Urinary and serum titanium: assessment as an indicator of exposure to ammonium citratoperoxotitanate (IV) and its influence on renal function

Ammonium citratoperoxotitanate IV (TAS-FINE) is a water-soluble titanium complex used to synthesize a photocatalytic titanium(IV) oxide film. This study was aimed to investigate the LD50, dose-response, time-course response, and renal toxicity of TAS-FINE using an animal model. Serum titanium (S-Ti) and its 24-h urinary excretion (U-Ti) were determined by inductively coupled plasma-argon emission spectrometry (ICPAES) after a single oral TAS-FINE administration to male Wistar rats. The LD50 of TAS-FINE was 7.97 g/kg body weight in 24 h, and its half-life was 3.78+/-1.28 d for S-Ti and 2.19+/-0.09 d for U-Ti. Although TAS-FINE was not easily absorbed in the gastrointestinal tract, it was distributed into the bloodstream in a dose-dependent manner. Within 24 h, 0.189% of administrated Ti was excreted via urine. It was speculated that TAS-FINE formed conjugates with serum constituents that resulted in nephrotoxicity resulting from an allergic reaction. The observed indices in this study were revealed to be good indicators for TAS-FINE exposure. The analytical method and animal model described in this study will help to further elucidate details about human exposure to TAS-FINE, which in recent times has become an occupational and environmental toxicant of concern.     

Salinity tolerances of four species of fish from the Murray-Darling River system

The salinity tolerances of four species of small fishes native to the Murray-Darling river system were measured. Slow acclimation LD₅₀s were 43.7 ± 1.7 g L⁻¹ for Craterocephalus stercusmuscarum Gunther, 38.0 ± 1.1 g L⁻¹ for Hypseleotris klunzingeri (Ogilby), 58.7 ± 0.9 g L⁻¹ for Retropinna semoni (Weber), and 29.8 ± 0.7 g L⁻¹ for Melanotaenia splendida (Castelnau). The salinity tolerance of M. splendida was also measured by direct transfer, providing an estimated LD₅₀ (infinite exposure time) of 20.8 g L⁻¹, ∼ 70% of the slow acclimation value. Results suggest that at least adults of the species studied are under no threat from present or foreseeable salinities in the Murray River.     

Upper temperature tolerance and the effects of temperature on byssus attachment in the queen scallop, Chlamys opercularis (L.)

The upper temperature tolerance ( LD₅₀-48 h) have been established for three size groups of Chlamys opercularis (L) at different acclimation temperatures In large and intermediate-sized scallops mortlaity was between 19 and 24°C, and in spat between 24 and 25°C. These mortalities varied according to the thermal history of the animals.The rate of byssus formation in spat was found to be maximum at 18°C.     

Increase in cold-shock tolerance by selection of cold resistant lines in Drosophila melanogaster

Abstract.

• 1 In Drosophila melanogaster, the cold-shock tolerance of adult flies at -7°C increased 22% after a prior 2h exposure to 4°C as measured by LD₅₀, the dose (degree minutes of exposure to subzero temperature) which resulted in 50% mortality.
• 2 Cold-shock tolerance was further significantly increased by selecting cold resistant lines by exposure of adults (1) to 4°C for 2 h (short-term chilling), or (2) to -7°C for 80–120 min (cold shock), or (3) to short-term chilling followed by cold-shock.
• 3 After ten generations of selection, the greatest increase in cold-shock tolerance was found in flies selected using the combined exposure of short-term chilling and cold shock. LD₅₀s increased 33% in comparison with the unselected control strain when no chilling pre-treatment was given prior to cold shock at -7°C.
• 4 The rapid cold-hardening response increased 82% in the line selected by the short-term chilling and cold-shock regime.
• 5 The enhanced cold-shock tolerance was relatively stable since no decrease was observed after four generations without selection.
• 6 This report shows the role of short-term adaptation as well as selection in the capacity to survive low temperatures in non-diapausing stages of insects.

     

Effects of extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs) on glioblastoma cells (U87)

The impact of extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs) at various frequencies and amplitudes was investigated on cell cycle, apoptosis and viability of the Glioblastoma Multiforme (GBM) cell line (U87), in vitro. The GBM is a malignant brain tumor with high mortality in humans and poorly responsive to the most common type of cancer treatments, such as surgery, chemotherapy and radiation therapy. U87 cells with five experimental groups (I–V) were exposed to various ELF-PEMFs for 2, 4 and 24 h, as follows: (I) no exposure, control; (II) 50 Hz 100 ± 15 G; (III) 100 Hz 100 ± 15 G; (IV) 10 Hz 50 ± 10 G; (V) 50 Hz 50 ± 10 G. The morphology properties, cell viability and gene expression of proteins involved in cell cycle regulation (Cyclin-D1 and P53) and apoptosis (Caspase-3) were investigated. After 24 h, the cell viability and Cyclin-D1 expression increased in Group II (30%, 45%), whereas they decreased in Groups III (29%, 31%) and IV (21%, 34%); P53 and Caspase-3 elevated only in Group III; and no significant difference was observed in Group V, respectively, compared with the control (p < 0.05). The data suggest that the proliferation and apoptosis of human GBM are influenced by exposure to ELF-PEMFs in different time-dependent frequencies and amplitudes. The fact that some of the ELF-PEMFs frequencies and amplitudes favor U87 cells proliferation indicates precaution for the use of medical devices related to the MFs on cancer patients. On the other hand, some other ELF-PEMFs frequencies and intensities arresting U87 cells growth could open the way to develop novel therapeutic approaches.     

Induction and Characterization of UV Resistant Calothrix braunii

A UV resistant mutant of Calothrix braunii has been isolated after repeated exposure to UV-C (254 nm) radiation. LD₅₀ for wild type against UV-B was 1.74 hand 100% lethality was achieved after 3.5 h. Whereas, UV resistant mutant showed LD₅₀ at 3.33 h and loss of complete survival after 5 h exposure. The growth rate of mutant was about 29 per cent greater than that of the wild type. The photosynthetic pigments, chlorophyll a and phycoerythrin were stimulated by 36.2 and 41.2 per cent, respectively over wild type. There were no differences in nitrogenase activity nitrate reductase activity, extracellular ammonia production and of plasmid DNA.     

Chromium intake and urinary chromium excretion of trauma patients

Glucose metabolism is altered after trauma and those factors that affect glucose metabolism often affect chromium (Cr) metabolism and excretion. To ascertain whether urinary Cr excretion is affected by the elevated serum glucose and other factors associated with trauma, the serum glucose and urinary Cr and Creatinine (Cre) excretion of seven severely traumatized patients were determined. The Cr concentration of intravenous (IV) fluids administered was determined and approximate Cr intake calculated. For all patients, urinary Cr concentration was high in the initial sample collected within 24 h of admission (10.3 ± 2.5 ng/mL, mean ± SEM) and decreased significantly (P < 0.05) by 42 h (2.0 ±0.6 ng/mL). The mean urinary Cr concentration 42 h following admission was 10 times greater than the urinary Cr concentration of normal, healthy subjects (0.2 ± 0.02 ng/mL). There was no significant change in urinary Cre concentration within 42 h of admission, therefore the ratio of urinary Cr to Cre (ng Cr:mg Cre) also decreased. Serum glucose concentration was elevated at admission (170 ± 18 mg/dL, mean ± SD) and decreased to 145 ± 10 mg/dL by 48 h post-admission. The intravenous fluids, dextrose and NaCl, were the lowest in Cr of the samples tested, range 0.02 to 0.20 ng/mL; lactated Ringer’s solution, with or without dextrose, contained 10-20 times more Cr and plasma protein fraction contained approximately 32 ng/mL. The mean calculated Cr intake for the first 24 h postadmission was 37.1 µg/d, significantly greater (P < 0.01) than intake from 24 to 48 h (0.12 µg/d) and 48-72 h (1.63 µg/d). The IV intake of Cr varied for trauma patients depending on fluids required during treatment, but for all patients the relatively high IV Cr intake was rapidly excreted in the urine. These data demonstrate that urinary Cr concentration is elevated several-fold within 24 h of trauma and that Cr contents of intravenous fluids administered in the days immediately following injury vary dramatically. The effects of trauma alone on Cr excretion are difficult to assess because of the variable intake of Cr from IV fluids.     

Comparison of the Virulence of Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus

The virulence of methicillin-resistant Staphylococcus aureus (MRSA) was compared with that of methicillin-sensitive S. aureus (MSSA), using 13 MRSA and 7 MSSA strains isolated from clinical specimens. The infectivity and lethality of the two groups were examined as to the inoculum required to infect 50% of guinea pigs (ID₅₀) and to kill 50% of mice (LD₅₀), respectively. The mean ID₅₀ [log₁₀ colony forming units (CFU)] for MRSA strains was 7.1 ± 0.60 standard deviation, which was 1.5 higher than that for MSSA strains (P < 0.001). The mean LD₅₀ (log₁₀ CFU) for MRSA strains was 9.0 ± 0.42, being 1.1 higher than that for MSSA strains (P = 0.001). Pretreatment of mice with cyclophosphamide decreased the mean LD₅₀ for MRSA strains more than that for MSSA strains, resulting in the difference in the mean LD₅₀ being insignificant (P = 0.502). These results indicate that MRSA is less virulent than MSSA in normal hosts, but that they are equally virulent in immunocompromised hosts. The growth of MRSA strains was much slower than that of MSSA strains in the lag phase, although their growth rates were almost the same in the exponential growth phase, suggesting that the difference in virulence between them may be at least partly due to such a difference in growth.     

Stabilized ferrous gluconate as iron source for food fortification

The iron bioavailability and acute oral toxicity in rats of a ferrous gluconate compound stabilized with glycine (SFG), designed for food fortification, was studied in this work by means of the prophylactic method and the Wilcoxon method, respectively. For the former studies, SFG was homogenously added to a basal diet of low iron content, reaching a final iron concentration of 20.1±2.4 mg Fe/kg diet. A reference standard diet using ferrous sulfate as an iron-fortifying source (19.0±2.1 mg Fe/kg diet) and a control diet without iron additions (9.3±1.4 mg Fe/kg diet) were prepared in the laboratory in a similar way. These diets were administered to three different groups of weaning rats during 23 d as the only type of solid nourishment. The iron bioavailability of SFG was calculated as the relationship between the mass of iron incorporated into hemoglobin during the treatment and the total iron intake per animal. This parameter resulted in 36.6±6.2% SFG, whereas a value of 35.4±8.0% was obtained for ferrous sulfate. The acute toxicological studies were performed in two groups of 70 female and 70 male Sprague-Dawley rats that were administered increasing doses of iron from SFG. The LD₅₀ values of 1775 and 1831 mg SFG/kg body wt were obtained for female and male rats, respectively, evidencing that SFG can be considered as a safe compound from a toxicological point of view.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Antimicrobial efficiency of titanium dioxide‐coated surfaces

Aims: Development and evaluation of an antimicrobially active titanium dioxide coating. Methods and results: For this purpose, titanium dioxide coatings were applied to glass slides by using a sol‐gel method and then exposed to a light source. The antimicrobial efficiency was determined by a count reduction test for selected test strains (Aspergillus niger, Bacillus atrophaeus, Kocuria rhizophila), which were homogenously sprayed onto surface. The bacterial count of K. rhizophila was reduced by up to 3·3 log₁₀ on titanium dioxide samples within 4 h of UV‐A light exposure. Experiments with spore formers did not lead to any significant log reduction. A further aspect of this work was to evaluate the effect of selected parameters (relative humidity, inoculation density, radiation intensity) on the antimicrobial efficiency to gain knowledge for further optimization procedures. At a high relative humidity (85% r.h.), increased inactivation was observed for K. rhizophila (up to 5·2 log₁₀). Furthermore, a dependency of the antimicrobial effect on the radiation intensity and the inoculation density was identified. Conclusions: Antimicrobial surfaces and coatings based on titanium dioxide have the potential to effectively inactivate vegetative micro‐organisms. Significance and impact of the study: Knowledge about the antimicrobial efficiency of titanium dioxide was gained. This is a prerequisite for industrial applications to improve hygiene, food quality and safety.     

Fumigant Toxicity of Plant Essential Oils to Plutella xylostella (Lepidoptera: Yponomeutidae) and Cotesia glomerata (Hymenoptera: Braconidae)

The fumigant toxicity of 66 plant essential oils to Plutella xylostella (L.) larvae and Cotesia glomerata (L.) adults was examined using a vapor-phase toxicity bioassay and compared with that of dichlorvos. Responses varied according to oil and insect species used. Based on 24 h LD₅₀ values, pennyroyal oil [10.77 mg/filter paper (4.25 cm diameter)] was the most toxic fumigant, followed by rosemary and sage (Dalmatin) oils (15.15 mg/paper). Potent fumigant toxicity was also produced from armoise, buchu leaf, cedarleaf, coriander, eucalyptus, howood, lavender, myrtle, niaouli, peppermint, and rosewood oils (LD₅₀, 21.29–27.31 mg/paper). All essential oils were less effective than dichlorvos (LD₅₀, 0.52 mg/paper). Against adult C. glomerata, dichlorvos (LD₅₀, 0.03 mg/paper) was the most toxic fumigant, whereas the LD₅₀ values of the 14 essential oils ranged from 1.59 to 8.51 mg/paper. Based on selective toxicity ratio (STR, P. xylostella LD₅₀/C. glomerata LD₅₀), the 14 essential oils (STR, 2.5–14.5) are more selective than dichlorvos (STR, 17.3). The essential oils tested merit further study as potential fumigants for the control of P. xylostella in greenhouses because of their selective toxicity to adult C. glomerata and their much greater activity as a fumigant.     

Characterization of a highly virulent and antimicrobial‐resistant Acinetobacter baumannii strain isolated from diseased chicks in China

Poultry husbandry is a very important aspect of the agricultural economy in China. However, chicks are often susceptible to infectious disease microorganisms, such as bacteria, viruses and parasites, causing large economic losses in recent years. In the present study, we isolated an Acinetobacter baumannii strain, CCGGD201101, from diseased chicks in the Jilin Province of China. Regression analyses of virulence and LD₅₀ tests conducted using healthy chicks confirmed that A. baumannii CCGGD201101, with an LD₅₀ of 1.81 (±0.11) × 10⁴ CFU, was more virulent than A. baumannii ATCC17978, with an LD₅₀ of 1.73 (±0.13) × 10⁷ CFU. Moreover, TEM examination showed that the pili of A. baumannii CCGGD201101 were different from those of ATCC17978. Antibiotic sensitivity analyses showed that A. baumannii CCGGD201101 was sensitive to rifampicin but resistant to most other antibiotics. These results imply that A. baumannii strain CCGGD201101 had both virulence enhancement and antibiotic resistance characteristics, which are beneficial for A. baumannii survival under adverse conditions and enhance fitness and invasiveness in the host. A. baumannii CCGGD20101, with its high virulence and antimicrobial resistance, may be one of the pathogens causing death of diseased chicks.     

Evaluating automated infrared thermography and vulva exposure tracking as components of an estrus detection platform in a commercial dairy herd

The primary objective of this study was to develop an automated infrared thermography platform (Estrus BenchMark) capable of measuring skin temperature and tail movements as a means of identifying cows in estrus. The secondary objective was to evaluate the accuracy of Estrus BenchMark to detect estrus compared to in-line milk progesterone (P₄) analysis (Herd Navigator System) in a commercial dairy herd managed under a robotic milking system. Data were collected on forty-six cows from 45 to 120 d after calving. Cows were flagged in estrus when milk P₄ fell below 5 ng/mL. The Estrus BenchMark true positive estrus alerts (Sensitivity; Se%) were compared to Herd Navigator System estrus alerts at different time-windows (±12 h, ±24 h, ±48 h, and ±72 h) relative to the Estrus BenchMark estrus alerts for all the estrus alerts (AE) and confidence-quality estrus (CQE; >80% quality) alerts identified by Herd Navigator System. The Estrus BenchMark captured skin temperature and tail movements resulting in vulva exposure (left tail movements, LTail; right tail movements, RTail; and pooled tail movements, PTail) for each milking event. Skin temperature tended to increase when the milk P₄ concentration (Least-Squares Means ± SE) dropped for AE (estrus day [d 0]; P₄; 3.51 ± 0.05 ng/mL, Skin temperature; 33.31 ± 2.38 °C) compared with d ?7 (P₄; 20.22 ± 0.73 ng/mL; Skin temperature: 32.05 ± 3.77 °C). The increase in skin temperature, however, was significant in cows with CQE > 80% at d 0 (32.75 ± 0.29 °C) compared to d ?7 (31.80 ± 0.28 °C). The prevalence of tail movements to expose vulva was greater (P = 0.01) in AE at d 0 (LTail: 62.50%; PTail; 68.75%; and RTail: 56.25%) compared with d ?7 (LTail: 18.75%; PTail: 9.37%: and RTail: 9.37%), and d +4 (LTail: 9.37%; PTail: 9.37%; and RTail: 12.5%). Moreover, the higher prevalence of tail movements at d 0 was observed in cows with CQE > 80% (LTail; 65%, PTail; 80%, and RTail; 70%) compared to those with CQE < 80%. The highest Estrus BenchMark Youden index (YJ; 0.45), diagnostic odds ratio (DOR; 9.04), and Efficiency (0.77) were achieved for AE in a ±48 h window and at ±72 h window for CQE (YJ; 0.66, DOR; 25.29, and Efficiency 0.76) relative to Herd Navigator System estrus alerts. The highest Estrus BenchMark resulted in 58% estrus detection rates for AE and 80% for cows with CQE compared to the Herd Navigator System.     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED₁₀₀; ED = effective dose) and a toxic (LD₂₅; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.     

Possible recovery from an acute envenomation in male rats with LD50 of Echis coloratus crude venom: I-A seven days hematological follow-up study

The effect of an acute LD₅₀ dose of Echis coloratus crude venom in male albino rats was tested on blood parameters: white blood cells (WBCs), red blood cells (RBCs), platelets count, hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC), also serum glucose, total protein, triglycerides with alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) enzyme activities. The effect of the LD₅₀ dose was monitored over a period of seven days, with time intervals of 1, 3, 6, 12, 24, 72 h. All of the tested parameters show fluctuations with time and with tendency to regain normal control level after 12 h. At 12–24 h it seems to be crucial for the process of physiological recovery, in spite of the irreversible damage and tissue distraction. The process of physiological adaptation and recovery from the lethal destructive venom effect seems to stabilize after one week, leaving the animal alive with several biochemical altered metabolisms and disturbed physiological profile.     

Supercooling ability and cold hardiness of the pollen beetle Meligethes aeneus

Supercooling point (SCP) and cold‐hardiness of the pollen beetle Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae) were investigated. Mature eggs from the oviduct were supercooled on average to ?28.0 °C and from oilseed rape buds to ?24.4 °C; first instars were supercooled to ?21.0 °C and second instars to ?16.8 °C. Despite their high supercooling ability, none of the eggs survived 24 h exposure to ?2.5 °C. The supercooling ability of adults varied significantly among feeding and non‐feeding beetles: high SCPs prevailed during the whole warm period, being about ?12 °C; low values of SCP of ?20 °C dominated in non‐feeding beetles. In spring and autumn, beetles displayed the same acclimation efficiency: after 1 week of exposure at 2.0 °C with no access to food their SCPs were depressed equally by about 3 °C. Meligethes aeneus beetles have a different response to low temperatures depending on the season. The lowest tolerance was found in reproductively active beetles after emergence from overwintering sites; the time needed to kill 50% of individuals (Ltime₅₀) was 56.2 h at ?7 °C and the lower lethal temperature needed to kill 50% (Ltemp₅₀) after 24 h exposure was ?8.6 °C. Cold hardiness increased from midsummer to midwinter; Ltime₅₀ was 80 h in August, 182.8 h in September, and 418.1 h in January. Lethal temperature after 24 h exposure was ?9.1 °C in August and ?9.8 °C in September. In February, after diapause, the beetles started to loose their cold tolerance, and Ltemp₅₀ was slightly increased to ?9.5 °C. Hibernating beetles tolerated long exposure at ?7 °C well, but mortality was high after short exposure if the temperature dropped below ?9 °C for 24 h. Despite the season, the beetles died at temperatures well above their mean SCP; consequently, SCP is not a suitable index for cold hardiness of M. aeneus.     

In vitro cytotoxicity testing for prediction of acute human toxicity

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD₅₀ values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD₅₀ values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC₅₀ values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD₅₀s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.Abbreviations DCP dichlorophenoxyacetic acid - DMEM Dulbecco's modified Eagles' medium - DMSO dimethylsulfoxide - IC inhibitory concentration - LD₅₀ lethal dose 50% - MEIC Multicenter Evaluation forIn Vitro Cytotoxicity - PI₅₀ protein inhibition 50% - PTG pollen tube growth - TCA trichloroacetic acid - TCE trichloroethane     

Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro

Dried axillary buds from plantlets of Asparagus lofficinalis L. grown in vitro were successfully cryopreserved. Single node segments (5mm in length) with axillary bud were taken from mature in vitro plantlets. The segments were precultured on solidfied Murashige-Skoog medium (1962) containing 0.7M sucrose at 25 °C in light for 2 days. Thereafter, these precultured segments were subjected to dehydration with silica gel at room temperature for 0 to 24 h. The axillary buds of precultured segments tolerated dehydration to about 14% water content(FW) with 50% lethality (LD₅₀) and the threshold water content at which the dried buds remained alive after exposure to liquid nitrogen was 16.9%(LD₅₀). The maximum rate of survival of cryopreserved buds was about 71% of untreated control. Surviving buds produced shoots and regenerated into plantlets. These results demonstrate the feasibility of cryopreserving dried axillary buds from in vitro plantlets.Abbreviations MS Murashige and Skoog medium(1962) - LN liquid nitrogen - FW fresh weight basis - LD₅₀ the water content at 50% lethality - ABA abscisic acid - NAA -naphthalene acetic acid - BA 6-benzyladenine - DTA differential thermal analysis     

Does Lighting Manipulation During Incubation Affect Hatching Rhythms and Early Development of Sole?

Light plays a key role in the development of biological rhythms in fish. Previous research on Senegal sole has revealed that both spawning rhythms and larval development are strongly influenced by lighting conditions. However, hatching rhythms and the effect of light during incubation are as yet unexplored. Therefore, the aim of this study was to investigate the impact of the light spectrum and photoperiod on Solea senegalensis eggs and larvae until day 7 post hatching (dph). To this end, eggs were collected immediately after spawning during the night and exposed to continuous light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white light (LD_W), blue light (LD_B; λ_peak?=?463?nm), or red light (LD_R; λ_peak?=?685?nm). Eggs exposed to LD_B had the highest hatching rate (94.5%?±?1.9%), whereas LD_R and DD showed the lowest hatching rate (54.4%?±?3.9% and 48.4%?±?4.2%, respectively). Under LD conditions, the hatching rhythm peaked by the end of the dark phase, but was advanced in LD_B (zeitgeber time 8 [ZT8]; ZT0 representing the onset of darkness) in relation to LD_W and LD_R (ZT11). Under DD conditions, the same rhythm persisted, although with lower amplitude, whereas under LL the hatching rhythm split into two peaks (ZT8 and ZT13). From dph 4 onwards, larvae under LD_B showed the best growth and quickest development (advanced eye pigmentation, mouth opening, and pectoral fins), whereas larvae under LD_R and DD had the poorest performance. These results reveal that developmental rhythms at the egg stage are tightly controlled by light characteristics, underlining the importance of reproducing their natural underwater photoenvironment (LD cycles of blue wavelengths) during incubation and early larvae development of fish. (Author correspondence: borja@um.es)