Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions

In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4–7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively.     

Asymmetric reduction of (4S)-(+)-carvone catalyzed by baker's yeast: A green method for monitoring the conversion based on liquid–liquid–liquid microextraction with polypropylene hollow fiber membranes

The α,β-unsaturated carbonyl compound (4S)-(+)-carvone was selectively reduced to (1R,2R,4S)-iso-dihydrocarveol using baker's yeasts. The conversion of the bioreduction reaction was monitored using a green hollow-fiber liquid–liquid–liquid microextraction (HF-LLLME) technique. Several parameters which may affect the bioreduction of (4S)-(+)-carvone, such as temperature, time, substrate/enzyme ratio, pH and buffer concentration, were evaluated. The effect of some additives, such as trehalose, DMSO and the ionic liquid [BMIm][PF₆], was also studied. The (1R,2R,4S)-iso-dihydrocarveol was recovered with 52.7% conversion and diastereoisomeric excess >99% after 48 h of reaction at 40 °C in an aqueous monophasic system, with 0.1 mol L^?1 buffer concentration (pH 7.5) and a substrate/yeast cell mass ratio of 8.0 mg g^?1. The HF-LLLME microextraction technique allowed the optimization of the reaction with a reduction of over 99.5% in relation to the use of organic solvents.     

Comparison of vitrification and slow cooling for umbilical tissues

The tissue cryopreservation maintains the cellular metabolism in a quiescence state and makes the conservation possible for an indefinite period of time. The choice of an appropriate cryopreservation protocol is essential for maintenance of cryopreserved tissue banks. This study evaluated 10 samples of umbilical cord, from which small fragments of tissue (Wharton’s jelly and cord lining membrane) were subjected to two protocols of cryopreservation: slow cooling and vitrification. The samples were frozen for a period of time ranging from 5 to 78 days. The efficiency of cryopreservation was evaluated by testing cell viability, histological analysis, cell culture, cytogenetic analysis and comparison with the results of the fresh samples. The results showed that the slow cooling protocol was more efficient than the vitrification for cryopreservation of umbilical cord tissue, because it has caused fewer changes in the structure of tissue (edema and degeneration of the epithelium) and, despite the significant decrease cell viability compared to fresh samples, the ability of cell proliferation in vitro was preserved in most samples. In conclusion, this study showed that it is possible to cryopreserve small fragments of tissue from the umbilical cord and, to obtain viable cells capable of proliferation in vitro after thawing, contributing to the creation of a frozen tissue bank.     

Aging in Mice Reduces the Ability to Sustain Sleep/Wake States

One of the most significant problems facing older individuals is difficulty staying asleep at night and awake during the day. Understanding the mechanisms by which the regulation of sleep/wake goes awry with age is a critical step in identifying novel therapeutic strategies to improve quality of life for the elderly. We measured wake, non-rapid eye movement (NREM) and rapid-eye movement (REM) sleep in young (2–4 months-old) and aged (22–24 months-old) C57BL6/NIA mice. We used both conventional measures (i.e., bout number and bout duration) and an innovative spike-and-slab statistical approach to characterize age-related fragmentation of sleep/wake. The short (spike) and long (slab) components of the spike-and-slab mixture model capture the distribution of bouts for each behavioral state in mice. Using this novel analytical approach, we found that aged animals are less able to sustain long episodes of wakefulness or NREM sleep. Additionally, spectral analysis of EEG recordings revealed that aging slows theta peak frequency, a correlate of arousal. These combined analyses provide a window into the mechanisms underlying the destabilization of long periods of sleep and wake and reduced vigilance that develop with aging.     

Growth and Airborne Transmission of Cell-Sorted Life Cycle Stages of Pneumocystis carinii

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.