Store-dependent and -independent modes regulating Ca2+ release-activated Ca2+ channel activity of human Orai1 and Orai3

We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, Orai1 induced a large inwardly rectifying Ca(2+)-selective current with Ca(2+)-induced slow inactivation. A point mutation of Orai1 (E106D) altered the ion selectivity of the induced Ca(2+) release-activated Ca(2+) (CRAC)-like current while retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with Orai1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively nonselective current in STIM1 and Orai3 co-expressing cells. 2-APB also induced Ca(2+) influx in Orai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monovalent current. A pore mutant of Orai3 inhibited store-operated Ca(2+) entry and did not carry significant current in response to either store depletion or addition of 2-APB. Analysis of a series of Orai1-3 chimeras revealed the structural determinant responsible for 2-APB-induced current within the sequence from the second to third transmembrane segment of Orai3. The Orai3 current induced by 2-APB may reflect a store-independent mode of CRAC channel activation that opens a relatively nonselective cation pore.     

CRMP2 is necessary for Neurofibromatosis type 1 related pain

Neurofibromatosis type 1 (NF1) is one of the most common genetic diseases, affecting roughly 1 in 3000 individuals. As a multisystem disorder, it affects cognitive development, as well as bone, nerve and muscle constitution. Peripheral neuropathy in NF1 constitutes a potentially severe clinical complication and is associated with increased morbidity and mortality. The discovery of effective therapies for Neurofibromatosis type 1 (NF1) pain depends on mechanistic understanding that has been limited, in part, by the relative lack of availability of animal models relevant to NF1 pain. We have used intrathecal targeted editing of Nf1 in rats to provide direct evidence of a causal relationship between neurofibromin and pain responses. We demonstrated that editing of neurofibromin results in functional remodeling of peripheral nociceptors characterized by enhancement of interactions of the tetrodotoxin-sensitive (TTX-S) Na⁺ voltage-gated sodium channel (NaV1.7) and the collapsin response mediator protein 2 (CRMP2). Collectively, these peripheral adaptations increase sensory neuron excitability and release of excitatory transmitters to the spinal dorsal horn to establish and maintain a state of central sensitization reflected by hyperalgesia to mechanical stimulation of the hindpaw. The data presented here shows that CRMP2 inhibition is sufficient to reverse the dysregulations of voltage-gated ion channels and neurotransmitter release observed after Nf1 gene editing. The concordance in normalization of ion channel dysregulation by a CRMP2-directed strategy and of hyperalgesia supports the translational targeting of CRMP2 to curb NF1-related pain.     

Rearing performances and environmental assessment of sea cage farming in Tunisia using life cycle assessment (LCA) combined with PCA and HCPC

Purpose

The present study aims to understand the influence of rearing practices and the contributions of production phases of fish farming to their environmental impacts and determine which practices and technical characteristics can best improve the farms’ environmental performance. Another objective is to identify the influence of variability in farming practices on the environmental performances of sea cage aquaculture farms of sea bass and sea bream in Tunisia by using principal component analysis (PCA) and hierarchical clustering on principal components (HCPC) methods and then combining the classification with life cycle assessment (LCA).

Methods

The approach consisted of three major steps: (i) of the 24 aquaculture farms in Tunisia, 18 were selected which follow intensive rearing practices in sea cages of European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) and then a typology was developed to classify the studied farms into rearing practice groups using HCPC; (ii) LCA was performed on each aquaculture farm and (iii) mean impacts and contributions of production phases were calculated for each group of farms. Impact categories included acidification, eutrophication, global warming, land occupation, total cumulative energy demand and net primary production use.

Results and discussion

Results revealed high correlation between rearing practices and impacts. The feed-conversion ratio (FCR), water column depth under the cages and cage size had the greatest influence on impact intensity. Rearing practices and fish feed were the greatest contributors to the impacts studied due to the production of fish meal and oil and the low efficiency of feed use, which generated large amounts of nitrogen and phosphorus emissions. It is necessary to optimise the diet formulation and to follow better feeding strategies to lower the FCR and improve farm performance. Water column depth greatly influenced the farms’ environmental performance due to the increase in waste dispersion at deeper depths, while shallow depths resulted in accumulation of organic matter and degradation of water quality. Cage size influences environmental performances of aquaculture farms. Thus, from an environmental viewpoint, decision makers should grant licences for farms in deeper water with larger cages and encourage them to improve their FCRs.

Conclusions

This study is the first attempt to combine the HCPC method and the LCA framework to study the environmental performance of aquacultural activity. The typology developed captures the variability among farms because it considers several farm characteristics in the classification. The LCA demonstrated that technical parameters in need of improvement are related to the technical expertise of farm managers and workers and to the location of the farm.

     

Intermediate filaments of the vimentin-type and the cytokeratin-type are distributed differently during mitosis

Immunofluorescence microscopy has been used to follow the rearrangement of intermediate-sized filaments during mitosis in rat kangaroo PtK2 cells. These epithelial cells express two different intermediate filament systems: the keratin-related tonofilament-like arrays typical of epithelial cells, and the vimentin-type filaments characteristic of mesenchymal cells in vivo, and of many established cell lines. The two filament systems do not appear to depolymerize extensively during mitosis, but show differences in their organization and display which may indicate different functions. The most striking rearrangements have been seen with the vimentin filaments, and in particular in prometaphase a transient cage-like structure of vimentin fibers surrounding the developing spindle is formed. In metaphase, this cage disappears, and vimentin fibers are found in an elliptical band surrounding the chromosomes and the interzone. In telophase, these bands separate, usually breaking first on the side closest to where the cleavage furrow has started to form. Double label experiments with tubulin and vimentin antibodies have indicated that the microtubules and the chromosomes are contained within the thick crescents of vimentin filaments and suggest that the vimentin intermediate filaments may be involved in the orientation of the spindle and/or the chromosomes during mitosis. In contrast, extensive arrays of cytokeratin filaments are present throughout mitosis on the substrate-attached side of the cell and also in other cellular areas, although they are usually not present in the spindle region. Thus the cytokeratin filaments probably continue to play a cytoskeletal role during mitosis and may be responsible for the flat shape that certain epithelial cells such as PtK2 cells continue to maintain during mitosis.     

Effects of transforming growth factor-beta on normal clonal bone cell populations.

Although transforming growth factor-beta (TGF-beta) has been implicated in the local regulation of bone growth and remodelling, its specific effects on different subpopulations of bone cells have not been elucidated. Cells derived from bone are known to be heterogeneous and include both cells of different lineages and osteoblastic populations with different levels of expression of osteoblast-associated properties. Consequently, we have isolated clonal populations of bone cells to examine more precisely the effects of TGF-beta on individual subpopulations. Several clonal populations were isolated by limiting dilution from cells derived from 21-day-old fetal rat calvaria. Two of these clones, RCA 11 and RCB 2, were used here. While the two clones responded similarly to parathyroid hormone (PTH) and isoproterenol (ISP) with increases in intracellular cAMP, prostaglandin E2 (PGE2) elicited a 10-fold higher response in RCB 2 cells compared with RCA 11. RCB 2 cells expressed a 10-fold higher alkaline phosphatase activity compared with RCA 11. Both clones synthesized a variety of bone matrix associated proteins, but only RCA 11 synthesized SPP-1 (osteopontin) constitutively. TGF-beta stimulated growth of RCB 2 cells after 24 and 48 h of treatment, but had no effect on growth of RCA 11. TGF-beta supported anchorage-independent growth of RCB 2 cells, but not that of RCA 11. A 24-h exposure to TGF-beta decreased cAMP responsiveness to PTH and ISP slightly in both clones, but had no effect on PGE2 responses. Significant reductions in alkaline phosphatase activity were seen in both clones after 24- and 48-h treatments with TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal sequence of interleukin 1α—mediated stimulation and inhibition of bone formation by isolated fetal rat calvaria cells in vitro

Cytokines released at sites of inflammation and infection may alter normal bone remodeling processes resulting in pathologic bone destruction or bone formation. Interleukin 1, an inflammatory mediator, has been shown to stimulate as well as inhibit parameters associated with bone formation. In this study we have examined temporal aspects of the biphasic effects of recombinant interleukin 1 alpha (IL 1 alpha) on the differentiation of osteoprogenitor cells into bone-forming osteoblasts (bone nodules) in vitro. A dose-dependent stimulation of bone formation over a concentration range of 0.5 to 50 U/mL (1.4 x 10(-12) to 1.4 x 10(-10) M) was observed when preconfluent, primary cultures of fetal rat calvaria (RC) cells were pulsed with IL 1 alpha for 72 to 96 hr from the beginning of the culture period. This was correlated with a stimulation of cell proliferation and alkaline phosphatase activity measured during the late log phase of growth. In contrast, continuous exposure to IL 1 alpha or exposure to IL 1 alpha after confluency resulted in inhibition of bone nodule formation and alkaline phosphatase activity. IL 1 alpha-stimulated prostaglandin E2 (PGE2) production until the RC cells became multilayered, but the addition of the cyclooxygenase inhibitor indomethacin had no effect in reducing the IL 1 alpha-mediated stimulation of cell proliferation or bone nodule formation. However, in cultures continuously exposed to IL 1 alpha, added indomethacin partially reduced the inhibition of bone formation, suggesting that prostaglandin production may play a role in the inhibitory effects of IL 1 alpha on bone formation.     

The odontoblast process extends to the dentinoenamel junction: an immunocytochemical study of rat dentine

The length and extent of the odontoblast cell process in dentine has been the subject of controversy for many years. Here an immunofluorescence technique has been applied at the light microscope level to rat coronal dentine to localize the intracellular components actin and tubulin. Adult rats were perfused with periodate-lysine-paraformaldehyde fixative, teeth were extracted, the molar crowns were demineralized, dehydrated, wax embedded, and 6 micron sections were prepared. The sections were postfixed in -20 degrees C acetone and then incubated with affinity-purified rabbit anti-actin or anti-tubulin antibodies, followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. Intratubular immunofluorescence labeling for tubulin extended to the dentinoenamel junction, whereas labeling for actin, although extending to the dentinoenamel junction, was more prominent in the pulpal third of the rat dentine. Areas in which odontoblast processes are known not to occur, i.e., the atubular dentine, were not labeled by either antibody. The presence of actin- and tubulin-containing structures extending to the dentinoenamel junction is consistent with the hypothesis that the odontoblast process traverses the dentine for up to 3-4 mm, all the way to the dentinoenamel junction. Furthermore, the different staining patterns for actin-containing microfilaments as compared to tubulin-containing microtubules suggest that these two filamentous systems may have different roles in the function of the odontoblast process.     

Calorimetric study of water in muscle tissue

Differential scanning calorimetry has been used to investigate the state of water in intact single muscle fibers of the giant barnacle, Balanus nubilus. The shapes of the melting curves suggest the presence of three types of water: unfrozen (or bound), free (or bulk) and intermediate water. The amount of unfrozen water per g protein was constant within experimental error. An increase in water content changed almost exclusively the amounts of free water. The amount of intermediate water varies only slightly with the fiber water content.     

Survival of white-tailed deer fawns on Marine Corps Base Quantico

Some jurisdictions in the eastern United States have reduced harvest of white-tailed deer (Odocoileus virginianus) because of perceived declines in recruitment and population size over the last decade. Although the restoration of American black bears (Ursus americanus) and the colonization of coyotes (Canis latrans) have increased fawn predation in some areas, limited information exists on how temporally dynamic resources and weather influence fawn survival. Therefore, we evaluated fawn survival probability, cause specific mortality, and if factors such as oak (Quercus spp.) mast abundance, winter severity, precipitation, and landscape composition influenced mortality risk on Marine Corps Base Quantico in northern Virginia, USA, from 2008 to 2019. We tracked 248 fawns outfitted with very high frequency radio-collars and predation was the leading cause of mortality (n = 42; 45%). We estimated survival to 133 days and survival pooling all years (2008–2019) was 0.50 (95% CI = 0.42–0.60). Increased annual red oak (Quercus spp.) mast abundance from the previous fall reduced mortality hazard for fawns. The longevity of our study revealed a link between fawn survival and a specific maternal resource (red oak mast) only available during gestation. Our results highlight the importance of oak mast in eastern deciduous forests and, more broadly, overwinter maternal condition on white-tailed deer recruitment.