Antibiotic resistance evolved via inactivation of a ribosomal RNA methylating enzyme.

Modifications of the bacterial ribosome regulate the function of the ribosome and modulate its susceptibility to antibiotics. By modifying a highly conserved adenosine A2503 in 23S rRNA, methylating enzyme Cfr confers resistance to a range of ribosome-targeting antibiotics. The same adenosine is also methylated by RlmN, an enzyme widely distributed among bacteria. While RlmN modifies C2, Cfr modifies the C8 position of A2503. Shared nucleotide substrate and phylogenetic relationship between RlmN and Cfr prompted us to investigate evolutionary origin of antibiotic resistance in this enzyme family. Using directed evolution of RlmN under antibiotic selection, we obtained RlmN variants that mediate low-level resistance. Surprisingly, these variants confer resistance not through the Cfr-like C8 methylation, but via inhibition of the endogenous RlmN C2 methylation of A2503. Detection of RlmN inactivating mutations in clinical resistance isolates suggests that the mechanism used by the in vitro evolved variants is also relevant in a clinical setting. Additionally, as indicated by a phylogenetic analysis, it appears that Cfr did not diverge from the RlmN family but from another distinct family of predicted radical SAM methylating enzymes whose function remains unknown

Assessment of the nucleotide modifications in the high-resolution cryo-electron microscopy structure of the Escherichia coli 50S subunit.

Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides' positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible

Evaluation of Periodontal Status in HIV Infected Persons in Croatia

A number of periodontal changes have been associated with human immunodeficiency virus (HIV) infection, however our knowledge of the epidemiology, microbiology, host response and natural history of these conditions remains limited. Therefore, the aim of our study was the assessment of possible differences in periodontal status of HIV infected subjects when compared with healthy controls matched for age, gender and smoking habit in Croatian population. Assessment included measurement of plaque accumulation using aproximal plaque index, measurement of gingival inflammation by use of sulcus bleeding index, pocket depth, gingival recession as well as the number of decayed, missing and filled teeth in 25 HIV infected subjects (age range 22–61, X=40.8 years) in comparison with 25 healthy controls (age range 20–62, X=40.9 years). Statistical analysis was performed by use of descriptive statistics and Mann-Whitney U test showed significantly increased level of inflammation of the marginal gingiva in HIV infected subjects when compared to the controls (p<0.002). Significantly increased mean values of periodontal pockets (p<0.002) and the deepest periodontal pocket (p<0.003) were also observed when HIV infected subjects were compared to the healthy controls. In HIV infected subjects there was significant increase in the number of decayed, missing and decrease in the number of filled teeth (p<0.002; p<0.002; p<0.009, respectively). The results of this study once again highlight the need for more prevalent periodontal check-ups and treatments in HIV infected subjects

Evaluation of Periodontal Status in HIV Infected Persons in Croatia

A number of periodontal changes have been associated with human immunodeficiency virus (HIV) infection, however our knowledge of the epidemiology, microbiology, host response and natural history of these conditions remains limited. Therefore, the aim of our study was the assessment of possible differences in periodontal status of HIV infected subjects when compared with healthy controls matched for age, gender and smoking habit in Croatian population. Assessment included measurement of plaque accumulation using aproximal plaque index, measurement of gingival inflammation by use of sulcus bleeding index, pocket depth, gingival recession as well as the number of decayed, missing and filled teeth in 25 HIV infected subjects (age range 22–61, X=40.8 years) in comparison with 25 healthy controls (age range 20–62, X=40.9 years). Statistical analysis was performed by use of descriptive statistics and Mann-Whitney U test showed significantly increased level of inflammation of the marginal gingiva in HIV infected subjects when compared to the controls (p<0.002). Significantly increased mean values of periodontal pockets (p<0.002) and the deepest periodontal pocket (p<0.003) were also observed when HIV infected subjects were compared to the healthy controls. In HIV infected subjects there was significant increase in the number of decayed, missing and decrease in the number of filled teeth (p<0.002; p<0.002; p<0.009, respectively). The results of this study once again highlight the need for more prevalent periodontal check-ups and treatments in HIV infected subjects

Evaluation of periodontal status in HIV infected persons in Croatia [Procjena parodontološkog statusa oboljelih od HIV-a u Republici Hrvatskoj]

A number of periodontal changes have been associated with human immunodeficiency virus (HIV) infection, however our knowledge of the epidemiology, microbiology, host response and natural history of these conditions remains limited. Therefore, the aim of our study was the assessment of possible differences in periodontal status of HIV infected subjects when compared with healthy controls matched for age, gender and smoking habit in Croatian population. Assessment included measurement of plaque accumulation using approximal plaque index, measurement of gingival inflammation by use of sulcus bleeding index, pocket depth, gingival recession as well as the number of decayed, missing and filled teeth in 25 HIV infected subjects (age range 22-61, X = 40.8 years) in comparison with 25 healthy controls (age range 20-62, X = 40.9 years). Statistical analysis was performed by use of descriptive statistics and Mann-Whitney U test showed significantly increased level of inflammation of the marginal gingiva in HIV infected subjects when compared to the controls (p < 0.002). Significantly increased mean values of periodontal pockets (p < 0.002) and the deepest periodontal pocket (p < 0.003) were also observed when HIV infected subjects were compared to the healthy controls. In HIV infected subjects there was significant increase in the number of decayed, missing and decrease in the number of filled teeth (p < 0.002; p < 0.002; p < 0.009, respectively). The results of this study once again highlight the need for more prevalent periodontal check-ups and treatments in HIV infected subjects

Synthesis of radiolabeled nicotinamide cofactors from labeled pyridines:versatile probes for enzyme kinetics

(14)C-labeled nicotinamide cofactors are widely employed in biomedical investigations, e.g. to delineate metabolic pathways, to elucidate enzymatic mechanisms, or as substrates in kinetic isotope effect (KIE) experiments. The (14)C-label has generally been located remote from the reactive position, frequently at the adenine ring. Rising costs of commercial precursors, and disruptions in the availability of enzymes required for established syntheses, have recently made the preparation of labeled nicotinamides such as [Ad-(14)C]-NADPH inviable. Here, we report the syntheses and characterization of several alternatives: [carbonyl-(14)C]-NADPH, 4R-[carbonyl-(14)C, 4-(2)H]-NADPH, and [carbonyl-(14)C, 4-(2)H(2)]-NADPH. The new procedures utilize [carbonyl-(14)C]-nicotinamide as starting material, as it is significantly cheaper than other commercial (14)C-precursors of NADPH, and require only one commercially available enzyme to prepare NAD(P)(+) and NAD(P)H. The proximity of carbonyl-(14)C to the reactive center raises the risk of an inopportune (14)C-isotope effect. This concern has been alleviated via competitive KIE measurements with Escherichia coli dihydrofolate reductase (EcDHFR), that use this specific carbonyl-(14)C NADPH. A combination of binding isotope effect and KIE measurements yielded no significant (12)C/(14)C isotope effect at the amide carbonyl (KIE = 1.003 ± 0.004). The reported procedure provides a high-yield, high-purity and cost-effective alternative to labeled nicotinamide cofactors synthesized by previously published routes

High-resolution cryo-electron microscopy structure of the Escherichia coli 50S subunit and validation of nucleotide modifications

ABSTRACT Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides’ positions, a task that has previously been accomplished by X-ray crystallography. Here we present a cryo-electron microscopy (cryo-EM) structure of Escherichia coli ( E. coli ) 50S subunit at an average resolution of 2.2Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg 2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for identification of post-transcriptional rRNA modifications using a cryo-EM map. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible

The advantages of video-assisted thoracoscopic surgery compared to thoracic drainage in the treatment of primary spontaneous pneumothorax

Introduction/Objective. The aim of the study is to analyze the treatment of spontaneous pneumothorax (PSP) through our 10-year experience. Methods. The study included 67 patients with PSP treated with video-assisted thoracoscopic surgery (VATS) or with thoracic drainage (TD) in the Clinic for Chest Surgery at the Military Medical Academy in Belgrade, Serbia in the 2008–2017 period. Results. PSP patients with VATS were younger (33.2 ± 16.4 vs. 45.5 ± 21.5 years, p = 0.010), and both groups consisted mainly of males (69.2% vs. 78%). VATS-treated patients were hospitalized shorter and wore drains (p < 0.001, p < 0.002). Recurrence after treatment was more common after TD (61% vs. 3.8%) and in most cases it was treated with VATS (92%). The incidence of intraoperative complications is similar between groups (p = 0.599, p = 0.636, p = 0.311, p = 0.388, p = 0.388, respectively). Pain was more common in TD (p < 0.001). The early complications in the group of patients treated with TD occurred more often (p < 0.001, p < 0.001), without significant difference in the incidence of pleura infections and intercostal blockade between groups (p = 0.388, p = 0.388, respectively). Patients treated for PSP with the VATS method came to the control follow-up later, compared to patients treated with TD (p < 0.001). Conclusion. VATS proved to be efficient, which was reflected in the optimal duration of surgery, length of hospitalization, tolerable postoperative pain and satisfactory cosmetic effect, and postsurgical relapse in only one case