Skeletal Muscle - one year on

Background: Skeletal muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia – the age-related loss of skeletal muscle mass and major cause of frailty. One important cause for impaired regeneration is increased TGF-beta accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in ageing which led us hypothesize their potential contribution to impaired regeneration in sarcopenia. Thus, we have further analysed the muscle differentiation-inhibition pathway by pro-inflammatory cytokines in human skeletal muscle cells Methods: We studied the modulation of human skeletal muscle cell (HuSKMCs) differentiation by pro-inflammatory cytokines IL-1alpha and TNF-alpha. Grade of differentiation was determined by either imaging (fusion index) or creatine kinase (CK) activity, a marker of muscle differentiation. Secretion of TGF-beta proteins during differentiation was assessed by using a TGF-beta responsive reporter gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored both in HuSKMC cultures as well as samples from a rat sarcopenia study by Western Blots and RT-PCR. Results: The pro-inflammatory cytokines IL-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires the activation of TAK-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFkappaB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway – ActRII/ALK/SMAD. Inhibition of Activin A signaling is able to rescue human myoblasts treated with IL-1alpha or TNF-α, resulting in normal differentiation into myotubes. Studies in aged rats as a model of sarcopenia confirmed that this pro-inflammatory cytokine pathway identified is activated during aging. Conclusions: This study demonstrates an unexpected connection between cytokine and Activin signaling, demonstrating a new mechanism by which cytokines affect skeletal muscle, establishing the physiologic relevance of this pathway in sarcopenia

DNA Polymerase III Holoenzyme From Thermus Thermophilus Identification Expression Purification of Components and use to Reconstitute a Processive Replicase

DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the β sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of α (pol III catalytic subunit), β (sliding clamp processivity factor), and the essential DnaX (τ/γ), δ and δ′ components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including δ-δ′ interaction, δδ′-τ/γ complex formation, and α-τ interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 °C, both with regard to initiation complex formation and processive DNA synthesis. The minimalTth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 °C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event

Taurolidine reduces the tumor stimulating cytokine interleukin-1beta in patients with resectable gastrointestinal cancer: a multicentre prospective randomized trial

Background The effect of additional treatment strategies with antineoplastic agents on intraperitoneal tumor stimulating interleukin levels are unclear. Taurolidine and Povidone-iodine have been mainly used for abdominal lavage in Germany and Europe. Methods In the settings of a multicentre (three University Hospitals) prospective randomized controlled trial 120 patients were randomly allocated to receive either 0.5% taurolidine/2,500 IU heparin (TRD) or 0.25% povidone-iodine (control) intraperitoneally for resectable colorectal, gastric or pancreatic cancers. Due to the fact that IL-1beta (produced by macrophages) is preoperatively indifferent in various gastrointestinal cancer types our major outcome criterion was the perioperative (overall) level of IL-1beta in peritoneal fluid. Results Cytokine values were significantly lower after TRD lavage for IL-1beta, IL-6, and IL-10. Perioperative complications did not differ. The median follow-up was 50.0 months. The overall mortality rate (28 vs. 25, p = 0.36), the cancer-related death rate (17 vs. 19, p = .2), the local recurrence rate (7 vs. 12, p = .16), the distant metastasis rate (13 vs. 18, p = 0.2) as well as the time to relapse were not statistically significant different. Conclusion Reduced cytokine levels might explain a short term antitumorigenic intraperitoneal effect of TRD. But, this study analyzed different types of cancer. Therefore, we set up a multicentre randomized trial in patients undergoing curative colorectal cancer resection. Trial registration : ISRCTN6647853

The heterotrimeric Thermus thermophilus Asp-tRNAAsn amidotransferase can also generate Gln-tRNAGln

AbstractThermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNAAsn amidotransferase (Asp-AdT) capable of forming Asn-tRNAAsn [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln-tRNAGln as well as Asn-tRNAAsn. Thus, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated

Derivation of gravity wave intrinsic parameters and vertical wavelength using a single scanning OH(3-1) airglow spectrometer

For the first time, we present an approach to derive zonal, meridional, and vertical wavelengths as well as periods of gravity waves based on only one OH* spectrometer, addressing one vibrational-rotational transition. Knowledge of these parameters is a precondition for the calculation of further information, such as the wave group velocity vector. OH(3-1) spectrometer measurements allow the analysis of gravity wave ground-based periods but spatial information cannot necessarily be deduced. We use a scanning spectrometer and harmonic analysis to derive horizontal wavelengths at the mesopause altitude above Oberpfaffenhofen (48.09∘ N, 11.28∘ E), Germany for 22 nights in 2015. Based on the approximation of the dispersion relation for gravity waves of low and medium frequencies and additional horizontal wind information, we calculate vertical wavelengths. The mesopause wind measurements nearest to Oberpfaffenhofen are conducted at Collm (51.30∘ N, 13.02∘ E), Germany, ca. 380 km northeast of Oberpfaffenhofen, by a meteor radar. In order to compare our results, vertical temperature profiles of TIMED-SABER (thermosphere ionosphere mesosphere energetics dynamics, sounding of the atmosphere using broadband emission radiometry) overpasses are analysed with respect to the dominating vertical wavelength

Microbe sampling by mucosal dendritic cells is a discrete, MyD88-independent stepin ΔinvG S. Typhimurium colitis

Intestinal dendritic cells (DCs) are believed to sample and present commensal bacteria to the gut-associated immune system to maintain immune homeostasis. How antigen sampling pathways handle intestinal pathogens remains elusive. We present a murine colitogenic Salmonella infection model that is highly dependent on DCs. Conditional DC depletion experiments revealed that intestinal virulence of S. Typhimurium SL1344 ΔinvG mutant lacking a functional type 3 secretion system-1 (ΔinvG)critically required DCs for invasion across the epithelium. The DC-dependency was limited to the early phase of infection when bacteria colocalized with CD11c+CX3CR1+ mucosal DCs. At later stages, the bacteria became associated with other (CD11c−CX3CR1−) lamina propria cells, DC depletion no longer attenuated the pathology, and a MyD88-dependent mucosal inflammation was initiated. Using bone marrow chimeric mice, we showed that the MyD88 signaling within hematopoietic cells, which are distinct from DCs, was required and sufficient for induction of the colitis. Moreover, MyD88-deficient DCs supported transepithelial uptake of the bacteria and the induction of MyD88-dependent colitis. These results establish that pathogen sampling by DCs is a discrete, and MyD88-independent, step during the initiation of a mucosal innate immune response to bacterial infection in vivo

Loss of oxidative defense and potential blockade of satellite cell maturation in the skeletal muscle of patients with cancer but not in the healthy elderly

Purpose: Muscle wasting in old age or cancer may result from failure of myofibre regeneration and /or accelerated apoptosis both of which may be up-regulated by oxidative stress or inflammation. The aim of this study was to determine from the transcriptome in human skeletal muscle whether there is evidence for oxidative stress and its relationship with satellite cell differentiation or apoptosis in the muscle of patients with cancer (weight-stable: CWS or weight-losing: CWL) or healthy elderly (HE) when compared with healthy middle aged controls (HMAC) . Design: 28 patients with resectable upper GI/pancreatic cancer (CWS: 14 and CWL14), 17 HE and 22 HMAC underwent biopsy of the quadriceps muscle. Markers of muscle regeneration, inflammation, oxidative stress and apoptosis were measured by qPCR. Results: The expression of transcription factors responsible for muscle regeneration (Pax3, Pax7 and MyoD) were increased in the skeletal muscle of CWS and HE when compared with HMAC (P<0.001). In contrast, the expression of myogenic differentiation markers (MyoG and Myh2) was reduced in CWS and CWL but increased in HE when compared with HMAC (P<0.0001). The expression of the pro-apoptotic gene Bax was significantly increased in CWS, CWL and HE compared with HMAC (P<0.0001). Pro-inflammatory cytokine expression was variable with increased expression of TNF in CWS and HE, increased Il-6 in CWS and increased Il-1 in CWL when compared with HMAC. Expression of the oxidative defense genes SOD2, GCLM, and NRF2 was decreased in CWS and CWL but increased in HE when compared with HMA (P<0.0001). Conclusion: There is evidence for blockade of satellite cell maturation, upregulation of apoptosis and reduced oxidative defense in the skeletal muscle of cancer patients. In contrast, in muscle from healthy elderly the potential for myotube differentiation and oxidative defense is maintained

Isotope Shift in the Dielectronic Recombination of Three-electron \u3csup\u3eA\u3c/sup\u3eNd⁵⁷⁺

Isotope shifts in dielectronic recombination spectra were studied for Li-like ANd57+ ions with A = 142 and A = 150. From the displacement of resonance positions energy shifts δE142 150(2s-2p1/2) = 40.2(3)(6) meV [(stat)(sys)] and δE142 150(2s - 2p3/2) = 42.3(12)(20)meV of 2s - 2pj transitions were deduced. An evaluation of these values within a full QED treatment yields a change in the mean-square charge radius of 142 150δ⟨ r2⟩ = -1.36(1)(3) fm2. The approach is conceptually new and combines the advantage of a simple atomic structure with high sensitivity to nuclear size

Clinical classification of cancer cachexia:phenotypic correlates in human skeletal muscle

Aim – To relate muscle phenotype to a range of current diagnostic criteria for cancer cachexia Methods – 41 patients with resectable upper gastrointestinal (GI) or pancreatic cancer underwent characterisation for cachexia based on weight-loss (WL) and / or low muscularity (LM). Four diagnostic criteria were used >5%WL, >10% WL, LM, and LM + >2%WL. Patients underwent biopsy of the rectus muscle. Analysis included immunohistochemistry for fibre size and type, protein and nucleic acid concentration, and Western blots for markers of autophagy, SMAD signalling, and inflammation. Results – Compared with non-cachectic cancer patients, if patients were classified by LM or LM + >2%WL, mean muscle fibre diameter was significantly reduced (p = 0.02 and p = 0.001) repectively. No difference in fibre diameter was observed if patients were classified with WL alone. Regardless of classification, there was no difference in fibre number or proportion of fibre type across all myosin heavy chain isoforms. Mean muscle protein content was reduced and the ratio of RNA/DNA decreased if patients were classified by either >5% WL or LM + >2%WL. Compared with non-cachectic patients, when patients were classified according to >5% WL, SMAD3 protein levels were increased (p=0.022) and with >10% WL, beclin (p = 0.05) and ATG5 (p = 0.01) protein levels were also increased. There were no differences in pNFkB or pSTAT3 levels across any of the groups. Conclusions – Whereas fibre type is not targeted selectively, muscle fibre size, biochemical composition and pathway phenotype can vary according to whether the criteria for cachexia include both a measure of low muscularity and weight loss