BioEnergetics (United States)

Genetically engineered porcine livers are being developed as a bridge therapy for acute liver failure, providing detoxification and restoration of hepatic protein synthesis. Severe xenograft-associated severe thrombocytopenia remains a major limitation, and human mechanistic data are scarce. Platelet kinetics were characterized in three human decedents undergoing extracorporeal cross-circulation with transgenic porcine livers. Platelet counts, transfusion requirements, and clearance patterns were assessed to distinguish consumption from marrow suppression or hypersplenism. Antibody- and complement-directed inhibitors were administered to test immune-mediated mechanisms. Mechanistic studies focused on porcine von Willebrand factor (pVWF)-dependent platelet activation, including ex vivo blockade with the anti-VWF nanobody caplacizumab, a vWF-directed antibody fragment that prevents vWF-platelet binding. A fourth decedent received caplacizumab during porcine liver perfusion. In all three initial cases, 80%-90% of circulating and transfused platelets were rapidly cleared, a pattern inconsistent with marrow suppression or hypersplenism. Antibody and complement inhibition failed to ameliorate thrombocytopenia. Recipient plasma induced robust pVWF-mediated platelet activation analogous to human Type IIb von Willebrand disease, which was completely abrogated ex vivo by caplacizumab. In a fourth decedent treated with caplacizumab, aberrant platelet activation was prevented, though full hematologic recovery was limited by pre-existing disseminated intravascular coagulation (DIC). Early thrombocytopenia during porcine liver xenotransplantation appears to be primarily driven by pVWF-mediated platelet activation rather than by classical immune or splenic mechanisms. Targeted VWF blockade with agents such as caplacizumab may mitigate platelet loss and improve the safety profile of extracorporeal porcine liver support in acute liver failure.

COX6B1 is a nuclear-encoded subunit of the human mitochondrial cytochrome c oxidase (cIV) located in its intermembrane space-facing region. The relevance of COX6B1 in mitochondrial physiopathology was highlighted by the missense pathogenic variants associated with cIV deficiency. Despite the assigned COX6B1 role as a late incorporation subunit, the COX6B1 human cell line KO exhibited a total loss of cIV. To get a deeper insight into the mechanisms driving the lack of cIV assembly or destabilization in the absence of COX6B1, we used the COX6B1 KO cell background to express alternative oxidase and COX6B1 pathogenic variants. These analyses uncovered that the COX6B1 subunit is indispensable for redox-sensitive early cIV assembly steps, besides its contribution to the stabilization of cIV in the late assembly stages. In addition, we have evidenced the incorporation of partially assembled cIV modules directly into supercomplex structures, supporting the "cooperative assembly" model for respiratory chain biogenesis.

Protein phosphorylation is critical to selective gene expression; the proteins that regulate transcription are often phosphorylated at multiple sites. Serine and threonine phosphorylation in transcription factors such as NF-κB has been studied, and specific serines are involved in transcriptional activation. Tyrosine phosphorylation of NF-κB p50 subunit can also facilitate the NF-κB-mediated expression of CD40 protein. This study seeks to determine whether tyrosine phosphorylations at positions not normally phosphorylated in vivo could nonetheless affect protein expression mediated by NF-κB. The alterations studied included p50 analogs having pTyr at positions 59 and 61, which do not contain Tyr naturally, and an analog containing a metabolically stable tyrosine methylene phosphonate at position 60. Additionally, to explore the structural basis for enhanced NF-κB binding to CD40 promoter DNA by tyrosine phosphorylation, an analog of NF-κB p50 containing pTyr at both positions 60 and 82 is prepared. The results reflected changes in the ability of the modified NF-κBs containing an altered p50 subunit to bind to CD40 promoter DNA in vitro, and to direct the synthesis of CD40 in cellulo.

Microglia exhibit targeted responses to different stimuli, including lipids, which can differ depending on the environmental conditions they encounter. These responses involve inflammatory mediators and are crucial for maintaining brain homeostasis. This study investigated whether inflammatory, metabolic, and phagocytic responses of microglia to the saturated fatty acid palmitate depend on extracellular glucose concentrations. BV2 microglial cells were cultured in low glucose (LG; 5.5mmol/L) or high glucose (HG; 25mmol/L) concentrations, and then exposed to palmitate (100 or 200µmol/L) or vehicle for 24h. Under HG, palmitate decreased cell viability, which was accompanied by an increase in inflammatory markers, which are associated with an activated state. Additionally, palmitate induced higher expression of genes related to lipid metabolism in both LG and HG, without affecting enzymes linked to glucose metabolism. HG condition led to an increase in the oxygen consumption rate (OCR) and glycolytic flux (i.e., extracellular medium acidification) compared to LG-cultured cells, with palmitate reducing OCR and glycolytic flux in both conditions. The short-chain fatty acid butyrate did not prevent palmitate-induced mitochondrial dysfunction in BV2 cells. In primary microglia, palmitate did not affect mitochondria density and cargo metabolism. Altogether, our results indicate that BV2 cells are prone to palmitate-induced stress on viability assays under HG but not LG in the medium.

Hypertriglyceridemia (HTG), particularly in combined hyperlipidemia, increases risk for atherosclerotic cardiovascular disease, but the underlying mechanisms remain incompletely understood. We sought to determine contributions of circulating monocytes to atherosclerosis associated with HTG in combined hyperlipidemia, created by transgenic expression of human apoCIII in Ldlr-/- mice (Ldlr-/-ApoCIIItg) fed Western high-fat diet (WD). Tissue culture with THP-1 and primary human monocytes was used to examine effects of triglyceride (TG)-rich lipoproteins on monocytes. Ldlr-/-ApoCIIItg mice were also treated with apoCIII antisense oligonucleotide (ASO) and examined for foamy monocytes and atherosclerosis. Compared to Ldlr-/- mice, Ldlr-/-ApoCIIItg mice fed WD had early and persistent increases in lipid accumulation within monocytes and enhanced atherosclerosis. Ldlr-/-ApoCIIItg mice versus Ldlr-/- mice had higher levels of CD11c, CD36, and cytokines in foamy monocytes, with increases in foamy monocyte adhesion to vascular cell adhesion molecule-1 and oxidized LDL uptake. Monocytes took up TG-rich lipoprotein invivo and invitro and changed phenotypes. Foamy monocytes infiltrated into atherosclerotic lesions, and specific and sustained depletion of CD11c+ (foamy) monocytes profoundly reduced atherosclerosis in Ldlr-/-ApoCIIItg mice on WD. Treatment with apoCIII ASO lowered plasma TG and cholesterol levels, improved foamy monocyte phenotypes, and reduced atherosclerosis in Ldlr-/-ApoCIIItg mice. In conclusion, HTG in combined hyperlipidemia accelerates atherosclerosis, in part, by increasing foamy monocyte formation and infiltration into atherosclerotic plaques. Treatment with apoCIII ASO is a potential new therapy for improving monocyte phenotypes and reducing atherosclerosis in combined hyperlipidemia.

<p>Individual complexes of the mitochondrial oxidative phosphorylation system (OXPHOS) are not linked solely by their function; they also share dependencies at the maintenance/assembly level, where one complex depends on the presence of a different individual complex. Despite the relevance of this ‘interdependence’ behavior for mitochondrial diseases, its true nature remains elusive. To understand the mechanism that can explain this phenomenon, we examined the consequences of the aberration of different OXPHOS complexes in human cells. We demonstrate here that complete disruption of each of the OXPHOS complexes resulted in a perturbation in energy deficiency sensing pathways, including the integrated stress response (ISR) pathway. The secondary decrease of complex I (cI) level was triggered by both complex IV and complex V deficiency, and it was independent of ISR signaling. On the other hand, we identified the unifying mechanism behind cI downregulation in the downregulation of mitochondrial ribosomal proteins and, thus, mitochondrial translation. We conclude that the secondary cI defect is due to mitochondrial protein synthesis attenuation, while the responsible signaling pathways could differ based on the origin of the OXPHOS defect.<i></i><i></i></p>

N-Methylated amino acids are constituents of natural bioactive peptides and proteins. Nα-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing Nα-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded Nα-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of Nα-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.

BackgroundNanoparticles represent one of the most important innovations in the medical field. Among nanocarriers, polymeric nanoparticles (PNPs) attracted much attention due to their biodegradability, biocompatibility, and capacity to increase efficacy and safety of encapsulated drugs. Another important improvement in the use of nanoparticles as delivery systems is the conjugation of a targeting agent that enables the nanoparticles to accumulate in a specific tissue. Despite these advantages, the clinical translation of therapeutic approaches based on nanoparticles is prevented by their interactions with blood proteins. In fact, the so-formed protein corona (PC) drastically alters the biological identity of the particles. Adsorbed activated proteins of the complement cascade play a pivotal role in the clearance of nanoparticles, making them more easily recognized by macrophages, leading to their rapid elimination from the bloodstream and limiting their efficacy. Since the mouse is the most used preclinical model for human disease, this work compared human and mouse PC formed on untargeted PNPs (uPNPs) and targeted PNPs (tPNPs), paying particular attention to complement activation.ResultsMouse and human serum proteins adsorbed differently to PNPs. The differences in the binding of mouse complement proteins are minimal, whereas human complement components strongly distinguish the two particles. This is probably due to the human origin of the Fc portion of the antibody used as targeting agent on tPNPs. tPNPs and uPNPs mainly activate complement via the classical and alternative pathways, respectively, but this pattern did not affect their binding and internalization in macrophages and only a limited consumption of the activity of the human complement system was documented.ConclusionsThe results clearly indicate the presence of complement proteins on PNPs surface but partially derived from an unspecific deposition rather than an effective complement activation. The presence of a targeting antibody favors the activation of the classical pathway, but its absence allows an increased activation of the alternative pathway. This results in similar opsonization of both PNPs and similar phagocytosis by macrophages, without an impairment of the activity of circulating complement system and, consequently, not enhancing the susceptibility to infection.Graphical abstract

BACKGROUND:Cardiac valve disease is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe cardiac valve disease require surgery. PROX1 (prospero-related homeobox transcription factor 1) and FOXC2 (Forkhead box C2 transcription factor) are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known.METHODS:We used histology, electron microscopy, and echocardiography to investigate the structure and functioning of heart valves from Prox1ΔVEC mice in which Prox1 was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models, and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse and aortic valve insufficiency.RESULTS:Histological analysis revealed that the aortic and mitral valves of Prox1ΔVEC mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of Prox1ΔVEC mice are stenotic. FOXC2 was downregulated and PDGF-B (platelet-derived growth factor-B) was upregulated in the VECs of Prox1ΔVEC mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of Prox1ΔVEC mice. PDGF-B was also increased in mice lacking FOXC2 and in human mitral valve prolapse and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of Prox1ΔVEC mice.CONCLUSIONS:PROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.