Longitudinal determination of BNT162b2 vaccine induced strongly binding SARS-CoV-2 IgG antibodies in a cohort of Romanian healthcare workers.

Mass vaccination against the disease caused by the novel coronavirus (COVID-19) was a crucial step in slowing the spread of SARS-CoV-2 in 2021. Even in the face of new variants, it still remains extremely important for reducing hospitalizations and COVID-19 deaths. In order to better understand the short- and long-term dynamics of humoral immune response, we present a longitudinal analysis of post-vaccination IgG levels in a cohort of 166 Romanian healthcare workers vaccinated with BNT162b2 with weekly follow-up until 35 days past the first dose and monthly follow-up up to 6 months post-vaccination. A subset of the patients continued with follow-up after 6 months and either received a booster dose or got infected during the Delta wave in Romania. Tests were carried out on 1694 samples using a CE-marked IgG ELISA assay developed in-house, containing S1 and N antigens of the wild type virus. Participants infected with SARS-CoV-2 before vaccination mount a quick immune response, reaching peak IgG levels two weeks after the first dose, while IgG levels of previously uninfected participants mount gradually, increasing abruptly after the second dose. Overall higher IgG levels are maintained for the previously infected group throughout the six month primary observation period (e.g. 36-65 days after the first dose, the median value in the previously infected group is 5.29 AU/ml, versus 3.58 AU/ml in the infection naïve group, p less than 0.001). The decrease of IgG levels is gradual, with lower median values in the infection naïve cohort even 7-8 months after vaccination, compared to the previously infected cohort (0.7 AU/ml versus 1.29 AU/ml, p = 0.006). Administration of a booster dose yielded higher median IgG antibody levels than post second dose in the infection naïve group and comparable levels in the previously infected group.

Extremely rare "daisy-like" crystals in urinary sediment can be due to a sampling artifact.

Manual urine sediment analysis of a sample obtained from a 5 year old child by our clinical diagnostics laboratory revealed abundant "daisy-like" crystals, which have been first described in 2004 and found to be extremely rare in a follow-up publication by the same research group. To date only 12 samples have been described in the literature containing such crystals. Upon further investigation on how the sample was obtained, we were able to reproduce the process without any biological specimen involved. We show that these crystals are in fact contaminants from the sample collection recipient itself, which was a glass recipient sterilized by the patient's family the night before sample collection, by boiling water with high calcium and magnesium content (hard water), and letting the recipient cool overnight with the water in it. The obtained abundant "daisy-like" crystals readily dissolve in acidic environment, and are composed most likely of mostly calcium carbonate. Sampling artifacts are therefore a possible explanation for at least some of the previously described "daisy-like" urinary crystals, as the formation of such crystals does not need to involve any biomolecules, only hard water and appropriate crystallization conditions for the limescale in it.

Packing of transmembrane domain 2 of carnitine palmitoyltransferase-1A affects oligomerization and malonyl-CoA sensitivity of the mitochondrial outer membrane protein.

The purpose of this study was to investigate the sequence-dependence of oligomerization of transmembrane domain 2 (TM2) of rat carnitine palmitoyltransferase 1A (rCPT1A), to elucidate the role of this domain in the function of the full-length enzyme. Oligomerization of TM2 was studied qualitatively using complementary genetic assays that facilitate measurement of helix-helix interactions in the Escherichia coli inner membrane, and multiple quantitative biophysical methods. The effects of TM2-mutations on oligomerization and malonyl-CoA inhibition of the full-length enzyme (expressed in the yeast Pichia pastoris) were quantified. Changes designed to disrupt close-packing of the GXXXG(A) motifs reduced the oligomeric state of the corresponding TM2 peptides from hexamer to trimer (or lower), a reduction also observed on mutation of the TM2 sequence in the full-length enzyme. Disruption of these GXXXG(A) motifs had a parallel effect on the malonyl-CoA sensitivity of rCPT1A, reducing the IC(50) from 30.3 ± 5.0 to 3.0 ± 0.6 µM. For all measurements, wild-type rCPT1A was used as a control alongside various appropriate (e.g., molecular mass) standards. Our results suggest that sequence-determined, TM2-mediated oligomerization is likely to be involved in the modulation of malonyl-CoA inhibition of CPT1A in response to short- and long-term changes in protein-protein and protein-lipid interactions that occur in vivo.

Self-association of transmembrane domain 2 (TM2), but not TM1, in carnitine palmitoyltransferase 1A: role of GXXXG(A) motifs.

Carnitine palmitoyltransferase 1 (CPT1) controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for beta-oxidation and has been reported to exist as an oligomer. We have investigated the in vivo oligomerization of full-length rat CPT1A (rCPT1A) along with those of the N-terminal truncation/deletion mutants Delta(1-82), Delta(1-18), and Delta(19-30) expressed in yeast mitochondria. The data indicate that in liver mitochondria in vivo CPT1A exists as a hexamer but that during preparation and storage of mitochondria the order of oligomerization is rapidly reduced to the trimer, such that a mixture of hexamer and trimer is observed in isolated mitochondria in vitro. Mutants bearing deletions of different segments of the N terminus (including the more N-terminal of the two transmembrane domains) have the same pattern of oligomerization when expressed in yeast mitochondria. The self-association of the individual rCPT1A transmembrane (TM) domains (TM1, TM2) was also studied using the TOXCAT assay (which measures TM self-association in the Escherichia coli inner membrane). There was minimal self-association of the sequence corresponding to TM1 but significant self-association of TM2 in TOXCAT. Chemical cross-linking and analytical ultracentrifugation of a TM2-derived synthetic peptide showed oligomerization with a similar trimer/hexamer equilibrium to that observed for native rCPT1A in isolated mitochondria. Therefore, there was a correlation between the oligomerization behavior of TM2 peptide and that of the full-length protein. In silico molecular modeling of rCPT1A TM2 highlighted the favorable orientation of GXXXG and GXXXA motifs in the formation of the TM2 hexamer.