With the recent success in determining membrane protein structures, further detailed understanding of the identity and function of the bound lipidome is essential. Using an approach that combines ...high-energy native mass spectrometry (HE-nMS) and solution-phase lipid profiling, this protocol can be used to determine the identity of the endogenous lipids that directly interact with a protein. Furthermore, this method can identify systems in which such lipid binding has a major role in regulating the oligomeric assembly of membrane proteins. The protocol begins with recording of the native mass spectrum of the protein of interest, under successive delipidation conditions, to determine whether delipidation leads to disruption of the oligomeric state. Subsequently, we propose using a bipronged strategy: first, an HE-nMS platform is used that allows dissociation of the detergent micelle at the front end of the instrument. This allows for isolation of the protein-lipid complex at the quadrupole and successive fragmentation at the collision cell, which leads to identification of the bound lipid masses. Next, simultaneous coupling of this with in-solution LC-MS/MS-based identification of extracted lipids reveals the complete identity of the interacting lipidome that copurifies with the proteins. Assimilation of the results of these two sets of experiments divulges the complete identity of the set of lipids that directly interact with the membrane protein of interest, and can further delineate its role in maintaining the oligomeric state of the protein. The entire procedure takes 2 d to complete.
•Circulating androgens decrease following capture in wild, breeding male birds.•Capture-induced declines in androgens depend on the stressor type and duration.•Handling and restraint have a stronger ...effect than capture in a mist net.•Androgen responses to capture stress depend, in part, on initial androgen levels.•Studies need to use methods that control for capture-induced variation in androgens.
Obtaining baseline hormone samples can be challenging because circulating hormone levels often change rapidly due to the acute stress of capture. Although field protocols are established for accurately sampling baseline glucocorticoid concentrations, fewer studies have examined how common sampling techniques affect androgens levels. Indeed, many studies focused on understanding the functional significance of baseline androgen levels use sampling methods known to activate the endocrine responses to stress. To understand how different field sampling protocols affect plasma androgen levels, we measured the androgen response to two types of capture stressors in a free-living tropical bird, the wire-tailed manakin (Pipra filicauda). First, we subjected males to a standardized capture and restraint protocol lasting either 15 or 30 min. Second, males were passively captured in nets that were filmed (to establish exact duration of time between capture and blood sampling) and checked every 30 min. The first study showed that circulating plasma androgen levels decreased significantly following both 15 and 30 min of restraint in a cloth bag, with a trend for the 30 min samples to be lower than the 15 min samples. Further, the change in androgen levels was dependent on an individual’s initial androgen levels, with the individuals with the highest initial levels registering the largest declines. The results of the second study suggest that hanging in a mist net for extended periods of time also leads to a decrease in circulating androgen levels, but this effect was weaker than that of capture and restraint in a cloth bag. Our findings demonstrate that, overall, circulating androgen levels decrease in response to common sampling techniques; a finding that has important implications for studies measuring baseline androgen levels in free-living birds. Future studies should prioritize sampling individuals immediately upon removal from the mist net, as handling and restraint have a strong negative effect on circulating androgen levels. When constant monitoring of the mist net is not possible, investigators should use video cameras to record the amount of time an individual spends in the net prior to blood sampling and then statistically control for the effect of this variable in analyses.
Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells, and ...human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes Dnmt1 and causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation, and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.
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•UHRF1 overexpression induces DNA hypomethylation•Liver-specific overexpression of UHRF1 in zebrafish causes senescence and HCC•Human HCCs with high UHRF1 are aggressive and inactivate TP53-mediated senescence•Our studies in zebrafish and human tumors identify UHRF1 as an oncogene in HCC
Mudbhary et al. show that overexpression of UHRF1, a critical DNA methylation regulator, in zebrafish causes DNA hypomethylation, p53-mediated senescence, and hepatocellular carcinoma (HCC) when senescence is bypassed. High UHRF1 expression in human HCC correlates with poor prognosis and TP53 mutation.
Age-dependent elevation in mitochondrial oxidative stress is widely posited to be a major factor underlying the loss of substantia nigra pars compacta (SNc) dopaminergic neurons in Parkinson's ...disease (PD). However, mechanistic links between aging and oxidative stress are not well understood. Sirtuin-3 (Sirt3) is a mitochondrial deacetylase that could mediate this connection. Indeed, genetic deletion of Sirt3 increased oxidative stress and decreased the membrane potential of mitochondria in SNc dopaminergic neurons. This change was attributable to increased acetylation and decreased activity of manganese superoxide dismutase (MnSOD). Site directed mutagenesis of lysine 68 to glutamine (K68Q), mimicking acetylation, decreased MnSOD activity in SNc dopaminergic neurons, whereas mutagenesis of lysine 68 to arginine (K68R), mimicking deacetylation, increased activity. Introduction of K68R MnSOD rescued mitochondrial redox status and membrane potential of SNc dopaminergic neurons from Sirt3 knockouts. Moreover, deletion of DJ-1, which helps orchestrate nuclear oxidant defenses and Sirt3 in mice led to a clear age-related loss of SNc dopaminergic neurons. Lastly, K68 acetylation of MnSOD was significantly increased in the SNc of PD patients. Taken together, our studies suggest that an age-related decline in Sirt3 protective function is a major factor underlying increasing mitochondrial oxidative stress and loss of SNc dopaminergic neurons in PD.
Precise monitoring of the rapidly changing immune status during the course of a disease requires multiplex analysis of cytokines from frequently sampled human blood. However, the current lack of ...rapid, multiplex, and low volume assays makes immune monitoring for clinical decision-making (e.g., critically ill patients) impractical. Without such assays, immune monitoring is even virtually impossible for infants and neonates with infectious diseases and/or immune mediated disorders as access to their blood in large quantities is prohibited. Localized surface plasmon resonance (LSPR)-based microfluidic optical biosensing is a promising approach to fill this technical gap as it could potentially permit real-time refractometric detection of biomolecular binding on a metallic nanoparticle surface and sensor miniaturization, both leading to rapid and sample-sparing analyte analysis. Despite this promise, practical implementation of such a microfluidic assay for cytokine biomarker detection in serum samples has not been established primarily due to the limited sensitivity of LSPR biosensing. Here, we developed a high-throughput, label-free, multiarrayed LSPR optical biosensor device with 480 nanoplasmonic sensing spots in microfluidic channel arrays and demonstrated parallel multiplex immunoassays of six cytokines in a complex serum matrix on a single device chip while overcoming technical limitations. The device was fabricated using easy-to-implement, one-step microfluidic patterning and antibody conjugation of gold nanorods (AuNRs). When scanning the scattering light intensity across the microarrays of AuNR ensembles with dark-field imaging optics, our LSPR biosensing technique allowed for high-sensitivity quantitative cytokine measurements at concentrations down to 5–20 pg/mL from a 1 μL serum sample. Using the nanoplasmonic biosensor microarray device, we demonstrated the ability to monitor the inflammatory responses of infants following cardiopulmonary bypass (CPB) surgery through tracking the time-course variations of their serum cytokines. The whole parallel on-chip assays, which involved the loading, incubation, and washing of samples and reagents, and 10-fold replicated multianalyte detection for each sample using the entire biosensor arrays, were completed within 40 min.
Dystrophin deficiency, which leads to severe and progressive muscle degeneration in patients with Duchenne muscular dystrophy (DMD), is caused by frameshifting mutations in the
dystrophin gene. A ...relatively new therapeutic strategy is based on antisense oligonucleotides (AONs) that induce the specific skipping of a single exon, such that the reading frame is restored. This allows the synthesis of a largely functional dystrophin, associated with a milder Becker muscular dystrophy phenotype. We have previously successfully targeted 20 different DMD exons that would, theoretically, be beneficial for >75% of all patients. To further enlarge this proportion, we here studied the feasibility of double and multiexon skipping. Using a combination of AONs, double skipping of exon 43 and 44 was induced, and dystrophin synthesis was restored in myotubes from one patient affected by a nonsense mutation in exon 43. For another patient, with an exon 46–50 deletion, the therapeutic double skipping of exon 45 and 51 was achieved. Remarkably, in control myotubes, the latter combination of AONs caused the skipping of the entire stretch of exons from 45 through 51. This in-frame multiexon skipping would be therapeutic for a series of patients carrying different DMD-causing mutations. In fact, we here demonstrate its feasibility in myotubes from a patient with an exon 48–50 deletion. The application of multiexon skipping may provide a more uniform methodology for a larger group of patients with DMD.
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