Location of the synaptosome-binding regions on botulinum neurotoxin B

The regions of botulinum neurotoxin B (BoNT/B) involved in binding to mouse brain synaptosomes (snps) were localized. Sixty 19-residue overlapping peptides (peptide C31 consisted of 24 residues) encompassing BoNT/B H chain (residues 442-1291) were synthesized and used to inhibit binding of (125)I-labeled BoNT/B to snps. Synaptosome-binding regions were noncompeting and existed on both H(N) and H(C) domains of neurotoxin. At 37 °C, inhibitory activities on H(N) resided, in decreasing order, in peptides 638-656 (26.7%), 596-614 (18.2%), 512-530 (13.9%), 778-796 (13.8%), and 526-544 (11.6%). On H(C), activity resided in decreasing order in peptides 1170-1188 (44.6%), 1128-1146 (21.6%), 1184-1202 (18.6%), 1156-1174 (13.0%), 946-964 (11.8%), 1114-1132 (11.2%), 1100-1118 (6.2%), 876-894 (6.1%), 1268-1291 (4.6%), and 1226-1244 (4.3%). The 45 remaining H(N) and H(C) peptides had no activity. At 4 °C, peptide C24 (1170-1188) remained quite active (inhibiting, 31.2%), while activities of peptides N15, C21, and C25 were little under 10%. The snp-binding regions contained sites that bind synaptotagmin II and gangliosides. Despite the low degree of sequence homology, BoNT/B and BoNT/A display significant structural homology and appeared to bind in part to the same snp-binding regions. Binding of each labeled toxin to snps was inhibited ~50% by the other toxin, 70-72% by its correlate H(C), and by the H(C) of the other toxin [29% (BoNT/A by H(C) of B) or 32% (BoNT/B by H(C) of A)]. In the three-dimensional structure of BoNT/B, the greater part of H(C), one H(N) face, and part of the belt on the same side interact with snps. Thus, BoNT/B binds to snps through the H(C) head and employs regions on one H(N) face and the belt, reserving flexibility for the belt's unbound part to release the light chain. Most snp-binding regions coincide or overlap with blocking antibody (Ab)-binding regions explaining how such Abs prevent BoNT/B toxicity.     

Mapping of the Synaptosome-Binding Regions on the Heavy Chain of Botulinum Neurotoxin A By Synthetic Overlapping Peptides Encompassing the Entire Chain

The purpose of this work was to map, on the heavy (H) chain of botulinum neurotoxin A (BoNT/A), the regions that bind to mouse brain synaptosomes (snps). We prepared 60 synthetic overlapping peptides that had uniform size and overlaps and encompassed the entire H chain (residues 499 to 1296) of BoNT/A. The ability of each peptide to inhibit the binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was studied. The binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was completely inhibited by free unlabeled BoNT/A, but not by unrelated proteins, indicating that the binding of BoNT/A to mouse brain snps was a specific event. Inhibition studies with the individual peptides showed that, on the H_N domain, inhibitory activities greater than 10% were exhibited, in decreasing order, by peptides 799–817, 659–677, 729–747, 533–551, 701–719, and 757–775. Lower inhibitory activities (between 5.6% and 8.7%) were exhibited by five other peptides, 463–481, 505–523, 519–537, 603–621 and 645–663. The remaining 18 H_N peptides had little or no inhibitory activity. In the H_C domain, peptides 1065–1083, 1163–1181 and 1275–1296 had the highest inhibitory activities (between 25% and 29%), followed (10–12% inhibitory activity) by peptides 1107–1125, 1191–1209 and 1233–1251. Two other peptides, 1079–1097 and 1177–1195, had very low (5.8% and 4.9 %) inhibitory activities. The remaining 23 H_C peptides had no inhibitory activity. Inhibition with mixtures of equimolar quantities of the most active 6 peptides of H_N, 5 of H_C or all 11 of H_N and H_C revealed that the peptides contain independent non-competing binding regions. Comparison of the locations of the snp-binding regions on the H-subunit with the regions that bind blocking mouse anti-BoNT/A Abs helped explain the protecting ability of these Abs. In the three-dimensional structure of BoNT/A, the snp-binding regions that completely coincide or significantly overlap with the antigenic regions occupy surface locations and most of them reside in the last half of the H_C domain. But some of the regions reside in the H_N domain and might play a role in the translocation event.     

Synaptotagmin II and Gangliosides Bind Independently with Botulinum Neurotoxin B but Each Restrains the Other

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37–63) or human SytII (hSytII 34–60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.     

Successful cryopreservation of Quercus robur plumules

Successful cryopreservation of Q. robur germplasm as plumules (i.e. shoot apical meristems of embryos) is described in this paper. After excision from the recalcitrant seeds and preliminary storage in 0.5 M sucrose solution (18 h), the plumules were subjected to cryoprotection (in 0.75 M sucrose, followed by 1.0 M sucrose and 1.5 M glycerol solutions), and next to desiccation (over silica gel or in nitrogen gas) and cooling (in slush at –210°C or in vials filled with liquid nitrogen, LN, −196°C), and were then cryostored for 24 h. High percentage of survival was obtained after cryostorage (21–67%, depending on pretreatment, assessed in vitro by greening plumules that increased in size). Desiccation of plumules over silica gel resulted in significantly higher survival after cryopreservation (58%) in comparison with desiccation in nitrogen gas (29%), with regrowth (shoots with leaves) 5–18%. The extent of plumule desiccation was comparable in both methods, in which drying of plumules for 20 min decreased the water content to 0.5–0.6 g H₂O g⁻¹ dry weight before LN exposure. The type of LN exposure did not significantly influence plumule survival and regrowth after cryostorage. Plumules isolated from acorns of four provenances survived cryostorage after cryoprotection followed by desiccation over silica gel and direct cooling in vials with LN (survival 51–76%, regrowth 8–20%). Normal plants developed from the recovered shoots after rooting. The presented protocol for Q. robur plumule cryopreservation may offer a potential approach for establishing germplasm conservation in gene banks for Quercus species.     

Impact of long-term nitrogen addition on carbon stocks in trees and soils in northern Europe

The aim of this study was to quantify the effects of fertiliser N on C stocks in trees (stems, stumps, branches, needles, and coarse roots) and soils (organic layer +0–10 cm mineral soil) by analysing data from 15 long-term (14–30 years) experiments in Picea abies and Pinus sylvestris stands in Sweden and Finland. Low application rates (30–50 kg N ha⁻¹ year⁻¹) were always more efficient per unit of N than high application rates (50–200 kg N ha⁻¹ year⁻¹). Addition of a cumulative amount of N of 600–1800 kg N ha⁻¹ resulted in a mean increase in tree and soil C stock of 25 and 11 kg (C sequestered) kg⁻¹ (N added) (“N-use efficiency”), respectively. The corresponding estimates for NPK addition were 38 and 11 kg (C) kg⁻¹ (N). N-use efficiency for C sequestration in trees strongly depended on soil N status and increased from close to zero at C/N 25 in the humus layer up to 40 kg (C) kg⁻¹ (N) at C/N 35 and decreased again to about 20 kg (C) kg⁻¹ (N) at C/N 50 when N only was added. In contrast, addition of NPK resulted in high (40–50 kg (C) kg⁻¹ (N)) N-use efficiency also at N-rich (C/N 25) sites. The great difference in N-use efficiency between addition of NPK and N at N-rich sites reflects a limitation of P and K for tree growth at these sites. N-use efficiency for soil organic carbon (SOC) sequestration was, on average, 3–4 times lower than for tree C sequestration. However, SOC sequestration was about twice as high at P. abies as at P. sylvestris sites and averaged 13 and 7 kg (C) kg⁻¹ (N), respectively. The strong relation between N-use efficiency and humus C/N ratio was used to evaluate the impact of N deposition on C sequestration. The data imply that the 10 kg N ha⁻¹ year⁻¹ higher deposition in southern Sweden than in northern Sweden for a whole century should have resulted in 2.0 ± 1.0 (95% confidence interval) kg m⁻² more tree C and 1.3 ± 0.5 kg m⁻² more SOC at P. abies sites in the south than in the north for a 100-year period. These estimates are consistent with differences between south and north in tree C and SOC found by other studies, and 70–80% of the difference in SOC can be explained by different N deposition.     

Microbial and decomposition efficiencies of monoculture and polyculture vermireactors,based on epigeic and anecic earthworms

Efforts have been made to evaluate the microbial and decomposition efficiency of three different vermireactors: (i) polyculture (introducing equal numbers of anecic and epigeic earthworms), (ii) monoculture (anecic) and (iii) monoculture (epigeic), designed by using earthworms of two different ecological categories i.e. anecic (Lampito mauritii Kinberg) and epigeic (Eisenia fetida (Savigny)). The microbial load of vermireactors was measured through substrate-induced respiration rate (SIR), microbial biomass N content and rate of dehydrogenase activity, while mineralization rate was evaluated measuring some chemical parameters of the substrate. Earthworms caused a decrease (as compared to initial value) in pH (41.9–80.7%), organic C (10.3–14.2%) and C:N ratio (41.9–80.7%) and an increase in total N (29.1–58.8%), NH₄-N (876.1–1485.7%), NO₃-N (29081.8–56792.6%), available P (16–19.4%) and exchangeable K (9.8–13.5%) contents of the substrate. The mineralization efficiency of the reactors was in the order: polyculture (epigeic + anecic) > monoculture (anecic) > monoculture (epigeic). The polyculture reactor showed the maximum rate of SIR (2.91 ± 0.2 mg CO₂ g⁻¹ substrate), microbial biomass N (3108.1 ± 289.2 mg N g⁻¹ substrate), and dehydrogenase activity (2453.3 ± 379.8 μg g⁻¹ substrate 24 h), while in the monoculture (epigeic) the lowest values of the same parameters were observed. It is concluded that the observed differences among reactors were due to different feeding behaviour and niche structures of epigeic and anecic earthworms. Data suggests that burrowing earthworms in waste-decomposing-system not only enhance the microbial efficiencies, but at the same time also accelerate the organic matter mineralization in a vermireactor. However, most of the previous studies were based on monoculture reactors (using epigeic earthworms) which have been recommended for waste decomposition operations, but this study revealed that polyculture vermicomposting (adding of burrowing worms with epigeic earthworms in vermicomposting system) might be beneficial for rapid decomposition of organic wastes.     

Susceptibility of <Emphasis Type="Italic">Candida</Emphasis> biofilms to histatin 5 and fluconazole

Candida-associated denture stomatitis has a high rate of recurrence. Candida biofilms formed on denture acrylic are more resistant to antifungals than planktonic yeasts. Histatins, a family of basic peptides secreted by the major salivary glands in humans, especially histatin 5, possess significant antifungal properties. We examined antifungal activities of histatin 5 against planktonic or biofilm Candida albicans and Candida glabrata. Candida biofilms were developed on poly(methyl methacrylate) discs and treated with histatin 5 (0.01–100 μM) or fluconazole (1–200 μM). The metabolic activity of the biofilms was measured by the XTT reduction assay. The fungicidal activity of histatin 5 against planktonic Candida was tested by microdilution plate assay. Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50% reduction in the metabolic activity; biofilm) of 4.6 ± 2.2, 6.9 ± 3.7 and 1.7 ± 1.5 μM, and IC₅₀ (planktonic cells) of 3.0 ± 0.5, 2.6 ± 0.1 and 4.8 ± 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were less susceptible to histatin 5, with 50% RMA of 31.2 ± 4.8 and 62.5 ± 0.7 μM, respectively. Planktonic C. glabrata was insensitive to histatin 5 (IC₅₀ > 100 μM). Biofilm-associated Candida was highly resistant to fluconazole in the range 1–200 μM; e.g. at 100 μM only ~20% inhibition was observed for C. albicans, and ~30% inhibition for C. glabrata. These results indicate that histatin 5 exhibits antifungal activity against biofilms of C. albicans and C. glabrata developed on denture acrylic. C. glabrata is significantly less sensitive to histatin 5 than C. albicans.     

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855–1296 with antitoxin antibodies from three host species

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced byClostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (H_C, residues 860–1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the H_C of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855–873, 939–957, 1079–1097/1093–1111 overlap, 1191–1209/1205–1223 overlap, 1261–1279 and 1275–1296. In addition, peptides 883–901, 911–929, 995–1013, 1023–1041/1037–1055 overlap, 1121–1139, and 1149–1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869–887, 925–943, 981–999, 995–1013, 1051–1069, and 1177–1195. In addition, lower amounts of Abs were bound by peptides 911–929, 939–957, 967–985, and the overlaps 1121–1139/1135–1153 and 1247–1265/1261–1279/1275–1296. With outbred mouse antisera, high amounts of Abs were bound by peptides 869–887, 1051–1069, and 1177–1195, while peptides 939–957, 995–1013, 1093–1111, and 1275–1296 bound lower amounts of Abs. The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.     

High survival during hibernation affects onset and timing of reproduction

The timing of reproduction is one of the most crucial life history traits, with enormous consequences for the fitness of an individual. We investigated the effects of season and timing of birth on local survival probability in a small mammalian hibernator, the common dormouse (Muscardinus avellanarius). Local monthly survival probability was lowest in the early active season (May–August, ϕ_adult = 0.75–0.88, ϕ_juvenile = 0.61–0.68), increased during the late active season (August–October), and highest during hibernation (October–May, ϕ_adult = 0.96–0.98, ϕ_juvenile = 0.81–0.94). Consequently, dormice had an extremely high winter survival probability. We observed two peaks in the timing of reproduction (June and August/September, respectively), with the majority of juveniles born late in the active season. Although early investment in reproduction seems the better life history tactic [survival probability until onset of reproduction: ϕ_{born early} = 0.46, 95% confidence interval (CI) 0.28–0.64; ϕ_{born late} = 0.19, 95% CI = 0.09–0.28], only females with a good body condition (significantly higher body mass) invest in reproduction early in the year. We suggest the high over-winter survival in dormice allows for a unique life history pattern (i.e., combining slow and fast life history tactics), which leads to a bimodal seasonal birth pattern: (1) give birth as early as possible to allow even the young to breed before hibernating, and/or (2) give birth as late as possible (leaving just enough time for these young to fatten) and enter directly into a period associated with the highest survival rates (hibernation) until maturity.     

Edible oil degradation by using yeast coculture of <Emphasis Type="Italic">Rhodotorula pacifica</Emphasis> ST3411 and <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> ST3412

To develop a microbial treatment of edible oil-contaminated wastewater, microorganisms capable of rapidly degrading edible oil were screened. The screening study yielded a yeast coculture comprising Rhodotorula pacifica strain ST3411 and Cryptococcus laurentii strain ST3412. The coculture was able to degrade efficiently even at low contents of nitrogen ([NH₄–N] = 240 mg/L) and phosphorus sources ([PO₄–P] = 90 mg/L). The 24-h degradation rate of 3,000 ppm mixed oils (salad oil/lard/beef tallow, 1:1 w/w) at 20°C was 39.8% ± 9.9% (means ± standard deviations of eight replicates). The highest degradation rate was observed at 20°C and pH 8. In a scaled-up experiment, the salad oil was rapidly degraded by the coculture from 671 ± 52.0 to 143 ± 96.7 ppm in 24 h, and the degradation rate was 79.4% ± 13.8% (means ± standard deviations of three replicates). In addition, a repetitive degradation was observed with the cell growth by only pH adjustment without addition of the cells.     

Comparative Tolerances of Various <Emphasis Type="Italic">Beauveria bassiana</Emphasis> Isolates to UV-B Irradiation with a Description of a Modeling Method to Assess Lethal Dose

The tolerances of 20 Beauveria bassiana isolates derived from host insects worldwide to UV-B irradiation were assessed quantitatively in multi-dose bioassays. Conidial suspensions of the isolates smeared on glass slides were exposed to the gradient UV-B doses of 0.1–1.6 J cm⁻² (D), which generated from 0.75 to 10.17 min irradiation of weighted 312-nm wavelength at 2.0–2.61 mW cm⁻². Irradiated conidia were then incubated for 24 h at 25°C under saturated humidity. The ratio of germination at each dose over that in the blank control was defined as survival index (I _s). For all isolates, the I _s − D observations fit well with the survival model I _s = 1/[1 + exp(a + bD)] (0.94 ≤ r ² ≤ 0.99) generated widely spanned lethal doses of 0.154–0.928, 0.240–1.139, and 0.383–1.493 J cm⁻² for their losses of 50%, 75%, and 95% viabilities, respectively. These were far below the solar UV-B dose of 2.439 J cm⁻² measured in a sunny day during the summer. The large variation of UV-B tolerance among the isolates indicates a necessity to select UV-tolerant candidates for formulations applied to insect control during summer. The highly efficient bioassay method was developed to measure accurately the UV-B tolerances of fungal biocontrol agents as lethal doses.     

N-removal performance and underlying bacterial taxa of upflow filter bioreactor system under different dissolved oxygen and internal recycle conditions

Biological N-removal treatment of piggery wastewater in the upflow anaerobic–anoxic–aerobic floating filter (UA³FF) bioreactor based on the concept of nitritation–denitritation was studied along with the changes in internal recycle ratio and dissolved oxygen concentration (DO). Consecutive changes in the recirculation ratio between the anoxic and aerobic reactors has resulted in abundance and composition shifts of N-cycling bacteria as well as other bacterial groups, reflecting different survival strategies across (bio/physico)chemical milieu. The DO concentration was optimized to achieve nitritation in the aerobic reactor and denitritation in the anoxic reactor. Optimal nitritation–denitritation (270 and 130 g NO₂ ⁻–N produced or reduced/m³ filter media/day) was obtained at DO of 1.0–1.5 mg/l, inter-reactor recirculation ratio of 1:1–2:1, HRT of 24 h, pH of 7.6 ± 0.3, and temperature of 28 ± 4 °C. Since only well known nitrifying and denitrifying taxa were found, nitritation–denitritation was likely carried out by these bacteria rather than the yet unidentified novel taxa. Archaeal nitrifiers recently discovered to be important in the global N-cycle were not detected.     

Nitrogen retention by peatland buffer areas at six forested catchments in southern and central Finland

We studied the nitrogen retention capacity of six peatland buffer areas constructed in forested catchments in southern and central Finland. The buffers (0.1–4.9% of the total catchment area) were either undrained mires or drained peatlands rewetted 4–7 years before the present study. The N retention capacity was studied by adding ammonium nitrate (NH₄NO₃–N) solution into the inflow waters of the buffers once (one area) or twice (five areas) during a period of 4–6 years. Except for the first N addition in one area, the three largest buffer areas (relative size > 1%) retained the added inorganic N almost completely; their retention efficiencies during the year of addition were >93% for both NO₃–N and NH₄–N. Two of the three small buffers (relative size < 0.25%) were also able to reduce inorganic N from the through-flow waters effectively; their retention capacities for inorganic nitrogen varied between 58 and 89%. However, one small buffer area had a retention capacity of only <20%. The factors contributing to efficient N retention were hydrological load during N addition, relative size of the buffer area, and its length, i.e. the distance between the inflow and outflow points. If there was any release of the added N, it mostly occurred within a relatively short-time period (<100 days) after the treatment. The buffer areas appeared to be efficient and long-term sinks for inorganic nitrogen because the release of N during the 2–4 years after N addition was minor.     

Production of Dissolved Organic Carbon in Canadian Forest Soils

To identify the controls on dissolved organic carbon (DOC) production, we incubated soils from 18 sites, a mixture of 52 forest floor and peats and 41 upper mineral soil samples, at three temperatures (3, 10, and 22°C) for over a year and measured DOC concentration in the leachate and carbon dioxide (CO₂) production from the samples. Concentrations of DOC in the leachate were in the range encountered in field soils (<2 to >50 mg l⁻¹). There was a decline in DOC production during the incubation, with initial rates averaging 0.03–0.06 mg DOC g⁻¹ soil C day⁻¹, falling to averages of 0.01 mg g⁻¹ soil C day⁻¹; the rate of decline was not strongly related to temperature. Cumulative DOC production rates over the 395 days ranged from less than 0.01 to 0.12 mg g⁻¹ soil C day⁻¹ (0.5–47.6 mg g⁻¹ soil C), with an average of 0.021 mg g⁻¹ soil C day⁻¹ (8.2 mg g⁻¹ soil C). DOC production rate was weakly related to temperature, equivalent to Q₁₀ values of 0.9 to 1.2 for mineral samples and 1.2 to 1.9 for organic samples. Rates of DOC production in the organic samples were correlated with cellulose (positively) and lignin (negatively) proportion in the organic matter, whereas in the mineral samples C and nitrogen (N) provided positive correlations. The partitioning of C released into CO₂–C and DOC showed a quotient (CO₂–C:DOC) that varied widely among the samples, from 1 to 146. The regression coefficient of CO₂–C:DOC production (log₁₀ transformed) ranged from 0.3 to 0.7, all significantly less than 1. At high rates of DOC production, a smaller proportion of CO₂ is produced. The CO₂–C:DOC quotient was dependent on incubation temperature: in the organic soil samples, the CO₂–C:DOC quotient rose from an average of 6 at 3 to 16 at 22°C and in the mineral samples the rise was from 7 to 27. The CO₂–C:DOC quotient was related to soil pH in the organic samples and C and N forms in the mineral samples.     

Treatment of domestic wastewater in an up-flow anaerobic sludge blanket reactor followed by moving bed biofilm reactor

The performance of a laboratory-scale sewage treatment system composed of an up-flow anaerobic sludge blanket (UASB) reactor and a moving bed biofilm reactor (MBBR) at a temperature of (22–35 °C) was evaluated. The entire treatment system was operated at different hydraulic retention times (HRT’s) of 13.3, 10 and 5.0 h. An overall reduction of 80–86% for COD_total; 51–73% for COD_colloidal and 20–55% for COD_soluble was found at a total HRT of 5–10 h, respectively. By prolonging the HRT to 13.3 h, the removal efficiencies of COD_total, COD_colloidal and COD_soluble increased up to 92, 89 and 80%, respectively. However, the removal efficiency of COD_suspended in the combined system remained unaffected when increasing the total HRT from 5 to 10 h and from 10 to 13.3 h. This indicates that, the removal of COD_suspended was independent on the imposed HRT. Ammonia-nitrogen removal in MBBR treating UASB reactor effluent was significantly influenced by organic loading rate (OLR). 62% of ammonia was eliminated at OLR of 4.6 g COD m⁻² day⁻¹. The removal efficiency was decreased by a value of 34 and 43% at a higher OLR’s of 7.4 and 17.8 g COD m⁻² day⁻¹, respectively. The mean overall residual counts of faecal coliform in the final effluent were 8.9 × 10⁴ MPN per 100 ml at a HRT of 13.3 h, 4.9 × 10⁵ MPN per 100 ml at a HRT of 10 h and 9.4 × 10⁵ MPN per 100 ml at a HRT of 5.0 h, corresponding to overall log₁₀ reduction of 2.3, 1.4 and 0.7, respectively. The discharged sludge from UASB–MBBR exerts an excellent settling property. Moreover, the mean value of the net sludge yield was only 6% in UASB reactor and 7% in the MBBR of the total influent COD at a total HRT of 13.3 h. Accordingly, the use of the combined UASB–MBBR system for sewage treatment is recommended at a total HRT of 13.3 h.     

Sucrose monolaurate synthesis with Protex 6L immobilized on electrospun TiO<Subscript>2</Subscript> nanofiber

TiO₂ nanofibers with uniform diameter about 125 nm were prepared based on sol–gel process and electrospinning technology. Protex 6L, an industrial alkaline protease, was covalently immobilized on TiO₂ nanofiber through γ-aminopropyltriethoxysilane modification and glutaraldehyde crosslinking. With 2 (v/v)% glutaraldehyde as crosslinker, the enzyme loading is about 201 mg (g nanofiber membrane)⁻¹, and the specific activity of the immobilized Protex 6L is 2.45 μmol h⁻¹ ml⁻¹ mg⁻¹ protein for synthesis of sucrose monolaurate from sucrose and vinyl laurate. The optimal condition for sucrose monolaurate production is 5% (v/v) water content in DMSO/2-methyl-2-butanol solvent mixture and 50°C. Under this condition, 97% conversion was achieved within 36 h by nanofibrous Protex 6L, which is corresponding to a productivity 34 times higher than that of most widely used Novozym 435. After 10 cycles reuse, nanofibrous Protex 6L retained 52.4% of its original activity.     

Biological activity of some plant essential oils against <Emphasis Type="Italic">Varroa destructor</Emphasis> (Acari: Varroidae), an ectoparasitic mite of <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera: Apidae)

This experiment was conducted to evaluate acaricidal activity of the essential oils of Thymus kotschyanus, Ferula assa-foetida and Eucalyptus camaldulensis against Varroa destructor under laboratory conditions. Moreover, fumigant toxicity of these oils was tested on Apis mellifera. After preliminary dose-setting experiments, mites and honey bees were exposed to different concentrations of the oil, with 10 h exposure time. Essential oil of T. kotschyanus appeared the most potent fumigant for V. destructor (LC₅₀ = 1.07, 95% confidence limit (CL) = 0.87–1.26 μl/l air), followed by E. camaldulensis (LC₅₀ = 1.74, 95% CL = 0.96–2.50 μl/l air). The lowest acaricidal activity (LC₅₀ = 2.46, 95% CL = 2.10–2.86 μl/l air) was attributed to essential oil of F. assa-foetida. Surprisingly, among the three oils tested, essential oil of T. kotschyanus had the lowest insecticidal activity against A. mellifera (LC₅₀ = 5.08, 95% CL = 4.54–5.06 μl/l air). These findings proved that essential oil of T. kotschyanus has potential of practical value for use as alternative acaricide in the management of varroa in apiaries.     

Torpor patterns,arousal rates,and temporal organization of torpor entry in wildtype and UCP1-ablated mice

In eutherian mammals, uncoupling protein 1 (UCP1) mediated non-shivering thermogenesis from brown adipose tissue (BAT) provides a mechanism through which arousal from torpor and hibernation is facilitated. In order to directly assess the magnitude by which the presence or absence of UCP1 affects torpor patterns, rewarming and arousal rates within one species we compared fasting induced torpor in wildtype (UCP1^+/+) and UCP1-ablated mice (UCP^−/−). Torpor was induced by depriving mice of food for up to 48 h and by a reduction of ambient temperature (T _a) from 30 to 18°C at four different time points after 18, 24, 30 and 36 h of food deprivation. In most cases, torpor bouts occurred within 20 min after the switch in ambient temperature (30–18°C). Torpor bouts expressed during the light phase lasted 3–6 h while significantly longer bouts (up to 16 h) were observed when mice entered torpor during the dark phase. The degree of hypometabolism (5–22 ml h⁻¹) and hypothermia (19.5–26.7°C) was comparable in wildtype and UCP1-ablated mice, and both genotypes were able to regain normothermia. In contrast to wildtype mice, UCP1-ablated mice did not display multiple torpor bouts per day and their peak rewarming rates from torpor were reduced by 50% (UCP1^+/+: 0.24 ± 0.08°C min⁻¹; UCP1^−/−: 0.12 ± 0.04°C min⁻¹). UCP1-ablated mice therefore took significantly longer to rewarm from 25 to 32°C (39 vs. 70 min) and required 60% more energy for this process. Our results demonstrate the energetic benefit of functional BAT for rapid arousal from torpor. They also suggest that torpor entry and maintenance may be dependent on endogenous rhythms.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.