational, supernatural and magical causes, as was the case with some patients in Colombia .ZCL patients think that living with the disease is a steady source of stress that forces them to develop anticipatory avoidance behaviour against rejection. They try to anticipate stigma primarily out of feelings of inferiority in society. These problems bring about a huge sense of injustice and anger to the point where there is little sympathy with other girls or women with ZCL. These difficulties can lead to depression and anxiety. The patients think their scars are degrading and this increases their feelings of shame. They describe their feeling of isolation, that is, they think subjectively that they are inferior to other members of society, that they are not fully fledged individuals, or that they have an "impaired identity" and suffer discrimination, which includes rejection and isolation from others. Anticipated stigma is a subjective experience that could negatively affect psychosocial and professional development and adjustment among patients. These results largely confirm the findings from similar studies conducted on patients with vitiligo .The psychological effects of ZCL tend to reduce as patients get older. The more educated a patient is, the more his knowledge of the disease develops, and the more he tends to analyse the consequences of the disease and display dramatic emotional reactions. A high level of education and high levels of knowledge are associated with greater cognitive sensitivity (perception of consequences) and emotional sensitivity .After calculating our correlation analysis, the results we obtained were consistent with the theoretical construct on illness perception in Leventhal’s model . We noteMoreover, there is a high level of consistency between perceptions of CL in the IPQ-R and stigma indices in the PLSI. Anticipation and avoidance of stress have a strong correlation with a patient’s perceptions of the "consequences", "coherence" and "emotional representation" of the disease. The more patients see ZCL as a disease with severe, unambiguous consequences that have harmful emotional effects, the more they tend to expect to be rejected by others, and the more they would be inclined to form avoidance attitudes towards others. These conclusions do not concern the relationships between these variables and the patient’s age, level of education, or knowledge of the disease.The correlation between the level of stigma and the number of lesions is not related to the location of these lesions on the face or elsewhere on the body. This rather surprising result confirms the work of Papadopoulos et al.  who repoOur results also suggest that the nature of stigma that patients experience are associated with certain general fears and anticipation of rejection, rather than the possible rejection that patient truly experience from society. However, this is not the case in some countries like Pakistan, where Afghan refugees with CL face stigma and are excluded from social groups , or in AThe respondents believe that ZCL has a generally negative impact on quality of life. But our results show that ZCL has differential impacts on the four domains of quality of life in the WHOQOL-26. The quality of life of ZCL patients is largely unsatisfactory in two domains: environmental quality of life and quality of mental life. The respondents are inclined to think that the environmental quality of life is the more degraded of the two. It includes availability of financial resources, freedom, security, access to and quality of housing, opportunities for acquiring new information and skills, opportunities for recreation and leisure, as well as the availability and quality of transport facilities. We are assuming that ZCL patients are not the only ones dissatisfied with this domain. The entire population shares this dissatisfaction with the environmental quality of life in the region of Sidi Bouzid.For the respondents, the quality of mental life is lower than physical quality of life and the quality of social life. It suggests that they are dissatisfied with their self-image and appearance, with the negative sentiment displayed towards them, with their self-esteem and their cognitive abilities . This latter result is frequently described in literature on skin infections .None of the domains on quality of life is closely correlated with the scores that reflect patients’ perception of ZCL in the IPQ-R. However, the quality of social life domain is significantly and negatively correlated to the "Anticipation and avoidance of stress" factor in the Psoriasis Life Stress Inventory (PLSI) questionnaire. The more a person expects to face rejection and stigma, the lower the quality of social life they have when it comes to personal relations and social support. These dynamics have been observed already in patients with psoriasis .ZCL got little attention for long because, apart from the aesthetic effects it has, the disease is neither contagious nor fatal, and causes no major health concerns. However, it can have adverse socio-economic effects. ZCL is a health problem in developing countries because of the social and economic challenges it causes . The disThese results show that public health officials, in planning control programmes, should pay attention to the gaps in knowledge perceptions, the vulnerability of women to cutaneous leishmaniasis, the ways of preventing their exposure and the psychological support to reduce the psychosocial effects .With the Leventhal model , we haveIt is crucial also to take preventive measures, given the importance of behavioural aspects in the onset of the disease. Byrne et al.  have desS1 Table(XLSX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.