The calm mouse: an animal model of stress reduction

Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a "calm mouse model" with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.     

Caged lipids for subcellular manipulation

We present recently developed strategies to manipulate lipid levels in live cells by light. We focus on photoremovable protecting groups that lead to subcellular restricted localization and activation and discuss alternative techniques. We emphasize the development of organelle targeting of caged lipids and discuss recent advances in chromatic orthogonality of caging groups for future applications.     

Fast laser-induced solute release from liposomes sensitized with photochromic lipid: effects of temperature, lipid host, and sensitizer concentration.

Liposomes of gel-phase phospholipid have been prepared containing a photochromic lipid sensitizer. A fast UV laser pulse isomerizes the sensitizer destabilizing the lipid bilayer structure and causing release of trapped solute. The kinetics of solute release have been investigated as a function of host lipid chain length, sensitizer concentration, and temperature, and the limits of liposome stability have been established. At low concentrations of sensitizer, pulsed laser irradiation induces some solute release when continuous UV illumination is ineffective. Although rates of solute release usually increase with temperature, at low sensitizer concentration in a rigid host, leakage at first increases but then decreases rapidly above a threshold temperature. The results presented are relevant to the design of photostimulated drug delivery systems and to potential applications of photosensitive liposomes as caging agents for biological effectors.     

Different fermentation strategies by Schizochytrium mangrovei strain pq6 to produce feedstock for exploitation of squalene and omega‐3 fatty acids

Schizochytrium mangrovei strain PQ 6 was investigated for coproduction of docosahexaenoic acid (C22: 6ω‐3, DHA ) and squalene using a 30‐L bioreactor with a working volume of 15 L under various batch and fed‐batch fermentation process regimes. The fed‐batch process was a more efficient cultivation strategy for achieving higher biomass production rich in DHA and squalene. The final biomass, total lipid, unsaponifiable lipid content, and DHA productivity were 105.25 g · L^?1, 43.40% of dry cell weight, 8.58% total lipid, and 61.66 mg · g^?1 · L^?1, respectively, after a 96 h fed‐batch fermentation. The squalene content was highest at 48 h after feeding glucose (98.07 mg · g^?1 of lipid). Differences in lipid accumulation during fermentation were correlated with changes in ultrastructure using transmission electron microscopy and Nile Red staining of cells. The results may be of relevance to industrial‐scale coproduction of DHA and squalene in heterotrophic marine microalgae such as Schizochytrium .     

Factors affecting the uncaging efficiency of 500 nm light-activatable BODIPY caging group

Photoremovable protective groups, or caging groups, enable us to regulate the activities of bioactive molecules in living cells upon photoirradiation. Nevertheless, requirement of UV light for activating caging group is a significant limitation due to its cell toxicity and its poor tissue penetration. Our group previously reported a 500?nm light-activatable caging group based on BODIPY scaffold, however, its uncaging efficiency was lower than those of conventional caging groups. Here we show that the uncaging quantum yield (QY) of BODIPY caging group depends upon the driving force of photo-induced electron transfer (PeT). We also found that the uncaging QY increased in less polar solvents. We applied these findings to develop BODIPY-caged capsaicin, which is well localized to low-polarity intracellular compartments, as a tool to stimulate TRPV1 in live cells in response to blue-green light.     

Nitrogen retention and partitioning at the initiation of lipid accumulation in nitrogen‐deficient algae

Nitrogen (N) deficiency promotes lipid accumulation in many oleaginous algae, but we have a poor understanding of the associations between the initiation of lipid accumulation and algal N retention and partitioning. Here, we report on total cell N, five bulk pools of N in the cell (protein, free amino acids, DNA, RNA, chl), and lipids from N saturation to growth cessation in three species. While the maximum level of N uptake differed among species, the ratio of minimum retained N to N retained at the initiation of lipid accumulation was consistent among species at 0.5 ± 0.04. This suggests that the cellular initiation of lipid accumulation was associated with a common magnitude of N deficiency among species. Concerning the partitioning of N, the concentration of RNA and the protein to RNA ratio were most similar among species at the initiation of lipid accumulation with averages of 3.2 ± 0.26 g · L^?1 (8.2% variation) and 16 ± 1.5 (9.2% variation), respectively. All other pools and physiologically relevant ratios were considerably more variable. The species commonalities in RNA and protein show a similar reduction in general cellular function due to N deficiency before cellular initiation of lipid accumulation. These results provide insight into the physiological drivers for lipid accumulation in N‐deficient algae and data for modeling these associations.     

Nonlethal sampling for stable isotope analysis of juvenile Chinese sturgeon (Acipenser sinensis): Comparing δ13C and δ15N signatures in muscle and fin tissues

We compared δ¹³C and δ¹⁵N values of muscle with fin from juvenile Chinese sturgeon (Acipenser sinensis), to evaluate the feasibility of using nonlethal (fin) as an alternative to lethal (muscle) sampling. Size and lipid effect on the relationship between fin and muscle were also investigated. Dorsal muscle (DM) and fin clip (FC) were collected from A. sinensis with different body length (120–373 mm) in the Yangtze Estuary for isotope analysis. The result showed that (1) muscle isotope values could estimated by the values of fin, from either use the regression model (δ¹³C_DM = 0.939 × FC ? 2.577; δ¹⁵N_DM = 0.737 × FC + 4.638) or constants factors (δ¹³C_DM = δ¹³C_FC ? 1.27; δ¹⁵N_DM = δ¹⁵N_FC + 0.59); (2) no size‐based relationships with δ¹³C and δ¹⁵N from either fin or muscle; (3) lipid extraction significantly improving the fin and muscle regression model fit for both δ¹³C and δ¹⁵N values. Therefore, this study support the use of nonlethal fin tissues for isotope analysis of juvenile A. sinensis, and will allow trophic studies to avoid the effect of lipid accumulation from muscle.     

Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics

It is generally assumed that type A lantibiotics primarily kill bacteria by permeabilization of the cytoplasmic membrane. As previous studies had demonstrated that nisin interacts with the membrane-bound peptidoglycan precursors lipid I and lipid II, we presumed that this interaction could play a role in the pore formation process of lantibiotics. Using a thin-layer chromatography system, we found that only nisin and epidermin, but not Pep5, can form a complex with [¹⁴C]-lipid II. Lipid II was then purified from Micrococcus luteus and incorporated into carboxyfluorescein-loaded liposomes made of phosphatidylcholine and cholesterol (1:1). Liposomes supplemented with 0.05 or 0.1 mol% of lipid II did not release any marker when treated with Pep5 or epilancin K7 (peptide concentrations of up to 5 mol% were tested). In contrast, as little as 0.01 mol% of epidermin and 0.1 mol% of nisin were sufficient to induce rapid marker release; phosphatidylglycerol-containing liposomes were even more susceptible. Controls with moenomycin-, undecaprenol- or dodecaprenolphosphate-doped liposomes demonstrated the specificity of the lantibiotics for lipid II. These results were correlated with intact cells in an in vivo model. M. luteus and Staphylococcus simulans were depleted of lipid II by preincubation with the lipopeptide ramoplanin and then tested for pore formation. When applied in concentrations below the minimal inhibitory concentration (MIC) and up to 5–10 times the MIC, the pore formation by nisin and epidermin was blocked; at higher concentrations of the lantibiotics the protective effect of ramoplanin disappeared. These results demonstrate that, in vitro and in vivo , lipid II serves as a docking molecule for nisin and epidermin, but not for Pep5 and epilancin K7, and thereby facilitates the formation of pores in the cytoplasmic membrane.     

Comparison of body composition and sensory quality of wild and farmed whitefish (Coregonus macrophthalmus [Nüsslin, 1882])

This study compared the body composition (fillet yield, chemical composition and lipid quality of fillets) and sensory quality of captured wild (by gillnet in August 2011) with experimentally raised farmed (reared in concrete flow‐through raceways, average water temperature 10 ± 3°C) whitefish, Coregonus macrophthalmus, from Lake Constance. The study was conducted in 2011 using 28 wild and 24 farmed market‐sized fish of approximately equal total lengths (25.1 ± 1.21 and 25.6 ± 1.28 cm). Farmed female Coregonus macrophthalmus exhibited a 3.3% lower fillet yield resulting from larger gonads and a shallower body shape. The protein contents of farmed and wild fillets were equal (17.5% vs. 17.4%), but farmed fish fillets contained less moisture (76.1% vs.77.4%), less ash (1.2% vs. 1.6%), and more lipid (5.1% vs. 4.1%) than wild‐caught specimens. Levels of monounsaturated fatty acids (MUFA) were higher in farmed fish (42.0 vs. 31.0 g/100 g lipid), as well as the levels of polyunsaturated fatty acids (PUFA; 26.0 vs. 19.7 g/100 g lipid), in particular 22:6n?3 (docosahexaenoic acid, DHA; 7.3 vs. 2.1 g/100 g lipid). Conversely, wild fish fillets contained significantly more 20:4n?6 (arachidonic acid, AA; 1.6 vs. 0.4 g/100 g lipid) and 20:5n?3 (eicosapentaenoic acid, EPA; 4.1 vs. 3.2 g/100 g lipid). Sensory evaluation of odour, colour, texture and flavour by an experienced six‐person panel revealed a significant preference for the colour (79% vs. 21%) of farmed fish fillets. In conclusion, farmed whitefish can be strong competitors to wild whitefish in terms of product quality, with higher levels of healthy fatty acids and a more attractive fillet colour. Farmed Coregonus macrophthalmus thus represents a promising and pragmatic approach, compensating for the current capture decline in the whitefish fisheries of Lake Constance.     

Nucleobase‐caged peptide nucleic acids: PNA/PNA duplex destabilization and light‐triggered PNA/PNA recognition

The 2‐(o‐nitrophenyl)‐propyl (NPP) group is used as caging group to mask the nucleobases adenine and cytosine in N‐(2‐aminoethyl)glycine peptide nucleic acids (aeg‐PNA). The adeninyl and cytosinyl nucleo amino acid building blocks Fmoc‐a^NPP‐aeg‐OH and Fmoc‐c^NPP‐aeg‐OH were synthesized and incorporated into PNA sequences by Fmoc solid phase synthesis relying on high stability of the NPP nucleobase protecting group toward Fmoc‐cleavage, coupling, capping, and resin cleavage conditions. Removal of the nucleobase caging group was achieved by UV‐LED irradiation at 365 nm. The nucleobase caging groups provided sterical crowding effecting the Watson–Crick base pairing, and thereby, the PNA double strand stabilities. Duplex formation can completely be suppressed for complementary PNA containing caging groups in both strands. PNA/PNA recognition can be completely restored by UV light‐triggered release of the photolabile protecting group. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and photoreactivity of caged blockers for glutamate transporters

L-TBOA (L-threo-beta-benzyloxyaspartate) is, so far, the most potent non-transportable blocker for glutamate transporters. We synthesized alpha-CMCM-L-TBOA (1a) possessing [7-(carboxymethoxy)coumarin-4-yl]methyl ester as a caging group. alpha-CMCM-L-TBOA (1a) is biologically inactive until UV irradiation and the photolysis of 1a immediately released L-TBOA to show glutamate uptake inhibition. The photoreactivity of the coumarin-type caging group was superior to that of the o-nitrobenzyl-type caging group.     

Optopharmacological control of TRPC channels by coumarin-caged lipids is associated with a phototoxic membrane effect

Photouncaging of second messengers has been successfully employed to gain mechanistic insight of cellular signaling pathways. One of the most enigmatic processes of ion channel regulation is lipid recognition and lipid-gating of TRPC channels, which represents pivotal mechanisms of cellular Ca~(2+) homeostasis. Recently, optopharmacological tools including caged lipid mediators became available, enabling an unprecedented level of temporal and spatial control of the activating lipid species within a cellular environment. Here we tested a commonly used caged ligand approach for suitability to investigate TRPC signaling at the level of membrane conductance and cellular Ca~(2+) handling. We report a specific photouncaging artifact that is triggered by the cage structure coumarin at UV illumination. Electrophysiological characterization identified a light-dependent membrane effect of coumarin. UV light(340 nm) as used for photouncaging, initiated a membrane conductance specifically in the presence of coumarin as low as 30 μmol L~(-1) concentrations. This conductance masked the TRPC3 conductance evoked by photouncaging, while TRPC-mediated cellular Ca~(2+) responses were largely preserved. The observed light-induced membrane effects of the released caging moiety may well interfere with certain cellular functions, and prompt caution in using coumarin-caged second messengers in cellular studies.     

Lipid biomarker and phylogenetic analyses to reveal archaeal biodiversity and distribution in hypersaline microbial mat and underlying sediment

This study has utilized the tools of lipid biomarker chemistry and molecular phylogenetic analyses to assess the archaeal contribution to diversity and abundance within a microbial mat and underlying sediment from a hypersaline lagoon in Baja California. Based on abundance of ether-linked isoprenoids, archaea made up from 1 to 4% of the cell numbers throughout the upper 100 mm of mat and sediment core. Below this depth archaeal lipid was two times more abundant than bacterial. Archaeol was the primary archaeal lipid in all layers. Relatively small amounts of caldarchaeol (dibiphytanyl glyceroltetraether) were present at most depths with phytanyl to biphytanyl molar ratios lowest (~10 : 1) in the 4–17 mm and 100–130 mm horizons, and highest (132 : 1) in the surface 0–2 mm. Lipids with cyclic biphytanyl cores were only detected below 100 mm. A novel polar lipid containing a C₃₀ isoprenoid (squalane) moiety was isolated from the upper anoxic portion of the core and partially characterized. Hydrocarbon biomarker lipids included pentamethylicosane (2–10 mm) and crocetane (primarily below 10 mm). Archaeal molecular diversity varied somewhat with depth. With the exception of samples at 0–2 mm and 35–65 mm, Thermoplasmatales of marine benthic group D dominated clone libraries. A significant number of phylotypes representing the Crenarchaeota from marine benthic group B were generally present below 17 mm and dominated the 35–65 mm sample. Halobacteriaceae family made up 80% of the clone library of the surface 2 mm, and consisted primarily of sequences affiliated with the haloalkaliphilic Natronomonas pharaonis .     

Digestion and absorption of a pure triacylglycerol and a free fatty acid by Clupea harengus L. larvae

The digestion, absorption and post absorptive metabolism of a radiolabelled triacylglycerol (TAG; triolein) and a free fatty acid (FFA; oleic acid), delivered by tube feeding, was studied in herring Clupea harengus larvae, using metabolic chambers and video analysis. In general, a large amount of the delivered lipid was evacuated. Most of the evacuation occurred between 2 and 6 h after tube feeding although a group of larvae responded by rapidly evacuating the lipid (>50% before 2 h). The volume of the tube‐fed lipid affected its utilization. A small volume of triolein (9·2 nl, representing c . 6% of gut filling capacity) resulted in a lower proportion of fast evacuating larvae and improved utilization (lower evacuation and higher absorption: body incorporation and catabolism) compared with 50·6 nl ( c . 17% of gut filling capacity). Increases in the volume of tube fed triolein enhanced only marginally label absorption and led to a steep rise in evacuation. At a comparable high volume (50·6 nl), oleic acid, which does not require digestion, was better absorbed and less evacuated than triolein. The video observation of the lipid digestive process revealed a considerable gut contractile activity that appeared effective in processing the tube fed lipid. Also, the gut wall seemed very sensitive to physical pressure. Signs of chemical degradation during lipid digestion were also noted. The metabolic studies, together with video image analysis, suggested that the limiting step for the utilization of high dietary lipid levels may have been the lipid absorption into the enterocytes and transport into the body, rather than lipid digestion. The results support the notion that the rate of lipid digestion and absorption in fishes is slower than that of mammals.     

Urologic syndrome associated with wire caging in AKR mice

Individually housed male AKR/NCrlBR mice used in a chronic inhalation experiment were noted to develop a severe obstructive genitourinary condition. The mouse urologic syndrome (MUS) had one or more of the following features: bladder distension; peripreputial urine staining, alopecia, and edema; paraphimosis; urethral blockage; ulcerative balanophosthitis; hydronephrosis; pyelonephritis; rectal prolapse; and perineal ulcerative dermatitis. MUS was less severe and less prevalent in similarly housed B6C3F1/CrlBR and NIH-Swiss mice used in the same experiment. Epidemiologic evidence within the animal facility restricted the syndrome to the inhalation toxicology area. The effects of intermittent water deprivation as well as wire caging on the development of MUS were studied because these conditions were only utilized in the inhalation facility. Male AKR/NCrlBR mice, caged individually in suspended wire caging or kept isolated in polystyrene shoebox style cages containing wire floorwalk bottoms, all developed MUS within 16 weeks. Mice which were housed directly on hardwood bedding in identical plastic caging remained free of the syndrome, as did castrated males which were kept in suspended wire cages. Water deprivation was not found to be a major contributing factor to the development of the condition, but was found to augment its severity. We concluded that although MUS is probably multifactorial in etiology, housing susceptible animals on wire bottom caging may exacerbate the incidence and severity of the condition in certain strains of male mice.     

An evaluation of condition indices and predictive models for noninvasive estimates of lipid mass of migrating Common Yellowthroats, Ovenbirds, and Swainson's Thrushes

ABSTRACT.   Noninvasive methods of measuring lipid mass in birds are widely used, but not frequently evaluated. I evaluated the ability of three noninvasive indicators of fat content (fat scores, body mass, body mass/wing chord) and regression models to predict lipid mass in two migratory songbirds previously unexamined in this context—Common Yellowthroat ( Geothlypis trichas ) and Ovenbird ( Seiurus aurocapillus ). I also examined the accuracy of these methods for Swainson's Thrushes ( Catharus ustulatus ) for comparison to a previous study. Fat score, body mass, and body mass/wing chord were highly correlated with chemically extracted lipid mass in each species. In all three species, birds with no visible subcutaneous fat possessed considerable quantities of fat, ranging from 9.8 to 19.7% of total dry body mass. Forward-selected regression models explained 69−87% of lipid mass variation, with prediction errors of 14.6−27.5%. An existing predictive model for the Swainson's Thrush overestimated lipid mass by an average of 92%. Fat score, body mass, and the regression models generated here are reliable predictors of lipid mass in two of the three migrating species examined. The accuracy of the methods, in addition to their low cost and simplicity, justifies their continued use in field studies of birds.     

Lipid mobilization and locomotor stimulation in Gryllus bimaculatus by topically applied adipokinetic hormone

Abstract.  Walking activity of 3-day-old adult female Gryllus bimaculatus (de Geer) (Ensifera, Gryllidae) was measured over 24 h. A high level of locomotor activity during the scotophase was found, which was two- to three-fold higher than that during the photophase. The titre of lipid in the haemolymph was relatively low 2 h after lights on, increased significantly 2 h after lights off, although, 2 h after lights on in the next photophase, the lipid titre had decreased to the basal level. Topical application of homologous Grybi-adipokinetic hormone (AKH) (100 pmol in 20% 2-propanol) led to a significant increase in haemolymph lipids, comparable with the maximal increase caused by injection of AKH (3 pmol in water). Topical application of AKH also stimulated locomotor activity in crickets (maximal stimulation 1.8-fold with 100 pmol Grybi-AKH). The results suggest that AKH penetrates the cuticle quickly. It is assumed that AKH stimulates locomotory activity at least in part via the increase of haemolymph lipid titres; however, the stimulation of locomotor activity via a direct neuromodulatory effect of AKH cannot be excluded.     

Effects of Increased Spatial Complexity on Behavioural Development and Task Performance in Lister Hooded Rats

Enhancing laboratory animal welfare, particularly in rodents, has been achieved through environmental enrichment in caging systems. Traditional enrichment such as adding objects has shown to impact development, reproductive and maternal performance as well as cognition. However, effects of increased spatial complexity as part of larger novel caging systems have not been investigated. While adoption of caging systems with increased spatial complexity seems uncontroversial from a welfare perspective, effects of such housing on the development and task performance of experimental animals remains unclear. In this study, we investigate differences in key behaviours and cognitive performance between Lister Hooded rats housed in traditional (single-shelf) cages (‘basic’) and those housed in larger cages with an additional shelf (‘enriched’). We found minor differences in maternal behaviour, such as nursing and offspring development. Further, we compared task performance in females, using a hippocampus-dependent task (T-maze) and a hippocampus-independent task (Novel Object Recognition, NOR). While in the T-maze no differences in either the rate of learning or probe trial performance were found, in the NOR task females housed in enriched cages performed better than those housed in basic cages. Our results show that increased spatial complexity does not significantly affect development and maternal performance but may enhance learning in females for a non-spatial task. Increased spatial complexity does not appear to have the same effects on behaviour and development as traditional enrichment. Thus, our results suggest no effect of housing conditions on the development of most behaviours in experimental animals housed in spatially enriched caging systems.     

棉蚜饲养技术——笼罩法

在长期的试虫饲养过程中 ,摸索出一种新的棉蚜饲养方法———笼罩法。利用A4幅面的透明胶片和纱网制成笼罩。并于室内催芽 ,培育棉花种苗 ,可利用自制笼罩于光照培养箱内隔离饲养棉蚜 ,经与琼脂叶片法和自制Blackmanbox方法比较 ,笼罩法具有省时省力、不受季节限制、棉蚜生长条件好以及取食活体植株等许多突出的优点。