Chlorococcum sp. GD on the flocculation, growth, and lipid accumulation in wastewater with different ammonia nitrogen concentrations was investigated. It was revealed that relative high ammonia nitrogen concentration (20–50 mg·L−1) was beneficial to the flocculation of Chlorococcum sp. GD, and the highest flocculating efficiency was up to 84.4%. It was also found that the highest flocculating efficiency occurred in the middle of the culture (4–5 days) regardless of initial ammonia concentration in wastewater. It was speculated that high flocculating efficiency was likely related to the production of extracellular proteins. 20 mg·L−1 of ammonia was found to be a preferred concentration for both biomass production and lipid accumulation. 92.8% COD, 98.8% ammonia, and 69.4% phosphorus were removed when Chlorococcum sp. GD was cultivated in wastewater with 20 mg·L−1 ammonia. The novelty and significance of the investigation was the integration of flocculation, biomass production, wastewater treatment, and lipid accumulation, simultaneously, which made Chlorococcum sp. GD a potential candidate for wastewater treatment and biodiesel production if harvested in wastewater with suitable ammonia nitrogen concentration.The performance of a self-flocculating microalga  The integration of microalgae-based wastewater treatment and lipid production has major advantages for both industries ,2. NeverChlorella sorokiniana with more than 99% efficiency. Bioflocculant production from Solibacillus silvestris W01 shows 90% flocculating efficiency on Nannochloropsis oceanica [Currently, microalgal harvesting mainly involves physical-, chemical-, and biological-based methods. For example, efficiencies higher than 90% are reached by applying ultrasound waves to harvest microalgae . Xu et aoceanica . Some otoceanica . HoweverAnkistrodesmus falcatus, C. vulgaris JSC-7, Ettlia texensis, Scenedesmus obliquus AS-6-1, Scenedesmus sp. BH, and Tetraselmis suecica, have been screened [Scenedesmus sp. BH is up to 92.3% [Self-flocculating microalgae are a kind of microalgae that can aggregate together by themselves. The aggregate can be easily harvested by sedimentation. Currently, some self-flocculating microalgae, such as screened ,13,14,15to 92.3% . Compareto 92.3% , there iFor microalgal cultivation in wastewaters, ammonia is one important form of nitrogen and can influence microalgal growth and nutrient uptake ,19. The At present, the flocculating ability of self-flocculating microalgae under different growth phasees, temperatures, and pHs has been investigated. It has been demonstrated that the suitable growth phase, temperature, and pH are beneficial to flocculation of self-flocculating microalgae ,20. NeveChlorococcum sp. GD belonging to green microalgae has been reported to exhibit excellent self-flocculating capability in secondary effluent [C. vulgaris JSC-7, E. texensis, and S. obliquus AS-6-1 [Chlorococcum sp. GD on the flocculation with different ammonia nitrogen concentrations is investigated. The response of growth to varied ammonia concentrations is also analyzed. The novelty and significance of the study is to evaluate the possibility of the integration of sewage treatment, biomass production, and microalgal harvesting of Chlorococcum sp. GD, simultaneously, by altering ammonia nitrogen concentration in wastewater.effluent , which ws AS-6-1 ,12,13. IChlorococcum sp. GD was used in the study. It was isolated from the moss Entodon obtusatus of Shanxi Province, China [A strain of e, China  and exhie, China .4Cl 38.21–191.04, KH2PO4 21.97, NaHCO3 100, NaCl 64, MgSO4 90, FeSO4 5, CaCl2 25, H3BO3 2.86, MnCl2·4H2O 1.86, ZnSO4·7H2O 0.22, Na2MoO4·2H2O 0.39, CuSO4·5H2O 0.08, and Co(NO3)2·6H2O 0.05. The COD of the synthetic wastewater was around 325 mg·L−1 and total phosphorus concentration was 5 mg·L−1. The ammonia concentration varied from 10 to 50 mg·L−1. In the study, synthetic wastewater was sterilized (121 °C for 20 min) before experiments.In the present study, synthetic wastewater was formulated to simulate municipal wastewater. The exact concentration of COD and phosphorus of the synthetic wastewater was derived from the average concentration of municipal wastewater in Shanxi Province, China. The composition of macro element of the synthetic wastewater was provided by Aslan and Kapdan  with somChlorococcum sp. GD was firstly cultivated in BG11 for 13 days. After that, microalgal suspension was centrifuged at 5000 rpm for 5 min. The pellet was then washed with deionized water and centrifuged at 5000 rpm for 5 min again. The washed pellet was distributed in synthetic wastewater and inoculated into 500 mL transparent conical flasks with an initial concentration of 20 mg·L−1. In order to investigate the effect of different ammonia nitrogen concentrations, four samples with initial ammonia nitrogen of 10, 20, 30, and 50 mg·L−1 were prepared. Cultures were grown at a constant temperature of 25 °C in a shaker with a shaking rate of 160 revolutions per minute (rpm). The cultures were illuminated with fluorescent lamps, providing an incident light intensity of 3000 lux under 14 h:10 h light:dark cycle. After daily sampling, pH of cultures was adjusted to 7.5 by supplementing HCl or NaOH solution. All these experiments were performed in triplicate.In the study, 680) of the aliquot before and after flocculation.During the process of microalgal cultivation, 25 mL culture was harvested and distributed in 25 mL cylindrical glass tubes. The culture was gently mixed for 1 min at room temperature. After that, an aliquot of the culture was withdrawn at a height of two-thirds from the bottle when the culture had settled for 3 h. The optical density of the aliquots was then measured at 680 nm. The flocculating ability was calculated using the following Equation (1):EPS mainly consisted of proteins and polysaccharides. The EPS were extracted according to procedures of Yang and Li  with somMicroalgal biomass concentration was measured according to the method of suspended solid (SS) measurement . The spe1 and t2, mg·L−1; t1 and t2 are the cultivation time, d. In Equations (2) and (3), For the measurement of water quality, the microalgal suspension was centrifuged at 6000 rpm for 5 min, and the supernatant was filtered through 0.22 μm membranes. The filtered supernatant was then used for the determination of chemical oxygen demand (COD), ammonia nitrogen, and total phosphorous. COD, ammonia, and total phosphorus were analyzed by dichromate method, Nessler’s reagent spectrophotometry, and ammonium molybdate spectrophotometric method, respectively .max is the maximal ammonia nitrogen removal rate, S is the ammonia nitrogen concentration, and Km is the ammonia nitrogen concentration at which ammonia removal rate reaches half-maximum.Michaelis–Menten kinetic can be used to evaluate the ammonia nitrogen removal capability of microalgae .(5)R=Rmm and Vmax [−1 DW·d−1) is the specific ammonia nitrogen removal rate per unit microalgal biomass; S0 (mg·L−1) is the initial ammonia nitrogen concentration; and Vmax (mg N·g−1 DW·d−1) is the maximal ammonia nitrogen removal rate.Equation (5) can be linearized in double reciprocal form as in Equation (6) to determine the ammonia nitrogen removal kinetic coefficients Kand Vmax .(6)1/RX−1 Nile red (9-diethylamino-5H-benzo(α)phenoxazine-5-one, dissolved in DMSO, 1 μL), and the mixture was incubated for 10 min at 37 °C. The fluorescence of the mixture was then determined using a microplate reader Tecan Infinite 200 Pro  with a 96-well plate. The fluorescence of microalgae alone was measured. The fluorescence of Nile red alone was also measured. The fluorescence intensity of microalgal lipid was obtained when the autofluorescence of microalgae and Nile red was subtracted. The excitation and emission wavelengths were 543 and 598 nm, respectively. Each experiment was performed in triplicate.During the cultivation of microalgae, a suspension of microalgae (240 μL) was stained with 0.5 mg·mL−1 DW biomass), FI is the total Nile Red fluorescence intensity of 240 μL microalgal suspension (a.u.), and DW is biomass dry weight in 240 μL microalgal suspension.The specific Nile Red fluorescence intensity was calculated as following:v/v) and was quantified gravimetrically [At the end of the culture, the total lipid was extracted with a chloroform/methanol solution , and there was a significant relationship when p < 0.05.The measured value was expressed as the mean ± standard deviation. If necessary, data were analyzed by analysis of variance (ANOVA) conducting by Statistical Product and Service Solutions (SPSS) software . There was a statistically significant difference when Chlorococcum sp. GD cultivated with different ammonia nitrogen concentrations is presented. Throughout the culture period, the flocculating ability of Chlorococcum sp. GD ranged from 54.3% to 84.4%. More specifically, the flocculating ability of Chlorococcum sp. GD cultivated with 10 mg·L−1 ammonia nitrogen decreased sharply from 74.6% to 54.3% after 3 days of culture. It then increased to 79% on the 4th day. Afterwards, flocculation efficiency gradually decreased to 62.7%. For Chlorococcum sp. GD cultivated with 20 mg·L−1 ammonia nitrogen, the highest flocculating ability was 79.4% on the 4th day. Then, the flocculating ability slightly decreased and maintained at around 72% until the end of cultivation. For Chlorococcum sp. GD cultivated with 30 mg·L−1 ammonia nitrogen, the flocculating ability remained at about 70%, except for a significant decrease to 60.6% on the 2nd day. The flocculating ability of Chlorococcum sp. GD cultivated with 50 mg·L−1 ammonia nitrogen was highest and maintained at about 80% at the end of the culture. Generally speaking, the flocculating ability of Chlorococcum sp. GD was affected by ammonia nitrogen concentration in wastewater. Chlorococcum sp. GD had an excellent and stable flocculating performance when cultivated in wastewater with 50 mg·L−1 ammonia nitrogen. Chlorococcum sp. GD cultivated with 10 mg·L−1 ammonia nitrogen exhibited the worst flocculation ability. It was also found that the flocculating ability of Chlorococcum sp. GD was dynamic when cultivated in wastewater with different ammonia nitrogen concentration. The highest flocculating efficiency occurred in the middle of the culture (4–5 days) regardless of initial ammonia nitrogen concentration in wastewater.In Chlorococcum sp. GD cultivated in different ammonia nitrogen concentrations is measured  when Chlorococcum sp. GD was cultivated in wastewater with 20 mg·L−1 ammonia nitrogen. That is to say, flocculation might be positively related to extracellular protein content in EPS.As shown in Chlorococcum sp. GD with different ammonia nitrogen concentrations is shown in Chlorococcum sp. GD could well adapt in synthetic wastewater with different ammonia nitrogen concentrations. After 11 days of cultivation, the biomass production increased and ranged from 138 to 190.1 mg·L−1. The biomass production was highest when ammonia nitrogen concentration in synthetic municipal wastewater was 20 mg·L−1. The specific growth rate of Chlorococcum sp. GD cultivated in wastewater with different ammonia nitrogen concentrations is also calculated as shown in p < 0.05). Chlorococcum sp. GD cultivated in 20 mg·L−1 ammonia nitrogen had higher specific growth rate (0.24 d−1) than other ammonia nitrogen concentrations (0.19–0.21 d−1)  . The meashortest . All res−1·d−1, respectively. Chlorococcum sp. GD showed excellent COD removal performance, and COD removal did not seem to be affected by ammonia nitrogen concentration. Conversely, the ammonia nitrogen removal efficiency was related to the ammonia nitrogen concentration. The ammonia nitrogen removal efficiency was around 98% when the ammonia nitrogen concentration of wastewater was 10 and 20 mg·L−1. The removal efficiency decreased to 43.9–78.0% when the initial ammonia nitrogen concentration was more than 20 mg·L−1 0 as shown in Chlorococcum sp. GD were Vmax = 15.2 mg NH4-N·g−1 DW·d−1 and Km = 13.4 mg NH4-N·L−1.The data on ammonia nitrogen removal of Chlorococcum sp. GD cultivated with different ammonia nitrogen concentrations is shown in Chlorococcum sp. GD was cultivated with 10 and 20 mg·L−1 ammonia nitrogen, the lipid accumulation firstly decreased and then increased. The difference was that the turning point for Chlorococcum sp. GD cultivated with 10 mg·L−1 ammonia nitrogen was in the 7th day, whereas the turning point for Chlorococcum sp. GD cultivated with 20 mg·L−1 ammonia nitrogen was in the 4th day. When Chlorococcum sp. GD was cultivated with 30 and 50 mg·L−1 ammonia nitrogen, the lipid accumulation decreased during the whole culture period. In conclusion, after 11 days of culture, the highest lipid accumulation of Chlorococcum sp. GD occurred when cultivated with 10 mg·L−1 ammonia nitrogen, followed by 20 and 50 mg·L−1 ammonia nitrogen. Chlorococcum sp. GD had lowest lipid accumulation when cultivated with 30 mg·L−1 ammonia nitrogen. The lipid content of Chlorococcum sp. GD cultivated with different ammonia nitrogen concentrations is shown in −1 ammonia nitrogen, Chlorococcum sp. GD had the highest lipid content at 47.5%, which was significantly higher than that of Chlorococcum sp. GD cultivated with wastewater of 20, 30, and 50 mg·L−1 ammonia nitrogen (p < 0.05). The lipid content of Chlorococcum sp. GD cultivated with 20, 30, and 50 mg·L−1 ammonia nitrogen was of the same order of magnitude.The lipid accumulation of Chlorococcum sp. GD cultivated in wastewater with high ammonia nitrogen concentration (20–50 mg·L−1) had high concentration of extracellular proteins at the end of the culture. Nitrogen is an essential nutrient that is required in microalgal growth and is used to synthesize a variety of biological substances, such as peptides, proteins, etc. [Chlorococcum sp. GD and protein content. Salim et al. [E. texensis was due to extracellular proteins patched to the cell surface. As reported by Díaz-Santos et al. [Saccharomyces bayanus var. uvarum and could effectively induce flocculation of Chlamydomonas reinhardtii and Picochlorum sp. HM1. Díaz-Santos et al. [FLO5 gene  from S. bayanus by cotransformation in C. reinhardtii. C. reinhardtii transformants exhibited self-flocculation abilities between 2- and 3.5-fold higher than the control untransformed strain. All these cases demonstrated the importance of extracellular proteins in microalgal flocculation, which was proved again in this study. Based on the analysis and discussion above, it was speculated that nitrogen indeed affected the flocculation efficiency of Chlorococcum sp. GD, which was likely related to the production of extracellular proteins. It was found that the flocculating ability of Chlorococcum sp. GD was affected by ammonia nitrogen. Chlorococcum sp. GD had an excellent flocculating performance when ammonia nitrogen concentration of wastewater was more than 10 mg·L−1. Chlorococcum sp. GD had the highest flocculating efficiency in the middle of the culture (4–5 days) regardless of initial ammonia nitrogen concentration in wastewater. The phenomenon likely came from the maximum extracellular protein content and high degradation efficiency of pollutants of Chlorococcum sp. GD synchronously in the middle of the culture (4–5 days). Of course, the extracellular proteins content of Chlorococcum sp. GD was obviously higher than the extracellular polysaccharides content, which was similar to self-flocculating microalga E. texensis, since it had high extracellular proteins content [In the present study, ns, etc. . Durmaz ns, etc.  found thm et al.  found ths et al. , a solubs et al.  expresse content ,15.Chlorococcum sp. GD. The optimal ammonia nitrogen concentration for its growth was 20 mg·L−1, which was consistent with when Tam and Wong [C. vulgaris grew well in cultures containing 20 mg·L−1 ammonia nitrogen. Of course, many microalgae could grow well in cultures with more than 20 mg·L−1 ammonia nitrogen [−1 of microalgal biomass was low compared to the common biomass levels of Chlorococcum sp. in BG11 (360–4140 mg·L−1) [It was found that ammonia nitrogen affected the growth of and Wong  showed tnitrogen ,33, whicnitrogen ,35. In t−1. Commonly, the elementary composition ratio of microalgal cells gave a hint of the optimal N/P ratio in wastewaters. The empirical elementary composition for microalgae was C106H263O110N16P (N/P ratio = 7.2:1). In our work, the optimal N/P ratio for pollutants removal was 4:1, which was relatively lower than the empirical value. Some investigations also found that Klebsormidium sp. and Pseudanabaena sp. had excellent nitrogen and phosphorus removal capability with high N/P ratio of 7–10 and 7–20 [Chlorococcum sp. GD were Vmax = 15.2 mg NH4-N·g−1 DW·d−1 and Km = 13.4 mg NH4-N·L−1, which was lower than those of Klebsormidium sp. and Pseudanabaena sp. [max is an indicator of the high potential capability of removing pollutants, while a low Km meant that the microalga could reach its highest pollutant removal rate at low pollutant concentrations [Chlorococcum sp. GD has a competitive advantage in wastewaters with relatively low ammonia nitrogen concentration.The pollutants removal was also affected by ammonia nitrogen, and pollutants removal efficiency was the best when the ammonia nitrogen concentration was 20 mg·Land 7–20 , which waena sp. . Normalltrations . Based oChlorococcum sp. GD was influenced by ammonia nitrogen. Nitrogen deprivation was commonly the main trigger for lipid accumulation of microalgae [Chlorococcum sp. GD cultivated with 30 and 50 mg·L−1 ammonia nitrogen, there was still a large amount of nitrogen not being degraded at the end of the culture. Microalgal cells consequently did not accumulate large amounts of lipids, and the lipid content was relatively low. For low nitrogen concentration groups (10 and 20 mg·L−1), almost all ammonia nitrogen was degraded within 7 days of culture. Afterwards, Chlorococcum sp. GD was in the stage of nitrogen deprivation, and thus accumulated high content of lipid. Of course, the lipid content in 10 mg·L−1 nitrogen concentration group was significantly higher than that in 20 mg·L−1 nitrogen concentration group. Nevertheless, the total lipid content in 20 mg·L−1 nitrogen concentration group  was almost the same as that in 10 mg·L−1 nitrogen concentration group  owing to high biomass production of Chlorococcum sp. GD cultivated with 20 mg·L−1 ammonia nitrogen. High pollutants removal efficiency also occurred in wastewater with 20 mg·L−1 ammonia nitrogen. Therefore, it was suggested that Chlorococcum sp. GD was cultivated with 20 mg·L−1 ammonia nitrogen for lipid production. In the study, 26.3% of lipid content was achieved when Chlorococcum sp. GD was cultured with 20 mg·L−1 ammonia nitrogen, which was comparable to lipid content of microalgae cultivated in wastewaters in former literatures [Chlorococcum sp. GD, high efficient accumulation of lipid occurred at the end of the culture, while optimum efficiency of pollutants degradation and microalgal harvesting occurred in the middle of the culture. Therefore, it was worth paying attention to how to solve this contradiction in the future.In this work, it was found that the lipid content of croalgae . Becauseeratures ,40. InteChlorococcum sp. GD was affected by ammonia nitrogen concentration in wastewater. Relative high ammonia nitrogen concentration (20–50 mg·L−1) was beneficial to flocculation, and the highest flocculating efficiency was in the middle of the culture (4–5 days) regardless of ammonia nitrogen concentration in wastewater. It was speculated that high flocculating efficiency was likely related to the production of extracellular proteins. Relative high ammonia nitrogen concentration (20 mg·L−1) was also beneficial to biomass production and lipid accumulation of Chlorococcum sp. GD. Pollutants removal efficiency was the best when the ammonia nitrogen concentration was 20 mg·L−1. A better understanding of the response of Chlorococcum sp. GD to ammonia nitrogen is beneficial for cultivating and harvesting self-flocculating microalga from wastewater.In the study, it was found that the flocculating ability of


Ornithonyssus sylviarum , infestations can stress birds, impairing welfare and causing substantial economic losses. A study was undertaken to determine the efficacy of an ectoparasiticide solution  for oral administration in the treatment of mite-infested hens.Northern fowl mite, n = 132), approximately 32 weeks of age, were ranked by Day -9 mite vent counts and randomized among 12 study pens, each to hold one of four treatment groups. Three groups received fluralaner-medicated water by oral gavage at dose rates of 0.25, 0.5 or 1.0 mg/kg on Days 0 and 7; one group was an untreated control (three pens for each group). Five naturally infested untreated birds were included in each pen to act as mite-infested source birds. Thus each pen, treated and control, had six non-source birds for assessment of efficacy, plus five source birds to provide ongoing challenge. Primary efficacy assessments were based on mean O. sylviarum vent counts from non-source birds in the control and treated group pens on Days 1, 2, 6, 8, 12, 15, 19, 22 and 26.Clinically healthy, naturally mite-infested laying hens (P ≤ 0.013). Relative to the control group, mean O. sylviarum counts were reduced by at least 90% from Day 6 through Days 19, 22 and 22 in the fluralaner 0.25, 0.5 and 1.0 mg/kg groups, respectively. On Day 19, mean mite counts were lower in the 0.5 and 1.0 mg/kg groups compared with the 0.25 mg/kg group (P ≤ 0.018), and in the 1.0 mg/kg compared with the 0.5 mg/kg group (P = 0.014). There were no adverse events in treated birds.Source-birds maintained infestations throughout the study, validating the challenge to study birds. On Days 1 through 22, mean control group mite counts were significantly greater than those of the treated groups (O. sylviarum infestations despite continuous challenge from infested birds. By eliminating mites, this fluralaner solution has the potential to improve bird health and productivity, and to eliminate the burden of topical pesticide application.A fluralaner solution administered twice by gavage to laying hens with a one-week between-treatment interval was safe and effective in quickly controlling  Ornithonyssus sylviarum , infests a wide variety of domestic fowl and wild birds and has been described as the most important ectoparasite of poultry in the United States and as a serious pest, especially on chickens  [The northern fowl mite (NFM), reeders) –4. Mite reeders) , 6.Current treatments involve the use of topically applied pesticides, including organophosphates, pyrethroids and carbamates , 8. ApplTo address that need, fluralaner has been developed as an orally administered systemic ectoparasiticide for the treatment and control of mites in laying hens. Fluralaner, an isoxazoline, is a potent inhibitor of γ-aminobutyric acid (GABA)-gated chloride channels and L-glutamate-gated chloride channels, which are widely expressed in arthropod central nervous and peripheral neuromuscular systems , 11. ThiO. sylviarum. Three dose rates were investigated, each administered by oral gavage as two single administrations, 7 days apart. By quickly killing adults and immature stages, the first dose would remove the source of mite egg production. The second dose, based on the fluralaner half-life in poultry of 5 days, would ensure sufficient duration of activity to eliminate live mites that had hatched from eggs laid prior to the first dose [A study was undertaken to determine the efficacy of a drinking-water formulation of fluralaner in the treatment of hens naturally infested with rst dose .This was a randomized, assessor-blinded, block-design study with an untreated control group, masked to all study personnel except the principal investigator who took no part in study assessments. The data were collected in compliance with the study protocol, the FDA/CVM Good Clinical Practice Guidance Document #85, May 9, 2001, and applicable regulatory requirements .O. sylviarum infestations, were sourced from a commercial flock. To be eligible for inclusion in the study, selected birds could not have been treated with an acaricide or anthelmintic within the previous 60 days. Each bird was individually identified with metal leg bands and colored numbered leg bands coordinated with the treatments and the bird’s assigned pens. Staff performing study assessments, including mite counts, were masked to the treatment assignments corresponding to each leg band color.One hundred and fifty clinically healthy laying hens, ISA Browns, approximately 32 weeks of age with confirmed Prior to randomization and allocation to treatment groups, the birds were comingled in pens. Following treatment group allocation, birds were assigned to twelve enclosed pens (1.2 × 1.8 × 1.8 m), with 11 birds per pen. The birds were held under natural lighting, initially with 11 h and 49 min of indirect daylight, declining in the next month to 10 h and 30 min, with exposure to ambient temperature and humidity. No artificial lighting was provided at night. Clean wood shavings were provided as bedding on an as-needed basis. Hens were provided ad libitum access to the standard post-peak layer diet used by the UGA Poultry Research Center, free of antibiotics, coccidiostats, anthelmintics and other medications. The feed exceeded 1994 National Research Council nutrient recommendations. Fresh feed and water were provided daily via typical poultry feeders and gravity-fed nipple waterers. Routine cleaning was performed according to standard facility practices. Birds were checked daily to ensure safety and health. No concomitant medications were administered to any of the test birds.Mites infesting hens were counted by visually inspecting the area near the vent , the area known to contain the vast majority of mites on domestic hens . This prBased on Day -9 mite vent counts, the 150 hens were ranked from highest to lowest mite numbers and the 132 hens with the highest counts were placed into 11 blocks of 12 birds (the 18 birds with the lowest mite counts were retained to be used as substitutes if needed). Within each block, SAS version 9.3 was used to randomly assign birds to the 12 study pens. Approximately 1 week prior to the first treatment day, the 12 pens were randomly allocated to either one of three fluralaner treatment groups  or to an untreated control group, resulting in three pens per group. Following assignment, five birds from each pen, including the control pens, were randomly selected and designated as “source birds” that would remain untreated. Thus, each of the 11 pens consisted of six “non-source” birds, to be used in efficacy determination, plus five untreated mite-infested source birds. The objective of this design was to maintain blinding of study assessors, to stock the same density of birds in each pen of each group, and to ensure a consistent and similar challenge across groups for the duration of the study.The fluralaner solution (10 mg/ml) was diluted with tap water to a concentration of 0.2 mg/ml and administered to treatment-group birds on Days 0 and 7 at dose rates of 0.25, 0.5 or 1.0 mg/kg body weight, based on weights obtained on Day -1 and Day 5 using scales checked for accuracy prior to and after each day of weighing. Dose volumes were calculated for each bird individually. The respective volume of the treatment solution was administered using a previously described avian gavage technique . A 10 mlTo minimize fluralaner exposure across birds in the different treatment groups, materials and equipment used were assigned to a treatment group at the start of the study. The materials for any group were then maintained separately from equipment assigned to other treatment groups.From Day -9 through Day -1 birds were observed for general health. Following treatment on Day 0, birds were observed daily for general health and adverse events on Days 0 through 26.O. sylviarum mite counts from the six non-source birds in each of the control and fluralaner-treated group pens. The mite vent count data for each bird were transformed prior to analysis using the Y = ln(x + 1) transformation and analyzed using a mixed linear model with treatment as the fixed effect; block, pen(block) and treatment*pen(block) as the random effects. Data were analyzed on each count day separately. The primary software used for statistical calculations was SAS version 9.3.The primary effectiveness variable was based on adult The null hypothesis tested was that the six non-source birds in each fluralaner treatment pen would have the same mean mite counts as the six non-source birds in each control group pen, versus the alternative that the mite counts of the two groups would be different. The hypothesis was tested using a t-test at α = 0.05 (two-sided) significance level. Effectiveness was also calculated using arithmetic means.Percent efficacy was calculated using the Abbott’s formula:C - MT) / MC.Efficacy (%) = 100 ×  maintained natural mite infestation challenge throughout the study Fig. , Table 1Assay results of the fluralaner treatment solution showed that the average actual dose rates administered to individual birds in each of the three treated groups on Days 0 and 7 were: 0.26 and 0.24 mg/kg (for the 0.25 mg/kg group); 0.52 and 0.47 mg/kg (for the 0.50 mg/kg group); and 1.04 and 0.94 mg/kg (for the 1.0 mg/kg dose group).P ≥ 0.532). At all assessments from Days 1 through 22 the least squares means mite counts for the control group were significantly greater than the counts for each of the fluralaner groups (Table P ≥ 0.085). Relative to the control group, mean O. sylviarum counts were reduced by at least 90% from Days 6 through 19, 22 and 22 in the fluralaner 0.25, 0.5 and 1.0 mg/kg groups, respectively (Table t-test: t(66) = 5.0, P < 0.0001) and the 0.5 mg/kg groups (t-test: t(66) = 2.5, P = 0.014), and significantly lower in the 0.5 mg/kg group than in the 0.25 mg/kg group (t-test: t(66) = 2.4, P = 0.018). Linear and quadratic effects were both significant on Days 1, 2, 6, 8, 12, 15, 19 and 22 (P ≤ 0.0436).On Day -9 there were no significant differences between any of the groups of non-source birds P ≥ 0.53. At all There was one post-treatment abnormal health observation (a leg laceration) in a source bird in the control group that was recorded as an adverse event. There were no abnormal health observations in treated birds.Sarcoptes scabiei [The potent effect of fluralaner on mites has been demonstrated in dogs, applied either orally or topically, with a single treatment demonstrating complete (100%) elimination of  scabiei , 21. In  scabiei .O. sylviarum vent counts were present between all groups.Following treatment, early onset of action was observed at all fluralaner dose rates, with statistically significant reductions in mite vent counts, relative to the control group, observed in all treated groups from Day 1. For all treated groups, the significantly lower mite numbers, compared with the control group, were maintained through all assessments to Day 22. On Day 19, significant differences in the mean Dermanyssus gallinae  [These results demonstrate the potential for fluralaner to provide a substantial leap forward in the safe and effective management of northern fowl mite infestations. In addition, Maximum Residue Limits have now been approved in Europe at two single doses of 0.5 mg/kg, 7 days apart, for the treatment of poultry red mites, r, 1778) . FluralaO. sylviarum. Despite continued mite exposure by penning untreated, infested birds with fluralaner-treated birds, dose rates of 0.25, 0.5 and 1.0 mg/kg produced significant reductions in mean mite vent counts, relative to untreated controls, from Day 1 through Day 22. All dose rates maintained efficacy greater than 90% through Day 19, indicating that by providing an unprecedented level of northern fowl mite control fluralaner has the potential to improve the efficiency of poultry production, improve animal health and welfare, and eliminate the burden of topical pesticide application.A water-soluble formulation of fluralaner administered by oral gavage to laying hens on two occasions, with a one-week between-treatment interval, was effective in quickly controlling infestations with the northern fowl mite,


Diabetes mellitus is a very rampant metabolic disorder, particularly type II. It has many complications such as the septic foot. Diabetic septic foot (DSF) patients are at high risk for coagulation abnormalities as well as surgical hazards. Owing to the potential sequelae of coagulation and vascular abnormalities, this work aimed at studying the hemostatic state and platelet indices in diabetes type II patients with septic foot.A case-control study was conducted during the period from July to September 2017 at Dr. Awaad medical center, Red Sea State, Sudan. 57 diabetic patients with septic foot, aged between 17 and 78 years along with 57 non-diabetic subjects as control were enrolled. Sociodemographic data were collected using a structured questionnaire. Venipuncture blood was taken with necessary safety measures. Diabetes profile, coagulation studies as well as platelet indices were estimated. Data was analyzed using SPSS version 24.0 for windows. Ethical approval was considered and written consent from each participant was obtained.P < 0.009) was found to be significantly shorter in DSF compared to control. Plasma activated partial thromboplastin time (APTT) value was significant in diabetic septic foot , and thrombin time (TT) did not changed in DSF. Mean platelet volume (MPV), platelet distribution width (PDW), and platelet large cell ratio (P-LCR) values were significantly decreased in DSF compared to control . PDW values were positively correlated with PT, APTT, and D-Dimer (DD) . FVIII activity  was insignificant with DSF, as the DD was also insignificant (P < 0.081).The mean age of diabetic patients with septic foot and healthy controls were 48.49 ± 15.8 and 32.77 ± 14.0, respectively. The duration of the diabetes onset was 10.43 ± 9.5 years. Plasma prothrombin time (PT) value (12.61 ± 2.6 vs 13.67 ± 1.5, Diabetes mellitus is associated with prothrombotic tendency. Hypercoagulable state in DSF is indicated by shortened PT finding. PDW is a manifesting evidence that proves the presence of more reactive and aggregable platelets in DSF patients. Globally, diabetes mellitus is rising, the prevalence is steadily increasing everywhere, mostly in the middle-income countries . The preA case- control study was carried out among patients with diabetes type II presented with diabetic septic foot (DSF) at the Dr. Awaad Medical Centre (private sector clinic), Red Sea State, Sudan. 57 patients confirmed diabetic with septic foot were recruited in the study along with 57 non-diabetic healthy individual. Diabetic patient with septic foot for a long duration at least one month was enrolled in this study. Diabetic subjects who had clinical or laboratory signs of malignancy, coagulation disorders, liver impairment, cardiovascular diseases, or receiving medication that could affect the platelets and coagulation system were excluded. Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), factor VIII, D-Dimer (DD), fasting blood glucose (FBG), glycosylated hemoglobin (Hb A1c) and platelet indices were screened in this study.A structured questionnaire was utilized to collect the data from diabetic septic foot patients . The queOpen Epi calculator for case-control study was used to determine the sampling size. The standard normal variant for the power of the study is 80% where, the control exposure was measured at 23% . The min2-EDTA) according to the international committee (council) of standardization in Hematology (ICSH) recommends. 3 ml was collected in fluoride oxalate vacutainer for (FBS) measured by URIT 810 semi-automated chemistry analyzer. 2.7 ml was dispensed into vacutainer bottle containing tri-sodium citrate (0.109 M) as the third sample. An aliquot of plasma was transferred into another plastic tube and promptly performed the PT, APTT, and TT . The remainder was frozen in − 80%°C until assayed for factor VIII and D-Dimer levels. Platelets indices were measured immediately with semi-automated hematology counter (Sysmex KX-21). Glycosylated hemoglobin was estimated by NycoCard® method using NycoCard® READER II .A venipuncture vacuum tube method was used to collect blood sample in the morning between 8.00–9.00 a.m. after at least 12 h fasting. 3 ml added to vacutainer bottle containing di-potassium ethylene diamine tetra acetic acid  was used for the statistical analysis.r = 0.39/p < 0.003, r = − 0.33/p < 0.014, r = 0.27/p < 0.043, r = 0.30/p < 0.024, r = 0.31/p < 0.019, and r = 0.27/p < 0.040, respectively).Fifty-seven DFS patients 33 (57.9%) were males and 24 42.1%) were females, with the mean age being 48.49 ± 15.8 years. Among the 57 controls 44 (77.2%) were males and 14 (22.8%) were females with the mean age being 32.77 ± 14.0 years. The laboratory characteristics for all participants, including diabetes profile, platelet indices, markers of coagulation and fibrinolysis are presented in Table % were feP < 0.000) and positively correlated with APTT  and negatively correlated with PT, TT, FVIII, DD, PLT, MPV, PDW, PCT and P-LCR .In the present study, FBG and HbA1c values were found to be significantly higher in DSF patients when compared to control . Furthermore, the MPV, PDW, and P-LCR were lower in DSF than the control. While PLT and PCT were higher in DSF as compared with the controls . Platelet count also correlated positively with MPV, PDW, PCT, and P-LCR  and negatively with PT, APTT, TT, VIII, and DD . PLT and PCT values were increased in DSF patients as compared with the non-diabetic subjects.PDW value has been recognized as an evidence of platelet activity and was significantly correlated with PT, APTT, DD, and PLT (P < 0.009 and 0.000). Whereas the thrombin time coagulation test was insignificant (0.801). 50.9% of the DSF patients had shortened PT (< 12.0 s) indicating that there is a significant effect of the disease on PT results. PT value was correlated positively with APTT, PDW, P-LCR , and MPV . The statistical details of the coagulation tests are illustrated in Table In this study, the plasma prothrombin time and activated partial thromboplastin time levels were significant in diabetic septic foot compared to the control (ed PT < 1.0 s indiP < 0.951) as depicted in Table However, the concentration of DD showed decreased levels for DSF patients in comparison with the control (130.81 ± 81.54 vs 150.61 ± 52.98). In contrast, factor VIII concentration demonstrated increased levels in DSF (121.86 ± 174.8 vs 98.66 ± 31.83), although it was insignificant (Diabetes impairs the coagulation homeostasis which cause vascular thrombotic events . In this2 and serotonin than the small platelet [With regard to platelet indices which represent as new insight in diagnosis and prediction of the platelet activity, the MPV, PDW, PCT, and P-LCR values were observed statistically significant in this study. The MPV denotes the platelet average size and activity . Large pplatelet . The MPVplatelet . In factplatelet , 17. Higplatelet . Howeverplatelet . With replatelet  reportedplatelet . Moreoveplatelet  and Jindplatelet  and contplatelet . Howeverplatelet .The short result of prothrombin time and activated partial thromboplastin time predisposes the patients to high risk of thrombosis . So, theFVIII has been reported to be increased in patient with type II diabetes. This augmentation may be relevant to the presence of endothelial dysfunction or inflammatory condition . 95% of It is important to know that diabetes is associated with prothrombotic state. Hypercoagulability was indicated in DSF by shortened PT. PDW is a manifesting evidence that proves the presence of more reactive and aggregable platelets in DSF patients.


Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma sp., Aspergillus sp., Schizophyllum commune, and Volvariella volvacea  supplemented with 60 mg mL−1 geneticin. Briefly, 2 mL from the cell bank was inoculated into 18 mL of culture medium and incubated for 24 h at 28°C and 180 rpm. The preinoculum was used to inoculate 180 mL of fresh culture medium and incubated for 24 h at 28°C and 180 rpm. Finally, the inoculum was used to inoculate 1000 mL of fresh culture medium at the bioreactor. After 15 h culture, 1200 mL of fresh medium was added to reach a final volume of 2.4 L and cultured during additional 96 h at 28°C, 400 to 800 rpm, and pH 6.0.Theβ-D-cellobioside  in sodium acetate buffer 50 mM, pH 5.0. One enzyme unit was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per min, and the activity was expressed as U L−1.The CBHII activity was carried out as previously described , 19, usiKM and Vmax) were estimated for the crude extract by using pNPC between 0 and 6.5 mM and fitting the experimental data to a Michaelis-Menten model using the software GraphPad PRISM 6.0.The CBHII enzyme extract was evaluated at different pH and temperature conditions, using pNPC. To evaluate the effect of pH and temperature on enzyme activity, the enzyme activity assay was performed at 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 ± 0.2 and at 30, 40, 50, 60, and 70°C for 1 h. The reactions were stopped and read as described above. Apparent kinetic parameters  cellulolytic and hemicellulolytic extract from a Penicillium sp. culture (hereafter Ce-Hem extract), (2) concentrated recombinant CBHII extract from W. anomalus 54-A (hereafter rCBHII), and (3) a 1 : 1 Ce-Hem : rCBHII extracts mixture. Control culture was carried out by using 40 mL of distilled water. All cultures were carried out with 10 mM sodium azide, 0.1% tween 80, and pH 5.0. The response variable was the concentration of reducing sugars as measured by DNS method [−1 of reducing sugars after subtraction of the results obtained with the control cultures, and normalized by the initial unis of CBHII present in each extract. Production of Ce-Hem extract was carried out by using a Penicillium sp. strain (Collection of Microorganisms of Pontificia Universidad Javeriana). For this purpose, 20 mL of a 106 conidia mL−1 suspension was inoculated in 200 mL of a rice straw medium without nitrogen source and incubated for 8 days at 28°C and 120 rpm. The crude extract was obtained by centrifugation and sequential filtrations through Whatman paper Number 42 and 0.45 and 0.22 μm polyether sulphone membranes . Concentrated recombinant CBHII extract from W. anomalus 54-A was produced by using a 2.4 L culture of the W. anomalus 54-A Celo-3.2 clone, as described above. Culture medium was centrifuged at 8000g and filtered sequentially through Whatman paper Number 42 and 0.45 and 0.22 μm polyether sulphone membranes . Permeate was ultrafiltered through a 30 kDa cut-off membrane , until reaching a final volume of 20 mL. CBHII activity in both extracts was assayed as described above. The β-glucosidase (BG) activity in both extracts was assayed using 5 mM 4-nitrophenyl β-D-glucuronide (Sigma-Aldrich) as previously described [μmol of p-nitrophenol per min, and the activity was expressed as U L−1. The endoglucanase (EG) activity in both extracts was assayed using 2% carboxymethylcellulose (Sigma-Aldrich) [μmol of reducing sugars, equivalent to glucose, per min [−1.The effect of recombinant CBHII on cellulose hydrolysis was evaluated by usingescribed , 19, witS method . Resultsescribed , 19. OneAldrich) , 19 and Aldrich) . One EG p < 0.05) was considered significant. All analyses were performed using the software GraphPad PRISM 6.0.Differences between groups were tested for statistical significance by using one-way ANOVA. An error level of 5%   and MALDI-TOF analysis . W. anomalus is a yeast that have been isolated from different sources, such as food- and feed-related systems, insects, and human clinical samples. This yeast has a wide robustness to environmental stresses conditions like extreme pH or low water activity [ W. anomalus strains include biopreservation agent due to antimicrobial activity against variety of microorganisms in fruits and cereals and production of enzymes , biofuels, and bioremediation [In this study, we evaluated the production of the recombinant CBHII in the native yeast strain 54-A, previously isolated from sugar cane bagasse from Puerto López, Meta, Colombia. This strain showed a carbon assimilation profile  different from that observed foranomalus ) with a activity . Biotechediation , 15. How W. anomalus 54-A with the vector pKS2-ST::CBHII , which were confirmed by PCR. The ADH2 gene promoter is repressed in the presence of glucose, since this metabolite inhibits the expression of the alcohol dehydrogenase regulator protein (Adr1) that is an activator of the ADH2 gene. On the other hand, under glucose depletion conditions, the expression of Adr1 is increased leading to the induction of the ADH2 gene [ W. anomalus 54-A pKS2-ST::CBHII clones, to establish the beginning of the induction phase. We observed, for all the evaluated clones, that, at the final culture step, the glucose was consumed within the first 6 h of incubation . In this sense, this point was considered as the beginning of the induction phase, which agrees with previous reports of the production of recombinant proteins in yeast under the control of the ADH2 gene promoter [ W. anomalus 54-A Celo-3.2 clone, reaching a final enzyme activity of 5.7 U L−1 after 96 h of induction  prepared from Avicel PH-101 as substrate [ W. anomalus 54-A. However, a crude culture broth from Penicillium sp. containing wild-type cellulase and hemicellulase enzymes (see Chrysanthemum Wastes Degradation Assay) showed a CBHII activity of 8.5 U L−1 using the pNPC substrate, which was similar to that observed for the recombinant CBHII produced in W. anomalus 54-A. Nevertheless, these results are lower than those observed for erlenmeyer flask cultures of Pleurotus ostreatus, which showed an activity of 445.3 ± 27.6 U L−1 using the pNPC substrate [As was mentioned above,revisiae , 9, P. ppastoris , 11, andubstrate , while rubstrate , 19. W. anomalus 54-A Celo-3.2 showed that recombinant CBHII has an apparent molecular weight of about 66 kDa ( T. reesei (50–58 kDa) [ W. anomalus 54-A has a molecular weight similar to that reported for a CBHII produced in P. pastoris X33 and Y. lipolytica POld (~63–65 kDa) [ P. pastoris GS115 (~58 kDa) [SDS-PAGE analysis of extracellular fraction oft 66 kDa . This re–58 kDa) . However–65 kDa)  but high~58 kDa) . Differe~58 kDa) .To improve the production of recombinant CBHII, two glucose and glycerol concentrations were evaluated. First, 1% w/v glucose was evaluated, which is lower than the concentration suggested by the vector manufacturer (2% w/v). We hypothesize that 1% w/v glucose would be depleted faster than 2% w/v, allowing a reduction in the induction time and a longer and higher enzyme production . In addi−1) and enzyme activity normalized by biomass (U g−1 dry weight of cells). A similar activity profile was observed between volumetric and normalized enzyme activities, suggesting that the production of recombinant CBHII was associated with yeast growth. In fact, the profile of recombinant CBHII production was similar to that previously reported for the ADH2 promoter, with a complete induction in the late stationary phase (>36 h culture) [p < 0.001). Finally, the highest CBHII activity was observed at 1% (w/v) glucose, 1% (w/v) glycerol, although this was not statistically different regarding the levels obtained with 2% (w/v) glucose (p > 0.05). However, 1% (w/v) glucose, 1% (w/v) glycerol, allowed an earlier detection of the enzyme activity than that observed with 2% (w/v) glucose. In this sense, 1% (w/v) glucose, 1% (w/v) glycerol, was selected to produce recombinant CBHII at 2.4 L scale. The reasons of the effect of glycerol on CBHII activity are unknown, and further investigations should be carried out to clarify this aspect. Nevertheless, it is important to mention that there is a wide variation among yeasts, and even strains, regarding glycerol metabolism  [ S. cerevisiae, limited information is available for other yeasts [ W. anomalus 54-A.culture) . The resabolism) . Althougr yeasts . In this−1). This activity significantly increased after 10 h (2.6 ± 0.09 U L−1) and continuously kept increasing to reach a final enzyme activity of 14.5 ± 0.86 U L−1, which was 2.7-fold higher than that observed at 55 mL scale. These results might suggest that, under controlled culture conditions, the strict repression of the ADH2 promoter might be affected allowing the expression of the recombinant protein even in the presence of residual glucose.The production of recombinant CBHII enzyme at 2.4 L scale was carried out for 117 h after the final feeding step .  W. anomalus 54-A was evaluated. In this work, crude extract rather than the purified recombinant CBHII was used for characterization, since this crude extract would be used in the production of bioethanol by the degradation of Chrysanthemum residues [p < 0.001), while the highest enzyme activities were observed at pH 5.0 and 6.0 ± 0.2 at all evaluated temperatures (p < 0.001). On the other hand, at pH 3.0 and 8.0 ± 0.2, a marked decrease in the enzymatic activity was observed regardless of the reaction temperature.The effect of temperature and pH on the enzyme activity of the recombinant CBHII produced inresidues . As obse P. pastoris showed an optimum reaction pH and temperature of 5.0 ± 0.2 and 50°C, respectively. In addition, this recombinant enzyme showed a significant loss of activity at pH of 3.0, 7.0, and 8.0 and at 30°C and temperatures above 65°C [ P. pastoris and Y. lipolytica showed an optimum pH between 5.0 and 6.0 and at a temperature of 60°C. Recombinant CBHII produced in P. pastoris and Y. lipolytica also showed rapid inactivation at pH and temperatures above 7.0 and 60°C, respectively, while at temperatures below 40°C, an 80% loss in the enzyme activity was observed [ T. reesei CBHII produced in W. anomalus 54-A has a reaction pH profile similar to that reported for recombinant CBHII enzymes expressed in other yeast. Nevertheless, it seems that the optimum temperature of reaction differs among the recombinant CBHII enzymes, which might be associated with the substrate used for the enzyme activity assay or the posttranslational modifications carried out for each host. It is noteworthy that recombinant CBHII produced in W. anomalus 54-A only showed 30% reduction in enzyme activity at 30°C. Functionality at 30°C might allow the use of this enzyme in SSF processes for the production of bioethanol, since this temperature is also the optimum growth temperature for S. cerevisiae that is the preferable microorganism used during the fermentation stage of bioethanol production [Using microcrystalline cellulose PH101 as substrate, recombinant CBHII produced inobserved . In thisoduction . T. reesei CBHII produced in W. anomalus 54-A showed KM and Vmax of 2.73 mM (est. error 0.26) and 23.1 µM min−1 (est. error 0.89), respectively. This KM value agrees with those reported for wild-type CBHII enzymes (EC 3.2.1.91) isolated from different microorganisms, which vary between 0.1 and 3.1 mM, depending on the source of the enzyme [ T. reesei CBHII enzymes there are no reports of kinetic parameters using pNPC substrate. Nevertheless, a wide variation in KM values is observed , depending on the substrate used for this estimation [Kinetic parameters for the crude extract were estimated using pNPC as substrate . Recombinante enzyme . In the timation . Chrysanthemum wastes degradation assay was performed using three enzyme extracts: (1) a crude culture broth from Penicillium sp. containing wild-type cellulase and hemicellulase enzymes (Ce-Hem extract), (2) a concentrated recombinant CBHII extract from W. anomalus 54-A (rCBHII extract), and (3) a 1 : 1 Ce-Hem : rCBHII extracts mixture. After treatment, 0.2 ± 0.02, 0.4 ± 0.01, and 1.25 ± 0.1 g L−1 of reducing sugars were observed for Ce-Hem, rCBHII, and 1 : 1 Ce-Hem : rCBHII extracts, respectively. However, when reducing sugar concentration was normalized by the CBHII units present in each extract at the beginning of the experiment (p > 0.05). Noteworthy, the use of a 1 : 1 Ce-Hem : rCBHII extracts mixture allowed an increase of about 5-fold, in the reducing sugars per CBHII unit, in comparison with the results obtained by using the Ce-Hem or concentrated recombinant CBHII extracts alone, suggesting a synergistic effect of the combination of these two enzymatic extracts. To understand the reasons of this synergistic effect, we measured the activity of β-glucosidase (BG) and endoglucanase (EG) in the extracts , manganese peroxidases, cellulases, and xylanases [−1), showing the importance of lignin degradation to increase biomass digestibility [ W. anomalus 54-A carrying a recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi, showing the potential of W. anomalus 54-A as a host to produce recombinant cellulases.g sugars . The higylanases –32. The ylanases , 33. In ylanases  or steamylanases  pretreattibility . Neverth T. reesei CBHII in W. anomalus 54-A strains. The results showed production of recombinant CBHII in W. anomalus 54-A, with enzyme activity levels of up to 14.5 U L−1. Recombinant CBHII showed optimum pH and temperature reaction of 5.0–6.0 ± 0.2 and 40°C, respectively, which were similar to those reported for other recombinant T. reesei CBHII enzymes. Km of this recombinant CBHII was between the ranges of values reported for other CBHII enzymes. The results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi, showing the potential of W. anomalus 54-A as a host to produce recombinant cellulases. Noteworthily, this is the first report on the use of W. anomalus as a host to produce heterologous proteins. Further studies should be focused on the consolidation of W. anomalus as a platform to produce recombinant proteins such as the design of specific expression vectors and the generation of auxotrophic strains.In conclusion, we reported the production of the recombinantSupplementary materials show: (1) Comparison of S. cerevisiae and W. anomalus codon usage, (2) Expression vector pKS2-ST::CBHII, (3) Wickerhamomyces anomalus 54-A identification by ITS amplification and MALDI-TOF, (4) Residual glucose during the culture of W. anomalus 54-A pKS2-ST::CBHII clones, and (5) Estimation of kinetic parameters for the recombinant T. reesei CBHII.


Candida albicans, Aspergillus niger, Aspergillus oryzae and Fusarium oxysporum. The Cu-BTC MOF can effectively inhibit the growth rate of C. albicans and remarkably inhibit the spore growth of A. niger, A. oryzae and F. oxysporum. This finding shows the potential of using Cu-BTC MOF as a strong biocidal material against representative yeasts and moulds that are commonly found in the food and agricultural industries.Although metal-organic frameworks (MOFs) or porous coordination polymers have been widely studied, their antimicrobial activities have not yet been fully investigated. In this work, antifungal activity of copper-based benzene-tricarboxylate MOF (Cu-BTC MOF), which is water stable and industrially interesting, is investigated against  A binuclear Cu2 paddle wheel unit is connected into a three-dimensional structure of an octahedron shape with square-shaped pores (9 Å by 9 Å) [2 g−1), large pore volume (approx. 0.7 cm3 g−1) and good thermal stability (up to 350°C) [Metal-organic frameworks (MOFs) or porous coordination polymers are a group of highly porous crystalline materials consisting of metal clusters interconnected with organic linkers –6. Owing by 9 Å) . Cu-BTC  by 9 Å)  and memb by 9 Å)  because o 350°C) . While CIt is necessary to note here that Cu ions have a powerful antibacterial activity on both Gram-negative and Gram-positive bacteria as well as antifungal activity ,14. The Candida sp., Aspergillus sp. and Fusarium sp. cause serious and common infections. Candida albicans is a dimorphic fungus that can form both yeast and filamentous cells, typically contaminating foods leading to foodborne disease and food spoilage. In some certain clinical situations, C. albicans may become virulent [Aspergillus species. Aspergillus niger and A. oryzae are usually found in bakery products, intermediate-moisture food products, cheeses, preserved fruits and grains. As a result, C. albicans, A. niger and A. oryzae were selected as the infectious fungi to be tested with the Cu-BTC. In addition, a fungus namely Fusarium oxysporum, typically found in soils leading to common adulteration in agricultural products, was also chosen as a representative of fungi because it has a huge impact on the environment. This fungus can grow on organic matter in soils and the rhizosphere in plants, which can give rise to saprophytic plant pathogens [Fungi are a member of eukaryotic organisms including yeasts and moulds. Among various resourceful fungal pathogens, virulent  because athogens .C. albicans, A. niger, A. oryzae and F. oxysporum, HKUSR-1 MOF can inhibit Saccharomyces cerevisiae [C. albicans, A. niger, A. oryzae and F. oxysporum in the culture media.Although there is no previous report on the antifungal activity of Cu-BTC against revisiae . Our res2.2.1.N,N-dimethylformamide , ethanol , Sabouraud Dextrose Agar , Sabouraud Dextrose Broth  and Potato Dextrose Agar  were of analytical reagent grade. All chemical compounds were diluted with deionized water .Copper nitrate trihydrate , trimesic acid , 2.2.3)2·3H2O was dissolved in deionized water (25 ml) and stirred for 15 min. Trimesic acid ligand (8 mM) was dissolved in DMF (25 ml). The two solutions were then mixed together by stirring at room temperature (25°C) for 10 min. The mixture was transferred to a Teflon-lined stainless-steel autoclave and heated at 110°C for 24 h. After the reaction, it was cooled naturally to room temperature. Blue crystals of Cu-BTC were recovered by washing with ethanol and deionized water for five times and dried in a vacuum oven at 100°C for 24 h. The as-synthesized material was characterized by field-emission scanning electron microscopy , transmission electron microscopy , Fourier transform infrared  and X-ray diffraction . To confirm that Cu-BTC is water stable, 2 g Cu-BTC was immersed in 50 ml deionized water for a week and filtered by a vacuum filtration. The element in the filtered water was measured by wavelength-dispersive X-ray fluorescent spectroscopy . The details of WDXRF measurement can be found in the electronic supplementary material.Cu-BTC (HKUST-1) was synthesized by a conventional hydrothermal method previously reported ,21. Brie2.3.Candida albicans (TISTR 5779), a yeast, was obtained from a culture collection at Thailand Institute of Scientific and Technological Research. The assay was prepared by cultivating the microorganism in SDB medium under shaking at 150 r.p.m. at 25°C for 16–18 h. The culture was sterile diluted in a saline solution to reach a concentration of approximately 1.5 × 108 colony forming units (CFU) per millilitre. For the fungicidal rate test, the Cu-BTC suspension (100–500 ppm) was mixed with the fungal suspension at a volume ratio of 1 : 2. The culture suspension was shaken at 150 r.p.m. at 25°C for 15, 30, 45 and 60 min, respectively after which 100 µl of culture suspension was spread on SDA plates. The number of viable fungal colonies was counted after incubation for 48 h at 25°C. Note, to confirm that Cu-BTC is stable in the culture, the culture with 500 ppm Cu-BTC was washed by vacuum filtration five times. The element in the filtered water was measured by WDXRF.A. niger, A. oryzae and F. oxysporum were also selected to test the antifungal activity of Cu-BTC. The antifungal activity assay was determined by a poison food technique because those fungi are normally found contaminating foods [dc is the colony diameter (millimetres) of the control; dt is the colony diameter (millimetres) of the treated disc.At the same time, three moulds including ng foods . BrieflyThe percentage inhibition was reported based on an average value of three replicates.2.4.C. albicans was cultivated in SDB medium under shaking at 150 r.p.m. at 25°C for 16–18 h. The culture was sterile diluted in a saline solution to a concentration of approximately 1.5 × 108 CFU per millilitre. The culture suspension was mixed and inoculated with Cu-BTC at a concentration of 500 ppm under shaking at 150 r.p.m. at 25°C for 60 min. Next, the suspension was filtered and dehydrated with different concentrations of ethanol  for 15–20 min at each step and put into a critical point dryer using liquid CO2. Finally, the dehydrated samples were coated with gold–palladium for SEM analysis .For yeast, 2. Finally, the dehydrated samples were coated with gold–palladium for SEM analysis.For moulds, the Petri plates with an 8 mm diameter mycelium at the centre of the disc were inoculated with PDA mixed with Cu-BTC at different concentrations (100–500 ppm) at 25°C for 7 days. After inoculation, the peripheral mycelial disc was taken for SEM analysis. The samples were dehydrated with different concentrations of ethanol  for 15–20 min at each step and put into a critical point dryer using liquid CO3.3.1.After 2 g Cu-BTC was immersed in 50 ml deionized water for a week and then filtered by vacuum filtration, the WDXRF of the filtered water shows no copper indicating that the Cu-BTC is water stable. The morphology of the as-synthesized Cu-BTC was investigated by FE-SEM and TEM as shown in a. All observed major peaks at 9.5° (220), 11.7° (222), 13.5° (400), 14.7° (331), 16.5° (422), 17.5° (511), 19.1° (440) and other minor peaks are the characteristics of pure Cu-BTC with a face centre cubic (FCC) structure, which are in good agreement with other previous work [X-ray diffraction pattern (XRD) analysis was carried out to study the crystalline structure of the as-synthesized material as shown in figure 2at 9.5° 2, 11.7°  also confirms the characteristics of Cu-BTC. Two peaks observed at 590 and 734 cm−1 are attributed to the vibrational modes of Cu-O. The peaks between 1390 and 1659 cm−1 represent the stretching modes of the BTC ligand [−1 can be assigned to the vibrational mode of the carboxylic group in trimesic acid [−) is coordinated with two Cu(II) ions by bridging via a syn‒syn configuration [−1 relate to the epoxy/peroxide and oxygen groups [−1 is due to the coordinated water.The FTIR spectrum b also coC ligand ,26. A smsic acid . This caguration . The pean groups ,30. The 3.2.C. albicans, which is a representative of yeasts, the dilution plate count technique was used. Different concentrations of Cu-BTC  were incubated for 60 min and spread on SDA plates as shown in C. albicans by approximately 96% at 300 ppm and up to 100% at 500 ppm.To study the antifungal properties of Cu-BTC against C. albicans. The number of C. albicans colonies after treatment with Cu-BTC at different concentrations and incubation time were counted at 25°C. It was found that 500 ppm of Cu-BTC and the incubation time of 60 min are the most effective conditions for inhibiting the growth of C. albicans when compared with other conditions and other previously related work [C. albicans, which is a dimorphic fungus, e.g. yeast and filamentous cell [In addition, the incubation time is another important factor for inhibiting the growth of ted work . It can ous cell , can be C. albicans after treatment with Cu-BTC was compared with the control sample, the untreated C. albicans. The control sample is shown in figure 4a to present a smooth surface of C. albicans, as seen in another previous report [figure 4b), C. albicans has many wrinkles with a rough surface. This can be attributed to the damage of the cell membrane thanks to the leakage of intracellular compounds leading to complete cell disruption. This result is rather similar to other previous reports using other antimicrobial nanomaterials such as ZnO nanoparticles, Ag nanoparticles, Ag–ZnO nanocomposites and reduced graphene oxide [To further confirm the result of the antifungal assay, the morphology of s report . After tne oxide –34, indi3.3.F. oxysporum, A. oryzae and A. niger), which are normally found in contaminated foods, were selected as the representatives of the moulds [F. oxysporum  and aluminium to inhibit F. oxysporum but not A. niger [To investigate the antifungal activity of Cu-BTC on moulds, the poison food technique or assay was used. In this experiment, three species of fungi  of F. oxysporum after treatment with Cu-BTC was shrunk when compared with the untreated morphology (figure 8a). For Aspergillus species including A. Oryzae (figure 8c) and A. niger (figure 8e), the hyphae surface after treatment with Cu-BTC becomes rough with granules on the surfaces of A. Oryzae (figure 8d) and A. niger (figure 8f), respectively. Note, the result here is quite similar to the inhibition of Trichophyton rubrum by allicin and garlic extracts previously reported [To further confirm the antifungal activities of Cu-BTC against the mycelial growth of reported , where i4.C. albicans, A. niger, A. oryzae and F. oxysporum. The Cu-BTC can damage the cell membrane resulting in the leakage of intracellular compounds leading to complete cell disruption and eventually death. As the Cu-BTC concentration increases, the inhibitory efficiency also increases. An amount of 500 ppm Cu-BTC and an incubation time of 60 min are found to be the most effective conditions for inhibiting the growth of C. albicans. For A. niger, A. oryzae and F. oxysporum, the Cu-BTC does inhibit the growth process of spores. The antifungal mechanism of Cu-BTC is most possibly based on the ROS generated via the oxygen reduction of oxygen gas by the Cu-BTC. The finding in this work may lead a new antifungal application of Cu-BTC. By contrast, it may lead to awareness of using Cu-BTC, which may have a negative impact on useful microorganisms in the environment. Also, further investigation on the antifungal mechanism of Cu-BTC and other water stable MOFs is needed.Cu-BTC, which is water stable, shows a strong inhibitory activity against


Drosophila model of Aβ (amyloid beta) toxicity, we show that genetic overexpression of a glucose transporter, specifically in neurons, rescues lifespan, behavioral phenotypes, and neuronal morphology. This amelioration of Aβ toxicity is associated with a reduction in the protein levels of the unfolded protein response (UPR) negative master regulator Grp78 and an increase in the UPR. We further demonstrate that genetic downregulation of Grp78 activity also protects against Aβ toxicity, confirming a causal effect of its alteration on AD-related pathology. Metformin, a drug that stimulates glucose uptake in cells, mimicked these effects, with a concomitant reduction in Grp78 levels and rescue of the shortened lifespan and climbing defects of Aβ-expressing flies. Our findings demonstrate a protective effect of increased neuronal uptake of glucose against Aβ toxicity and highlight Grp78 as a novel therapeutic target for the treatment of AD.Glucose hypometabolism is a prominent feature of the brains of patients with Alzheimer’s disease (AD). Disease progression is associated with a reduction in glucose transporters in both neurons and endothelial cells of the blood-brain barrier. However, whether increasing glucose transport into either of these cell types offers therapeutic potential remains unknown. Using an adult-onset  •Drosophila Aβ toxicity modelOverexpression of glucose transporter Glut1 rescues a •Glut1 overexpression reduces Grp78 protein levels and induces the UPR•DrosophilaA Grp78 dominant-negative mutant also rescues Aβ toxicity in •Metformin rescues Aβ toxicity and leads to reduced Grp78 expression Drosophila Alzheimer’s disease model. This was associated with a reduction in Grp78 levels and induction of the unfolded protein response. This rescue can be mimicked by using metformin, a drug known to induce glucose uptake.Niccoli et al. show that overexpression of the glucose transporter Glut1 in neurons rescues Aβ toxicity in a  This drGlucose does not freely cross cell membranes and is, instead, actively shuttled by transporters. In humans, there are 12 glucose transporters, with different expression patterns and affinities. In the brain, Glut1 is expressed mainly in glia and endothelial cells, whereas Glut3 is expressed in neurons . A reducWhether impaired neuronal glucose metabolism plays a causal role in neurodegeneration in AD awaits investigation. The drop in glucose metabolism could contribute to disease progression in several ways. It could lead to a reduction in ATP in neurons, since glucose is the main source of energy. Downregulation of the hexosamine pathway, which relies on glucose for GlcNAc production, would lead to a reduction in Tau GlcNAcylation, which, in turn, could drive up toxic Tau phosphorylation, since the two are negatively correlated . HypometDrosophila melanogaster [Drosophila has proved to be an excellent model system in which to study neurodegenerative diseases. The fly has a distinct brain structure with cell types analogous to the human brain, as well as a blood-brain barrier (BBB), and is, therefore, ideal for studying the neurodegenerative process in a complex tissue. The metabolic coupling between glia and neurons observed in mammalian brains is also conserved in flies [To begin to experimentally test the role of glucose transport and metabolism in AD pathogenesis, we used a model of Aβ toxicity in the fruit fly nogaster . Drosophin flies . The flyin flies  exclusivin flies .If lowered glucose metabolism in neurons is part of the pathogenic cascade from toxic Aβ, then experimentally increasing glucose metabolism in neurons should ameliorate the pathogenesis in the AD model. Therefore, we assessed the effect of overexpressing a glucose transporter, Glut1, in the neurons of the AD flies. We found that this partially rescued the Aβ phenotypes, without affecting the expression level of the toxic Aβ peptide. Glut1 overexpression led to downregulation of the expression of Grp78 (glucose-regulated protein 78/BiP), the negative master regulator of the UPR. This, in turn, increased the UPR, in association with an improvement in protein homeostasis. Interestingly, feeding the flies the drug metformin, which increases glucose transport, also caused a drop in Grp78 levels and an increase in lifespan, suggesting a possible pharmacological therapeutic avenue.Drosophila melanogaster has two glucose transporters: Glut1 and Glut3. Glut3 is expressed only in testes, while Glut1 is expressed ubiquitously [uitously . In ordeNext, we overexpressed Glut1 in the neurons of adult flies, using an inducible elavGS  driver and confirmed the overexpression by qPCR in fly heads B. OverexDrosophila brain  cycle at constant humidity and on standard sugar-yeast-agar (SYA) medium . Adult-onset, neuron-specific expression of UAS constructs was achieved as described elsewhere  and obtaAll transgenes were backcrossed into the w1118 background to ensure a homogeneous genetic background between transgenic lines. All experiments were carried out on mated females, unless otherwise stated.Flies were raised at standard density in 200-ml bottles. After eclosion, flies were allowed to mate for 24–48 hr. At least 110–150 females of the appropriate genotype were split into groups of 15 and housed in vials containing SYA medium with or without drugs. Deaths were scored, and flies tipped onto fresh food three times a week. Data are presented as cumulative survival curves, and survival rates were compared using log-rank tests or Cox proportional hazards performed in JMP (version 9.0) software (SAS Institute). All lifespans were performed at 25°C unless otherwise stated.The climbing assay in Protein samples were prepared by homogenizing in 2× SDS Laemmli sample  and boiled at 95°C for 5 min. Samples were separated on pre-cast 4%–12% Invitrogen Bis-Tris gels (NP0322) and blotted onto PVDF (polyvinylidene fluoride) or nitrocellulose membrane (for Grp78) in Tris-glycine buffer supplemented with 10% ethanol. Membranes were blocked in 5% milk and 1% BSA in TBS-T  for 1 hr at room temperature (RT) and then incubated with primary antibodies in block. Ubiquitin westerns were blocked in 1% BSA. Primary antibody dilutions used were as follows: anti-Grp78, 1:1,000 ; anti-actin, 1:10,000 ; anti-Ubiquitin, 1:1,000 ; and anti-eIF2A-phospho, 1:1,000 . Secondaries used were anti-rabbit and anti-mouse  at 1:10,000 dilutions for 1 hr at RT. Bands were visualized with Luminata Forte (Millipore) and imaged with ImageQuant LAS4000 . Quantification was carried out with ImageQuant software or ImageJ.g for 10 min at 4°C, and the supernatant was collected as the Triton X soluble fraction. The pellet was re-suspended in 50 μl SDS extraction buffer and spun again, and the supernatant was collected as the SDS soluble fraction. Samples were stored at −80°C and run as described earlier.The method was adapted from . BrieflyTotal RNA was extracted from heads or whole flies for  and convg for 5 min at 4°C, and cleared supernatant was retained as the total fly Aβ42 sample. Aβ42 was measured with an ELISA kit , according to the manufacturer’s instructions, and protein was measured with a Bradford assay (Bio-Rad protein assay reagent), and the amount of Aβ42 in each sample was expressed as a ratio of the total protein content . Data are expressed as the mean ± SEM obtained from three biological repeats for each genotype.Five fly heads were homogenized in 50 ml GnHCl extraction buffer  and centrifuged at 21,000 × Brains were dissected in 4% paraformaldehyde in PBS with Tween 20 (PBST), incubated for 20 min, rinsed in PBST, mounted in Vectashield with DAPI, and imaged on a Zeiss LSM510 inverted confocal microscope. Images were taken on the 20× or 40× objective as stacks and are shown as maximum intensity projections of the complete stack. The same size stacks were taken for experimental and control samples. All images for one experiment were taken at the same settings. For Xbp1 fluorescence in C, total Drosophila Activity Monitoring System; TriKinetics) as described previously [Individual, 21-day-old, mated female flies were placed in glass tubes (65 mm × 5 mm) containing standard 1× SYA, and activity was recorded using the DAM System (eviously . Flies weviously . Sleep weviously . All behFlies were tipped into vials containing 1% agar, 1.5% sucrose, and 10 mg/l of tunicamycin. Dead flies were scored at regular intervals, and lifespan curves were compared with log-rank test.Conceptualization, T.N. and L.P.; Methodology, T.N., A.T., F.K., and L.P.; Investigation, T.N., M.C., A.T., F.K., L.L., C.T.W., D.C., L.B., and A.J.V.; Writing – Original Draft, T.N.; Writing – Review & Editing, T.N. and L.P.; Funding Acquisition, L.P.; Resources, S.G. and J.D.; Supervision, T.N. and L.P.