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Transplantation Direct Jun 2024Four-factor prothrombin complex concentrate (PCC) is a plasma product that contains factors II, VII, IX, X, protein C, and protein S. PCC can be used off-label to treat...
BACKGROUND
Four-factor prothrombin complex concentrate (PCC) is a plasma product that contains factors II, VII, IX, X, protein C, and protein S. PCC can be used off-label to treat coagulopathy during orthotopic liver transplantation (OLT). However, its use comes with safety concerns regarding thrombosis. The purpose of our study is to determine the safety of PCC in OLT.
METHODS
We conducted a retrospective cohort study of patients who received 4-factor PCC during OLT at our institution from January 1, 2018, to May 1, 2022, with a 1:1 match of 83 patients who received PCC and 83 patients who did not. We evaluated 30-d mortality, 1-y mortality, prevalence of thrombotic complications (portal vein thrombosis, deep venous thrombosis, myocardial infarction, and pulmonary embolus), and postoperative intensive care (ICU) length of stay (LOS).
RESULTS
There was no significant difference in 30-d mortality (odds ratio [OR] 5; 95% confidence interval [CI], 0.58-42.8; = 0.14), 1-y mortality (OR 3; 95% CI, 0.61-14.86; = 0.18), or ICU LOS (OR -13.8; 95% CI, -39.2 to 11.6; = 0.29). There was no increased incidence of thrombotic complications among patients receiving PCC 90 d after surgery, including portal vein thrombosis (OR 1.5; 95% CI, 0.42-5.32; = 0.53), pulmonary embolus (OR 1; 95% CI, 0.14-7.1; = 0.99), deep venous thrombosis (OR 0.67; 95% CI, 0.11-3.99; = 0.66), and myocardial infarction (OR 1.67; 95% CI, 0.4-6.97; = 0.48).
CONCLUSIONS
Although there was a statistically insignificant increase in mortality after PCC administration during OLT, we did not see a significant increase in perioperative complications, including thrombotic events and increased ICU LOS.
PubMed: 38769975
DOI: 10.1097/TXD.0000000000001637 -
Journal of Biotechnology Jul 2024Nutrient signaling pathways play a pivotal role in regulating the balance among metabolism, growth and stress response depending on the available food supply. They are...
Nutrient signaling pathways play a pivotal role in regulating the balance among metabolism, growth and stress response depending on the available food supply. They are key factors for the biotechnological success of the yeast Saccharomyces cerevisiae during food-producing fermentations. One such pathway is Retrograde Response, which controls the alpha-ketoglutarate supply required for the synthesis of amino acids like glutamate and lysine. Repressor MKS1 is linked with the TORC1 complex and negatively regulates this pathway. Deleting MKS1 from a variety of industrial strains causes glycerol to increase during winemaking, brewing and baking. This increase is accompanied by a reduction in ethanol production during grape juice fermentation in four commercial wine strains. Interestingly, this does not lead volatile acidity to increase because acetic acid levels actually lower. Aeration during winemaking usually increases acetic acid levels, but this effect reduces in the MKS1 mutant. Despite the improvement in the metabolites of oenological interest, it comes at a cost given that the mutant shows slower fermentation kinetics when grown in grape juice, malt and laboratory media and using glucose, sucrose and maltose as carbon sources. The deletion of RTG2, an activator of Retrograde Response that acts as an antagonist of MKS1, also results in a defect in wine fermentation speed. These findings suggest that the deregulation of this pathway causes a fitness defect. Therefore, manipulating repressor MKS1 is a promising approach to modulate yeast metabolism and to produce low-ethanol drinks.
Topics: Glycerol; Saccharomyces cerevisiae; Ethanol; Fermentation; Wine; Saccharomyces cerevisiae Proteins; Up-Regulation; Repressor Proteins; Gene Expression Regulation, Fungal; Transaminases
PubMed: 38768686
DOI: 10.1016/j.jbiotec.2024.05.007 -
Platelets Dec 2024Small molecule drugs play a major role in the study of human platelets. Effective action of a drug requires it to bind to one or more targets within the platelet (target...
Small molecule drugs play a major role in the study of human platelets. Effective action of a drug requires it to bind to one or more targets within the platelet (target engagement). However, although assays with isolated proteins can be used to determine drug affinity to these targets, additional factors affect target engagement and its consequences in an intact platelet, including plasma membrane permeability, intracellular metabolism or compartmentalization, and level of target expression. Mechanistic interpretation of the effect of drugs on platelet activity requires comprehensive investigation of drug binding in the proper cellular context, i.e. in intact platelets. The Cellular Thermal Shift Assay (CETSA) is a valuable method to investigate target engagement within complex cellular environments. The assay is based on the principle that drug binding to a target protein increases that protein's thermal stability. In this technical report, we describe the application of CETSA to platelets. We highlight CETSA as a quick and informative technique for confirming the direct binding of drugs to platelet protein targets, providing a platform for understanding the mechanism of action of drugs in platelets, and which will be a valuable tool for investigating platelet signaling and function.
Topics: Humans; Blood Platelets; Protein Binding
PubMed: 38767506
DOI: 10.1080/09537104.2024.2354833 -
ELife May 2024A protein's genetic architecture - the set of causal rules by which its sequence produces its functions - also determines its possible evolutionary trajectories. Prior...
A protein's genetic architecture - the set of causal rules by which its sequence produces its functions - also determines its possible evolutionary trajectories. Prior research has proposed that the genetic architecture of proteins is very complex, with pervasive epistatic interactions that constrain evolution and make function difficult to predict from sequence. Most of this work has analyzed only the direct paths between two proteins of interest - excluding the vast majority of possible genotypes and evolutionary trajectories - and has considered only a single protein function, leaving unaddressed the genetic architecture of functional specificity and its impact on the evolution of new functions. Here, we develop a new method based on ordinal logistic regression to directly characterize the global genetic determinants of multiple protein functions from 20-state combinatorial deep mutational scanning (DMS) experiments. We use it to dissect the genetic architecture and evolution of a transcription factor's specificity for DNA, using data from a combinatorial DMS of an ancient steroid hormone receptor's capacity to activate transcription from two biologically relevant DNA elements. We show that the genetic architecture of DNA recognition consists of a dense set of main and pairwise effects that involve virtually every possible amino acid state in the protein-DNA interface, but higher-order epistasis plays only a tiny role. Pairwise interactions enlarge the set of functional sequences and are the primary determinants of specificity for different DNA elements. They also massively expand the number of opportunities for single-residue mutations to switch specificity from one DNA target to another. By bringing variants with different functions close together in sequence space, pairwise epistasis therefore facilitates rather than constrains the evolution of new functions.
Topics: Epistasis, Genetic; Evolution, Molecular; Transcription Factors; DNA; Mutation; Protein Binding
PubMed: 38767330
DOI: 10.7554/eLife.88737 -
BioRxiv : the Preprint Server For... May 2024The mammalian PAS-domain protein PERIOD (PER) and its orthologue LIN-42 have been proposed to constitute an evolutionary link between two distinct, circadian and...
The mammalian PAS-domain protein PERIOD (PER) and its orthologue LIN-42 have been proposed to constitute an evolutionary link between two distinct, circadian and developmental, timing systems. However, while the function of PER in animal circadian rhythms is well understood molecularly and mechanistically, this is not true for the function of LIN-42 in timing rhythmic development. Here, using targeted deletions, we find that the LIN-42 PAS domains are dispensable for the protein's function in timing molts. Instead, we observe arrhythmic molts upon deletion of a distinct sequence element, conserved with PER. We show that this element mediates stable binding to KIN-20, the CK1δ/ε orthologue. We demonstrate that CK1δ phosphorylates LIN-42 and define two conserved helical motifs, CK1δ-binding domain A (CK1BD-A) and CK1BD-B, that have distinct roles in controlling CK1δ-binding and kinase activity . KIN-20 and the LIN-42 CK1BD are required for proper molting timing . These interactions mirror the central role of a stable circadian PER-CK1 complex in setting a robust ~24-hour period. Hence, our results establish LIN-42/PER - KIN-20/CK1δ/ε as a functionally conserved signaling module of two distinct chronobiological systems.
PubMed: 38766223
DOI: 10.1101/2024.05.09.593322 -
BioRxiv : the Preprint Server For... May 2024Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the...
Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood. We found that the yeast membrane protein Yet3, the homolog of human BAP31, is localized to multiple ER contact sites. We show that Yet3 interacts with all the enzymes of the post-squalene ergosterol biosynthesis pathway and recruits them to create sterol-rich domains. Increasing sterol levels at ER contacts causes its depletion from the plasma membrane leading to a compensatory reaction and altered cell metabolism. Our data shows that Yet3 provides on-demand sterols at contacts thus shaping organellar structure and function. A molecular understanding of this protein's functions gives new insights into the role of BAP31 in development and pathology.
PubMed: 38766039
DOI: 10.1101/2024.05.09.593285 -
Cureus Apr 2024Splenic infarction is a rare and likely underdiagnosed complication of Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). Here, we describe an...
Splenic infarction is a rare and likely underdiagnosed complication of Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). Here, we describe an 18-year-old Guyanese male with persistent severe left-sided abdominal pain found to be EBV positive and have a large splenic infarct, along with a transient decrease in protein C, protein S, and antithrombin III activity levels. He was treated with supportive care and anticoagulated with heparin and apixaban. We review prior reports and perspectives on underlying pathophysiology, diagnosis, and the management of these cases, which likely do not require anticoagulation but may be considered on a per-case basis.
PubMed: 38756285
DOI: 10.7759/cureus.58414 -
Nucleic Acids Research Jun 2024Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We...
Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We investigated two Pif1 variants (F723A and T464A) that have each lost one site of interaction of the protein with the DNA substrate. Both variants exhibit minor reductions in affinity for DNA and ATP hydrolysis but have impaired DNA unwinding activity. However, these variants translocate on single-stranded DNA faster than the wildtype enzyme and can slide on the DNA substrate in an ATP-independent manner. This suggests they have lost their grip on the DNA, interfering with coupling ATP hydrolysis to translocation and unwinding. Yeast expressing these variants have increased gross chromosomal rearrangements, increased telomere length, and can overcome the lethality of dna2Δ, similar to phenotypes of yeast lacking Pif1. However, unlike pif1Δ mutants, they are viable on glycerol containing media and maintain similar mitochondrial DNA copy numbers as Pif1 wildtype. Overall, our data indicate that a tight grip of the trailing edge of the Pif1 enzyme on the DNA couples ATP hydrolysis to DNA translocation and DNA unwinding. This tight grip appears to be essential for the Pif1 nuclear functions tested but is dispensable for mitochondrial respiratory growth.
Topics: Adenosine Triphosphate; Binding Sites; Cell Nucleus; DNA Helicases; DNA, Mitochondrial; DNA, Single-Stranded; Hydrolysis; Mitochondria; Mutation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 38752483
DOI: 10.1093/nar/gkae403 -
Scientific Reports May 2024During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of...
During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.
Topics: Chemokine CXCL10; Humans; Spike Glycoprotein, Coronavirus; SARS-CoV-2; COVID-19; Macrophages; Leukocytes, Mononuclear; NF-kappa B; THP-1 Cells
PubMed: 38750069
DOI: 10.1038/s41598-024-61906-6 -
BioRxiv : the Preprint Server For... May 2024The monomeric heme protein myoglobin (Mb), traditionally thought to be expressed exclusively in cardiac and skeletal muscle, is now known to be expressed in...
UNLABELLED
The monomeric heme protein myoglobin (Mb), traditionally thought to be expressed exclusively in cardiac and skeletal muscle, is now known to be expressed in approximately 40% of breast tumors. While Mb expression is associated with better patient prognosis, the molecular mechanisms by which Mb limits cancer progression are unclear. In muscle, Mb's predominant function is oxygen storage and delivery, which is dependent on the protein's heme moiety. However, prior studies demonstrate that the low levels of Mb expressed in cancer cells preclude this function. Recent studies propose a novel fatty acid binding function for Mb via a lysine residue (K46) in the heme pocket. Given that cancer cells can upregulate fatty acid oxidation (FAO) to maintain energy production for cytoskeletal remodeling during cell migration, we tested whether Mb-mediated fatty acid binding modulates FAO to decrease breast cancer cell migration. We demonstrate that the stable expression of human Mb in MDA-MB-231 breast cancer cells decreases cell migration and FAO. Site-directed mutagenesis of Mb to disrupt Mb fatty acid binding did not reverse Mb-mediated attenuation of FAO or cell migration in these cells. In contrast, cells expressing Apo-Mb, in which heme incorporation was disrupted, showed a reversal of Mb-mediated attenuation of FAO and cell migration, suggesting that Mb attenuates FAO and migration via a heme-dependent mechanism rather than through fatty acid binding. To this end, we show that Mb's heme-dependent oxidant generation propagates dysregulated gene expression of migratory genes, and this is reversed by catalase treatment. Collectively, these data demonstrate that Mb decreases breast cancer cell migration, and this effect is due to heme-mediated oxidant production rather than fatty acid binding. The implication of these results will be discussed in the context of therapeutic strategies to modulate oxidant production and Mb in tumors.
HIGHLIGHTS
Myoglobin (Mb) expression in MDA-MB-231 breast cancer cells slows migration.Mb expression decreases mitochondrial respiration and fatty acid oxidation.Mb-dependent fatty acid binding does not regulate cell migration or respiration.Mb-dependent oxidant generation decreases mitochondrial metabolism and migration.Mb-derived oxidants dysregulate migratory gene expression.
PubMed: 38746370
DOI: 10.1101/2024.04.30.591659