Practical three color live cell imaging by widefield microscopy

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.     

Muscle receptor organs do not mediate load compensation during body roll and defense response extensions in the crayfish Cherax destructor.

It has been proposed that the abdominal muscle receptor organ (MRO) of decapod crustaceans acts in a sensory feedback loop to compensate for external load. There is not yet unequivocal evidence of MRO activity during slow abdominal extension in intact animals, however. This raises the possibility that MRO involvement in load compensation is context-dependent. We recorded from MRO tonic stretch receptors (SRs) in freely behaving crayfish (Cherax destructor) during abdominal extension occurring during two different behaviors: body roll and the defense response. Abdominal extensions are similar in many respects in both behaviors, although defense response extensions are more rapid. In both situations, SR activity typically ceased when the abdominal extension commenced, even if the joint of the SR being monitored was mechanically prevented from extending by a block. Since extensor motor neuron activity increased when the abdomen was prevented from extending, we concluded that the load compensation occurring in these behaviors was not mediated by the MROs.     

Trends in the association of obesity and self-reported overall health in 30 years of the Integrated Health Interview Series

This research examines trends in the relationship between obesity based on self-report height and weight and self-perceived health over a 30-year period. Importantly, this period included the articulation of comprehensive public health campaigns on excess weight and thus provides opportunities for assessment of the efficacy of the campaign, as well as the broader psycho-social impact of excess weight. Using novel data from the Integrated Health Interview Series, odds ratios for the association between obesity and self-perceived health were estimated for repeated cross-sectional samples that are nationally representative of noninstitutionalized American adults aged 18-85 and older spanning 1976-2006. Our findings show that (i) there are weak associations between obesity and self-perceived poor health; (ii) these associations are particularly small among men, often to the point of being nonexistent; and (iii) weak relationships for both men and women have remained virtually unchanged over the past 30 years. Several reasons why the public health campaign around excess weight has had limited traction are discussed including the collective problem of excess weight in America and how this undermines current approaches in public health efforts addressing excess weight.     

Altered aggression in different sized groups of crayfish supports a dynamic social behaviour model

Living in groups with conspecifics can increase an animal's fitness in the wild. A social environment may also be imposed by commercial farming industries. One important measure of competition and group dynamics is the level of aggressive interaction. This can also influence the level of damage or injury in cultured populations, a commercial issue at point of sale. There is considerable research into this issue in commercial species such as pigs, cattle and chickens but less is known about aquatic communal species such as decapod crustaceans. Here we manipulated group size in the freshwater crayfish Cherax destructor, a species that forms social groups in the wild and one that is also commercially farmed. Aggressive behaviour was scored during 1 h of observation in replicates of groups of 4, 16 and 36 animals to analyse 11 variables of fight dynamics that ensued. The number of fights per crayfish (4.0 ± 0.8 to 1.9 ± 0.2 fights, P = 0.017) and the time each crayfish was involved in a fight (113.9 ± 32.6 to 21.6 ± 2.6 s, P = 0.011) decreased as group size increased. Conversely, the number of failed tailflips elicited per crayfish increased from 0 to 0.08 ± 0.03 tailflips in the largest groups (P = 0.011). Together, the data suggest that despite C. destructor's different biology and habitat, compared to prior work that manipulates group size, the crayfish adjusts its fighting strategy when social circumstances change. Theory has proposed aggressive behaviour could change in groups of animals and our data indicates that this applies more broadly across species and more dynamically than previously demonstrated.     

Iodine-mediated glycosylation en route to mucin-related glyco-aminoacids and glycopeptides

The use of iodine-DDQ as a promoter for glycosylation of Fmoc-Ser-OBn and Fmoc-Thr-OBn with phenylseleno 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranoside in toluene-dioxane gave 49% and 73% yields, respectively, of the corresponding alpha-glycosides as the sole glycoside products. Reductive acetylation of the azide groups and cleavage of the benzyl esters by hydrogenolysis gave building blocks that were used in solid-phase synthesis to prepare triglycosylated tetrapeptides (Ac(3)GalNAc-alpha-Ser)(3)-Gly and (Ac(3)GalNAc-alpha-Thr)(3)-Gly in 27% and 49% overall yield, respectively.     

A novel approach identified the FOLR1 gene, a putative regulator of milk protein synthesis

This study has utilised comparative functional genomics to exploit animal models with extreme adaptation to lactation to identify candidate genes that specifically regulate protein synthesis in the cow mammary gland. Increasing milk protein production is valuable to the dairy industry. The lactation strategies of both the Cape fur seal (Artocephalus pusillus pusillus) and the tammar wallaby (Macropus eugenii) include periods of high rates of milk protein synthesis during an established lactation and therefore offer unique models to target genes that specifically regulate milk protein synthesis. Global changes in mammary gene expression in the Cape fur seal, tammar wallaby, and the cow (Bos taurus) were assessed using microarray analysis. The folate receptor α (FOLR1) showed the greatest change in gene expression in all three species [cow 12.7-fold (n = 3), fur seal 15.4-fold (n = 1), tammar 2.4-fold (n = 4)] at periods of increased milk protein production. This compliments previous reports that folate is important for milk protein synthesis and suggests FOLR1 may be a key regulatory point of folate metabolism for milk protein synthesis within mammary epithelial cells (lactocytes). These data may have important implications for the dairy industry to develop strategies to increase milk protein production in cows. This study illustrates the potential of comparative genomics to target genes of interest to the scientific community.     

A Serological Survey of Ruminant Livestock in Kazakhstan During Post-Soviet Transitions in Farming and Disease Control

The results of a serological survey of livestock in Kazakhstan, carried out in 1997–1998, are reported. Serum samples from 958 animals (cattle, sheep and goats) were tested for antibodies to foot and mouth disease (FMD), bluetongue (BT), epizootic haemorrhagic disease (EHD), rinderpest (RP) and peste des petits ruminants (PPR) viruses, and to Brucella spp. We also investigated the vaccination status of livestock and related this to changes in veterinary provision since independence in 1991. For the 2 diseases under official surveillance (FMD and brucellosis) our results were similar to official data, although we found significantly higher brucellosis levels in 2 districts and widespread ignorance about FMD vaccination status. The seroprevalence for BT virus was 23%, and seropositive animals were widespread suggesting endemicity, despite the disease not having being previously reported. We found a few seropositives for EHDV and PPRV, which may suggest that these diseases are also present in Kazakhstan. An hierarchical model showed that seroprevalence to FMD and BT viruses were clustered at the farm/village level, rather than at a larger spatial scale. This was unexpected for FMD, which is subject to vaccination policies which vary at the raion (county) level.     

Local community attitudes to wildlife utilisation in the changing economic and social context of Mongolia

In recent years illegal hunting has increased in Mongolia, putting considerable pressure on large mammals populations. The causes for this phenomenon lie in increasing rural poverty, ineffective policies to regulate hunting, as well as a ready market for many wildlife products in the Chinese medicine markets of east Asia. It is now accepted that biodiversity is ultimately lost or conserved at the local level and it is therefore imperative that the perspectives of the local people are better understood if wildlife management programmes are to be sustainable. This research uses a grounded theory approach to investigate local community attitudes to wildlife utilisation and to explore what local people consider as a sustainable wildlife management strategy in remoter regions of Mongolia. The findings clearly suggest that the current situation is not conducive to sustainability either of wildlife populations or human livelihoods. Important changes are needed if sustainability is to be achieved, including alterations to property rights, greater government support, and improved marketing skills and employment opportunities from wildlife. Linked to all of the above points is the need to address the serious information deficit experienced in rural Mongolia to enable local people to examine critically the issues at hand and to participate actively in solutions.     

Effect of treatment with progesterone and oestradiol when starting treatment with an intravaginal progesterone releasing insert on ovarian follicular development and hormonal concentrations in Holstein cows

Ovarian follicular development and concentrations of gonadotrophin and steroid hormones were studied in non-lactating Holstein cows following administration of progesterone (P(4)) or oestradiol benzoate (ODB) at the start of treatment with an intravaginal progesterone releasing insert (IVP(4)) in a 2 by 2 factorial experiment. Cows were treated at random stages of the oestrous cycle with an IVP(4) device (Day 0) and either no other treatment (n=8), 200 mg of P(4) IM (n=9), 2.0 mg of ODB IM (n=8) or both P(4) and ODB (n=9). Seven days later devices were removed and PGF(2alpha) was administered. Twenty-four hours later 1.0mg of ODB was administered IM. Oestrus was detected in 97.1% and ovulation in 64.7% (effect of treatment, P=0.41) of cows within 96 h of removing inserts. In the cows that ovulated, day of emergence of the ovulatory follicle was delayed (P<0.01) and more precise (P<0.05) in cows treated with ODB compared to the cows treated with P(4). Interval from wave emergence to ovulation and the diameter of the ovulatory follicle was less (P<0.05) in cows treated with ODB compared to cows treated with P(4). Combined treatment with P(4) and ODB at the time of starting treatment with an IVP(4) device did not significantly change the pattern of ovarian follicular development compared to treatment with ODB alone. Concentrations of LH and FSH in plasma were less in cows treated with ODB between Days 0 and 4 (P<0.05) while treatment with P(4) increased concentrations of FSH in plasma between Days 0 and 4 (P<0.05). When anovulatory cows were compared to ovulatory cows, diameters of follicles (P<0.001) and growth rate of follicles (P<0.01) were less in anovulatory cows between Days 7 and 9, while concentrations of FSH in plasma were greater (P<0.01), concentrations of LH similar (P>0.90) and concentrations of oestradiol were less (P=0.01) in the anovulatory cows between Days 4 and 10. Our findings support a hypothesis that ovarian follicular development following administration of P(4) or ODB at the start of treatment with an IVP(4) device differs. Anovulatory oestrus may have been associated with reduced maturity and/or later emergence of ovarian follicles.