Objective-An estimated 200 million patients worldwide have surgery each year. Anesthesia and surgery have been reported to facilitate emergence of Alzheimer's disease (AD). The commonly used inhalation anesthetic isoflurane has previously been reported to induce apoptosis and to increase levels and aggregation of AD-associated amyloid β-protein (Aβ) in cultured cells. However, the in vivo relevance has not been addressed.Methods-We therefore set out to determine effects of isoflurane on caspase activation, levels of BACE and Aβ in naïve mice, employing Western blot, immunohistochemistry and RT-PCR.Results-Here we show for the first time that a clinically relevant isoflurane anesthesia (1.4% isoflurane for two hours) leads to caspase activation and modest increases in levels of the β-site APP-cleaving enzyme (BACE) six hours after anesthesia in mouse brain. Isoflurane anesthesia induces caspase activation, increases levels of BACE and Aβ up to 24 hours after anesthesia. Isoflurane may increase BACE levels by reducing BACE degradation. Moreover, the Aβ aggregation inhibitor, clioquinol, was able to attenuate isoflurane-induced caspase-3 activation in vivo.Interpretation-Given that transient insults to brain may lead to long term brain damage, these findings suggest that isoflurane may promote AD neuropathogenesis and, as such, have implications for use of isoflurane in humans, pending on human study confirmation.

Cachectic muscle wasting is a frequent complication of many inflammatory conditions, due primarily to excessive muscle catabolism. However, the pathogenesis and intervention strategies against it remain to be established. Here, we tested the hypothesis that Toll-like receptor 4 (TLR4) is a master regulator of inflammatory muscle catabolism. We demonstrate that TLR4 activation by lipopolysaccharide (LPS) induces C2C12 myotube atrophy via up-regulating autophagosome formation and the expression of ubiquitin ligase atrogin-1/MAFbx and MuRF1. TLR4-mediated activation of p38 MAPK is necessary and sufficient for the up-regulation of atrogin1/MAFbx and autophagosomes, resulting in myotube atrophy. Similarly, LPS up-regulates muscle autophagosome formation and ubiquitin ligase expression in mice. Importantly, autophagy inhibitor 3-methyladenine completely abolishes LPS-induced muscle proteolysis, while proteasome inhibitor lactacystin partially blocks it. Furthermore, TLR4 knockout or p38 MAPK inhibition abolishes LPS-induced muscle proteolysis. Thus, TLR4 mediates LPS-induced muscle catabolism via coordinate activation of the ubiquitin-proteasome and the autophagy-lysosomal pathways.

To identify a rare (e.g., diseased or foreign) cell in a complex mixture, or to understand the proteomic complexity [1,2] of cells, one needs to be able to measure simultaneously and quantitatively a large number of proteins or other biomarkers that may be present in a complex sample. This is a difficult task and is beyond the reach of current capabilities. To address a problem of this complexity, we have begun to develop a high-sensitivity assay [3][4][5][6] based upon elemental tags that will enable the simultaneous measurement of many proteins in a single sample. The advantage of this approach lies in the large number of available elements and isotopes (potentially greater than 79) found in low abundance in biological systems, which will allow multiple tags to be used simultaneously. Inductively coupled plasma mass spectrometry (ICP-MS) is an ideal technique for detecting and quantifying these tags, as ICP-MS provides excellent resolution between the tag masses and an exceptional dynamic range (nine orders of magnitude). [7] This method allows one to overcome some of the limitations of currently available fluorescent tagging approaches.[8] These limitations arise from the spectral overlap of different dyes and the difficulty in measuring simultaneously targets that differ in abundance by an order of magnitude or more. Other benefits of ICP-MS detection include the high sensitivity, which is comparable to that of radioimmunoassays or chemiluminescent assays, [3] insensitivity of elemental tags to photobleaching and storage time, as well as the stability of the tagged sample so that it can be stored or shipped for analysis. We discuss herein the development of a new class of elemental tags for ICP-MS detection and their use for tagging of antibodies chosen to allow specific recognition of distinguishing cell surface markers. By using this technique it should be possible to achieve detection limits on the order of parts per billion, which will allow the detection of cell surface markers with copy numbers as low as 100.Our experimental design is presented in Figure 1. The assay is based upon the concept of a water-soluble polymer bearing multiple metal-chelating ligands. The polymer contains a terminal maleimide group for coupling to cysteine -SH groups on the Fc portion of an antibody. It is now well established that attaching tags to antibodies through -SH groups (generated by selective reduction of disulfide bonds) is much more likely to preserve antibody activity than, for example, the random covalent attachment of tags to the amino group of lysines. The chelating ligand is chosen to form high-affinity complexes with lanthanide (Ln 3+ ) ions. These elements satisfy our requirement for low natural abundance and a wide selection of elements and isotopes. The use of a metal-chelating polymeric tag allows us to incorporate multiple numbers of a given ion, which leads to an increase in the sensitivity of the method, since the ICP-MS signal increases linearly with the number of atoms of a given element. Another impo...

Cachexia, characterized by muscle wasting, is a major contributor to cancer-related mortality. However, the key cachexins that mediate cancer-induced muscle wasting remain elusive. Here, we show that tumor-released extracellular Hsp70 and Hsp90 are responsible for tumor’s capacity to induce muscle wasting. We detected high-level constitutive release of Hsp70 and Hsp90 associated with extracellular vesicles (EVs) from diverse cachexia-inducing tumor cells, resulting in elevated serum levels in mice. Neutralizing extracellular Hsp70/90 or silencing Hsp70/90 expression in tumor cells abrogates tumor-induced muscle catabolism and wasting in cultured myotubes and in mice. Conversely, administration of recombinant Hsp70 and Hsp90 recapitulates the catabolic effects of tumor. In addition, tumor-released Hsp70/90-expressing EVs are necessary and sufficient for tumor-induced muscle wasting. Further, Hsp70 and Hsp90 induce muscle catabolism by activating TLR4, and are responsible for elevation of circulating cytokines. These findings identify tumor-released circulating Hsp70 and Hsp90 as key cachexins causing muscle wasting in mice.

BackgroundCachexia and muscle atrophy are common consequences of cancer and chemotherapy administration. The novel hormone ghrelin has been proposed as a treatment for this condition. Increases in food intake and direct effects on muscle proteolysis and protein synthesis are likely to mediate these effects, but the pathways leading to these events are not well understood.MethodsWe characterized molecular pathways involved in muscle atrophy induced by Lewis lung carcinoma (LLC) tumour implantation in c57/bl6 adult male mice and by administration of the chemotherapeutic agent cisplatin in mice and in C2C12 myotubes. The effects of exogenous ghrelin administration and its mechanisms of action were examined in these settings.ResultsTumour implantation and cisplatin induced muscle atrophy by activating pro-inflammatory cytokines, p38-C/EBP-β, and myostatin, and by down-regulating Akt, myoD, and myogenin, leading to activation of ubiquitin-proteasome-mediated proteolysis and muscle weakness. Tumour implantation also increased mortality. In vitro, cisplatin up-regulated myostatin and atrogin-1 by activating C/EBP-β and FoxO1/3. Ghrelin prevented these changes in vivo and in vitro, significantly increasing muscle mass (P < 0.05 for LLC and P < 0.01 for cisplatin models) and grip strength (P = 0.038 for LLC and P = 0.001 for cisplatin models) and improving survival (P = 0.021 for LLC model).ConclusionGhrelin prevents muscle atrophy by down-regulating inflammation, p38/C/EBP-β/myostatin, and activating Akt, myogenin, and myoD. These changes appear, at least in part, to target muscle cells directly. Ghrelin administration in this setting is associated with improved muscle strength and survival.

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Upregulation of ubiquitin ligase atrogin1/MAFbx and muscle wasting are hallmarks of cancer cachexia; however, the underlying mechanism is undefined. Here, we describe a novel signalling pathway through which Lewis lung carcinoma (LLC) induces atrogin1/MAFbx upregulation and muscle wasting. C2C12 myotubes treated with LLC-conditioned medium (LCM) rapidly activates p38 MAPK and AKT while inactivating FoxO1/3, resulting in atrogin1/MAFbx upregulation, myosin heavy chain loss, and myotube atrophy. The p38α/β MAPK inhibitor SB202190 blocks the catabolic effects. Upon activation, p38 associates with C/EBPβ resulting in its phosphorylation and binding to a C/EBPβ-responsive cis-element in the atrogin1/MAFbx gene promoter. The promoter activity is stimulated by LCM via p38β-mediated activation of the C/EBPβ-responsive cis-element, independent of the adjacent FoxO1/3-responsive cis-elements in the promoter. In addition, p38 activation is observed in the muscle of LLC tumour-bearing mice, and SB202190 administration blocks atrogin1/MAFbx upregulation and muscle protein loss. Furthermore, C/EBPβ(-/-) mice are resistant to LLC tumour-induced atrogin1/MAFbx upregulation and muscle wasting. Therefore, activation of the p38β MAPK-C/EBPβ signalling pathway appears a key component of the pathogenesis of LLC tumour-induced cachexia.

This study suggests that mandatorily quarantined individuals are more likely to have mental distress and negative cognitions related to COVID-19 than their nonquarantined counterparts. Attention should be paid to the heightened perceived discrimination as it was associated with both mandatory quarantine status and negative mental health status. The findings demonstrate the need to develop interventions to meet the psychological needs of people in quarantine during the COVID-19 pandemic.