Rapid fibroblast growth factor-induced increases in protein phosphorylation and ornithine decarboxylase activity: regulation by heparin and comparison to nerve growth factor-induced increases.

Fibroblast growth factors (FGFs), like nerve growth factor (NGF), induce morphological differentiation of PC12 cells. This activity of FGF is regulated by glycosaminoglycans. To further understand the mechanisms of FGF and glycosaminoglycan actions in PC12 cells, we studied the regulation of protein phosphorylation and ornithine decarboxylase (ODC) activity by FGF in the presence and absence of heparin. As with NGF, aFGF and bFGF increased the incorporation of radioactive phosphate into the protein tyrosine hydroxylase (TH). The increase in TH phosphorylation was localized to the tryptic peptide, T3. Both T3 and T1 phosphorylations occur in response to NGF, but there was no evidence that aFGF or bFGF stimulated the phosphorylation of the T1 peptide. This result suggests differential regulation of second messenger systems by NGF and FGF in PC12 cells. Heparin, at a concentration that potentiated aFGF-induced neurite outgrowth 100-fold (100 micrograms/ml), did not alter the ability of aFGF to increase S6 phosphorylation or ODC activity. One milligram per milliliter of heparin, a concentration that inhibited bFGF-induced neurite outgrowth, also inhibited bFGF-induced increases in S6 phosphorylation and ODC activity. These observations suggest (i) that acidic and basic FGF activate a protein kinase, possibly protein kinase C, resulting in the phosphorylation of peptide T3 of TH; (ii) that the FGFs and NGF share some but not all second messenger systems; (iii) that heparin potentiates aFGF actions and inhibits bFGF actions in PC12 cells via distinct mechanisms; (iv) that heparin does not potentiate the neurite outgrowth promoting activity of aFGF by enhancing binding to its PC12 cell surface receptor; and (v) that heparin may coordinately regulate several activities of bFGF (induction of protein phosphorylation, ODC and neurite outgrowth) via a common mechanism, most likely by inhibiting the productive binding of bFGF to its PC12 cell surface receptor.     

Distribution of microtubules in prolactin cells of lactating rats

The intracellular distribution of microtubules was studied using serial sections of prolactin cells in anterior pituitary glands from lactating rats. Numerous microtubules were present in these cells following fixation with glutaraldehyde and osmium tetroxide. The greatest number of microtubules were present in the Golgi complex, situated around the perimeter and in association with the cisternae, vesicles and developing secretory granules. Microtubules were found in channels between groups of parallel cisternae of rough surfaced endoplasmic reticulum and in close proximity to small vesicles. They were also located adjacent to mitochondria, the plasmalemma, the nuclear envelope, and among mature secretory granules. Due to their orientation within the cell, it is suggested that the microtubules may act to direct the movement of organelles from one region of the cell to another and to give internal support to the cell.     

Operation Crayweed: Ecological and sociocultural aspects of restoring Sydney’s underwater forests

Operation Crayweed focuses on the restoration of underwater forests that disappeared from the coastline of Sydney, Australia’s largest city, 40 years previously. We show how a combination of science, hands‐on restoration, community engagement and art has helped the project to reach its goals as well as raise awareness about the importance of underwater kelp forests that are experiencing global decline.     

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD’s localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of I_Na by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.     

The mitochondria targeted antioxidant MitoQ protects against fluoroquinolone-induced oxidative stress and mitochondrial membrane damage in human Achilles tendon cells

Tendinitis and tendon rupture during treatment with fluoroquinolone antibiotics is thought to be mediated via oxidative stress. This study investigated whether ciprofloxacin and moxifloxacin cause oxidative stress and mitochondrial damage in cultured normal human Achilles’ tendon cells and whether an antioxidant targeted to mitochondria (MitoQ) would protect against such damage better than a non-mitochondria targeted antioxidant. Human tendon cells from normal Achilles’ tendons were exposed to 0–0.3 mm antibiotic for 24 h and 7 days in the presence of 1 µm MitoQ or an untargeted form, idebenone. Both moxifloxacin and ciprofloxacin resulted in up to a 3-fold increase in the rate of oxidation of dichlorodihydrofluorescein, a marker of general oxidative stress in tenocytes (p<0.0001) and loss of mitochondrial membrane permeability (p<0.001). In cells treated with MitoQ the oxidative stress was less and mitochondrial membrane potential was maintained. Mitochondrial damage to tenocytes during fluoroquinolone treatment may be involved in tendinitis and tendon rupture.     

Effects of seasonal snow on the growing season of temperate vegetation in China

Variations in seasonal snowfall regulate regional and global climatic systems and vegetation growth by changing energy budgets of the lower atmosphere and land surface. We investigated the effects of snow on the start of growing season (SGS) of temperate vegetation in China. Across the entire temperate region in China, the winter snow depth increased at a rate of 0.15 cm yr^?1 (P = 0.07) during the period 1982–1998, and decreased at a rate of 0.36 cm yr^?1 (P = 0.09) during the period 1998–2005. Correspondingly, the SGS advanced at a rate of 0.68 day yr^?1 (P < 0.01) during 1982–1998, and delayed at a rate of 2.13 day yr^?1 (P = 0.07) during 1998–2005, against a warming trend throughout the entire study period of 1982–2005. Spring air temperature strongly regulated the SGS of both deciduous broad‐leaf and coniferous forests, whereas the winter snow had a greater impact on the SGS of grassland and shrubs. Snow depth variation combined with air temperature contributed to the variability in the SGS of grassland and shrubs, as snow acted as an insulator and modulated the underground thermal conditions. In addition, differences were seen between the impacts of winter snow depth and spring snow depth on the SGS; as snow depths increased, the effect associated went from delaying SGS to advancing SGS. The observed thresholds for these effects were snow depths of 6.8 cm (winter) and 4.0 cm (spring). The results of this study suggest that the response of the vegetation's SGS to seasonal snow change may be attributed to the coupling effects of air temperature and snow depth associated with the underground thermal conditions.     

Monocytes Loaded with Indocyanine Green as Active Homing Contrast Agents?Permit Optical Differentiation of Infectious and Non-Infectious Inflammation

Distinguishing cutaneous infection from sterile inflammation is a diagnostic challenge and currently relies upon subjective interpretation of clinical parameters, microbiological data, and nonspecific imaging. Assessing characteristic variations in leukocytic infiltration may provide more specific information. In this study, we demonstrate that homing of systemically administered monocytes tagged using indocyanine green (ICG), an FDA-approved near infrared dye, may be assessed non-invasively using clinically-applicable laser angiography systems to investigate cutaneous inflammatory processes. RAW 264.7 mouse monocytes co-incubated with ICG fluoresce brightly in the near infrared range. In vitro, the loaded cells retained the ability to chemotax toward monocyte chemotactic protein-1. Following intravascular injection of loaded cells into BALB/c mice with induced sterile inflammation (Complete Freund’s Adjuvant inoculation) or infection (Group A Streptococcus inoculation) of the hind limb, non-invasive whole animal imaging revealed local fluorescence at the inoculation site. There was significantly higher fluorescence of the inoculation site in the infection model than in the inflammation model as early as 2 hours after injection (p<0.05). Microscopic examination of bacterial inoculation site tissue revealed points of near infrared fluorescence, suggesting the presence of ICG-loaded cells. Development of a non-invasive technique to rapidly image inflammatory states without radiation may lead to new tools to distinguish infectious conditions from sterile inflammatory conditions at the bedside.     

DNA Barcode Identification of Podocarpaceae—The Second Largest Conifer Family

We have generated matK, rbcL, and nrITS2 DNA barcodes for 320 specimens representing all 18 extant genera of the conifer family Podocarpaceae. The sample includes 145 of the 198 recognized species. Comparative analyses of sequence quality and species discrimination were conducted on the 159 individuals from which all three markers were recovered (representing 15 genera and 97 species). The vast majority of sequences were of high quality (B ₃₀ = 0.596–0.989). Even the lowest quality sequences exceeded the minimum requirements of the BARCODE data standard. In the few instances that low quality sequences were generated, the responsible mechanism could not be discerned. There were no statistically significant differences in the discriminatory power of markers or marker combinations (p = 0.05). The discriminatory power of the barcode markers individually and in combination is low (56.7% of species at maximum). In some instances, species discrimination failed in spite of ostensibly useful variation being present (genotypes were shared among species), but in many cases there was simply an absence of sequence variation. Barcode gaps (maximum intraspecific p–distance > minimum interspecific p–distance) were observed in 50.5% of species when all three markers were considered simultaneously. The presence of a barcode gap was not predictive of discrimination success (p = 0.02) and there was no statistically significant difference in the frequency of barcode gaps among markers (p = 0.05). In addition, there was no correlation between number of individuals sampled per species and the presence of a barcode gap (p = 0.27).     

Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial–mesenchymal transition

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague–Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial–mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.     

Transmissibility of the Monkeypox Virus Clades via Respiratory Transmission: Investigation Using the Prairie Dog-Monkeypox Virus Challenge System

Monkeypox virus (MPXV) is endemic within Africa where it sporadically is reported to cause outbreaks of human disease. In 2003, an outbreak of human MPXV occurred in the US after the importation of infected African rodents. Since the eradication of smallpox (caused by an orthopoxvirus (OPXV) related to MPXV) and cessation of routine smallpox vaccination (with the live OPXV vaccinia), there is an increasing population of people susceptible to OPXV diseases. Previous studies have shown that the prairie dog MPXV model is a functional animal model for the study of systemic human OPXV illness. Studies with this model have demonstrated that infected animals are able to transmit the virus to naive animals through multiple routes of exposure causing subsequent infection, but were not able to prove that infected animals could transmit the virus exclusively via the respiratory route. Herein we used the model system to evaluate the hypothesis that the Congo Basin clade of MPXV is more easily transmitted, via respiratory route, than the West African clade. Using a small number of test animals, we show that transmission of viruses from each of the MPXV clade was minimal via respiratory transmission. However, transmissibility of the Congo Basin clade was slightly greater than West African MXPV clade (16.7% and 0% respectively). Based on these findings, respiratory transmission appears to be less efficient than those of previous studies assessing contact as a mechanism of transmission within the prairie dog MPXV animal model.