N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca²⁺ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His₁₀) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.     

Response of foundation macrophytes to near‐natural simulated marine heatwaves

Marine heatwaves have been observed worldwide and are expected to increase in both frequency and intensity due to climate change. Such events may cause ecosystem reconfigurations arising from species range contraction or redistribution, with ecological, economic and social implications. Macrophytes such as the brown seaweed Fucus vesiculosus and the seagrass Zostera marina are foundation species in many coastal ecosystems of the temperate northern hemisphere. Hence, their response to extreme events can potentially determine the fate of associated ecosystems. Macrophyte functioning is intimately linked to the maintenance of photosynthesis, growth and reproduction, and resistance against pathogens, epibionts and grazers. We investigated morphological, physiological, pathological and chemical defence responses of western Baltic Sea F. vesiculosus and Z. marina populations to simulated near‐natural marine heatwaves. Along with (a) the control, which constituted no heatwave but natural stochastic temperature variability (0HW), two treatments were applied: (b) two late‐spring heatwaves (June, July) followed by a summer heatwave (August; 3HW) and (c) a summer heatwave only (1HW). The 3HW treatment was applied to test whether preconditioning events can modulate the potential sensitivity to the summer heatwave. Despite the variety of responses measured in both species, only Z. marina growth was impaired by the accumulative heat stress imposed by the 3HW treatment. Photosynthetic rate, however, remained high after the last heatwave indicating potential for recovery. Only epibacterial abundance was significantly affected in F. vesiculosus. Hence both macrophytes, and in particular F. vesiculosus, seem to be fairly tolerant to short‐term marine heatwaves at least at the intensities applied in this experiment (up to 5°C above mean temperature over a period of 9 days). This may partly be due to the fact that F. vesiculosus grows in a highly variable environment, and may have a high phenotypic plasticity.     

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.     

An unusual form of phase walk in a system of coupled oscillators

The authors describe an unusual form of phase walk (i.e., a progressive change in phase angle between coupled oscillators) using the 10-Hz rhythmic discharges of the inferior cardiac and vertebral postganglionic sympathetic nerves (CN and VN, respectively) in hypercapnic, baroreceptor-denervated, and vagotomized cats anesthetized with urethane. Unlike phase walk ascribable to weakened coupling (desynchronization of oscillators), the phase walk of VN 10- Hz activity relative to CN10-Hz activity 1) recurred on the time scale of the respiratory cycle, 2) was bidirectional with CN-VN phase angle increasing during expiration and decreasing during inspiration, and 3) occurred over a range equivalent to one-half the period of the 10-Hz rhythm rather than a full cycle. Moreover, this form of phase walk occurred during strong coupling of the 10-Hz oscillators, as reflected by CN-VN coherence values approaching 1.0. The authors propose that the bidirectional phase walk reflects a state of strong coupling of the 10-Hz oscillators controlling the CN and VN, the angle of which is reset from cycle to cycle by the continuously changing level of activity in their respiratory inputs. In addition, the data demonstrate that frequency and amplitude modulation of sympathetic nerve discharge can be independently regulated by respiratory inputs.     

Inhibition of myostatin in adult mice increases skeletal muscle mass and strength

A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.     

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.     

Geographic variation in fitness‐related traits of the bladderwrack Fucus vesiculosus along the Baltic Sea‐North Sea salinity gradient

In the course of the ongoing global intensification and diversification of human pressures, the study of variation patterns of biological traits along environmental gradients can provide relevant information on the performance of species under shifting conditions. The pronounced salinity gradient, co‐occurrence of multiple stressors, and accelerated rates of change make the Baltic Sea and its transition to North Sea a suitable region for this type of study. Focusing on the bladderwrack Fucus vesiculosus, one of the main foundation species on hard‐bottoms of the Baltic Sea, we analyzed the phenotypic variation among populations occurring along 2,000 km of coasts subjected to salinities from 4 to >30 and a variety of other stressors. Morphological and biochemical traits, including palatability for grazers, were recorded at 20 stations along the Baltic Sea and four stations in the North Sea. We evaluated in a common modeling framework the relative contribution of multiple environmental drivers to the observed trait patterns. Salinity was the main and, in some cases, the only environmental driver of the geographic trait variation in F. vesiculosus. The decrease in salinity from North Sea to Baltic Sea stations was accompanied by a decline in thallus size, photosynthetic pigments, and energy storage compounds, and affected the interaction of the alga with herbivores and epibiota. For some traits, drivers that vary locally such as wave exposure, light availability or nutrient enrichment were also important. The strong genetic population structure in this macroalgae might play a role in the generation and maintenance of phenotypic patterns across geographic scales. In light of our results, the desalination process projected for the Baltic Sea could have detrimental impacts on F. vesiculosus in areas close to its tolerance limit, affecting ecosystem functions such as habitat formation, primary production, and food supply.     

Association between Common Allergic Symptoms and Cancer in the NHANES III Female Cohort

Background

Previous epidemiological studies have investigated the association between allergic symptoms and cancer occurrence. However, the role of allergy in cancer has been elusive, especially for the female population.

Methods

We examined the relationship between cancer prevalence and common allergic symptoms of rhinoconjunctivitis (RC) and wheezing (WZ) among NHANES III female participants.

Results

Among 4600 people, 36.3% (n = 1669) did not have any allergic symptoms (NO), while 47.6% (n = 2188) reported RC, and 16.2% (n = 743), WZ. The proportion of cancer among NO groups was 5.43% (91/1669), among RC group, 7.63% (167/2188), and among WZ group, 11.23% (83/743) (RC group- OR 1.44 with 95% CI 1.00–2.08; p = 0.05 while for WZ group- OR 2.20 with 95%CI 1.27–3.80; p = 0.01). After adjusting for all the possible confounding variables including age, smoking, or COPD, having symptoms of RC (AOR 1.49 with 95%CI 1.12–2.36; p = 0.01) or WC (AOR 2.08 with 95%CI 1.11–3.89; p = 0.02) demonstrated consistent strong association with cancer. Among nonsmokers (n = 2505, 54.5%) only symptoms of RC showed association with cancer (AOR 1.51 with 95%CI 1.00–2.28; p = 0.05). Among former or current smokers (n = 2094, 45.5%), only symptoms of WZ demonstrated association with cancer (AOR 2.38 with 95%CI 1.16–4.87; p = 0.02). Among different types of cancers, odds of having breast cancer among participants with symptoms of RC or WZ were approximately twice the odds of having breast cancer among participants without any of these symptoms. AOR for RC group was 1.89 with 95%CI 1.04–3.42 and p = 0.04 while AOR for WC group was 2.08 with 95%CI 0.90–4.78 and p = 0.08.

Conclusions

In summary, we found associations between common allergic symptoms like rhinitis/conjunctivitis and wheezing and prevalence of cancer, specifically between rhinitis/conjunctivitis and breast cancer that were not found in previous studies. Larger prospective studies are required to validate our findings.     

Heat waves and their significance for a temperate benthic community: A near‐natural experimental approach

Climate change will not only shift environmental means but will also increase the intensity of extreme events, exerting additional stress on ecosystems. While field observations on the ecological consequences of heat waves are emerging, experimental evidence is rare, and lacking at the community level. Using a novel “near‐natural” outdoor mesocosms approach, this study tested whether marine summer heat waves have detrimental consequences for macrofauna of a temperate coastal community, and whether sequential heat waves provoke an increase or decrease of sensitivity to thermal stress. Three treatments were applied, defined and characterized through a statistical analysis of 15 years of temperature records from the experimental site: (1) no heat wave, (2) two heat waves in June and July followed by a summer heat wave in August and (3) the summer heat wave only. Overall, 50% of the species showed positive, negative or positive/negative responses in either abundance and/or biomass. We highlight four possible ways in which single species responded to either three subsequent heat waves or one summer heat wave: (1) absence of a response (tolerance, 50% of species), (2) negative accumulative effects by three subsequent heat waves (tellinid bivalve), (3) buffering by proceeding heat waves due to acclimation and/or shifts in phenology (spionid polychaete) and (4) an accumulative positive effect by subsequent heat waves (amphipod). The differential responses to single or sequential heat waves at the species level entailed shifts at the community level. Community‐level differences between single and triple heat waves were more pronounced than those between regimes with vs. without heat waves. Detritivory was reduced by the single heat wave while suspension feeding was less common in the triple heat wave regime. Critical extreme events occur already today and will occur more frequently in a changing climate, thus, leading to detrimental impacts on coastal marine systems.     

Discovery of 7-azaindole based anaplastic lymphoma kinase (ALK) inhibitors: Wild type and mutant (L1196M) active compounds with unique binding mode

We have identified a novel 7-azaindole series of anaplastic lymphoma kinase (ALK) inhibitors. Compounds 7b, 7m and 7n demonstrate excellent potencies in biochemical and cellular assays. X-ray crystal structure of one of the compounds (7k) revealed a unique binding mode with the benzyl group occupying the back pocket, explaining its potency towards ALK and selectivity over tested kinases particularly Aurora-A. This binding mode is in contrast to that of known ALK inhibitors such as Crizotinib and NVP-TAE684 which occupy the ribose binding pocket, close to DFG motif.