Combined whole-body vibration, resistance exercise, and sustained vascular occlusion increases PGC-1α and VEGF mRNA abundances

We previously reported that high load resistance exercise with superimposed whole-body vibration and sustained vascular occlusion (vibroX) markedly improves cycling endurance capacity, increases capillary-to-fibre ratio and skeletal muscle oxidative enzyme activity in untrained young women. These findings are intriguing, since increases in oxidative muscle phenotype and endurance capacity are typically induced by endurance but not heavy resistance exercise. Here, we tested the hypothesis that vibroX activates genes associated with mitochondrial biogenesis and angiogenesis. Eight healthy, recreationally resistance-trained young men performed either vibroX or resistance exercise (RES) in a randomised, cross-over design. Needle biopsies (M. vastus lateralis) were obtained at rest and 3h post-exercise. Changes in relative gene expression levels were assessed by real-time quantitative PCR. After vibroX, vascular endothelial growth factor and peroxisome proliferator-activated receptor-γ coactivator 1α mRNA abundances increased to 2- and 4.4-fold, respectively, but did not significantly change above resting values after RES. Other genes involved in mitochondrial biogenesis were not affected by either exercise modality. While vibroX increased the expression of hexokinase II, xanthine dehydrogenase, and manganese superoxide dismutase mRNA, there were no changes in these transcripts after RES. This study demonstrates that high load resistance exercise with superimposed whole-body vibration and sustained vascular occlusion activates metabolic and angiogenic gene programs, which are usually activated after endurance but not resistance exercise. Thus, targeted modification of high load resistance exercise by vibration and vascular occlusion might represent a novel strategy to induce endurance-type muscle adaptation

Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 angstrom resolution. The hexagonal-shaped crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 angstrom, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

No Evidence for a Boost in Psychosocial Functioning in Older Age After a 6-Months Physical Exercise Intervention

The beneficial effects of physical exercise on physical health and cognitive functioning have been repeatedly shown. However, evidence of its effect on psychosocial functioning in healthy adults is still scarce or inconclusive. One limitation of many studies examining this link is their reliance on correlational approaches or specific subpopulations, such as clinical populations. The present study investigated the effects of a physical exercise intervention on key factors of psychosocial functioning, specifically well-being, stress, loneliness, and future time perspective. We used data from healthy, previously sedentary older adults (N = 132) who participated in a 6-month at-home intervention, either engaging in aerobic exercise or as part of a control group who participated in foreign language-learning or reading of selected native-language literature. Before and after the intervention, comprehensive cardiovascular pulmonary testing and a psychosocial questionnaire were administered. The exercise group showed significantly increased fitness compared to the control group. Contrary to expectations, however, we did not find evidence for a beneficial effect of this fitness improvement on any of the four domains of psychosocial functioning we assessed. This may be due to pronounced stability of such psychological traits in older age, especially in older adults who show high levels of well-being initially. Alternatively, it may be that the well-documented beneficial effects of physical exercise on brain structure and function, as well as cognition differ markedly from beneficial effects on psychosocial functioning. While aerobic exercise may be the driving factor for the former, positive effects on the latter may only be invoked by other aspects of exercise, for example, experiences of mastery or a feeling of community.Peer Reviewe

DOTA-NOC, a high-affinity ligand of somatostatin receptor subtypes 2, 3 and 5 for labelling with various radiometals

Earlier studies have shown that modification of the octapeptide octreotide in positions 3 and 8 may result in compounds with increased somatostatin receptor affinity that, if radiolabelled, display improved uptake in somatostatin receptor-positive tumours. The aim of a recent research study in our laboratory was to employ the parallel peptide synthesis approach by further exchanging the amino acid in position 3 of octreotide and coupling the macrocyclic chelator DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to these peptides for labelling with radiometals like gallium-67 or -68, indium-111, yttrium-90 and lutetium-177. The purpose was to find radiopeptides with an improved somatostatin receptor binding profile in order to extend the spectrum of targeted tumours. A first peptide, [111In,90Y-DOTA]-1-Nal3-octreotide (111In,90Y-DOTA-NOC), was isolated which showed an improved profile. InIII-DOTA-NOC exhibited the following IC50 values (nM) when studied in competition with [125I][Leu8, d-Trp22, Tyr25]somatostatin-28 (values for YIII-DOTA-NOC are shown in parentheses): sstr2, 2.9±0.1 (3.3±0.2); sstr3, 8±2 (26±1.9); sstr5, 11.2±3.5 (10.4±1.6). Affinity towards sstr1 and 4 was very low or absent. InIII-DOTA-NOC is superior to all somatostatin-based radiopeptides having this particular type of binding profile, including DOTA-lanreotide, and has three to four times higher binding affinity to sstr2 than InIII,YIII-DOTA-Tyr3-octreotide (InIII,YIII-DOTA-TOC). In addition, [111In]DOTA-NOC showed a specific and high rate of internalization into AR4-2J rat pancreatic tumour cells which, after 4h, was about two times higher than that of [111In]DOTA-TOC and three times higher than that of [111In]DOTA-octreotide ([111In]DOTA-OC). The internalized radiopeptides were externalized intact upon 2h of internalization followed by an acid wash. After 2-3h of externalization a plateau is reached, indicating a steady-state situation explained by reactivation of the receptors followed by re-endocytosis. Biodistribution studies in CA 20948 tumour-bearing rats showed rapid clearance from all sstr-negative tissues except the kidneys. At 4h the uptake of [111In]DOTA-NOC in the tumour and sstr-positive tissues, such as adrenals, stomach and pancreas, was three to four times higher than that of [111In]DOTA-TOC. Differential blocking studies indicate that this is at least partially due to the uptake mediated by sstr3 and sstr5. These very promising preclinical data justify the use of this new radiopeptide for imaging and potentially internal radiotherapy studies in patient

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1-/-, or Sphk2-/- mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2-/- renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2-/- cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease. Keywords: HIF-2α, erythropoietin; Sphk1; Sphk2; anemia; renal fibroblasts; sphingosine; sphingosine 1-phosphate

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells.

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1-/-, or Sphk2-/- mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2-/- renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2-/- cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease

Combined whole-body vibration, resistance exercise, and sustained vascular occlusion increases PGC-1α and VEGF mRNA abundances

We previously reported that high load resistance exercise with superimposed whole-body vibration and sustained vascular occlusion (vibroX) markedly improves cycling endurance capacity, increases capillary-to-fibre ratio and skeletal muscle oxidative enzyme activity in untrained young women. These findings are intriguing, since increases in oxidative muscle phenotype and endurance capacity are typically induced by endurance but not heavy resistance exercise. Here, we tested the hypothesis that vibroX activates genes associated with mitochondrial biogenesis and angiogenesis. Eight healthy, recreationally resistance-trained young men performed either vibroX or resistance exercise (RES) in a randomised, cross-over design. Needle biopsies (M. vastus lateralis) were obtained at rest and 3 h post-exercise. Changes in relative gene expression levels were assessed by real-time quantitative PCR. After vibroX, vascular endothelial growth factor and peroxisome proliferator-activated receptor-γ coactivator 1α mRNA abundances increased to 2- and 4.4-fold, respectively, but did not significantly change above resting values after RES. Other genes involved in mitochondrial biogenesis were not affected by either exercise modality. While vibroX increased the expression of hexokinase II, xanthine dehydrogenase, and manganese superoxide dismutase mRNA, there were no changes in these transcripts after RES. This study demonstrates that high load resistance exercise with superimposed whole-body vibration and sustained vascular occlusion activates metabolic and angiogenic gene programs, which are usually activated after endurance but not resistance exercise. Thus, targeted modification of high load resistance exercise by vibration and vascular occlusion might represent a novel strategy to induce endurance-type muscle adaptations

Evaluation of butorphanol, medetomidine and midazolam as a reversible narcotic combination in free-ranging African lions (Panthera leo)

Objective To evaluate the effects of the combination butorphanol, medetomidine and midazolam (BMM) and its reversibility in lions. Study design Prospective clinical trial. Animals Thirty free-ranging lions, 10 male and 20 female, weighing 81–210 kg. Methods Lions were immobilised with butorphanol mean 0.31 ± SD 0.034 mg kg-1, medetomidine 0.052 ± 0.006 mg kg-1, midazolam 0.21 ± 0.024 mg kg-1 and hyaluronidase 1250 IU administered intramuscularly with a dart gun. Upon recumbency, physiological parameters and anaesthetic depth were monitored 10–15 minutes after darting (T1) and repeated every 10 minutes for a further 30 minutes (T2, T3, T4). Arterial blood gas analyses were performed at T1 and T4. At the end of the procedure, 45–60 minutes after initial darting, immobilisation was reversed with naltrexone 0.68 ± 0.082 mg kg-1, atipamezole 0.26 ± 0.031 mg kg-1, and flumazenil 0.0032 ± 0.0007 mg kg-1 administered intravenously and subcutaneously. Results The BMM combination rapidly induced immobilisation and lateral recumbency was reached within 7.25 ± 2.3 minutes. Median induction score [scored 1 (excellent) to 4 (poor)] was 1.4 (range 1–2). Cardio-respiratory parameters were stable. Heart rate varied from 32 to 72 beats per minute, respiratory rate from 14 to 32 breaths minute-1 and rectal temperature from 36.6 to 40.3 C. No sudden arousals were observed. Arterial blood gas analyses revealed a mean pH of 7.33, PaCO2 of 33 mmHg and PaO2 of 87 mmHg. Mild to moderate hypoxemia was seen in four lions. Recovery was smooth and lions were walking within 4.4 ± 4.25 minutes. Median recovery score [scored 1 (excellent) to 4 (poor)] was 1.3 (range 1–2). Conclusion and clinical relevance The drug combination proved to be effective in immobilising freeranging healthy lions of both sexes with minimal cardio-respiratory changes

Viral vector-mediated reprogramming of the fibroblastic tumor stroma sustains curative melanoma treatment.

The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas. Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells

Adaptation Strategies for Personalized Gait Neuroprosthetics

Personalization of gait neuroprosthetics is paramount to ensure their efficacy for users, who experience severe limitations in mobility without an assistive device. Our goal is to develop assistive devices that collaborate with and are tailored to their users, while allowing them to use as much of their existing capabilities as possible. Currently, personalization of devices is challenging, and technological advances are required to achieve this goal. Therefore, this paper presents an overview of challenges and research directions regarding an interface with the peripheral nervous system, an interface with the central nervous system, and the requirements of interface computing architectures. The interface should be modular and adaptable, such that it can provide assistance where it is needed. Novel data processing technology should be developed to allow for real-time processing while accounting for signal variations in the human. Personalized biomechanical models and simulation techniques should be developed to predict assisted walking motions and interactions between the user and the device. Furthermore, the advantages of interfacing with both the brain and the spinal cord or the periphery should be further explored. Technological advances of interface computing architecture should focus on learning on the chip to achieve further personalization. Furthermore, energy consumption should be low to allow for longer use of the neuroprosthesis. In-memory processing combined with resistive random access memory is a promising technology for both. This paper discusses the aforementioned aspects to highlight new directions for future research in gait neuroprosthetics.Peer ReviewedPostprint (published version