IMPORTANCEFewer than 50% of kidney transplant recipients (KTRs) develop antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Preliminary data suggest that a heterologous vaccination, combining mRNA and viral vector vaccines, may increase immunogenicity.OBJECTIVE To assess the effectiveness of a third dose of an mRNA vs a vector vaccine in KTRs who did not have antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. DESIGN, SETTING, AND PARTICIPANTSThis was a single center, single-blinded, 1:1 randomized clinical trial of a third dose of vaccine against SARS-CoV-2, conducted from June 15 to August 16, 2021, in 201 KTRs who had not developed SARS-CoV-2 spike protein antibodies after 2 doses of an mRNA vaccine. Data analyses were performed from August 17 to August 31, 2021.INTERVENTIONS mRNA (BNT162b2 or mRNA-1273) or vector (Ad26COVS1) as a third dose of a SARS-CoV-2 vaccine. MAIN OUTCOMES AND MEASURESThe primary study end point was seroconversion after 4 weeks (29-42 days) following the third vaccine dose. Secondary end points included neutralizing antibodies and T-cell response assessed by interferon-γ release assays (IGRA). In addition, the association of patient characteristics and vaccine response was assessed using logistic regression, and the reactogenicity of the vaccines was compared. RESULTS Among the study population of 197 kidney transplant recipients (mean [SD] age, 61.2 [12.4] years; 82 [42%] women), 39% developed SARS-CoV-2 antibodies after the third vaccine. There was no statistically significant difference between groups, with an antibody response rate of 35% and 42% for the mRNA and vector vaccines, respectively. Only 22% of seroconverted patients had neutralizing antibodies. Similarly, T-cell response assessed by IGRA was low with only 17 patients showing a positive response after the third vaccination. Receiving nontriple immunosuppression (odds ratio [OR], 3.59; 95% CI, 1.33-10.75), longer time after kidney transplant (OR, 1.44; 95% CI, 1.15-1.83, per doubling of years), and torque teno virus plasma levels (OR, 0.92; 95% CI, 0.88-0.96, per doubling of levels) were associated with vaccine response. The third dose of an mRNA vaccine was associated with a higher frequency of local pain at the injection site compared with the vector vaccine, while systemic symptoms were comparable between groups.CONCLUSIONS AND RELEVANCE This randomized clinical trial found that 39% of KTRs without an immune response against SARS-CoV-2 after 2 doses of an mRNA vaccine developed antibodies against the SARS-CoV-2 spike protein 4 weeks after a third dose of an mRNA or a vector vaccine. The heterologous vaccination strategy with a vector-based vaccine was well tolerated and safe but not significantly better than the homologous mRNA-based strategy.

Creatine kinase (CK) isoenzymes, specifically located at places of energy demand and energy production, are linked by a phosphocreatine/creatine (PCr/Cr) circuit, found in cells with intermittently high energy demands. Cytosolic CKs, in close conjunction with Ca(2+)-pumps, play a crucial role for the energetics of Ca(2+)-homeostasis. Mitochondrial Mi-CK, a cuboidal-shaped octamer with a central channel, binds and crosslinks mitochondrial membranes and forms a functionally coupled microcompartment with porin and adenine nucleotide translocase for vectorial export of PCr into the cytosol. The CK system is regulated by AMP-activated protein kinase via PCr/Cr and ATP/AMP ratios. Mi-CK stabilizes and cross-links cristae- or inner/outer membranes to form parallel membrane stacks and, if overexpressed due to creatine depletion or cellular energy stress, forms those crystalline intramitochondrial inclusions seen in some mitochondrial cytopathy patients. Mi-CK is a prime target for free radical damage by peroxynitrite. Mi-CK octamers, together with CK substrates have a marked stabilizing and protective effect against mitochondrial permeability transition pore opening, thus providing a rationale for creatine supplementation of patients with neuromuscular and neurodegenerative diseases.

ABSTRACT:The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents -NO 2 , -CN, and CH 3 CO-), 3S-C 8 -, and 3′-dephospho-3S-C 8 CoA. These analogues lack a C-H and cannot undergo R, -dehydrogenation. Instead they deprotonate at RC-H at pH g 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pK a s of the corresponding phenylacetone analogues were determined as ≈13.6 (-NO 2 ), ≈14.5 (-CN), and ≈14.6 (CH 3 CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo RC-H deprotonation. While the extent of deprotonation varies, the anionic products form charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (K d ) of p-NO 2 -phenylacetyl-CoA (4NPA-CoA), 3S-C 8 -CoA, and 3′-dephospho-3S-C 8 CoA, four pK a s at ≈5, ≈6, ≈7.3, and ≈8 were identified. They were assigned to the following ionizations: (a) pK a ≈5, ligand (L-H) in the MCADH∼ligand complex; (b) pK a ≈6, Glu376-COOH in uncomplexed MCADH; (c) pK a ≈7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK ≈8, Glu99-COOH in the MCADH∼4NPA-CoA complex. The pK a ≈6 (b) is not significantly affected in the MCADH∼4NPA-CoA complex, but it is increased by g1 pK unit in that with 3S-C 8 CoA and further in the presence of C 8 -CoA, the best substrate. The RC-H pK a s of 4NPA-CoA, of 3S-C 8 -CoA, and of 3′-dephospho-3S-C 8 CoA in the complex with MCADH are ≈5, ≈5, and ≈6. Compared to those of the free species these pK a values are therefore lowered by 8 to g11 pH units (50 to g 65 kJ mol -1 ) and are close to the pK a of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2′-OH of FAD and Glu376-NH. It is concluded that the pK a shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9-13 pK units. With 4NPA-CoA and MCADH, RC-H abstraction is fast (k app ≈55 s -1 at pH 7.5 and 25°C, deuterium isotope effect ≈1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6-9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pK a s of ≈5 and ≈7.3, which correspond to those determined in static experiments.Acyl-CoA dehydrogenases catalyze the R, -dehydrogenation of fatty acid acyl-CoA conjugates to their corresponding enoyl-CoA products; the redox equivalents formed in this reaction are transferred to electron transferring flavoprotein and further to the respiratory chain (1, 2). A peculiarity of the R, -dehydrogenation reaction is that it involves the concomitant fission of two kinetically stable C-H bonds. In the past, studies with medium-chain acyl-CoA dehydrogenase ...

Creatine kinase (CK), catalyzing the reversible trans-phosphorylation between ATP and creatine, plays a key role in the energy metabolism of cells with high and fluctuating energy requirements. We have solved the X-ray structure of octameric human ubiquitous mitochondrial CK (uMtCK) at 2.7 A resolution, representing the first human CK structure. The structure is very similar to the previously determined structure of sarcomeric mitochondrial CK (sMtCK). The cuboidal octamer has 422 point group symmetry with four dimers arranged along the fourfold axis and a central channel of approximately 20 A diameter, which extends through the whole octamer. Structural differences with respect to sMtCK are found in isoform-specific regions important for octamer formation and membrane binding. Octameric uMtCK is stabilized by numerous additional polar interactions between the N-termini of neighboring dimers, which extend into the central channel and form clamp-like structures, and by a pair of salt bridges in the hydrophobic interaction patch. The five C-terminal residues of uMtCK, carrying positive charges likely to be involved in phospholipid-binding, are poorly defined by electron density, indicating a more flexible region than the corresponding one in sMtCK. The structural differences between uMtCK and sMtCK are consistent with biochemical studies on octamer stability and membrane binding of the two isoforms.