A case of giant cell arteritis associated with culture-proven Coxiella burnetii aortitis.

A case of proven Coxiella burnetii aortitis, possibly associated with giant cell arteritis (GCA), is reported. A 72-year-old man, who is a hunter, presented with weight loss, fever, jaw claudication, and hardened temporal arteries associated with a persistent inflammatory syndrome and arteritis of the whole aorta, including the brachiocephalic arteries, as seen on 18F-fluorodeoxyglucose positron emission tomography/computed tomography. The diagnosis of GCA was retained, and treatment with prednisolone was started. Given the aneurysm of the abdominal aorta, the patient underwent replacement of the abdominal aorta with an allograft. Histology showed intense chronic arteritis attributed to atherosclerosis with dissection. However, Coxiella burnetii infection was confirmed by serology and then by culture and molecular biology on the surgical specimen. A combination of hydroxychloroquine and doxycycline was added to tapered prednisolone and the outcome was favourable.

Evaluation of HIV protease inhibitor use and the risk of sudden death or nonhemorrhagic stroke.

Concerns have arisen about possible effects of protease inhibitors (PIs) on cardiac conductivity. We found no significant association between current or recent PI exposure and sudden death or nonhemorrhagic stroke (adjusted rate ratio, 1.22; 95% confidence interval, .95-1.57), whereas cumulative exposure to PIs was associated with an increased risk (adjusted rate ratio, 1.06 per year of exposure; 95% confidence interval, 1.01-1.11).

Rates of cardiovascular disease following smoking cessation in patients with HIV infection: results from the D:A:D study(*).

OBJECTIVES: The aim of the study was to estimate the rates of cardiovascular disease (CVD) events after stopping smoking in patients with HIV infection. METHODS: Patients who reported smoking status and no previous CVD prior to enrolment in the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study were included in this study. Smoking status is collected at each visit as current smoker (yes/no) and ever smoker (yes/no). Time since stopping smoking was calculated for persons who had reported current smoking during follow-up and no current smoking subsequently. Endpoints were: myocardial infarction (MI); coronary heart disease (CHD: MI plus invasive coronary artery procedure or death from other CHD); CVD (CHD plus carotid artery endarterectomy or stroke); and all-cause mortality. Event rates were calculated for never, previous and current smokers, and smokers who stopped during follow-up. Incidence rate ratios (IRRs) were determined using Poisson regression adjusted for age, sex, cohort, calendar year, family history of CVD, diabetes, lipids, blood pressure and antiretroviral treatment. RESULTS: A total of 27 136 patients had smoking status reported, with totals of 432, 600, 746 and 1902 MI, CHD, CVD and mortality events, respectively. The adjusted IRR of CVD in patients who stopped smoking during follow-up decreased from 2.32 within the first year of stopping to 1.49 after >3 years compared with those who never smoked. Similar trends were observed for the MI and CHD endpoints. Reductions in risk were less pronounced for all-cause mortality. CONCLUSION: The risk of CVD events in HIV-positive patients decreased with increasing time since stopping smoking. Smoking cessation efforts should be a priority in the management of HIV-positive patients.

Linezolid contributed to Clostridium difficile colitis with fatal outcome.

Linezolid, the first of a new class of antibacterial drugs, the oxazolidinones, has inhibitory activity against a broad range of gram-positive aerobic cocci and also against certain anaerobes. Although diarrhea is one of the most frequently encountered adverse effects of linezolid, Clostridium difficile-related complications are very uncommon. One case of fatal C. difficile colitis in a patient with spondylodiscitis, who had received a long-term course of linezolid therapy, is presented. Colitis was able to be exclusively assigned to linezolid. Factors contributing to the colitis are discussed.

Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages.

The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Q(beta), which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5' terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3'-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3' end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3' UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3' UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Q(beta) suggests that, in an evolutionary sense, AP205 should be placed between Q(beta) and MS2. A phylogenetic tree for the ssRNA phages is presented.

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: implications for binding requirements.

The coat protein of bacteriophage MS2 is known to bind specifically to an RNA hairpin formed within the MS2 genome. Structurally this hairpin is built up by an RNA double helix interrupted by one unpaired nucleotide and closed by a four-nucleotide loop. We have performed crystallographic studies of complexes between MS2 coat protein capsids and four RNA hairpin variants in order to evaluate the minimal requirements for tight binding to the coat protein and to obtain more information about the three-dimensional structure of these hairpins. An RNA fragment including the four loop nucleotides and a two-base-pair stem but without the unpaired nucleotide is sufficient for binding to the coat protein shell under the conditions used in this study. In contrast, an RNA fragment containing a stem with the unpaired nucleotide but missing the loop nucleotides does not bind to the protein shell.

Crystal structures of MS2 coat protein mutants in complex with wild-type RNA operator fragments.

In MS2 assembly of phage particles results from an interaction between a coat protein dimer and a stem-loop of the RNA genome (the operator hairpin). Amino acid residues Thr45, which is universally conserved among the small RNA phages, and Thr59 are part of the specific RNA binding pocket and interact directly with the RNA; the former through a hydrogen bond, the latter through hydrophobic contacts. The crystal structures of MS2 protein capsids formed by mutants Thr45Ala and Thr59Ser, both with and without the 19 nt wild-type operator hairpin bound, are reported here. The RNA hairpin binds to these mutants in a similar way to its binding to wild-type protein. In a companion paper both mutants are shown to be deficient in RNA binding in an in vivo assay, but in vitro the equilibrium dissociation constant is significantly higher than wild-type for the Thr45Ala mutant. The change in binding affinity of the Thr45Ala mutant is probably a direct consequence of removal of direct hydrogen bonds between the protein and the RNA. The properties of the Thr59Ser mutant are more difficult to explain, but are consistent with a loss of non-polar contact.

Base complementarity in helix 2 of the central pseudoknot in 16S rRNA is essential for ribosome functioning.

Helix 2 of the central pseudoknot structure in Escherichia coli 16S rRNA is formed by a long-distance interaction between nt 17-19 and 918-916, resulting in three base pairs: U17-A918, C18-G917and A19-U916. Previous work has shown that disruption of the central base pair abolishes ribosomal activity. We have mutated the first and last base pairs and tested the mutants for their translational activity in vivo , using a specialized ribosome system. Mutations that disrupt Watson-Crick base pairing result in strongly impaired translational activity. An exception is the mutation U916-->G, creating an A.G pair, which shows almost no decrease in activity. Mutations that maintain base complementarity have little or no impact on translational efficiency. Some of the introduced base pair substitutions substantially alter the stability of helix 2, but this does not influence ribosome functioning, neither at 42 nor at 28 degrees C. Therefore, our results do not support models in which the pseudoknot is periodically disrupted. Rather, the central pseudoknot structure is suggested to function as a permanent structural element necessary for proper organization in the center of the 30S subunit.

The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions.

Crystal structures of two complexes between recombinant MS2 capsids and RNA operator fragments have been determined at 2.7 A resolution. The coat protein of the RNA bacteriophage MS2 is bifunctional; it forms the icosahedral virus shell to protect the viral nucleic acid and it acts as a translational repressor by binding with high specificity to a unique site on the RNA, a single stem-loop structure, containing the initiation codon of the gene for the viral replicase. In order to determine the structure of these protein-RNA complexes, we have used chemically synthesized variants of the stem-loop fragment and soaked them into crystals of recombinant capsids. The RNA stem-loop, as bound to the protein, forms a crescent-like structure and interacts with the surface of the beta-sheet of a coat protein dimer. It makes protein contacts with seven phosphate groups on the 5' side of the stem-loop, with a pyrimidine base at position -5, which stacks onto a tyrosine, and with two exposed adenine bases, one in the loop and one at a bulge in the stem. Replacement of the wild-type uridine with a cytosine at position -5 increases the affinity of the RNA to the dimer significantly. The complex with RNA stem-loop having cytosine at this position differs from that of the wild-type complex mainly by having one extra intramolecular RNA interaction and one extra water-mediated hydrogen bond.

Crystal structures of MS2 capsids with mutations in the subunit FG loop.

The loop between the F and G beta strands (FG loop) of the bacteriophage MS2 coat protein subunit forms inter-subunit contacts around the 5-fold and 3-fold (quasi 6-fold) axes of the T=3 protein shell. In capsids, the loop is found in two very different conformations, one in B subunits, which form the 5-fold contact, and one in A and C subunits, which form the quasi 6-fold contact. One proline residue, Pro78, is strictly conserved in the coat protein of all related bacteriophages, and in the case of MS2 this proline residue is preceded by a cis peptide bond in the B subunit. In order to probe the role of the FG loop in capsid assembly, we have determined the crystal structures of two MS2 capsids, formed by coat proteins with mutations at two positions in the FG loop, P78N or E76D. These mutants show conformational changes in the FG loops that explain the reduced temperature stability of the capsids. The P78N mutant has a normal trans peptide bond at position 78.