Demographic and clinical characteristics of primary care patients vaccinated against COVID-19 in spring 2021.

AIM: The aim of this study was to examine the age distribution and comorbidities of individuals vaccinated in primary care practices in April and May 2021. MATERIALS AND METHODS: The analysis was based on data from the IQVIA Disease Analyzer database and included 245,948 patients who received their first COVID-19 vaccination from one of 820 family medicine practices in April or May 2021. RESULTS: 93.6% of individuals received a vaccination based on general indication, 6.2% based on occupational indication, and 0.2% were nursing home residents. Men were 3.5 years younger on average than women (59.2 vs. 62.7 years). 54% of women and 52% of men younger than 60 years had at least one diagnosis from the priority list. Hypertension was the most common diagnosis (23.6% of men and 20.7% of women). In men, chronic respiratory diseases such as COPD or asthma were the second most common diagnosis (11.0%), while in women, depression (17.0%) was the second most common diagnosis. CONCLUSION: In the first 2 months of vaccination in general practices, most patients vaccinated were either elderly or chronically ill. Further studies comparing the characteristics of vaccinated and unvaccinated individuals would also be of great epidemiological relevance.

Crystal structures of SnoaL2 and AclR: two putative hydroxylases in the biosynthesis of aromatic polyketide antibiotics.

SnoaL2 and AclR are homologous enzymes in the biosynthesis of the aromatic polyketides nogalamycin in Streptomyces nogalater and cinerubin in Streptomyces galilaeus, respectively. Evidence obtained from gene transfer experiments suggested that SnoaL2 catalyzes the hydroxylation of the C-1 carbon atom of the polyketide chain. Here we show that AclR is also involved in the production of 1-hydroxylated anthracyclines in vivo. The three-dimensional structure of SnoaL2 has been determined by multi-wavelength anomalous diffraction to 2.5A resolution, and that of AclR to 1.8A resolution using molecular replacement. Both enzymes are dimers in solution and in the crystal. The fold of the enzyme subunits consists of an alpha+beta barrel. The dimer interface is formed by packing of the beta-sheets from the two subunits against each other. In the interior of the alpha+beta barrel a hydrophobic cavity is formed that most likely binds the substrate and harbors the active site. The subunit fold and the architecture of the active site in SnoaL2 and AclR are similar to that of the polyketide cyclases SnoaL and AknH; however, they show completely different quaternary structures. A comparison of the active site pockets of the putative hydroxylases AclR and SnoaL2 with those of bona fide polyketide cyclases reveals distinct differences in amino acids lining the cavity that might be responsible for the switch in chemistry. The moderate degree of sequence similarity and the preservation of the three-dimensional fold of the polypeptide chain suggest that these enzymes are evolutionary related. Members of this enzyme family appear to have evolved from a common protein scaffold by divergent evolution to catalyze reactions chemically as diverse as aldol condensation and hydroxylation.

Biochemical coupling of the two nucleotide binding domains of ClpB: covalent linkage is not a prerequisite for chaperone activity.

ClpB cooperates with the DnaK chaperone system in the reactivation of protein from aggregates and is a member of the ATPases associated with a variety of cellular activities (AAA+) protein family. The underlying disaggregation reaction is dependent on ATP hydrolysis at both AAA cassettes of ClpB but the role of each AAA cassette in the reaction cycle is largely unknown. Here we analyze the activity of the separately expressed and purified nucleotide binding domains of ClpB from Thermus thermophilus. The two fragments show different biochemical properties: the first construct is inactive in ATPase activity assays and binds nucleotides weakly, the second construct has a very high ATPase activity and interacts tightly with nucleotides. Both individual fragments have lost their chaperone function and are not able to form large oligomers. When combined in solution, however, the two fragments form a stable heterodimer with oligomerization capacities equivalent to wild-type ClpB. This non-covalent complex regains activity in reactivating protein aggregates in cooperation with the DnaK chaperone system. Upon complex formation the ATPase activity of fragment 2 is reduced to a level similar to wild-type ClpB. Hence functional ClpB can be reassembled from its isolated AAA cassettes showing that covalent linkage of these domains is not a prerequisite for the chaperone activity. The observation that the intrinsically high ATPase activity of AAA2 is suppressed by AAA1 allows a hypothetical assignment of their mechanistic function. Whereas the energy gained upon ATP hydrolysis at the AAA2 is likely to drive a conformational change of the structure of ClpB, AAA1 might function as a regulator of the chaperone cycle.

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit.

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.

A chaperone network for the resolubilization of protein aggregates: direct interaction of ClpB and DnaK.

The molecular chaperones ClpB (Hsp104) and DnaK (Hsp70) co-operate in the ATP-dependent resolubilization of aggregated proteins. A sequential mechanism has been proposed for this reaction; however, the mechanism and the functional interplay between both chaperones remain poorly defined. Here, we show for the first time that complex formation of ClpB and DnaK can be detected by using various types of affinity chromatography methods. The finding that the DnaK chaperone of Escherichia coli is not co-operating with ClpB from Thermus thermophilus further strengthens the specificity of this complex. The affinity of the complex is weak and interaction between both chaperones is nucleotide-dependent. The presence of ADP, which is shown to cause dissociation of ClpB(Tth), as well as ClpB deletion mutants incapable of oligomer formation prevent ClpB-DnaK complex formation. The experiments presented indicate a correlation between the oligomeric state of ClpB and its ability to interact with DnaK. The chaperone complex described here might facilitate transfer of intermediates between ClpB and DnaK during refolding of substrates from aggregates.

The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity.

ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system. The mechanism of protein reactivation and interaction with the DnaK system remains unclear. ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior. The role of the N-terminal domain that precedes the first nucleotide binding domain is largely unknown. We purified and characterized an N-terminal shortened ClpB variant (ClpBDeltaN; amino acids 140-854), which remained active in refolding assays with three different substrate proteins. In addition the N-terminal truncation did not significantly change the nucleotide binding affinities, the nucleotide-dependent oligomerization, and the allosteric behavior of the protein. In contrast casein binding and stimulation of the ATPase activity by kappa-casein were affected. These results suggest that the N-terminal domain is not essential for the chaperone function, does not influence the binding of nucleotides, and is not involved in the formation of intermolecular contacts. It contributes to the casein binding site of ClpB, but other substrate proteins do not necessarily interact with the N terminus. This indicates a substantial difference in the binding mode of kappa-casein that is often used as model substrate for ClpB and other possibly more suitable substrate proteins.