steady state levels
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2022 ◽  
pp. 94-122
Author(s):  
Shreyas Anantray Bhatt

Life on the Earth has evolved in the cold environments. Such cold habitats pose special challenges to the microbes in cold ecosystems, such as minimum metabolic activities, very limited nutrient availability, and often extreme conditions such as pH and salinity apart from temperature. Microbial communities surviving under these extreme conditions must have evolved complex structural and functional adaptations. Prokaryotic adaptations to cold environments are through physiological adaptations by increasing membrane fluidity through large amount of unsaturated fatty acids. These microbes also possess some cold adapted proteins whose steady state levels are maintained. They also produce certain compounds such as polyamines, sugars, polyols, amino acids, and some antifreeze proteins to protect themselves under freezing conditions. They also produce exopolymeric substances that promote adhesion of microbes to moist surfaces to induce biofilm formation which helps getting nutrients and protect the cells from harsh conditions. Antioxidants help destroying toxic reactive oxygen species.


PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0258682
Author(s):  
Declan Williams ◽  
Mohadeseh Mehrabian ◽  
Hamza Arshad ◽  
Shehab Eid ◽  
Christopher Sackmann ◽  
...  

The prion protein (PrP) is best known for its ability to cause fatal neurodegenerative diseases in humans and animals. Here, we revisited its molecular environment in the brain using a well-developed affinity-capture mass spectrometry workflow that offers robust relative quantitation. The analysis confirmed many previously reported interactions. It also pointed toward a profound enrichment of Na,K-ATPases (NKAs) in proximity to cellular PrP (PrPC). Follow-on work validated the interaction, demonstrated partial co-localization of the ATP1A1 and PrPC, and revealed that cells exposed to cardiac glycoside (CG) inhibitors of NKAs exhibit correlated changes to the steady-state levels of both proteins. Moreover, the presence of PrPC was observed to promote the ion uptake activity of NKAs in a human co-culture paradigm of differentiated neurons and glia cells, and in mouse neuroblastoma cells. Consistent with this finding, changes in the expression of 5’-nucleotidase that manifest in wild-type cells in response to CG exposure can also be observed in untreated PrPC-deficient cells. Finally, the endoproteolytic cleavage of the glial fibrillary acidic protein, a hallmark of late-stage prion disease, can also be induced by CGs, raising the prospect that a loss of NKA activity may contribute to the pathobiology of prion diseases.


Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1810
Author(s):  
Luka A. Clarke ◽  
Vanessa C. C. Luz ◽  
Szymon Targowski ◽  
Sofia S. Ramalho ◽  
Carlos M. Farinha ◽  
...  

Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation; (2) differences in abundance between the 3′ and 5′ regions of CFTR mRNA, even following PTC readthrough or NMD inhibition; and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 858-858
Author(s):  
Justin J Yoo ◽  
Sara H Graciaa ◽  
Jayre A Jones ◽  
Zoey Zuo ◽  
Connie M Arthur ◽  
...  

Abstract Background: Sickle cell disease (SCD) affects millions of individuals worldwide with substantial morbidity and mortality. The sickle hemoglobin (HbS) polymerizes upon deoxygenation, causing rigid and adhesive red blood cells (RBCs), triggering vascular occlusion, greatly shortened RBC lifespan, and chronic hemolysis. Amongst acute complications in SCD, vaso-occlusive pain crisis (VOC) is the leading cause of hospitalization, with supportive care being the primary approach to management. We and others have recently demonstrated important contributions of complement to the pathophysiology of SCD. When the complement pathway (CP) is activated during SCD crises, inhibition at C5 using eculizumab, has been successful in treating various acute complications in SCD (Chonat et al, Haematologica). In this study, we prospectively analyzed the extent of CP activation among children with SCD presenting with VOC. Methods: Patients aged 0-21 years old managed at Children's Healthcare of Atlanta with homozygous sickle cell (SS) or S beta zero thalassemia genotypes were enrolled in an IRB-approved research study. Inclusion criteria included those requiring intravenous opioids for VOC, and excluded those with chronic pain, >6 VOC admission in the previous 12 months or on chronic transfusions. Blood samples were collected within 48 hours of VOC presentation, and steady-state levels were obtained at a 4-week clinic follow-up. Data was analyzed using a paired t-test, and receiver operator characteristic (ROC) curves were generated comparing intra-person complement levels during acute VOC versus respective steady-state levels. Results: Sixty-four patients have been enrolled thus far, of which 43 (67%) had steady-state samples collected. The majority of patients (90.5%) have SS genotype with a mean (SD) patient age of 14.15 (4.68) years. Fifty-three (84.1%) patients reported taking hydroxyurea (HU). Fifty-nine (93.7%) patients had at least one VOC admission in the past 12 months, with an average of 2.98 (1.67) VOC admissions. Pain Score reported on 55 patients averaged 4.93 (4.78) on a pain scale of 0 to 10. Mean values during VOC and steady-state of hemoglobin (Hb) were 8.12 and 9.01 g/dL, platelet count 431 and 511, and lactate dehydrogenase (LDH) 549 and 483 U/L, respectively. Seventeen patients had complement work-up performed during acute and steady-state, and 4 of them had additional samples collected during subsequent VOC. Complement protein levels C3, C4, C5, properdin, factor B, and complement regulatory proteins factor H and I were unremarkable during VOC and steady-state. However, complement activation markers, specifically anaphylatoxins C3a, C5a and Bb were significantly elevated during VOC compared to steady-state (see Table 1) suggesting activation of alternative CP during VOC (see Table 1 and Figure 1A-C). Terminal complement complex (C5b9) was not statistically different between VOC and steady-state (Figure 1D, red dotted lines signify normal ranges). Remarkably, patients who re-presented with acute VOC exhibited similar increases in their C3a/C5a (Figure 1E-F), substantiating the increases related to their VOC. Hemoglobin and LDH (Figure 1G-H) were similarly significant, suggestive of intravascular hemolysis. Three (7.1%) patients developed acute chest syndrome, two of whom experienced respiratory failure requiring intensive care management, and all exhibited significant CP activation. The area under the curve (AUC) of the ROC curve was analyzed to determine the ability of complement biomarkers to differentiate VOC from steady-state. Based on the AUC of these biomarkers, complement anaphylatoxins C3a and C5a exhibited the highest AUC of 0.76 and 0.87, respectively. Discussion: To our knowledge, this is the first prospective and comprehensive evaluation of CP in patients with SCD during VOC and steady-states. These preliminary findings suggest CP activation is present in a large proportion of patients during VOC, with increased activation of alternative and common CP, associated with intravascular hemolysis. Minimal increase in C5b9 could be explained by a significant proportion (> 80%) of our patients being on HU therapy, similar to prior data (Roumenina et al, AJH). Specifically, C3a/C5a, along with other biomarkers, could not only predict disease activity in patients during VOC, but provide pharmacological targets in VOC, which need further validation. Figure 1 Figure 1. Disclosures Stowell: Alexion: Consultancy; Argenx: Speakers Bureau; Grifols: Speakers Bureau. Chonat: Alexion: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Takeda: Consultancy, Research Funding.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3193-3193
Author(s):  
Roberta Gualtierotti ◽  
Carla Valsecchi ◽  
Chiara Suffritti ◽  
Eugenia Biguzzi ◽  
Sara Arcudi ◽  
...  

Abstract Background Patients with severe hemophilia A may develop inhibitors against factor VIII (FVIII) in around 30% of cases. Recently, the introduction of non-replacement therapies such as emicizumab, a FVIII-mimicking agent administered as a subcutaneous injection, has revolutionized the treatment of patients with inhibitors. However, although rarely, some patients may develop antibodies against this drug. If neutralizing, these antibodies interfere with the activity of the drug, making it ineffective. Mim8 (Novo Nordisk®) is a novel experimental FVIII-mimetic human bispecific antibody that has a similar function as emicizumab, by bridging activated FIX (FIXa) and FX to activate FX, although with a different molecular structure compared to emicizumab. It is currently in phase II clinical trial for subcutaneous treatment of patients with hemophilia A with or without FVIII inhibitors (1, 2). It is currently unknown whether the antibodies developed against emicizumab by patients with hemophilia A could cross-react with Mim8. Aim Our aim was to study the cross-reactivity of anti-emicizumab antibodies developed by patients with hemophilia A against Mim8 with an in-house detection method. Methods We studied the serum of three patients who developed anti-emicizumab antibodies. Plasma from one patient with persistent inhibiting antibodies was collected both during the treatment (thus also containing emicizumab at steady-state levels) and two years after treatment discontinuation due to inefficacy (neutralizing persistent antibodies). Plasma from two patients who developed transient antibodies against emicizumab were also tested in the course of treatment with emicizumab (non-neutralizing transient antibodies). The plate was coated both with emicizumab and with Mim8 provided by the pharmaceutical companies for research purposes. Plasma samples, diluted 1/20, were loaded into the coated wells and incubated 90 min at 37°C. The cross-reactivity to Mim8 was evaluated also by using the affinity purified anti-emicizumab IgG, which was loaded at 5 ug/mL. A properly adapted ELISA method already described (3) was used as reference assay. Then, biotinylated-emicizumab (1.5 ug/mL) or biotinylated-Mim8 (at 2 and 4 ug/mL) were added and the plate incubated 1 hour at 37°C. Moreover, a competition test was performed by using a mixture of biotinylated-emicizumab (1.5 ug/mL) and an excess of Mim8 (at 4, 8 and 40 ug/mL) in the detection phase. Results The Mim8 molecule - either alone, or matched with emicizumab - used both in the capture phase and in the detection phase did not bind to neither patient's plasma antibodies nor to anti-emicizumab purified IgG, which were instead revealed with the reference assay. The binding of the anti-emicizumab antibodies to biotinylated-emicizumab was not inhibited by the addition of Mim8, even at 40 ug/mL. Conclusions Our in-house method showed that anti-emicizumab antibodies do not react with Mim8 in vitro. Observational studies to test whether Mim8 can be used safely in patients with anti-emicizumab antibodies are needed to confirm our findings in vivo as well. References 1. Østergaard et al. A FVIIIa-mimetic bispecific antibody (Mim8) ameliorates bleeding upon severe vascular challenge in hemophilia A mice. Blood. 2021;blood.2020010331. doi:10.1182/blood.2020010331. 2. Valsecchi C et al. J Thromb Haemost. 2021;19(3):711-718. Disclosures Peyvandi: Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.


2021 ◽  
Vol 22 (19) ◽  
pp. 10692
Author(s):  
Petra Van Damme

N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.


2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Marcos Fernández-Mora ◽  
Diego Sánchez-Popoca ◽  
Gloria Altamirano-Cruz ◽  
Grecia López-Méndez ◽  
Andrea Teresa Téllez-Galicia ◽  
...  

The S. Typhi leuO gene, which codes for the LysR-type transcriptional regulator LeuO, contains five forward promoters named P3, P1, P2, P5 and P4, and two reverse promoters, P6 and P7. The activity of the forward promoters was revealed by primer extension using gene reporter fusions in an S. Typhi hns lrp mutant strain. Likewise, the activity of the reverse promoters was revealed in an hns background. Derepression of the transcription of the chromosomal gene was confirmed by RT-PCR in the hns lrp mutant. The leuOP1 transcriptional reporter fusion, which contained only the major P1 promoter, had a lower expression in a relA spoT mutant strain, indicating that the steady-state levels of the (p)ppGpp alarmone positively regulate it. In contrast, the leuOP3, leuOP5P4, leuOP6 and leuOP7 transcriptional fusions were derepressed in the relA spoT background, indicating that the alarmone has a negative effect on their expression. Thus, the search for genetic regulators and environmental cues that would differentially derepress leuO gene expression by antagonizing the action of the H-NS and Lrp nucleoid-associated proteins, or that would fine-tune the expression of the various promoters, will further our understanding of the significance that multiple promoters have in the control of LeuO expression.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mateusz Jurga ◽  
Arwa A. Abugable ◽  
Alastair S. H. Goldman ◽  
Sherif F. El-Khamisy

AbstractR-loops are by-products of transcription that must be tightly regulated to maintain genomic stability and gene expression. Here, we describe a mechanism for the regulation of the R-loop-specific helicase, senataxin (SETX), and identify the ubiquitin specific peptidase 11 (USP11) as an R-loop regulator. USP11 de-ubiquitinates SETX and its depletion increases SETX K48-ubiquitination and protein turnover. Loss of USP11 decreases SETX steady-state levels and reduces R-loop dissolution. Ageing of USP11 knockout cells restores SETX levels via compensatory transcriptional downregulation of the E3 ubiquitin ligase, KEAP1. Loss of USP11 reduces SETX enrichment at KEAP1 promoter, leading to R-loop accumulation, enrichment of the endonuclease XPF and formation of double-strand breaks. Overexpression of KEAP1 increases SETX K48-ubiquitination, promotes its degradation and R-loop accumulation. These data define a ubiquitination-dependent mechanism for SETX regulation, which is controlled by the opposing activities of USP11 and KEAP1 with broad applications for cancer and neurological disease.


2021 ◽  
Author(s):  
Yvonne Nyathi ◽  
Jake Alfie Hill

Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects.  However, the DUBs that play a role in this process have not been identified.  In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5.  We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.


2021 ◽  
Author(s):  
Ioannis Nellas ◽  
K. Venkatesan Iyer ◽  
Juan M. Iglesias-Artola ◽  
André Nadler ◽  
Natalie A. Dye ◽  
...  

ABSTRACTEnergy production and utilization is critically important for animal development and growth. How it is regulated in space and time during tissue growth remains largely unclear. Toward this end, we used a FRET-based adenosine triphosphate (ATP) sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila wing disc. We discovered that steady-state levels of ATP are spatially uniform across the wing pouch. Pharmacologically inhibiting oxidative phosphorylation, however, reveals spatial heterogeneities in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog (Hh) signaling pathway can enhance ATP production by glycolysis. Collectively, our work reveals a positive feedback loop between Hh signaling and energy metabolism, advancing our understanding of the connection between conserved developmental patterning genes and energy production during animal tissue development.


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