Growth of the fungus Paecilomyces lilacinus with n-hexadecane in submerged and solid-state cultures and recovery of hydrophobin proteins

The filamentous fungus Paecilomyces lilacinus was grown on n-hexadecane in submerged (SmC) and solid-state (SSC) cultures. The maximum CO₂ production rate in SmC (V_max = 11.7 mg CO₂ L_g⁻¹ day⁻¹) was three times lower than in SSC (V_max = 40.4 mg CO₂ L_g⁻¹ day⁻¹). The P. lilacinus hydrophobin (PLHYD) yield from the SSC was 1.3 mg PLHYD g protein⁻¹, but in SmC, this protein was not detected. The PLHYD showed a critical micelle concentration of 0.45 mg mL⁻¹. In addition, the PLHYD modified the hydrophobicity of Teflon from 130.1 ± 2° to 47 ± 2°, forming porous structures with some filaments <1 μm and globular aggregates <0.25 μm diameter. The interfacial studies of this PLHYD could be the basis for the use of the protein to modify surfaces and to stabilize compounds in emulsions.     

Effect of modified MRS medium on production and purification of antimicrobial peptide ST4SA produced by Enterococcus mundtii

Highest antimicrobial activity of peptide ST4SA (51,200 AU/mL) was recorded after 14 h of growth in MRS broth with optimal production at pH 6.0 or 6.5. Growth of strain ST4SA in the presence of tryptone, yeast extract, or a combination of the two, yielded 102,400 AU/mL. An increase in production of peptide ST4SA to 102,400 AU/mL was recorded in the presence of 20.0 g/L fructose, but decreased to 25,600 AU/mL in the presence of lactose (20.0 g/L) or mannose (20.0 g/L) as sole carbon source. Lower activity (25,600 AU/mL) was recorded when 2.0 g/L K₂HPO₄ was replaced by 2.0 g/L KH₂PO₄ in MRS broth. An increase of K₂HPO₄ to 10.0 g/L and 20.0 g/L resulted in higher activity (102,400 AU/mL). Addition of glycerol to MRS broth had a negative effect on peptide ST4SA production. Production of peptide ST4SA required the presence of magnesium sulphate, manganese sulphate and 5.0 g/L sodium acetate. Exclusion of tri-ammonium citrate from the medium resulted in reduction of activity to 3,200 AU/mL. Maximum activity (102,400 AU/mL) was recorded in MRS supplemented with 1.0 ppm Vit. C, DL-6,8-thioctic acid or thiamine, respectively. Growth of Listeria ivanovii susbp. ivanovii ATCC 19119 in the presence of peptide ST4SA (12,800 AU/mL) resulted in 99% cell lysis after 18 h. Improved production of peptide ST4SA was recorded in MRS broth (Biolab) pre-treated with Amberlite XAD-1180. Precipitation with ammonium sulphate, followed by gel filtration chromatography, yielded the highest level of peptide ST4SA. This paper describes the partially deproteination of growth medium to facilitate peptide ST4SA purification.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

Use of Saccharum spontaneum (wild sugarcane) as biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS3

Saccharum spontaneum is a wasteland weed consists of 45.10 ± 0.35% cellulose and 22.75 ± 0.28% of hemicellulose on dry solid (DS) basis. Aqueous ammonia delignified S. spontaneum yielded total reducing sugars, 53.91 ± 0.44 g/L (539.10 ± 0.55 mg/g of substrate) with a hydrolytic efficiency of 77.85 ± 0.45%. The enzymes required for hydrolysis were prepared from culture supernatants of Aspergillus oryzae MTCC 1846. A maximum of 0.85 ± 0.07 IU/mL of filter paperase (FPase), 1.25 ± 0.04 IU/mL of carboxy methyl cellulase (CMCase) and 55.56 ± 0.52 IU/mL of xylanase activity was obtained after 7 days of incubation at 28 ± 0.5 °C using delignified S. spontaneum as carbon source under submerged fermentation conditions. Enzymatic hydrolysate of S. spontaneum was then tested for ethanol production under batch and repeated batch production system using “in-situ” entrapped Saccharomyces cerevisiae VS₃ cells in S. spontaneum stalks (1 cm × 1 cm) size. Immobilization was confirmed by the scanning electron microscopy (SEM). Batch fermentation of VS₃ free cells and immobilized cells showed ethanol production, 19.45 ± 0.55 g/L (yield, 0.410 ± 0.010 g/g) and 21.66 ± 0.62 g/L (yield, 0.434 ± 0.021 g/g), respectively. Immobilized VS₃ cells showed maximum ethanol production (22.85 ± 0.44 g/L, yield, 0.45 ± 0.04 g/g) up to 8th cycle during repeated batch fermentation followed by a gradual reduction in subsequent cycles of fermentation.     

A comparison of the kinetic properties of free and immobilized Aspergillus oryzae β-galactosidase

The objective of this work was to compare the properties of free and immobilized β-galactosidase (Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90 g/L and 140 g/L, respectively, a temperature of 35 °C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The K_m and V_m values of the free enzyme were 52.13 ± 2.8 mM and 2.56 ± 0.3 g_glucose/L min mg_enzyme, respectively, and were 60.30 ± 3.3 mM and 1032.07 ± 51.6 g_lactose/min m³_catalyst, respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH 4.5 and 55 °C. Alternatively, the immobilized form was most active at pH 5.0 at 60 °C. The free and immobilized enzymes presented activation energies of 6.90 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer.     

Hexokinase—A limiting factor in lipid production from fructose in Yarrowia lipolytica

Microbial biolipid production has become an important part of making biofuel production economically feasible. Genetic engineering has been used to improve the ability of Yarrowia lipolytica, an oleaginous yeast, to produce lipids using glucose-based media. However, few studies have examined lipid accumulation by Y. lipolytica׳s ability to utilize other hexose sugars, and as of yet, the rate-limiting steps in this process are unidentified. In this study, we investigated the de novo accumulation of lipids by Y. lipolytica when grown in glucose, fructose, and sucrose. Three Y. lipolytica wild-type (WT) strains of varied origin differed significantly in their lipid production, growth, and fructose utilization. Hexokinase (ylHXK1p) activity partially explained these differences. Overexpression of the ylHXK1 gene led to increased hexokinase activity (6.5–12 times higher) in the mutants versus the WT strains; a pronounced reduction in cell filamentation in mutants grown in fructose-based media; and improved biomass production, particularly in the mutant whose parent had shown the lowest growth capacity in fructose (French strain W29). All mutants showed improved lipid yield and production when grown on fructose, although the effect was strain dependent (23–55% improvement). Finally, we overexpressed ylHXK1 in a highly modified strain of Y. lipolytica W29 engineered to optimize oil production. This modification was combined with Saccharomyces cerevisiae invertase gene expression to evaluate the resulting mutant׳s ability to produce lipids using cheap industrial substrates, namely sucrose (a major component of molasses). Sucrose turned out to be a better substrate than either of its building blocks, glucose or fructose. Over its 96 h of growth in the bioreactors, this highly modified strain produced 9.15 g L⁻¹ of lipids, yielding 0.262 g g⁻¹ of biomass.     

Protocorm culture of Dendrobium candidum in balloon type bubble bioreactors

Protocorm cultures of Dendrobium candidum were established in balloon type bubble bioreactors using Murashige and Skoog (MS) medium with 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5% (w/v) sucrose, 5:25 mM NH₄:NO₃ and 1% (v/v) banana homogenate for the production of biomass and bioactive compounds. In 3 l bioreactor containing 2 l medium, a maximum protocorm biomass (21.0 g l⁻¹ dry biomass) and also optimum quantities of total polysaccharides (389.3 mg g⁻¹ DW), coumarins (18.0 mg g⁻¹ DW), polyphenolics (11.9 mg g⁻¹ DW), and flavonoids (4.5 mg g⁻¹ DW) were achieved after 7 weeks of culture. Based on these studies, 5 and 10 l bioreactor cultures were established to harvest 80 g and 160 g dry biomass. In 10 l bioreactors, the protocorms grown were accumulated with optimal levels of polysaccharides (424.1 mg g⁻¹ DW), coumarins (15.8 mg g⁻¹ DW), polyphenols (9.03 mg g⁻¹ DW) and flavonoids (4.7 mg g⁻¹ DW). The bioreactor technology developed here will be useful for the production of important bioactive compounds from D. candidum.     

Association Between Thyroid Function and Objective and Subjective Sleep Quality in Older Men: The Osteoporotic Fractures in Men (MrOS) Study

ObjectiveTo determine the association between thyroid hormone levels and sleep quality in community-dwelling men.MethodsAmong 5,994 men aged ≥ 65 years in the Osteoporotic Fractures in Men (MrOS) study, 682 had baseline thyroid function data, normal free thyroxine (FT₄) (0.70 ≤ FT₄ ≤ 1.85 ng/dL), actigraphy measurements, and were not using thyroid-related medications. Three categories of thyroid function were defined: subclinical hyperthyroid (thyroid-stimulating hormone [TSH] < 0.55 mIU/L), euthyroid (TSH, 0.55 to 4.78 mIU/L), and subclinical hypothyroid (TSH > 4.78 mIU/L). Objective (total hours of nighttime sleep [TST], sleep efficiency [SE], wake after sleep onset [WASO], sleep latency [SL], number of long wake episodes [LWEP]) and subjective (TST, Pittsburgh Sleep Quality Index score, Epworth Sleepiness Scale score) sleep quality parameters were measured. The association between TSH and sleep quality was examined using linear regression (continuous sleep outcomes) and log-binomial regression (categorical sleep outcomes).ResultsAmong the 682 men examined, 15 had subclinical hyperthyroidism and 38 had subclinical hypothyroidism. There was no difference in sleep quality between subclinical hypothyroid and euthyroid men. Compared to euthyroid men, subclinical hyperthyroid men had lower mean actigraphy TST (adjusted mean difference [95% confidence interval (CI)], − 27.4 [− 63.7 to 8.9] minutes), lower mean SE (− 4.5% [− 10.3% to 1.3%]), and higher mean WASO (13.5 [− 8.0 to 35.0] minutes]), whereas 41% had increased risk of actigraphy-measured TST < 6 hours (relative risk [RR], 1.41; 95% CI, 0.83 to 2.39), and 83% had increased risk of SL ≥ 60 minutes (RR, 1.83; 95% CI, 0.65 to 5.14) (all P > .05).ConclusionNeither subclinical hypothyroidism nor hyperthyroidism is significantly associated with decreased sleep quality. (Endocr Pract. 2014;20:576-586)     

Subcellular fractionation of Cu exposed oysters,Crassostrea virginica,and Cu accumulation from a biologically incorporated Cu rich oyster diet in Fundulus heteroclitus in fresh and sea water

In order to examine the effect of salinity on Cu accumulation from a naturally incorporated diet, oysters (Crassostrea virginica) were exposed in sea water for 96 days to four waterborne [Cu]: 2.9 ± 0.7 (control), 4.3 ± 0.6, 5.4 ± 0.5, and 10.7 ± 1.0 µg L^? 1. After 96 days, the control whole body [Cu] increased from 2.1 ± 0.5 to 9.1 ± 1.1 µg g^? 1 w.w. and the highest [Cu] was 163.4 ± 27.1 µg g^? 1 w.w. in the oysters. Despite large differences in tissue [Cu], there was no effect on the fraction of trophically available metal in the oyster suggesting that trophic transfer will correlate well with tissue [Cu]. The control and highest [Cu] oysters became diet for killifish (Fundulus heteroclitus) in fresh and seawater for 40 days. The two diets contained 84.7 ± 5.1 and 850.5 ± 8.8 µg Cu g^? 1 d.w. Fish were fed a combined diet of oyster and a pellet supplement (20.5 ± 1.0 µg Cu g^? 1 d.w.) both at 5% body mass day^? 1. In killifish, Cu increased ~ 7% in gills and 100% in intestines after 6 weeks of exposure to the high Cu diet. No other tissues accumulated Cu above control levels. An 11-fold difference free Cu²⁺ concentrations was predicted in intestinal fluid between fresh and sea water, but there was no corresponding effect of salinity on intestinal Cu accumulation suggesting that Cu is not accumulated as the free ion.     

Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence of the ratio of renewed volume to total volume (R), the urea feeding time (t_f) and the number of successive repeated fed-batch cycles on the maximum cell concentration (X_m), cell productivity (P_x), nitrogen-to-cell conversion yield (Y_x/n), maximum specific growth rate (μ_m) and protein content of dry biomass. The experimental results demonstrated that R = 0.80 and t_f = 6 d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (X_m = 2101 ± 113 mg L^?1, P_x = 219 ± 13 mg L^?1 d^?1 and Y_x/n = 10.3 ± 0.8 g g^?1).     

Peel of araticum fruit (Annona crassiflora Mart.) as a source of antioxidant compounds with α-amylase, α-glucosidase and glycation inhibitory activities

Annona crassiflora Mart., whose fruit is popularly known as araticum, is a member of the Annonaceae family found in the Brazilian Cerrado. Although this plant has several medicinal uses, its bioactive molecules are not fully understood. A bioguided assay was performed to identify the main bioactive compounds of A. crassiflora fruit peel from the ethanol extract fractions with antioxidant capacity and α-amylase, α-glucosidase and glycation inhibitory activities. Ethyl acetate and n-butanol fractions showed, respectively, higher antioxidant capacity (DPPH IC₅₀ 1.5 ± 0.1 and 0.8 ± 0.1 μg mL⁻¹, ORAC 3355 ± 164 and 2714 ± 79 μmol trolox eq/g, and FRAP 888 ± 16 and 921 ± 9 μmol trolox eq/g) and inhibitory activities against α-amylase (IC₅₀ 4.5 ± 0.8 and 1.7 ± 0.3 μg mL⁻¹), α-glucosidase (IC₅₀ 554.5 ± 158.6 and 787.8 ± 140.6 μg mL⁻¹) and glycation (IC₅₀ 14.3 ± 3.3 and 16.0 ± 4.2 μg mL⁻¹), and lower cytotoxicity, compared to the other fractions and crude ethanol extract. The HPLC-ESI-MS/MS analysis identified various biomolecules known as potent antioxidants, such as chlorogenic acid, (epi)catechin, procyanidins, caffeoyl-hexosides, quercetin-glucosides and kaempferol. The fruit peel of A. crassiflora, a specie from Cerrado, the Brazilian Savanna, provided a source of antioxidant compounds with properties to block carbohydrate digestive enzymes and formation of glycation products. Thus, there is potential to use the by-products of araticum in order to identify and isolate phytochemicals for application in nutraceutical supplements, food additives and pharmaceuticals products.     

Bioconversion of phenylpyruvic acid to l-phenylalanine by mixed-gel immobilization of Escherichia coli EP8-10

A mixed-gel of κ-carrageenan and gelatin was used in l-phenylalanine production. The mixed-gel, containing 87.5% κ-carrageenan and 12.5% gelatin [the total gel concentration was 4 wt%], showed the best performance and was selected for further study with Escherichia coli EP8-10. The optimum pH and temperature were 8.5 and 37 °C, respectively. The effects of trehalose and Mg²⁺ were studied in the mixed-gel immobilization. Their optimum concentrations were 5 × 10^?2 and 2 × 10^?3 mol/L, respectively. Under the optimal conditions, 98.3% of the phenylpyruvic acid (PPA) was converted to l-phenylalanine. The activity recovery of the transaminase enzyme in the mixed-gel immobilization was higher than that in single κ-carrageenan immobilization, which was 93.6%. The total PPA conversion rate was over 80% in all 15 batches, suggesting great sustainability in the mixed-gel immobilization. The maximum reaction rate (r_max) was calculated to be 4.75 × 10^?2 mol/(L g h).     

Synthesis,biological evaluation and molecular docking study of N-arylbenzo[d]oxazol-2-amines as potential α-glucosidase inhibitors

A novel series of N-arylbenzo[d]oxazol-2-amines (4a–4m) were synthesized and evaluated for their α-glucosidase inhibitory activity. Compounds 4f–4i, 4k and 4m displayed potent inhibitory activity against α-glucosidase with IC₅₀ values in the range of 32.49 ± 0.17–120.24 ± 0.51 μM as compared to the standard drug acarbose. Among all tested compounds, compound 4g having 4-phenoxy substitution at the phenyl ring was found to be the most active inhibitor of α-glucosidase with an IC₅₀ value of 32.49 ± 0.17 μM. Analysis of the kinetics of enzyme inhibition indicated that compound 4g is a noncompetitive inhibitor of α-glucosidase with a K_i value of 31.33 μM. Binding interaction of compound 4g with α-glucosidase was explored by molecular docking simulation.     

Effect of Zinc nanoparticles on oxidative stress-related genes and antioxidant enzymes activity in the brain of Oreochromis niloticus and Tilapia zillii

This study was carried out to determine the median lethal concentrations (LC₅₀) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii. The biochemical and molecular potential effects of ZnNPs (500 and 2000 μg L⁻¹) on the antioxidant system in the brain tissue of O. niloticus and T. zillii were investigated. Four hundred fish were used for acute and sub-acute studies. ZnNP LC₅₀ concentrations were investigated in O. niloticus and T. zillii. The effect of 500 and 2000 μg L⁻¹ ZnNPs on brain antioxidants of O. niloticus and T. zillii was investigated. The result indicated that 69 h LC₅₀ was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. Fish exposed to 500 μg L⁻¹ ZnNPs showed a significant increase in reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, fish exposed to 2000 μg L⁻¹ ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GST activity and gene expression. On the contrary, MDA levels significantly increased. It was concluded that, the 96 h LC₅₀ of ZnNPs was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. ZnNPs in exposure concentrations of 2000 μg/L induced a deleterious effect on the brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentrations of 500 μg L⁻¹ produced an inductive effect on the brain antioxidant system of O. nilotica and T. zillii.     

Volatile fatty acids accumulation and rhamnolipid generation in situ from waste activated sludge fermentation stimulated by external rhamnolipid addition

The solubilization and acidification of waste activated sludge (WAS) were apparently enhanced by external rhamnolipid (RL) addition. The maximum solute carbohydrate concentrations increased linearly from 48 ± 5 mg COD L⁻¹ in the un-pretreated WAS (blank) to 566 ± 19 mg COD L⁻¹, and protein increased from 1050 ± 8 to 3493 ± 16 mg COD L⁻¹ at RL dosage of 0.10 g g⁻¹ TSS. The highest VFAs concentration peaked at 3840 mg COD L⁻¹ at RL dosage of 0.04 g g⁻¹ TSS, which was 4.24-fold higher than the blank test. RL was generated in situ during WAS fermentation when external RL was added. It was detected that RL concentration was increased from initial 880 ± 92 mg L⁻¹ to 1312 ± 7 mg L⁻¹ at the end of 96 h with RL dosage of 0.04 g g⁻¹ TSS, which was increased to 1.49-fold. Meanwhile, methane production was notably reduced to a quite low level of 2.0 mL CH₄ g⁻¹ VSS, showing effective inhibition of methanogens by RL (58.8 mL CH₄ g⁻¹ VSS in the blank). In addition, the activity of hydrolytic enzymes (protease and α-glucosidase) was enhanced accordingly. VFAs accumulation and RL generation in situ demonstrated that the additional RL substantially performed enhanced biological effects for waste activated sludge fermentation.     

Application of an integrated statistical design to optimize the cold enzyme hydrolysis conditions for ethanol production

Cold enzyme hydrolysis was investigated on the ethanol production by Saccharomyces cerevisiae during simultaneous saccharification and fermentation (SSF) processing. An integrated statistical design, which incorporated single factor design, response surface methodology (RSM) and weighting coefficient method, was used to determine the optimum hydrolysis conditions leading to maximum biomass, ethanol concentration and starch utilization ratio. After the studied ranges of α-amylase, glucoamylase and liquefaction time were identified by single factor design, RSM was used to further optimize the hydrolysis conditions for each objective. The results showed that, under hydrolysis condition optimized with RSM, biomass, ethanol concentration and starch utilization ratio reached 4.401 ± 0.042 × 10⁸ cells/ml, 14.81 ± 0.23% (wt.%) and 94.52 ± 0.53%, respectively. Finally, multi-objective optimization (MOO) was applied to obtain a compromised result of three desirable responses by weighting coefficient methodology. Biomass of 4.331 ± 0.038 × 10⁸ cells/ml, ethanol concentration of 14.12 ± 0.21% (wt.%) and starch utilization ratio of 92.88 ± 0.21% were simultaneous obtained when hydrolysis at pH 5.9 for 114 min with 233 IU/g_starch α-amylase and 778 IU/g_starch glucoamylase. The optimized conditions were shown to be feasible and reliable through verification tests.     

Abundance and biomass of planktonic ciliates in the sea area around Zhangzi Island,Northern Yellow Sea

We investigated the abundance and biomass of planktonic ciliates in the sea area around Zhangzi Island, Northern Yellow Sea, from July 2009 to June 2010. Ciliates were sampled monthly from surface to bottom with a 10 m depth interval at 13 sample stations along three transects. A 1 L sample of water from each depth was collected with a 2.5 L Niskin water sampler and fixed in 1% acid Lugol’s iodine solution. Water samples were pre-concentrated using the Utermöhl method and observed using an Olympus IX51 inverted microscope at 100× or 200x. The dimensions of the ciliates were measured and the cell volume of each species was estimated using appropriate geometric shapes. The carbon:volume ratio used to calculate biomass was 0.19 pg C/μm³. Abundance and biomass of the ciliate in water column were calculated as the integral of the abundance and biomass from bottom to surface, respectively. The classification of tintinnids was based on taxonomic literature. The average abundance of non-loricate ciliates was 3066 ± 2805 ind/L, ranging from 165 ind/L (50 m depth of St. B6 in July) to 26,595 ind/L (surface of St. C1 in September). The average biomass of non-loricate ciliates was 2.88 ± 2.68 μg C/L, ranging from 0.05 μg C/L (10 m depth of St. A6 in July) to 20.51 μg C/L (surface of St. A5 in August). The average tintinnid abundance was 142 ± 273 ind/L, ranging from 0 ind/L (monthly) to 2756 ind/L (surface of St. A1 in July). The average tintinnid biomass was 0.84 ± 2.19 μg C/L, ranging from 0.00 μg C/L (every month) to 37.64 μg C/L (20 m depth of St. C5 in July). The results showed that the average abundance of total ciliates was 3208 ± 2828 ind/L, ranging from 166 ind/L (10 m depth of St. A6 in July) to 26,625 ind/L (surface of St. C1 in September); the average biomass of total ciliates was 3.73 ± 3.55 μg C/L, ranging from 0.05 μg C/L (10 m depth of St. A6 in July) to 38.29 μg C/L (20 m depth of St. C5 in July). Abundance and biomass were vertically homogeneous in February, November and December, but decreased dramatically from the surface down to the bottom in other months. 23 tintinnid species were identified, 12 of which were in genus Tintinnopsis. Tintinnid species were more abundant in February, July and August. Tintinnids occupied 6.6 ± 10.2% and 19.7 ± 23.3% of the total ciliate abundance and biomass, respectively, which increased during the warm season and at coastal stations, and decreased during the cold season and at offshore stations. Large non-loricate ciliate species were prevalent in spring, while smaller species dominated in summer and autumn. The average abundance of total ciliates in water column was 132 ± 72 × 10⁶ ind/m², with increases during spring and autumn. The average biomass of total ciliates in water column was 152.57 ± 93.10 mg C/m², with increases during spring and summer. The average abundance and biomass of total ciliates in water column were greater at offshore stations than at coastal stations during spring and autumn, and were lower during summer and winter. Non-loricate ciliates, tintinnids and total ciliates showed significant positive correlation with temperature and significant negative correlation with salinity (p < 0.01). Non-loricate ciliates and total ciliates showed significant positive correlation with Chl a concentration (p < 0.01); however, relationship between Chl a concentration and tintinnids was not significant.     

5-Nitro-2-furfuriliden derivatives as potential anti-Trypanosoma cruzi agents: Design,synthesis, bioactivity evaluation,cytotoxicity and exploratory data analysis

The anti-Trypanosoma cruzi activity of 5-nitro-2-furfuriliden derivatives as well as the cytotoxicity of these compounds on J774 macrophages cell line and FN1 human fibroblast cells were investigated in this study. The most active compounds of series I and II were 4-butyl-[N′-(5-nitrofuran-2-yl) methylene] benzidrazide (3g; IC₅₀ = 1.05 μM ± 0.07) and 3-acetyl-5-(4-butylphenyl)-2-(5-nitrofuran-2-yl)-2,3-dihydro,1,3,4-oxadiazole (4g; IC₅₀ = 8.27 μM ± 0.42), respectively. Also, compound 3g was more active than the standard drugs, benznidazole (IC₅₀ = 22.69 μM ± 1.96) and nifurtimox (IC₅₀ = 3.78 μM ± 0.10). Regarding the cytotoxicity assay, the 3g compound presented IC₅₀ value of 28.05 μM (SI = 26.71) against J774 cells. For the FN1 fibroblast assay, 3g showed IC₅₀ value of 98 μM (SI = 93.33). On the other hand, compound 4g presented a cytotoxicity value on J774 cells higher than 400 μM (SI >48), and for the FN1 cells its IC₅₀ value was 186 μM (SI = 22.49). Moreover, an exploratory data analysis, which comprises hierarchical cluster (HCA) and principal component analysis (PCA), was carried out and the findings were complementary. The molecular properties that most influenced the compounds’ grouping were C log P and total dipole moment, pointing out the need of a lipophilic/hydrophilic balance in the designing of novel potential anti-T. cruzi molecules.     

Oleanolic acid (OA) as an antileishmanial agent: Biological evaluation and in silico mechanistic insights

Although a worldwide health problem, leishmaniasis is considered a highly neglected disease, lacking efficient and low toxic treatment. The efforts for new drug development are based on alternatives such as new uses for well-known drugs, in silico and synthetic studies and naturally derived compounds. Oleanolic acid (OA) is a pentacyclic triterpenoid widely distributed throughout the Plantae kingdom that displays several pharmacological activities. OA showed potent leishmancidal effects in different Leishmania species, both against promastigotes (IC_{50 L. braziliensis} 30.47 ± 6.35 μM; IC_{50 L. amazonensis} 40.46 ± 14.21 μM; IC_{50 L. infantum} 65.93 ± 15.12 μM) and amastigotes (IC_{50 L. braziliensis} 68.75 ± 16.55 μM; IC_{50 L. amazonensis} 38.45 ± 12.05 μM; IC_{50 L. infantum} 64.08 ± 23.52 μM), with low cytotoxicity against mouse peritoneal macrophages (CC₅₀ 235.80 ± 36.95 μM). Moreover, in silico studies performed to evaluate OA molecular properties and to elucidate the possible mechanism of action over the Leishmania enzyme sterol 14α-demethylase (CYP51) suggested that OA interacts efficiently with CYP51 and could inhibit the ergosterol synthesis pathway. Collectively, these data indicate that OA is a good candidate as leading compound for the development of a new leishmaniasis treatment.