Marinobacter lipolyticus, showed advantages over other lipases, being an enzyme active over a wide range of pH values and temperatures. The immobilized LipBL derivatives obtained and tested in regio- and enantioselective reactions, showed an excellent behavior in the production of free polyunsaturated fatty acids (PUFAs). On the other hand, the extremely halophilic bacterium, Salicola marasensis sp. IC10 showing lipase and protease activities, was studied for its ability to produce promising enzymes in terms of its resistance to temperature and salinity.Hydrolases constitute a class of enzymes widely distributed in nature from bacteria to higher eukaryotes. The halotolerance of many enzymes derived from halophilic bacteria can be exploited wherever enzymatic transformations are required to function under physical and chemical conditions, such as in the presence of organic solvents and extremes in temperature and salt content. In recent years, different screening programs have been performed in saline habitats in order to isolate and characterize novel enzymatic activities with different properties to those of conventional enzymes. Several halophilic hydrolases have been described, including amylases, lipases and proteases, and then used for biotechnological applications. Moreover, the discovery of biopolymer-degrading enzymes offers a new solution for the treatment of oilfield waste, where high temperature and salinity are typically found, while providing valuable information about heterotrophic processes in saline environments. In this work, we describe the results obtained in different screening programs specially focused on the diversity of halophiles showing hydrolytic activities in saline and hypersaline habitats, including the description of enzymes with special biochemical properties. The intracellular lipolytic enzyme LipBL, produced by the moderately halophilic bacterium  Microbial life can be found over a wide range of extreme conditions . Hypersaline environments constitute typical examples of environments with extreme conditions due to their high salinity, exposure to high and low temperatures, low oxygen conditions and in some cases, high pH values. Bacteria and Archaea are the most widely distributed organisms in these environments .The classification of Kushner and Kamekura  defines +, Na+, Cl−) to balance the osmotic pressure of the medium, and they have developed specific proteins that are stable and active in the presence of salts. In contrast, moderate halophiles accumulate in the cytoplasm high amounts of specific organic osmolytes, which function as osmoprotectants, providing osmotic balance without interfering with the normal metabolism of the cell [Halophiles have developed two different adaptive strategies to cope with the osmotic pressure induced by the high NaCl concentration of the normal environments they inhabit ,4. The hthe cell .In recent years, halophilic microorganisms have been explored for their biotechnological potential in different fields . The appw) produces conformation changes in the enzymes affecting the catalytic activity due to the reduced hydration. However, halophilic enzymes are active and stable in media with low water activity because even at low aw enough water is present to retain suitable charge distribution at the active site maintaining the conformation of the enzyme [In general, low water activity  displaying lipolytic activity was isolated and characterized. Strain SM19T is a Gram-negative rod that grows optimally in culture media containing 7.5% NaCl under aerobic conditions and was classified in the genus Marinobacter as a novel species Marinobacter lipolyticus sp. nov. [Sánchez-Porro et al.  showed tronments ,13. Amonsp. nov. .et al. [Salicola, Salinibacter and Pseudomonas). The genera Salinibacter and Salicola have been previously reported to compete with halophilic members of the Archaea in crystallizer ponds of different solar salterns [Salicola strain IC10 was selected for further studies for two main reasons, the potential biotechnological applications due to its dual hydrolytic activity (lipase and protease) and its use as a model for fundamental studies aimed to unravel the adaptations of halophilic enzymes, which allow them to be stable and active at high salt concentrations.Moreno et al.  studied salterns ,17. The et al. [Salicola as the predominant genus among Gram-negative isolates, in contrast with the study of Sanchez-Porro et al. [Halomonas. Among the Gram-positive hydrolase-producing isolates, representatives of the genera Virgibacillus and Thalassobacillus were predominant. On the other hand, strains of the genus Salinivibrio were not found, probably due to the higher concentration of salt in Howz Soltan sea site in comparison with those saline habitats studied in Spain.In a hypersaline lake in Iran, Rohban et al.  investigo et al. , accordiBacillus, Paenibacillus, Halobacterium and Aeromonas were described. One species of Bacillus was found as the highest protease producer and it was further studied. Other study, concerning lipolytic activity, investigated the presence of halophilic bacteria producing lipases from a Maharlu salt lake in Iran [Bacillus and Staphylococcus. Ghasemi and coworkers  performe in Iran . All stret al. [Alcanivorax, Bacillus, Cobetia, Halomonas, Methylarcula, Micrococcus, Myroides, Paracoccus, Planococcus, Pseudomonas, Psychrobacter, Sporosarcina, Sufflavibacter and Wangia. Bacteria of the γ-Proteobacteria lineage, especially those from the Halomonas and Psychrobacter groups, dominated in the culturable bacteria assemblage. This predominance could indicate that this population is ubiquitous in marine sediments, at least in the west Pacific and the distinct distribution of the other bacterial groups might indicate that their distributions could be restricted by certain environmental conditions.Deep-sea sediments have also been considered interesting habitats as source of novel enzymes, due to their extreme conditions; however, they have been practically unexplored on earth ,22,23,24et al.  screenedet al. [et al. [According to the studies of Sánchez-Porro et al.  and More [et al. , the str [et al.  suggesteIn a similar study, Cojoc and colleagues  elucidatet al. [et al. [et al. [et al. [Bacillaceae, comprising species of the genera Bacillus, Halobacillus and Thalassobacillus. Only two isolates were related to the Gram-negative bacteria Pseudomonas halophila [Halomonas organivorans [Salinicoccus roseus [Recently, in heavy-metal-contaminated soils with extreme salt conditions from the Atacama Desert, Moreno et al.  carried  [et al.  and More [et al. . In the  [et al. ,15. As r [et al. , most enalophila  and Halonivorans  and the s roseus .et al. [Bacillus. Khunt et al.  isolatedBacillus in most habitats. This genus is well known as an extracellular enzyme producer and many industrial processes use species of this genus for commercial production of enzymes [In summary, we can conclude that there is a wide taxonomic diversity of microorganisms showing hydrolytic activity in saline environments and in most cases multiple activities are present. Aerial distribution of the dormant spores probably explains the occurrence of  enzymes .Although halophilic enzymes are considered a novel alternative for use as biocatalysts in different industries, there are relatively few studies on halophilic enzymes, with some being based on their isolation and others on their characterization. However, it is important to thoroughly study these enzymes in order to use them in biotechnological processes ,34. OnlyBacterial lipolytic enzymes are valuable biocatalysts due to their broad substrate specificity and high chemo-, regio- and stereoselectivity ,38,39,40Marinobacter lipolyticus SM19 was isolated and characterized [Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80 °C and the pH optimum determined at 25 °C was pH 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited optimal activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested. For improving the stability and for use in industrial processes LipBL was immobilized in different supports [An interesting intracellular lipolytic enzyme produced by the moderately halophilic bacterium cterized . This ensupports . The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. The fish oil used in the experiment contained 18.6% of EPA, increasing to 27% after the treatment with LipBL-CNBr immobilized derivative, representing an increase of 45.2% of the total concentration, however, the content in DHA only increase from 12% to 12.5%, representing an increase of 4.2%. These results indicated that LipBL showed more selectivity towards the hydrolysis of DHA increasing EPA, being a good candidate for use at an industrial scale for the production of fish oils enriched in polyunsaturated fatty acids (PUFAs) ,49. Due Pseudoalteromonas ruthenica [P. ruthenica CP76 was detected at the end of the exponential growth phase at 37 °C, in media containing 7.5% salt and supplemented with sucrose (50 mM). Protease CP1 was purified from P. ruthenica CP76 using ion exchange and gel filtration chromatography. The purified enzyme was biochemically characterized, showing optimal activity at 55 °C, pH 8.5 and high tolerance to a wide range of NaCl concentrations (0 to 4 M NaCl). It has a molecular mass of 38 kDa and the activity was strongly inhibited by ethylenediamunetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF) and Pefabloc [Halobacillus karajensis strain MA-2. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion of the enzyme occurred at 34 °C, pH 8.0–8.5, and in the presence of gelatin. The maximum enzyme activity was obtained at pH values ranged from 8.0 to 10.0, with 55% and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by PMSF, Pefabloc SC and EDTA; indicating that this enzyme probably belongs to the subclass of serine metalloproteases. These findings suggested that the protease secreted by Halobacillus karajensis presents a great potential for biotechnological applications when alkaline conditions are required [Proteases constitute one of the most important groups of industrial enzymes, comprising currently the majority of worldwide enzyme sales. They have been widely used in industry for a long time, especially in washing detergent, baking, brewing, cheese industry and tanning industry ,52. Due uthenica . The maxPefabloc ,54. AnotNesterenkonia sp. strain F [2+, Rb+, Li+, Cs+, Mg2+ and Hg2+, whereas Fe2+, Cu2+, Zn2+ and Al2+ strongly inhibited the enzyme. Moreover, this α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol [Amylases constitute a group of interesting enzymes from the biotechnological point of view . Most instrain F . This enoethanol . Thalassobacillus sp. LY18 [2+, but greatly inhibited by EDTA, indicating it was a metalloenzyme. Complete inhibition by diethyl pyrocarbonate and β-mercaptoethanol revealed that histidine residue and disulfide bond were essential for enzyme catalysis. Furthermore, it showed high activity and stability in the presence of water-insoluble organic solvents with log P (ow) ≥ 2.13 [An extracellular halophilic α-amylase was purified from sp. LY18 . This en) ≥ 2.13 .Salicola marasensis IC10 produces an extracellular protease, designated Salipro, and at least an intracellular lipase, which was named LipL [S. marasensis IC10 is mainly located in the cytoplasmatic fraction. This enzyme is active in presence of different compounds as substrates: p-nitrophenyl butyrate, p-nitrophenylvalerate, p-nitrophenylcaprilate and p-nitrophenyldecanoate as well as 4-methylumbelliferone and the enzyme production is maximal at the end of the exponential phase. The effect of different factors on the lipolytic activity was analyzed and the maximum values were obtained in the presence of 1 M NaCl, although the tolerance was found in the range from 0 to 4 M NaCl, with 6 mM of betaine. The lipolytic activity increased in the presence of organic solvents as 1-buthanol, 2-buthanol and acetone , EDTA 1% (v/v) and metal ions as Ni2+ and Ca2+.On the other hand, there are some hydrolases characterized from extremely halophilic bacteria. The extremely halophilic bacterium med LipL . The lipSalicola sp. IC10 during growth was performed, finding the optimal conditions at pH 8.0, 40 °C and a medium with 15%–20% (w/v) NaCl. Thus, there is a correlation between the optimal conditions for cultivation of the strain and the maximum production of the proteolytic enzyme. This protease showed the capability to effectively catalyze the hydrolysis of various proteins. The most specific substrate to the enzyme was egg albumin, followed by gelatine (97% relative activity) [To identify the optimal conditions for protease production, the characterization of the intracellular fraction of Therefore, we can conclude that enzymes produced by halophilic bacteria show interesting properties for use in different industries.


Prader-Willi syndrome (PWS) is a rare multisystem genetic disorder demonstrating great variability with changing clinical features during patient’s life. It is characterized by severe hypotonia with poor sucking and feeding difficulties in early infancy, followed by excessive eating and gradual development of morbid obesity in later infancy or early childhood. The phenotype is most probably due to hypothalamic dysfunction which is also responsible for growth hormone (GH) and thyroid-stimulating hormone (TSH) deficiencies, central adrenal insufficiency and hypogonadism. The multidimensional problems of patients with PWS can be managed with multidisciplinary approach. Reduced GH secretion, low peak GH response to stimulation, decreased spontaneous GH secretion and low serum IGF-1 levels in PWS patients have been documented in many studies. GH therapy has multiple beneficial effects on growth and body composition, motor and mental development in PWS patients. The recommended dosage for GH is 0.5-1 mg/m2/day. GH therapy should not be started in the presence of obstructive sleep apnea syndrome, adenotonsillar hypertrophy, severe obesity and diabetes mellitus. GH treatment should be considered for patients with genetically confirmed PWS in conjunction with dietary, environmental and life-style measures. Prader-Willi syndrome (PWS) was first defined in 1956 by Swiss scientists ,3,4. In Pediatric endocrinologists, using a holistic approach, need to manage the age-related worsening obesity and associated complications in addition to the central hypothyroidism due to hypothalamo-hypophyseal dysfunction, the central adrenal insufficiency, hypogonadotropic hypogonadism and growth hormone (GH) deficiency in PWS patients.The present review will focus on the characteristic findings of patients with PWS, with special reference to GH and GH treatment.Clinical FindingsThe clinical signs of PWS vary by age group. Prenatal period: Decreased fetal activity, polyhydramnios, breech presentation and abnormal position of the hands and feet on ultrasonography in the 3rd trimester (elbows in flexion and feet in dorsi extension) constitute the characteristic findings of PWS in this period of life , dermal and ocular depigmentation are the main findings , abnormal body composition , reduction in resting energy expenditure, short stature, behavior problems  are the main findings in this age group , lack of pubertal height gain, morbid obesity and related complications , osteoporosis, scoliosis (30%), behavior problems, epilepsy (25%) are the main findings in this age group  levels, in PWS patients, IGF-1 levels are also decreased ,12. The GH treatment in PWS was first applied in 1990s. Treatment outcomes and benefits for these patients were presented in the national conference of the Prader-Willi Syndrome Association which took place in 1998-1999 in the United States of America . The FedEffects of GH Therapy GH has beneficial effects on linear growth, body composition, basal energy consumption, muscle strength, exercise tolerance, decrease in free fat mass, bone density, lipid profile and physical and cognitive functions ,22,23,24The results of the Bakker et al’s study , conductIn a study that compared the final heights in PWS patients who received GH with those who did not, final height was 171±8 cm in boys and 158±4 cm in girls in the treatment group, whereas it was 154±9 cm in boys and 144±6 cm in girls in the group without treatment .Pretreatment EvaluationBefore starting GH therapy, one should perform anthropometric evaluation and systemic examination including height, height SD score (SDS), body weight, body mass index (BMI) SDS, waist/hip ratio, bone age and pubertal staging assessment. Moreover, central hypothyroidism and adrenal insufficiency should certainly be explored and L-thyroxine therapy should be started in case hypothyroidism is detected. Presence of adrenal insufficiency should be explored by estimation of serum adrenocorticotropic hormone (ACTH)-cortisol levels in blood samples obtained in the early morning and by low-dose ACTH stimulation test when needed ,7,8,9,10Efficacy of GH DosageThe best response to GH in PWS patient is observed in the first 12 months of treatment. Response to GH therapy changes according to age of starting treatment, degree of growth retardation and duration of treatment. Improvement in linear growth, bone density and body composition continues in those who receive therapy for 5 years. Nevertheless, it has been reported that long-term therapy may always not result in complete improvement in body composition, but stabilization is provided. Although early treatment is important for the improvement in body composition, generally, in practice, it is possible to start treatment only after age 2 years ,23,24,32Treatment can be started in a dose of 0.034 mg/kg/day (0.24 mg/kg/week) in infants and toddlers. IGF-1 and IGFBP-3 levels are used to specify the dose of GH therapy. The dose can be increased up to 1 mg/m2/day so that IGF-1 levels are between the ranges +1 SD and +2 SD depending on age ,9,33. ThBenefits of continuing GH therapy in adulthood remains unclear. However, a moderate improvement has been observed in body composition and cognitive functions in PWS patients who received treatment in adulthood but not in childhood, suggesting that beneficial effects are still present even after the epiphyses are fused 26,33,34). Similar to the dose used for GH deficiency in adults, a daily dose of 0.1-0.2 mg is recommended; it should be adjusted so that the IGF-1 level is maintained between 0 SDS and - 2 SDS to reduce the probability of adverse events ,26,33,34,33,34. SContraindications to GH TreatmentSevere obesity, uncontrolled diabetes mellitus, untreated severe OSA, active cancer and psychosis are the accepted contraindications for GH therapy in PWS patients ,4,7,9.Adverse Events and SafetyAs true for other patients receiving GH, patients with PWS should be monitored for potential adverse events by periodic complete blood count, thyroid function evaluation and evaluation of glucose metabolism. In addition, with the prediction that GH therapy will enhance adenotonsillar hypertrophy in PWS, the patients should be questioned for shortness of breath, snoring and sleep apnea and, if necessary, should be evaluated via polysomnography on the 3rd and 6th months after treatment. It should be kept in mind during monitoring that glucose metabolism disorders and type 2 diabetes are expected to be more common in PWS patients due to concomitant obesity. However, it has been reported that GH treatment showed no adverse effects on glucose parameters and lipid profile in PWS patients . ScoliosWorldwide, 28 deaths have been reported among PWS patients who had been receiving GH. Common characteristics of these patients were that they generally died due to respiratory problems or respiratory system infections in the first 9 months of treatment and that these patients were morbid obese ,9,28,35.Whilst GH therapy leads to adenotonsillar hypertrophy and obstructive apnea on the one hand, on the other hand, it improves central hypoventilation via direct effect on hypothalamic function ,21,32,35The patient should be monitored periodically over the course of GH therapy. Polysomnography and ear-nose-throat examination should be performed on the 6th week, 3rd month and on the 6th month of treatment and whenever necessary . Fasting blood glucose, OGTT, IGF-1, free thyroxine and thyroid-stimulating hormone testing should be performed on the first month of treatment and/or whenever necessary and orthopedic examination and X-ray imaging of the spine should be performed in case of necessity .In conclusion, GH treatment therapy appears to be of benefit in selected cases of PWS, provided that these patients are monitored carefully and precisely. Diet and physical activity should continue over the course of treatment. It will be reasonable to discontinue the treatment in the presence of infection and respiratory obstruction. Steroid replacement in stress doses needs to be given in case of infections.


The aim of our study was to investigate the relationship between cancer-related fatigue and clinical parameters, and the effect factors of fatigue for the prostate cancer patients. Long-term follow-up is performed using the Fatigue Symptom Inventory before treatment (A), at the end of intensity-modulated radiotherapy (B), and 3 months (C), 12 months (D), 24 months (E), 36 months (F), and 48 months (G) after the end of intensity-modulated radiotherapy. Three dimensions of fatigue are assessed during follow-up: severity, perceived interference with quality of life, and duration in the past week. In all, 97 patients with locally advanced prostate cancer were enrolled in the study. Median follow-up time was 43.9 months. The fatigue index was significantly higher in the prostate-specific antigen >20 ng/mL, Gleason score >8, the Eastern Cooperative Oncology Group scores, and the higher education. The most severe fatigue occurred at time points B and C. The score for duration of fatigue fluctuated across the time points, with significantly increased scores at time points D, E, and F.In conclusion, we show that cancer-related fatigue is the important symptom which affects the quality of life for the prostate cancer patients. For patients with locally advanced prostate cancer with a high Eastern Cooperative Oncology Group score, a Gleason score of >8 points, prostate-specific antigen levels of >20 ng/mL, and high education, attention should be paid to the interference of fatigue with quality of life, especially general level of activity, ability to concentrate, and mood, after radiotherapy combined with hormonal therapy. Our previous follow-up of patients with locally advanced prostate cancer for up to 8 years after radiotherapy combined with hormonal therapy found that fatigue was an important clinical symptom affecting QOL. The questionnaire can be used as a primary investigation method to make intuitive and effective analysis of survival in patients with prostate cancer. Fatigue has not been investigated in previous studies on QOL in patients with prostate cancer after radiotherapy.,8,9 In the present study, patients receiving radiotherapy combined with hormonal therapy were followed up by questionnaire, to evaluate the effect of this treatment on fatigue, and to investigate the relationship between cancer-related fatigue (CRF) and clinical parameters.Prostate cancer is the most frequent malignant cancer in American men, and also has an increasing incidence in developing countries, including China.22.1Patients with prostate cancer, confirmed by histopathology, who were admitted to Fuzhou General Hospital of Nanjing Military Command from February 2008 to December 2012, were selected. Among them, those who had an Eastern Cooperative Oncology Group (ECOG) score ≤2 points, no distant organ metastasis confirmed by imaging, and no previous history of cancer, and met any of the following criteria, were included: (1) a Gleason score of 8 to 10 points; (2) serum prostate-specific antigen (PSA) of ≥20 ng/mL; (3) prostate cancer staged as T3 or T4 by magnetic resonance imaging (MRI) , with or without regional lymph node metastasis.Approval for the study was given by the FuZhou General Hospital Research Ethics Committee. Eligible men were identified from the clinic 7 days before their appointment.2.2nd was defined as pelvic lymph node (minor axis≥1.0 cm). The following normal organs were also delineated: rectum, bladder, femoral head, small intestine, and colon. A total dose of PTV and GTVnd were 72.6 Gy/2.2 Gy/6+w. A total dose of prophylactic irradiation of the pelvic lymph node drainage area was 56.1 Gy/2.2 Gy/6+w.Supine position was used. Computed tomography (CT)-enhanced scan images (2.5 mm thick) were ranged from the edge of L2 to 10 cm below the edge of sciatic. The CT-scan images and pelvic MR images fused together. Clinical target volume included entire prostate, seminal vesicle, and pelvic lymph node drainage area. The pelvic lymph node drainage area was defined according to the recommendation of Radiation Therapy Oncology Group (RTOG). Gross tumor volume (GTV)2.3Hormonal therapy was performed from the first day of intensity-modulated radiotherapy (IMRT) with 50 mg of oral Casodex once daily and 3.6 mg of Zoladex via subcutaneous injection every 28 days for 30 months.2.4 The questionnaire included 13 items on 3 dimensions: severity, perceived interference with QOL, and duration in the past week. Severity was measured by assessing most, least, and average fatigue in the past week, and also current fatigue. Perceived interference with QOL was measured by assessing interference with general level of activity, ability to bathe and dress, normal work activity, ability to concentrate, relations with others, enjoyment of life, and mood. Duration was measured as the number of days in the past week that respondents felt fatigued, and also the extent of each day on average they felt fatigued. A higher score indicates more severe fatigue. The questionnaire was administered before treatment (A), the day when IMRT ended (B), and 3 months (C), 12 months (D), 24 months (E), 36 months (F), and 48 months (G) after the end of IMRT.The Fatigue Symptom Inventory (FSI) was used.2.5At time points A, B, and C, the questionnaire was completed by the doctor and patient together. At the other time points, the questionnaire was mailed to the patient. If a letter in reply was not received within 4 weeks, the questionnaire was completed by telephone. A questionnaire was excluded if none of the items in any dimension was scored. Patients who had organ metastasis or biochemical recurrence, were lost to follow-up, or died during follow-up were removed. If all items of the questionnaire were answered, scores for each dimension were calculated from the actual scores. If only part of the items were answered, scores for each dimension were calculated by averaging the nonmissing scores within the dimension.2.6t test. Changes in each dimension were assessed compared with time point A. The rate of change in a single sample was compared using the Wilcoxon rank-sum test. The scores at each time point were compared with those at time point A using the Mann–Whitney test. Two-tailed P < 0.05 was considered statistically significant. Line charts were produced using Graphpad Prism 5.All data were analyzed using SPSS 13.0. Clinical data were presented as mean ± standard deviation (SD). Scores for each dimension in the FSI questionnaire were presented as mean values. Normality tests were performed using the chi-square test. Differences between groups were determined by the descriptive 33.1A total of 126 patients with prostate cancer met the inclusion criteria were admitted to the hospital. Of them, 97 (76.9%) agreed to participate in the long-term questionnaire follow-up. General information and IMRT-related data of patients enrolled are shown in Table 3.2P > 0.05). No significant difference was noted in fatigue index between stages T3 and T4 (P > 0.05). The fatigue index was higher with PSA >20 ng/mL than PSA ≤20 ng/mL, indicating that fatigue is associated with PSA levels. The ECOG score was significantly positively correlated with the fatigue index (P < 0.05). The higher the Gleason score and education level, the higher the fatigue index. The relationship between fatigue index and clinical parameters is shown in Table No significant difference was noted in baseline assessment among the 3 age groups  2014 Practice Guidelines for CRF, it is a distressing, persistent, subjective sense of physical, emotional, and/or cognitive tiredness or exhaustion related to cancer or cancer treatment that is not proportional to recent activity that interferes with usual functioning. It must be managed and assessed at the beginning of treatment. PSA should not be used as a routine diagnostic screening tool for patients aged <50 years and >75 years, although it can provide a survival benefit for older men.–16 The median age of patients enrolled in this study was 64 years. Prostate cancer is mostly diagnosed in men aged >50 years, and locally advanced prostate cancer mostly presents at an older age. Age is a factor in development of prostate cancer, but it is not related to fatigue; and the higher the ECOG score, the more severe the fatigue, which is consistent with the conclusion of study by Blackhall et al. This indicates that fatigue is not related to body function degeneration, but merely is a subjective feeling. The ECOG score verifies the reliability of FSI indirectly. Physical condition is closely related to the fatigue index, and is also the main factor affecting QOL. Timely assessment and intervention of fatigue can improve physical condition, thus improving QOL. The prostate tumor burden increased with the progression of clinical stage, but no significant differences in the fatigue index was observed. However, there were significant differences in the stratified PSA levels and Gleason score. This is consistent with the findings about fatigue in breast cancer, but contrary to our previous findings about fatigue in nasopharynx cancer. The pathogenesis of fatigue in hormonal therapy-related tumors may be associated with hormone disorders. Solid tumors may be associated with physiological reactions caused by changes in tumor burden. It needs to be further verified whether CRF is related to disorders of inflammatory regulators and excessive release of inflammatory cytokines acting on the endocrine system, and whether severity of fatigue is related to androgen deprivation. Patients with higher education have more knowledge of prostate cancer, but do not deeply understand the pathogenesis and prognosis of the disease, which to some extent increases the possibility of fatigue. By contrast, patients with lower education have better treatment compliance, which, to some extent, reduces the psychological burden that may induce fatigue.Because of possible overdiagnosis, At present, studies on CRF focus on breast cancer and lung cancer, and the relevant large clinical research or meta-analysis focuses on how it can be treated.–26 Few studies on prostate CRF have been reported, and interference of fatigue with QOL have not been reported. A questionnaire was administered to patients with locally advanced prostate cancer at selected time points to investigate the interference of fatigue with QOL after radiotherapy combined with hormonal therapy. The severity of fatigue did not change significantly among each time point, but the most severe fatigue was observed 3 months and 1 year after the end of IMRT.In general, 60% to 100% of cancer patients have varying degrees of fatigue, a higher incidence in those receiving active anticancer treatment. Androgen deprivation may increase the severity of fatigue. It needs to be further verified whether the development of these fatigue-related symptoms is related to dysregulation of cortisol secretion after androgen deprivation. The patients enrolled experienced serious interference with QOL, especially general level of activity, ability to concentrate, and mood, from 1 year after treatment, until the end of follow-up. They experienced fluctuations in general level of activity, ability to concentrate, and mood due to the treatment of disease itself, and also hormonal dysfunction caused by castration level of testosterone after hormonal therapy. Intermittent hormonal therapy seems to provide a better QOL, including mood, compared with continuous hormonal therapy. More clinical data are required to investigate whether IMRT combined with intermittent hormonal therapy can improve fatigue, including changes in the FSI items.Our previous study found that hormonal function was a main factor affecting QOL in the first year after concurrent chemoradiotherapy.4.1The FSI mainly assesses interference of fatigue with QOL in the past week. Precise assessment of fatigue cannot be made during the long-term survival of patients with prostate cancer due to the poor timeliness and limited follow-up time points. The FSI items describe the specific impact of fatigue only, but do not allow stratification of fatigue. In addition, a total of 127 questionnaires were not fully completed during the follow-up of 48 months, which caused a deviation in the assessment of fatigue.5For patients with locally advanced prostate cancer with a high ECOG score, a Gleason score of >8 points, PSA levels of >20 ng/mL, and high education, attention should be paid to the interference of fatigue with QOL, especially general level of activity, ability to concentrate, and mood, after radiotherapy combined with hormonal therapy.


Lytechinus variegatus gut microbiome and its culture environment using NextGen amplicon sequencing of the V4 segment of the 16S rRNA gene, and downstream bioinformatics tools. Overall, the gut and tank water was dominated by Proteobacteria, whereas the feed consisted of a co-occurrence of Proteobacteria and Firmicutes at a high abundance. The gut tissue represented Epsilonproteobacteria as dominant, with order Campylobacterales at the highest relative abundance (>95%). However, the pharynx tissue was dominated by class Alphaproteobacteria. The gut digesta and egested fecal pellets had a high abundance of class Gammaproteobacteria, from which Vibrio was found to be the primary genus, and Epsilonproteobacteria, with genus Arcobacter occurring at a moderate level. At the class level, the tank water was dominated by Gammaproteobacteria, and the feed by Alphaproteobacteria. Multi-Dimensional Scaling analysis showed that the microbial community of the gut tissue clustered together, as did the pharynx tissue to the feed. The gut digesta and egested fecal pellets showed a similarity relationship to the tank water. Further analysis of Campylobacterales at a lower taxonomic level using the oligotyping method revealed 37 unique types across the 10 samples, where Oligotype 1 was primarily represented in the gut tissue. BLAST analysis identified Oligotype 1 to be Arcobacter sp., Sulfuricurvum sp., and Arcobacter bivalviorum at an identity level >90%. This study showed that although distinct microbial communities are evident across multiple components of the sea urchin gut ecosystem, there is a noticeable correlation between the overall microbial communities of the gut with the sea urchin L. variegatus culture environment.In this study, we have examined the bacterial community composition of the laboratory cultured sea urchin  Recent advancements in the discovery of gut microbial communities in the animal kingdom has offered a glimpse into the supportive role of various microbial taxa in growth, development, metabolism, and digestive physiology of the host, as well as protection from predators, and adaptive fitness to the environment they inhabit , and transported in seawater to a recirculating salt water system within the laboratory at the University of Alabama at Birmingham. Water conditions were maintained at 22 ± 2°C, with a pH of 8.2 ± 0.2 and a salinity of 32 ± 1 ppt. using synthetic sea salt  added to treated municipal water. Prior to use, municipal water was filtered by 5 micron sediment, charcoal, and reverse osmosis membranes, followed by an ion exchange resin, with the final addition of Instant Ocean sea salts to achieve the desired salinity of 32 ppt. Water was replaced in the recirculating seawater culture system at a rate of ca. 5% water exchange per day. Water quality was maintained using a dolomite mechanical gravel filter, followed by biological filtration using Bioballs biological media , and UV sterilization of water exiting the recirculating filter. The sea urchins were fed a formulated feed . Sample collection from each sea urchin began 22 ± 1 h after feeding. Prior to dissection, the sea urchins were relocated to a temporary container containing sterile (autoclaved at 121°C for 20 min at 103.42 kPa) sea water, from which the egested fecal pellets from each sea urchin were collected. After fecal pellet collection, the sea urchins were then removed from the water and dissected immediately. Briefly, an incision was made with sterilized scissors into the test surrounding the peristomial membrane, and a dissection was performed circumnavigating the mouth. The peristomial membrane, along with the nested mouth (the Aristotle's lantern) , which included the esophagus, stomach, and intestine Holland, , was the2O to a total reaction volume of 50 μL. The PCR cycling parameters were as follows: initial denature 94°C for 1 min; 32 cycles of amplification in which each cycle consisted of 94°C for 30 s, 50°C for 1 min, 65°C for 1 min; followed by final extension of 65°C for 3 min; then a final hold at 4°C. Following PCR amplification of the targeted gene, the entire PCR reaction was electrophoresed on a 1.0% (w/v) Tris-borate-EDTA/agarose gel. The PCR product (approximately 380 bp predicted product size) was visualized by UV illumination. The amplified DNA band was excised with a sterile scalpel, and purified from the agarose matrix using QIAquick Gel Extraction Kit according to manufacturer's instructions .Microbial community DNA was isolated using the Fecal DNA isolation kit from Zymo Research  following the manufacturer's instructions. Once the sample DNA was prepared, PCR was used with unique bar coded primers to amplify the hyper variable region 4 (V4) of the 16S rRNA gene, to create an amplicon library from metacommunity DNA samples . The FASTQ files were subjected to quality assessment using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), prior to merging and trimming of the raw sequence data, which was followed by quality filtering using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Since the overlap between the paired reads from each 16S fragment was approximately 245 bases, the overlapping paired end regions were merged to generate a single high quality read, using the “fastq_mergepairs” module of USEARCH   was trained using the Greengenes (v13.8) 16S rRNA database (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) . The raw sequence files from this study are deposited in the NCBI SRA (http://www.ncbi.nlm.nih.gov/sra), under the accession number SRP062365.The PCR products were sequenced using the NextGen sequencing Illumina MiSeq platform .Oligotyping utilizes informative nucleotide variations between similarly clustered reads to designate an oligotype identity . Discrete OTU counts per sample were standardized, and then transformed to the square root values  of the sea urchin microbiome and the culture environment was determined using QIIME (v1.7.0). The alpha-diversity was estimated using observed OTUs, Shannon diversity index Shannon, , and SimA total of 1,481,476 raw sequence reads of the V4 segment of the 16S rRNA gene from 10 samples of the two sea urchin (UR1 and UR2) gastrointestinal tracts, feeds, and tank water, were generated on an Illumina Miseq sequencing platform Table .Arcobacter, Mycoplana, and Vibrio appeared as the highly represented genera from phylum Proteobacteria. Phylum Firmicutes was represented by a high relative abundance of the genera Bacillus and Allobaculum.The relative abundances of taxa identified to the most resolvable taxa  across all 10 samples are elaborated in Figure Agarivorans, Arcobacter, Shewanella, and Vibrio, all of which belonging to phylum Proteobacteria. The bacterial composition in the egested fecal pellets consisted of many of the same taxa observed in the gut digesta. In the egested fecal pellets, Proteobacteria accounted for the highest abundance, and at the class level, Gammaproteobacteria was dominant. At the genus level, Agarivorans, Arcobacter, Shewanella, and Vibrio were detected as dominant taxa.The gut digesta consisted mainly of bacteria belonging to phylum Proteobacteria, with class Gammaproteobacteria being distinguishably elevated. The dominant genera were Agrobacterium, Acinetobacter, Limnohabitans, and Mycoplana were observed. From phylum Firmicutes, order Lactobacillales dominated in the feed, and at the genus level, Lactobacillus, Lactococcus, Leuconostoc, and Streptococcus were observed. The microbial composition of the tank water was found to be more diverse as compared to the other samples. Of the represented phyla, Proteobacteria was found to be dominant, followed by Chloroflexi, and to a lesser extent Bacteroidetes. Classes Gamma- and Alphaproteobacteria were dominant, and at the order level, Alteromonadales and Vibrionales were represented at relatively high abundances. In addition, significant abundances of genera Arcobacter, Agarivorans, Shewanella, Pseudoalteromonas, and Vibrio were identified within phylum Proteobacteria. For all samples, the taxonomic groups identified at the genus level have been elaborated in Supplementary Table The microbiota of the sea urchin feed consisted of phylum Proteobacteria, as well as Firmicutes at the highest abundance. Classes Alpha- and Betaproteobacteria were dominant in the feed, and at the genus level, Arcobacter sp. clone , Arcobacter bivalviorum , Sulfuricurvum sp. , and an uncultured bacterium clone  reached or approached a plateau, indicating that a sufficient sequencing depth was used to assess community diversity . In a previous study, Epsilonproteobacteria clones identified as Arcobacter sp. were found to be associated with marine organisms, including shrimp species (Rimicaris exoculata) and the Chilean oyster (Tiostrea chilensis) (Romero et al., Further analysis of the lower level of taxonomic groups within Campylobacterales showed 37 oligotypes across all ten samples, with Oligotype 1 displaying a dominant presence in the gut tissue Figure . This suVibrio of family Vibrionaceae (Figure Vibrio, Arcobacter and Agarivorans, genera later determined by NextGen sequencing using the Illumina MiSeq sequencing platform (Supplementary Figure Vibrio to the sea urchin Strongylocentrotus droebachiensis, specifically nitrogenase activity, which is necessary for nitrogen fixing in the assimilation of proteins in sea urchin gonad (Fong and Mann, As food enters the digestive tract of sea urchins, it is enveloped in a mucosal film that remains intact even after egestion, as a microbial-enriched fecal pellet (Sauchyn et al., e Figure . In as ee Figure . SimilarVibrio species, which were not observed to be significant in the gut tissue (Figure P. lividus (Meziti et al., L. variegatus gut ecosystem, as well as its culture environments, using NextGen sequencing and bioinformatics to achieve taxonomic coverage at the highest level. Future evaluation of the functional metagenomics of the gut microbiome of L. variegatus is warranted to establish the role of the microbial community associated with the digestive physiology, nutritional and other health benefits of this animal.Trends of microbial ecology in the sea urchin have been suggested by Guerinot and Patriquin , who proe Figure . Moreovee Figure . Moreovee Figure , suggeste Figure , a trende Figure  in P. liThe 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.


Although Colombia has witnessed an important decrease in malaria transmission, the disease remains a public health problem with an estimated ~10 million people currently living in areas with malaria risk and ~61,000 cases reported in 2012. This study aimed to determine and compare the level of knowledge, attitudes and practices (KAP) about malaria in three endemic communities of Colombia to provide the knowledge framework for development of new intervention strategies for malaria elimination.A cross-sectional KAP survey was conducted in the municipalities of Tierralta, Buenaventura and Tumaco, categorized according to high risk (HR) and moderate risk (MR) based on the annual parasite index (API). Surveys were managed using REDCap and analysed using MATLAB and GraphPad Prism.A total of 267 residents, mostly women (74%) were surveyed. Although no differences were observed on the knowledge of classical malaria symptoms between HR and MR regions, significant differences were found in knowledge and attitudes about transmission mechanisms, anti-malarial use and malaria diagnosis. Most responders in both regions  indicated use of insecticide-treated nets (ITNs) to protect themselves from malaria, and 75.5% of responders in HR indicated they did nothing to prevent malaria transmission outdoors. Despite a high level of knowledge in the study regions, significant gaps persisted relating to practices. Self-medication and poor adherence to treatment, as well as lack of both indoor and outdoor vector control measures, were significantly associated with higher malaria risk.Although significant efforts are currently being made by the Ministry of Health to use community education as one of the main components of the control strategy, these generic education programmes may not be applicable to all endemic regions of Colombia given the substantial geographic, ethnic and cultural diversity. Malaria causes over 207 million clinical cases and ~627,000 deaths worldwide every year representing an enormous global, social and economic burden. AlthougAlthough previous studies have indicated that malaria risk in both rural and urban endemic areas of Colombia is associated with factors such as age, number of people per household, occupation and level of education-8, regioPrevention and control activities developed by the National Malaria Control Programme (NMCP) of Colombia are mainly focused on early diagnosis and prompt treatment; distribution of long-lasting, insecticide-treated nets (LLINs); and education programmes. Despite these activities, the milestones proposed by the Ministry of Health (MoH) for reducing morbidity by 70% and eliminating urban malaria transmission by 2021 have notTwo of the study areas, Tierralta (HR) and Buenaventura (MR), are currently the subject of active control activities developed jointly between the NMCP and a project sponsored by the Global Fund for AIDS, Tuberculosis and Malaria (GFATM) where education is one of the major components. The ongoing education effort consists of distributing booklets, videos, shirts, school bags, and other materials targeting children; and games, murals, and advertisements that target adults.Because malaria is associated with poverty, most enIn view of the limited knowledge, attitudes and practices (KAP) studies in Colombia,8,14, anPlasmodium vivax (82.4%) followed by Plasmodium falciparum (17.4%), and 0.2% of mixed malaria infections. The sentinel sites selected in this region were Tuis Tuis and La Union.Sites selected for this survey included Tierralta, Buenaventura and Tumaco Figure . Tierralmestizo. Malaria occurs throughout the year, with two small seasonal transmission peaks from April to May and September to October[P. vivax (85%) followed by P. falciparum (15%). The sentinel sites selected in this region were Punta Soldado, Zacarias and La Delfina.Buenaventura is located on the Pacific coast in the department of Valle in the western part of Colombia, at 7 masl, covering an area of 6,078 sq km, with an average temperature of 28°C. The region consists of a tropical rainforest with a relative humidity of 85% and ~8,000 mm of annual rainfall. Its population of ~350,000 inhabitants is predominantly Afro-descendant (72.4%), with 13.4% considered white, 8.5% indigenous, and 5.7%  October. The prePlasmodium falciparum has a prevalence of 79.2% and 20.8% P. vivax. The sentinel sites selected in this region were Robles, Candelilla and Bucheli.Tumaco is located in the department of Nariño, in the south-western region of the country, near the border with Ecuador, at 2 masl, covering an area of 3,778 sq km, with an average temperature of 28°C. The population of Tumaco, estimated at 187,084 inhabitants, is composed mainly of Afro-descendants (88%). The population was divided into two groups according to the API recorded in the last five years (2008–2012), HR areas corresponded to API ≥10, whereas MR were areas with 10 > API > 1. Based on these categories, Tierralta (Córdoba) with API of 44.0 cases/1,000 habitants was considered an HR area, whereas Buenaventura  and Tumaco (Nariño), with API of 6.0 cases/1,000 habitants and 7.7 cases/1,000 habitants, respectively, were considered MR areas.The study corresponded to a cross-sectional survey, conducted between October and December 2011 in eight sentinel sites selected based on API and socio-demographic characteristics. The study was developed in two steps: the first, a door-to-door census of the entire population of each sentinel site; and the second, a survey using a structured questionnaire carried out by trained field workers. The survey was directed to heads of households in a subset of randomly selected houses. The same team of trained physicians accompanied the local teams to all eight sentinel sites. The trial was conducted according to Declaration of Helsinki principles, International Conference on Harmonization, ICH E-6 Guidelines for Good Clinical Practices and all pertinent Colombian regulations. The study protocol was approved by Institutional Review Boards (IRB) of the Malaria Vaccine and Drug Development Center–MVDC  (Number: 009). Written informed consent (IC) was obtained from each volunteer at enrollment.The questionnaire had a total of 41 questions divided as follows: 12 questions on knowledge of the malaria vector, symptoms, treatment, diagnosis, and prevention measures; ten questions on attitudes towards transmission, prevention and diagnosis; and, nine questions on practices such as the use of insecticide-treated nets (ITN), and thick blood smear (TBS) diagnosis and treatment. For assessment of socio-demographic characteristics, ten questions on public services, education level, ethnicity, and employment were included.Data entry: information was captured in the field on paper-based case report forms (CRF). Data were digitized using REDCap (version 4.1) and imported into MATLAB (version 2011b) for analysis. The number of CRFs per location was: Buenaventura, 105; Tierralta, 53; Tumaco, 109. The MATLAB script used for analysis was provided in Additional fileq less than the total number p of all CRFs for each location. Since during the QC procedure all errors found were corrected, it is possible to calculate the number of iterations of QC needed to bring the error below an acceptable threshold. During the first iteration of QC,q isn iterations of QC, the percentage of total error isTa represent now the target QA threshold of acceptable error. Then, the number n of iterations of QC needed to bring down the error to acceptable levels isTa for this study was established at 1%. While this procedure might seem unnecessary for such a small sample, it lays the foundation for more comprehensive studies that will be conducted in the near future as a continuation of the present study.The data quality assurance (QA) procedure consisted of setting a quality control (QC) sample of size Using descriptive statistics, the general characteristics of communities, families, and individuals admitted to the study were established. Measures of central tendency and dispersion were calculated for quantitative characteristics, whereas absolute frequencies as well as confidence intervals were used for qualitative characteristics.For each question, association tests were performed using the Chi-square test with significant differences set at p-value <0.05. Some questions in the CRFs were used to determine the correlation of malaria incidence and KAP. For the purpose of analysis, incidence was estimated taking into account the records of the NMCP, which were based on microscopic analyses of active and passive case detections.A total of 267 houses inhabited by 1,170 residents were surveyed; 214 houses were located in MR, and 53 in HR. In MR areas (Tumaco and Buenaventura), 13.2% of responders reported at least one malaria episode in the last year in the household, whereas in the HR area  this proportion was found to be 25.2%. This information was used for association analysis.The surveys were administered to heads of households, who in most cases were women . Most of the participants were housewives ; some were students or had other occupations Table . A smallA higher percentage of the population from MR areas (37.4%) than HR areas (9.4%) had some level of secondary education; 4.2% in MR areas, and 0% in HR areas had technical education  knew that malaria could be transmitted through blood transfusion. In both groups, fever, headache and chills were identified as the first symptoms detected in a person with malaria. Other recognized symptoms were myalgia and arthralgia, weakness and sweating; no significant differences were found between the two groups. Additionally, ~90% in both groups reported to have visited a local clinic with a microscopist or other health officer when they had malaria symptoms ; however a small proportion (2.8%) of responders from MR areas believed that home treatments were more suitable to cure the disease, while in HR areas nobody believed this; and 1.9% indicated that malaria treatment was not necessary. With respect to completing drug treatment in MR areas, 82.2% knew that interrupting malaria treatment may lead to death, whereas in HR areas only 15.1% knew this (p =0.000). In both areas, the community health promoter was identified by responders as responsible for malaria control  (p = 0.174). For 66.4% of responders from MR areas, the main source of knowledge on malaria was friends, compared with 34.0% in HR areas. In contrast, in HR areas it was found that family played an important role (52.8%) as a source of knowledge on malaria, while in MR areas this was less prevalent (29.4%). This factor was significantly different between the responders of the two areas (p = 0.001). Surprisingly, few responders felt that the government or health promoters had taught them about malaria.With regard to intervention measures for indoor prevention and vector control, a higher percentage of responders from HR areas (86.8%) mentioned the use of ITNs compared with responders from MR areas (68.2%). Despite MoH recommend to avoid indoor residual spraying (IRS) in areas with coverage of ITN, in MR areas, 40.2% of responders knew that indoor residual spraying (IRS) was a prevention measure compared with only 3.8% in HR areas (p < 0.000). Regarding outdoor vector control measures, a high percentage of people in MR areas knew that the presence of long grass (34.6%) and standing water (39.7%) should be avoided. Use of insecticide spraying (28.5%) in order to significantly reduce mosquito populations was recognized, whereas in HR areas knowledge of these three features was minimal . This knowledge was significantly different between the responders from the two areas. In HR areas 62.3% did not respond or did not know the availability of outdoor malaria prevention measures.In MR areas 45.8% of responders believed that getting malaria was normal, whereas only 11.3% in HR areas believed it was normal  indicated the use of INT for malaria protection; further, 26.2% of responders from MR areas, but none from HR areas, reported using home spray . Likewise, no correlation was found between malaria prevalence in the three municipalities and the knowledge about use of IRS to reduce malaria transmission. This knowledge was related to malaria prevalence in MR areas (p < 0.050).A high proportion of responders in both areas  reported using ITNs because they knew it prevented malaria transmission, however no significant differences were found between the use of ITNs and having malaria. Having ponds or standing water around the house was significantly associated with having suffered malaria over the last year (p < 0.050) in MR areas but not in HR areas: 36.4% of responders in MR areas and 4.8% in HR areas knew that avoiding standing water could reduce malaria transmission.This study was conducted to identify the KAP related to malaria in endemic areas of Colombia. Significant differences in the level of education in the two group categories were found. HR areas showed a higher proportion of illiteracy than in MR areas (p = 0.000). Furthermore, the level of primary education was higher in HR areas (64.2%) than in MR (47.7%) (p = 0.031); however, the overall education level was higher in MR areas. Correlation between the level of formal education and malaria risk was not found, probably due to the overall high knowledge level in both areas. Most people in both regions stated that they had learned about malaria from their families (33.6%) or friends (59.0%). Previous reports indicating that the level of literacy has a direct correlation with best malaria practices,17, and Similar to other reports-27, in tOther studies carried out in Latin American and Caribbean countries have investigated knowledge of the role of mosquitoes in malaria transmission. In a previous study in Haiti, despite the high level of knowledge about the role of mosquitoes in malaria transmission (61.8%), poor ownership and use of ITNs was observed; no significant association was found between education and correct knowledge. LikewisAbout half of the population interviewed in the present study identified fever as a major malaria symptom, results that are consistent with other studies,22,30-34vs 3.8%, p = 0.000), although overall results here are lower than those reported in a previous study carried out in Iran (64.6%)[Another relevant finding was that people from Buenaventura and Tumaco, both MR areas, had greater knowledge and better practices against the disease, probably due to permanent malaria education programmes. Tumaco was the subject of previous interventions by a GFATM for malaria control in the borders of Peru, Ecuador, Colombia, and Venezuela. This appears to be in agreement with the finding that knowledge about IRS was significantly higher in the MR area than in HR area (40.2  (64.6%). However (64.6%).The limitations of this study are associated with the design of data collection procedures. The cross-sectional design provides information about a particular point in time, but it is unable to determine rates of change or stochastic variation. In addition, the data were collected from households only; some information was not confirmed, for example, that related to ITN use.Despite high levels of knowledge in both regions, significant gaps relating to practices persist. There were significant differences in attitudes about buying medicine without a prescription, adherence to treatment and practices in both indoor and outdoor vector control prevention measures between respondents living in MR and HR areas. This study contrasts geographical regions of Colombia in their KAP on malaria and presents evidence that point to the need to develop educational programmes specific to each region based on the specific gaps found in this study. Education, control and elimination malaria programmes must take into account local characteristics in terms of the variables measured in this study for analysis, reporting and planning.Finally, it is evident that communities in high and moderate transmission settings of Colombia are aware about transmission, symptoms, diagnosis and prevention of malaria. Nevertheless, Ministry of Health programmes must be able to correct misconceptions about malaria diagnosis and treatment as well as knowledge about mosquito control with focused health education initiatives.The authors declare that they have no competing interests.SHV and MAR conceived and designed the study; PC, SH, MAR, AV, DF, and JG wrote the manuscript; YB and JG conducted data management and data analysis. All authors read and approved the final manuscript.The supplementary file is an analysis script in MATLAB to reproduce the results reported in the paper. Data are available upon request, provided that the protocol to access, compliant with the funding agency, is followed.Click here for file


One of the most important application spectrums of transcriptomic data is cancer phenotype classification. Many characteristics of transcriptomic data, such as redundant features and technical artifacts, make over-fitting commonplace. Promising classification results often fail to generalize across datasets with different sources, platforms, or preprocessing. Recently a novel differential network rank conservation (DIRAC) algorithm to characterize cancer phenotypes using transcriptomic data. DIRAC is a member of a family of algorithms that have shown useful for disease classification based on the relative expression of genes. Combining the robustness of this family’s simple decision rules with known biological relationships, this systems approach identifies interpretable, yet highly discriminate networks. While DIRAC has been briefly employed for several classification problems in the original paper, the potentials of DIRAC in cancer phenotype classification, and especially robustness against artifacts in transcriptomic data have not been fully characterized yet.In this study we thoroughly investigate the potentials of DIRAC by applying it to multiple datasets, and examine the variations in classification performances when datasets are (i) treated and untreated for batch effect; (ii) preprocessed with different techniques. We also propose the first DIRAC-based classifier to integrate multiple networks. We show that the DIRAC-based classifier is very robust in the examined scenarios. To our surprise, the trained DIRAC-based classifier even translated well to a dataset with different biological characteristics in the presence of substantial batch effects that, as shown here, plagued the standard expression value based classifier. In addition, the DIRAC-based classifier, because of the integrated biological information, also suggests pathways to target in specific subtypes, which may enhance the establishment of personalized therapy in diseases such as pediatric AML. In order to better comprehend the prediction power of the DIRAC-based classifier in general, we also performed classifications using publicly available datasets from breast and lung cancer. Furthermore, multiple well-known classification algorithms were utilized to create an ideal test bed for comparing the DIRAC-based classifier with the standard gene expression value based classifier. We observed that the DIRAC-based classifier greatly outperforms its rival.Based on our experiments with multiple datasets, we propose that DIRAC is a promising solution to the lack of generalizability in classification efforts that uses transcriptomic data. We believe that superior performances presented in this study may motivate other to initiate a new aline of research to explore the untapped power of DIRAC in a broad range of cancer types.The online version of this article (doi:10.1186/s12859-015-0737-3) contains supplementary material, which is available to authorized users. High-throughput genomic technologies have dramatically expanded the breadth of biological information available for the analysis and characterization of disease. One of the most important application fields of transcriptomic data is classification, where a multitude of promising results have been reported in classification literatures with near perfect classification accuracy for given datasets. Yet, despite this progress, the reliability of these results has been proven illusory, as the seemingly promising results often fail in reproducing, inextricable yet largely unachieved goal, in new datasets . This iset al. (2010) . This binary vector is then used to create a most likely binary vector termed the Rank Template. Each element of the Rank Template is 1 if the probability of the gene comparison at that position is greater than 50 % under a given condition, i.e., RTij= [ P(gi<gj|C)>0.5]. The Rank Template for each condition is then used in classification to generate a Rank Matching Score. This is effectively one minus the Hamming distance between an unknown sample’s pairwise gene comparison and the Rank Template, i.e., DIRAC is composed of a set of measures to quantify differential expression variability between conditions using subsets of genes. These subsets generally correspond to predefined gene networks or pathways. The rationale for composing the analysis using these subsets comes from the perspective of systems biology. The contextualization of expression changes in functional units is viewed as more informative than looking at gene-level changes in isolation. Briefly, DIRAC transforms the rank ordering of genes within a network into a binary vector of pairwise gene comparisons Fig. . A more . (2010) . Each elnot a classification algorithm. It falls within the realm of data transformation, i.e. project samples that are originally in the gene space to a much lower dimensional pathway space. The hope is that the difference between groups under investigation will be pronounced in the new space. The transformed data can be combined with any exiting classification algorithm.We find it necessary to point out that the DIRAC is sensitivity×specificity)/(sensitivity+specificity), is a harmonic mean of the former two. It ranges from 0 to 1, with one being the perfect classification.We first perform the pathway and GEV signatures identification for each cytogenetic subtype. Then, the signatures are used in later stage to evaluate their robustness against different preprocessing techniques and reproducibility in independent datasets in presence of batch effect, respectively. Performances of the two classifiers are measured by calculating their sensitivity, specificity and F-score. The F-score, that equals 2: RUNX1T1, POU4F1, CACNA2D2.inv(16): MN1, TM4SF1, MYH11, CLIP3, SPARC, AK5, PTRF, FAM171A1, AK022033, LOC100506388.t: STAB1, FGF13, LGALS12, PTGDS.NUP98.NSD1: VENTX, PRDM16, FAM92A1.CEPBA.dm: LRRC28, CNRIP1, ST8SIA1, MSS51, SLC14A1, LGSN.NPM1: HOXB-AS3, HOXB3, SMC4, SEPP1, LTBP1, CLIC2, HOXB4, TWISTNB, HIST1H2BC, SIAE, PIEZO2, HLF, ELL2, LOC100507520, HOXB2, FOXC1, HOXB6, HOXB5, EMR1, SNCAIP, RPL39L, USP44, BEX1, TTC27, PTPRC, HENMT1, AK027199, COL4A5, NAP1L5, TIAM1, NPDC1, CLEC11A, SCD5, CCL1, FTO, AK093529, ENSG00000184551, CDKN1B, FAM105A, PHLDA1, HOXA-AS5, LOC100506591, GMDS, TOM1L1, IL12A, DMXL2, SDPR, FOXF1.CBPA double mutations, NPM1 mutations are characterized by one or more tightly regulated MAP kinase pathways, P38MAPK, KERATINOCYTE and/or MAPK. This suggests that inhibition of MAP kinase activity within these pathways may be a way to effectively target these cells, which needs further biological validation studies. Another pathway of interest is the BIOPEPTIDES  pathway, which is characterized by the RAS, JAK and STAT signal transduction route. This pathway appears to be tightly regulated in subgroups characterized by t, Inv(16) and NUP98/NSD1. Surprisingly, AML with MLL rearrangements were not classified by any tightly regulated pathway, but rather by three loosely regulated pathways. This may be explained by the relative heterogeneity of this subgroup, which contains several MLL translocation partners, in particular AF6, AF9 and AF10, which have strikingly different clinical characteristics [DIRAC considers a pathway to be tightly regulated under a given condition if, for that condition, the rank ordering of the genes in that pathway are highly correlated; loosely regulated if they are less correlated. We noticed that, the pathway signatures Table  specificThese pathway signatures may be options for understanding specific disease mechanisms and provide a key for the design of new treatment. For example, one may seek network aware intervention to adjust the signature pathways in each subtype to its normal non-leukemic cell behavior and, consequently, to control clinical presentation.et al. (2011) [1 dataset and were used to predict subtype labels of the same dataset that has been preprocessed using six well-known methods. This is meant to be representative of a situation where a researcher intends to combine or compare transcriptomic profiles from two studies where the preprocessing methods were different and the original unprocessed data is not available. Ideally, one would have the original data and use the same preprocessing methods on those data sources, but this is not always an option. The six preprocessing methods are: M1) CEL file transformed to raw expression data using Robust multi-array average expression measure (RMA); M2) Raw data obtained with RMA and normalized with quantile normalization; M3) CEL file transformed to raw expression data using Affymetrix’s MAS 5.0 expression measure (MAS5); M4) Raw data obtained with MAS5 and normalized with scale normalization; M5) CEL file transformed to raw expression data using Robust multi-array average expression measure with help of probe sequence (GCRMA); M6) Raw data obtained with GCRMA and normalized with quantile normalization.Multiple preprocessing methods for gene expression data have been proposed . However. (2011)  reportedFigures Genomic data often contain batch effects, i.e. differences in sample sources and other technical variations. Many algorithms have been proposed to remove these artifacts, but this (often) comes along with the potential cost of removing between-group biological heterogeneity and consequently salient genomic signatures . Note th2 and the adult AML datasets. The GEV-based classifier relies on expression values and reflects commonly used practice when no additional knowledge is available. Both pathway and gene expression classifiers were trained using all samples in the pediatric 1 dataset and used to predict the subtype labels of the two independent datasets. We observed strong batch effects between the pediatric 1 and the two new independent datasets  subtypes.To demonstrate the robustness in translating trained predictor across independent datasets, we analyzed performance characteristics of the DIRAC-based classifier as well as the GEV-based classifier on the pediatric In order to better comprehend the prediction power of the DIRAC-based classifier in general, we conducted performance comparison using publicly available datasets from breast and lung cancer . Then, the rank matrix, instead of the original of gene expression values, was used to construct classifiers. Classification results are shown in Fig. Clearly, the DIRAC-based classifier is robust against most of the major ancillary sources of variation in the data. We attribute this to the robustness of relative expression compared to absolute expression values used  and the According to the no-free-lunch theorem  no classIn this study, we investigate the performance of the DIRAC-based classifier using multiple AML datasets. We believe that its superior performances presented in this study may motivate other to explore the untapped power of DIRAC in different types of cancer or may even open a new line of application.In summary, through this study we have demonstrated the robustness of DIRAC in classification, which has previously been undocumented and underestimated. While this is not the first multi-class DIRAC method proposed , we haveIn this study we investigate the potentials of DIRAC in multiple scenarios frequently appear in analyzing high-dimensional transcriptomic data. The number of conceivable applications of DIRAC is possibly large, hence we do not claim to provide exhaustive list. Instead, the focus is on classification problem that has been the subject of bioinformatics research for long time. Reported experimental results from multiple real-world datasets serve two ends: 1) to illustrate the benefits of the DIRAC-based classifier, 2) to clarify what makes the DIRAC-based classifier superior than exiting approaches. In the following sections we describe the datasets that are considered in this study, and the experimental setups, i.e. how we compare performance of the DIRAC-based classifier with exiting approaches in an unbiased way.1 AML dataset, which was generated in our lab, the raw data (CEL files) were also available [Three publicly available acute myeloid leukemia (AML) datasets Table  were usevailable . Details1 and adult AML datasets, 57 % in the pediatric 2 AML dataset. Note that, for the DIRAC-based classifier only those genes that belong to the retained pathways were used. For the GEV-based classifier the full list of genes, not just the pathway mapped subsets, was used. Note that, the number of genes used for the GEV-based classifier is far more exceeds the number of pathways used for the DIRAC-based classifier. This means, the former has a larger search space to find the discriminative features compared to the latter.In this study, 217 manually curated (comprised of 1279 unique genes) BioCarta pathways from the Molecular Signatures Database  were used to construct the DIRAC-based classifier. For each dataset, genes were grouped according to their presence in these pathways. To reliably represent the pathways, only those pathways that have at least 30 % of associated genes presented in a given dataset were retained. The minimum fraction of genes that are present both in individual pathways and gene expression data is: 70 % in the pediatric 1 AML dataset  [We used the pediatric et Table  to gener. (2011) . SpecifiTo obtain reliable results, we performed 100 outer-cross-validation (OCV) and 100 inner-cross-validation (ICV) on the discovery set Fig. . In eachThe selected features (50 genes/pathways) were processed in following ways to obtain reduced set of GEV and pathway signatures. For each subtype, the GEV (pathways) signatures were ranked (from best to worst) according to their association with the subtype via the global test . Subsequet al. (2011) [At the end of 100 ICV iterations, a signature list of smallest size that renders highest prediction sensitivity was determined. Among the three optimal lists generated via 3-fold CV using the discovery set, the one with the median prediction sensitivity was taken as the optimal signature list for one OCV iteration. The final classifier was trained using the signature list that produced the highest prediction sensitivity in 100 OCV iterations and subsequently used to predict the labels of the validation set. Balgobind . (2011)  argued tEthics approval is not required for this study.Additional file 1Supplementary material. This document includes additional information not included in the paper. (PDF 1239 kb)


Pseudomonas aeruginosa. The redox-active tricyclic zwitterion has been shown to have a number of potential effects on various organ systems in vitro, including the respiratory, cardiovascular, urological, and central nervous systems. It has been shown that a large number of the effects to these systems are via the formation of reactive oxygen species. The limitations of studies are, to date, focused on the localized effect of the release of pyocyanin (PCN). It has been postulated that, given its chemical properties, PCN is able to readily cross biological membranes, however studies have yet to be undertaken to evaluate this effect. This review highlights the possible manifestations of PCN exposure; however, most studies to date are in vitro. Further high quality in vivo studies are needed to fully assess the physiological manifestations of PCN exposure on the various body systems.Pyocyanin has recently emerged as an important virulence factor produced by  Pseudomonas aeruginosa is a Gram-negative bacterium responsible for severe nosocomial infections in a number of body systems including the respiratory tract, the vascular system, the urinary tract, and the central nervous system (CNS) [P. aeruginosa produces a number of secreted virulence factors known as phenazines. One such compound is pyocyanin (PCN) [em (CNS) ,4,5,6,7.in (PCN)  which hain (PCN) .P. aeruginosa into the local environment by a type II secretion system [P. aeruginosa [PCN is a nitrogen-containing aromatic compound belonging to the tricyclic phenazine class of compounds ,13,14,15n system . Signifiruginosa ,21,22. TP. aeruginosa in cystic fibrosis (CF), the majority of research on PCN to date has primarily been focused on its effect on human airway. However, recently there have been a number of studies demonstrating the broader consequences of PCN exposure, especially in the urinary tract [To date, numerous studies have shown the potential importance of PCN in the virulence and pathogenicity of pseudomonal infections and its potential toxic effects ,23,24. Pry tract ,28, the ry tract , and thery tract ,31. In l2·−) [2O2) [2O2 and O2·− and are formed by cyclic non-enzymatic reduction by NAD(P)H [Oxidative stress is a major contributing factor to the cytotoxicity displayed by PCN ,33, a re2·−) ,25,34,35−) [2O2) . These i NAD(P)H ,17,34 wi NAD(P)H ,11,12. T NAD(P)H , NAD(P)H NAD(P)H .P. aeruginosa that overproduce PCN produce greater levels of oxidative stress, subsequent cell lysis, and a significant increase in extracellular DNA (eDNA) [P. aeruginosa, through an oxidative stress-dependent mechanism, PCN has then been shown to intercalate with eDNA to promote cell-to-cell interactions between P. aeruginosa cells by influencing their cell surface properties and physicochemical interactions. It has, therefore, been suggested that PCN may also contribute to biofilm formation by the promotion of eDNA [P. aeruginosa via the above mechanism.In further support of the integral role of oxidative stress in PCN’s ability to induce cytotoxicity, it has been demonstrated that strains of A (eDNA) . To furt of eDNA . This stP. aeruginosa, and subsequent secretion of PCN, has been shown to have a dramatic effect on the cellular protective mechanisms of the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidise (GSH-Px) [2·− to H2O2, thereby acting as the first protective mechanism against PCN induced oxidative stress [2O2 generated from both PCN and SOD, is then in turn partially removed by the enzyme CAT [2·− and H2O2 [Infection by (GSH-Px) . When ce(GSH-Px) ; however(GSH-Px) , indicate stress . Subsequzyme CAT  or by thzyme CAT ,46. PCN zyme CAT . It has and H2O2 ,38 and a2O2 levels, as described earlier [A second protective mechanism against free radical damage is through the redox action of the thiol compounds such as GSH , whereby earlier . During  earlier . Treatme earlier . This fuP. aeruginosa [Recently, the effects of PCN-induced free radical formation on various cell signaling pathways have been assessed in a number of studies. It has been reported that PCN induced the oxidative stress-dependent induction of MUC2 and MUC5AC, both genes encoding for mucin secretion, through a mechanism involving epidermal growth factor receptor/extracellular signal-regulated kinase (EGFR/ERK) activation. This resulted in increased mucous secretion in the respiratory tract further aiding in the colonization of the airways by ruginosa . The undruginosa . A subseruginosa .P. aeruginosa. Further studies are needed, however, to fully elucidate the cell signaling pathways involved in these effects.The extensive studies on the free radical effects of PCN show the relevance and importance of this property to the virulence and pathogenicity of Studies have been undertaken assessing the impact of PCN on respiratory, urological, neurological, cardiovascular, and hepatic models, both in vitro and in vivo ,48,50,51Numerous studies have investigated the effects of PCN on the host immune function in in vivo and in vitro studies. However, to date, no studies have assessed the inflammatory effects in humans ,37,52. TP. aeruginosa from the immune system. PCN concentrations as low as 40 µM (0.08 mg/mL) have been shown to be toxic to lymphocytes, which are consistent with reported physiological concentrations [In vivo studies have predominantly investigated immunological effects in the respiratory tract of animals and will be discussed in further detail below. PCN has, however, been shown to alter the expression and release of a number of cytokines in vitro ,37,52. Ptrations .4 (LTB4) [In addition to effects on lymphocytes, PCN also has toxic effects on neutrophils, predominantly through apoptotic pathways . Neutrop4 (LTB4) , and ind4 (LTB4) . PCN exp4 (LTB4) , through4 (LTB4) . This pa4 (LTB4) . Recent 4 (LTB4) ,59. Neut4 (LTB4) ,55,60,614 (LTB4) .P. aeruginosa infection, stimulated neutrophils secrete myeloperoxidase (MPO) and H2O2, both of which are essential components of a peroxidase system and are known causes of oxidative stress [Neutrophils are additionally responsible for the release of large amounts of elastase and other proteases ,63, as we stress . Bianchie stress . This ide stress .P. aeruginosa and, ultimately, further contributing to tissue damage. Interestingly, PCN has been shown to have little direct effect on inducing monokines, including IL-1 and tumor necrosis factor (TNF) by monocytes, but significantly enhances their lipopolysaccharide (LPS)-stimulated production [P. aeruginosa infection, due to the bacterium’s ability to produce LPS. These effects lead to the suppression of defense mechanisms and enhance harmful inflammatory reactions of the host during infection with P. aeruginosa [PCN has been shown to exert various other effects on inflammation including its ability to decrease Regulated upon Activation, Normal T Expressed and Secreted (RANTES) , an impooduction . This isruginosa , potentiThe respiratory effects of PCN have been extensively studied due to the high prevalence of pseudomonal infections in respiratory conditions such as CF and bronchiectasis . The damP. aeruginosa with detectable concentrations of PCN was shown to significantly slow ciliary beating in all nine cases [A limited number of human studies have evaluated the effects of PCN on the respiratory tract ,70. The ne cases . Furtherne cases . A seconIn addition to the human studies, a limited number of in vivo studies have been conducted evaluating the effects of PCN on the respiratory tract ,50,71,72A recent study suggests that PCN suppresses FoxA2, a transcriptional repressor of goblet cell hyperplasia and mucous production, via the upregulation of Stat6 and EGFR . It is s2O2 in a dose-dependent manner and that the senescence-inducing effect of PCN could be overcome in the presence of glutathione at concentrations of 0.5 mM above [The majority of studies investigating the effects of PCN in respiratory cells have been done in in vitro models and have evaluated the effects of PCN on oxidative stress and inflammatory-mediated damage. One such study showed that when both A549 and primary NHBE respiratory cells were exposed to ≥5 µM concentrations of PCN over at least 12 h, the activity of catalase decreased significantly . SimilarmM above .P. aeruginosa colonization in CF airways [Recently, oxidative stress has been causally linked to PCN-induced toxicity in human respiratory A549 cells . It was  airways .Investigations into the effects of PCN on pro-inflammatory mediators and the chemotaxis of polymorphonuclear (PMN) cells have been undertaken . After 2The studies outlined above highlight the importance of the free radical effects PCN produces and highlights the importance of this mechanism as major cause of the pathogenicity of this compound.P. aeruginosa is implicated in a large percentage of nosocomial urinary tract infections, particularly in patients with urethral catheters, and is commonly associated with high levels of antibiotic resistance [sistance . Chronicsistance . To dateA recent study by McDermott et al. (2012) was the first to assess the effects of PCN on the urothelial cells. They reported that PCN significantly reduced RT4 urothelial cell viability at concentrations of 25 µM and greater . FurtherP. aeruginosa urinary tract infections may not be associated with enhanced urothelial ATP release.This study also provided evidence that PCN reduced baseline and stimulated adenosine triphosphate (ATP) release . This su2 (PGE2) production in RT4 urothelial cells in a concentration-dependent manner, with statistically significant increases observed at 100 μM. Furthermore, it was found that 100 μM of PCN increased IL-6 levels in the same cell line [P. aeruginosa [A second study on the effects of PCN on the urinary tract investigated the inflammatory effects. It was found that PCN increased prostaglandin Eell line . This struginosa .P. aeruginosa, though rare, is associated with high mortality. There is currently limited evidence to support mechanisms underlying this toxicity, with all available studies currently using in vitro models. A preliminary study by McFarland et al. (2012) was the first to determine the effect of PCN in a neurological model, identifying that PCN treatment induced toxicity in 1321N1 astrocytoma cells, and correlated with the formation of acidic vesicular organelles (AVOs) [Infection of the CNS with s (AVOs) . This fis (AVOs) .2 and were observed with both cell lines, implicating PCN-induced inflammation as a possible contributing factor to the pathophysiology of CNS pseudomonal infections. This study established that the toxic effects of PCN in these cell lines was independent of oxidative stress, hence further supporting the role of autophagy-mediated death in PCN-treated 1321N1 and SH-SY5Y cells [A second follow-up study, comparing the effects of PCN on both 1321N1 astrocytoma cells and SH-SY5Y neuroblastoma cells, found SH-SY5Y cells to be significantly more resistant to the toxic effects of PCN . Of part5Y cells . FurtherP. aeruginosa is a rare cause of infective endocarditis and is seen primarily in injection drug users and patients undergoing cardiac procedures [P. aeruginosa sepsis, a life-threatening systemic infection that is becoming more prevalent in society [Few studies have evaluated the effects of PCN on the vascular system to date, and those available are limited to in vitro studies ,77,78. Hocedures ,4,5,6. T society . PCN, at society ,77 and - society . A recenTo date, only a small number of in vivo studies have been undertaken assessing the effects of PCN on hepatic function ,51. PCN P. aeruingona organisms in CF, this mechanism may provide an insight into the associated pathophysiology, however, further studies are needed to confirm these effects. A second potential mechanism by which PCN may impact on the development of diabetes, in CF patients in particular, is through oxidative stress pathways. PCN, as outlined previously, is a highly redox-active compound that causes many of its cytotoxic effects through oxidative stress. Studies have shown that oxidative stress, in particular superoxide, may play an important role in the onset of diabetes and have been reviewed extensively in the past . Given iewed in  it is poiewed in . PCN inht manner ,87. GiveCaenorhabditis elegans as a model, have shown phenazines produced by P. aeruginosa to mediate its toxic effects through the generation of ROS [C. elegans to pathogenic P. aeruginosa, found that surveillance pathways controlling core cellular activities of the organism allow detection of pathogens with the capability to produce virulence factors and undermine vital host functions [C. elegans that resulted in aversion behavior and was found to be mediated through a mitochondrial pathway resulting in unfolded protein responses [In vivo studies, using n of ROS . A furthunctions . This, tunctions . Disruptunctions . Given Punctions ,91. PCN esponses ,91. ThesP. aeruginosa infection. Further studies assessing the observed effects are warranted in vivo and in humans, particularly given the studies suggesting that aversion behavior exists in C. elegans exposed to PCN. To date very few in vivo studies have investigated the effects of PCN, however, within the limited number of studies undertaken significant cellular damage was reported. Given the highly toxic nature of PCN, future studies investigating strategies to minimize cellular damage associated with PCN exposure should be investigated. Such strategies include modifying either the production of PCN by P. aeruginosa or by preventing the toxic effects using pharmacological modulators. To date, there have been a small number of studies assessing the effects of pharmacological agents on the production of PCN from P. aeruginosa in vitro. However, these have not been assessed in vivo [As highlighted in the studies outlined above, PCN has been shown to impact on the cellular function of many body systems. However, these are predominantly limited to in vitro studies. These studies have suggested that PCN elicits its toxic effects through ROS and inflammatory mediated pathways. This provides us with the basic mechanisms of cellular toxicity associated with PCN exposure and highlights possible pathways to target in the search for therapies to minimize toxicities associated with  in vivo ,96,97,98 in vivo . Both ofP. aeruginosa are warranted to assess the impact of this exposure, physiologically.Numerous studies have been undertaken assessing the cellular effects of PCN exposure in various body systems, both in vitro and in vivo. A diverse range of toxic effects have been observed in animals and cells exposed to PCN which have been shown to be predominantly mediated by free radical and pro-inflammatory cytokine production. Given the nature of pseudomonal infections, the effects of PCN on the respiratory tract are the most widely studied phenomenon. Recent in vitro studies have, however, shown that PCN has deleterious effects on cells in numerous other body systems, including the CNS, the urolological and the vascular system, with mechanisms other than oxidative stress implicated in a number of circumstances. In light of this, further studies investigating these effects in vivo and, ultimately, in a human population infected with PCN-producing