Analysis of the Pseudouridimycin Biosynthetic Pathway Provides Insights into the Formation of C-nucleoside Antibiotics.

Pseudouridimycin (PUM) is a selective nucleoside-analog inhibitor of bacterial RNA polymerase with activity against Gram-positive and Gram-negative bacteria. PUM, produced by Streptomyces sp. ID38640, consists of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 5'-aminopseudouridine. We report the characterization of the PUM gene cluster. Bioinformatic analysis and mutational knockouts of pum genes with analysis of accumulated intermediates, define the PUM biosynthetic pathway. The work provides the first biosynthetic pathway of a C-nucleoside antibiotic and reveals three unexpected features: production of free pseudouridine by the dedicated pseudouridine synthase, PumJ; nucleoside activation by specialized oxidoreductases and aminotransferases; and peptide-bond formation by amide ligases. A central role in the PUM biosynthetic pathway is played by the PumJ, which represents a divergent branch within the TruD family of pseudouridine synthases. PumJ-like sequences are associated with diverse gene clusters likely to govern the biosynthesis of different classes of C-nucleoside antibiotics.

Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells.

Objectives: To characterize NAI-107 and related lantibiotics for their in vitro activity against Gram-negative pathogens, alone or in combination with polymyxin, and against non-dividing cells or biofilms of Staphylococcus aureus. NAI-107 was also evaluated for its propensity to select or induce self-resistance in Gram-positive bacteria. Methods: We used MIC determinations and chequerboard experiments to establish the antibacterial activity of the examined compounds against target microorganisms. Time-kill assays were used to evaluate killing of exponential and stationary-phase cells. The effects on biofilms (growth inhibition and biofilm eradication) were evaluated using biofilm-coated pegs. The frequency of spontaneous resistant mutants was evaluated by either direct plating or by continuous sub-culturing at 0.5 × MIC levels, followed by population analysis profiles. Results: The results showed that NAI-107 and its brominated variant are highly active against Neisseria gonorrhoeae and some other fastidious Gram-negative pathogens. Furthermore, all compounds strongly synergized with polymyxin against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, and showed bactericidal activity. Surprisingly, NAI-107 alone was bactericidal against non-dividing A. baumannii cells. Against S. aureus, NAI-107 and related lantibiotics showed strong bactericidal activity against dividing and non-dividing cells. Activity was also observed against S. aureus biofilms. As expected for a lipid II binder, no significant resistance to NAI-107 was observed by direct plating or serial passages. Conclusions: Overall, the results of the current work, along with previously published results on the efficacy of NAI-107 in experimental models of infection, indicate that this lantibiotic represents a promising option in addressing the serious threat of antibiotic resistance.

Allocyclinones, hyperchlorinated angucyclinones from Actinoallomurus.

A screening program on a limited number of strains belonging to the Actinoallomurus genus yielded a series of new angucyclinones. NMR and MS analyses established that these compounds are characterized by an unusual lactone ring and present up to four halogens per molecule, with one congener representing the first natural product containing a trichloromethyl substitution on an aromatic system. Remarkably, this family of metabolites seems to be produced by phylogenetically distinct Actinoallomurus isolates. Because of the unique structural features and wide distribution among Actinoallomurus, we have designated these angucyclinones as allocyclinones. Allocyclinones possess interesting activity against different Gram-positive bacteria, including antibiotic-resistant strains, with antibacterial potency increasing with the number of chlorine substituents. The tetrachlorinated compound is the most abundant congener in the allocyclinone complex.

Brominated Variant of the Lantibiotic NAI-107 with Enhanced Antibacterial Potency.

We identified an Actinoallomurus strain producing NAI-107, a chlorinated lantibiotic effective against multidrug-resistant Gram-positive pathogens and previously reported from the distantly related genus Microbispora. Inclusion of KBr in the production medium of either the Actinoallomurus or the Microbispora producer readily afforded brominated variants of NAI-107, which were designated as NAI-108. The other post-translational modifications naturally occurring in this lantibiotic family (i.e., hydroxylation of Pro-14 and C-terminal decarboxylation) were unaffected by the presence of a brominated tryptophan. In addition to being the first example of a bromine-containing lantibiotic, NAI-108 displayed a small but consistent improvement in antibacterial activity against all tested strains. The brominated lantibiotic maintained the same rapid bactericidal activity as NAI-107 but at reduced concentrations, consistent with its increased potency and with the role played by the hydrophobicity of the first lanthionine ring. NAI-108 thus represents an interesting addition to a promising family of potent and effective lantibiotics.

A Derivative of the Thiopeptide GE2270A Highly Selective against Propionibacterium acnes.

A chemical derivative of the thiopeptide GE2270A, designated NAI003, was found to possess a substantially reduced antibacterial spectrum in comparison to the parent compound, being active against just a few Gram-positive bacteria. In particular, NAI003 retained low MICs against all tested isolates of Propionibacterium acnes and, to a lesser extent, against Enterococcus faecalis. Furthermore, NAI003 showed a time- and dose-dependent killing of both a clindamycin-resistant and a clindamycin-sensitive P. acnes isolate. Gel shift experiments indicated that, like the parent compound, NAI003 retained the ability to bind to elongation factors Tu (EF-Tus) derived from Escherichia coli, E. faecalis, or P. acnes, albeit with reduced efficiency. In contrast, EF-Tus derived from the NAI003-insensitive Staphylococcus aureus or Streptococcus pyogenes did not bind this compound. These results were confirmed by in vitro studies using a hybrid translation system, which indicated that NAI003 can inhibit most efficiently protein synthesis driven by the P. acnes EF-Tu. P. acnes mutants resistant to NAI003 were isolated by direct plating. With one exception, all analyzed strains carried mutations in the tuf gene, encoding EF-Tu. Because of its selective effect on P. acnes in comparison to resident skin flora, NAI003 represents a promising candidate for the topical treatment of acne, which has already completed a phase 1 clinical study.

Characterization of the congeners in the lantibiotic NAI-107 complex.

NAI-107, a lantibiotic produced by Microbispora sp. 107891, shows potent activity against multi-drug-resistant bacterial pathogens. It is produced as a complex of related molecules, which is unusual for ribosomally synthesized peptides. Here we describe the identification, characterization, and antibacterial activity of the congeners produced by Microbispora sp. 107891 and by the related Microbispora corallina NRRL 30420. These molecules differ by the presence of two, one, or zero hydroxyl groups at Pro-14, by the presence of a chlorine at Trp-4, and/or by the presence of a sulfoxide on the thioether of the first lanthionine.

Pharmacological properties of NAI-603, a well-tolerated semisynthetic derivative of ramoplanin.

NAI-603 is a ramoplanin derivative designed to overcome the tolerability issues of the parent drug as a systemic agent. NAI-603 is highly active against aerobic and anaerobic Gram-positive bacteria, with MICs ranging from 0.008 to 8 µg/ml. MICs were not significantly affected by pH (range, 6 to 8), by inoculum up to 10(8) CFU/ml, or by addition of 50% human serum. Against staphylococci and enterococci, NAI-603 was rapidly bactericidal, with minimum bactericidal concentration (MBC)/MIC ratios never exceeding 4. The frequency of spontaneous resistance was low at 2× to 4× MIC (≤1×10(-6) to ≤1×10(-8)) and below the detection limit (about ≤1×10(-9)) at 8×MIC. Serial subcultures at 0.5×MIC yielded at most an 8-fold increase in MICs. In a systemic infection induced by methicillin-resistant Staphylococcus aureus (MRSA), the 50% effective dose (ED50) of intravenous (i.v.) NAI-603 was 0.4 mg/kg, lower than that of oral (p.o.) linezolid (1.4 mg/kg) and subcutaneous (s.c.) teicoplanin (1.4 mg/kg) or vancomycin (0.6 mg/kg). In neutropenic mice infected with vancomycin-resistant enterococci (VRE), the ED50s for NAI-603 were 1.1 to 1.6 mg/kg i.v., compared to 0.5 mg/kg i.v. of ramoplanin. The bactericidal activity was confirmed in vivo in the rat granuloma pouch model induced by MRSA, where NAI-603, at 40 mg/kg i.v., induced about a 2- to 3-log10-reduction in viable bacteria in the exudates, which persisted for more than 72 h. The pharmacokinetic (PK) profiles of NAI-603 and ramoplanin at 20 mg/kg show similar half-lives (3.27 and 3.80 h, respectively) with the maximum concentration (Cmax) markedly higher for NAI-603 (207 µg/ml versus 79 µg/ml). The favorable pharmacological profile of NAI-603, coupled with the absence of local tolerability issues, supports further investigation.

An Unusual Case of Mammary Paget's Disease Diagnosed Using Dynamic Contrast-Enhanced MRI.

Mammary Paget's disease is a rare presentation of breast cancer. At clinical examination, it is characterized by skin lesions of the nipple-areola complex, almost always a sign of malignancy. In fact, it is often associated with an underlying mammary ductal carcinoma in situ (DCIS) or invasive carcinoma. An underlying carcinoma is also common in women with negative mammography and ultrasound (US); in these cases, magnetic resonance imaging (MRI) is a diagnostic tool useful in the detection of occult cancer. We described an unusual case of mammary Paget's disease with underlying DCIS, in a patient without nipple-areola complex alterations and/or palpable lump. On suspicion of Paget's disease, the patient underwent MRI examination that proved useful for an accurate diagnosis. Biopsy confirmed dynamic MRI findings.

Isolation and characterization of NAI-802, a new lantibiotic produced by two different Actinoplanes strains.

Lantibiotics are biologically active peptides produced by Gram-positive bacteria. Starting from fermentation broth extracts preselected from a high-throughput screening program for discovering cell-wall inhibitors, we successfully isolated a new lantibiotic produced by Actinoplanes sp., designated as NAI-802. MS and NMR analysis together with explorative chemistry established that NAI-802 consists of 21 amino acids, 19 of which are identical to those present in the class II lantibiotic actagardine. Interestingly, NAI-802 carries one extra alanine and one extra arginine at the N- and C-termini, respectively. As expected from the overall higher positive charge, NAI-802 was slightly more active than actagardine against staphylococci and streptococci. Further improvement of its antibacterial activity was achieved by adding one additional positive charge through conversion of the C-terminal carboxylate into the corresponding basic amide. NAI-802 thus represents a novel promising candidate for treating Gram-positive infections caused by multidrug-resistant pathogens.

Hybrid SPECT/CT Imaging in the Evaluation of Coronary Stenosis: Role in Diabetic Patients.

Purpose. Our purpose was to combine the results of the MDCT (multidetector computed tomography) morphological data and the SPECT (single-photon emission computed tomography) data using hybrid imaging to overcome the limits of the MDCT in the evaluation of coronary stenosis in diabetic patients with large amount of calcium in the coronary arteries. Method and Materials. 120 diabetic patients underwent MDCT examination and SPECT examination. We evaluated 324 coronary arteries. After the examinations, we merged CT and SPECT images. Results. CT evaluation: 52 (32.8%) coronaries with stenosis ≥ 50%, 228 (70.4%) with stenosis < 50%, and 44 (13.6%) with a doubtful evaluation. SPECT evaluation: 80 (24.7%) areas with hypoperfusion, 232 (71.6%) with normal perfusion, and 12 (3.7%) with a doubtful evaluation. Of 324 coronary arteries and corresponding areas, the hybrid SPECT/CT evaluation showed 92 (28.4%) areas with hypoperfusion, and 232 (71.6%) with normal perfusion. Conclusion. Hybrid CT/SPECT imaging could be useful in the detection of significant coronary stenosis in patients with large amount of coronary calcifications.

Chrolactomycins from the actinomycete actinospica.

Examination of the metabolites produced by an Actinospica strain led to the identification of 6-hydroxychrolactomycin (compound 1), which is produced along with minor amounts of chrolactomycin (compound 2). The structure of 1 was established on the basis of extensive spectroscopic analysis, including one- and two-dimensional NMR. Compound 1 showed antimicrobial activity against Gram-positive bacteria, although it was generally less active than 2.

Correlation between US-PSV and 64-Row MDCTA with Advanced Vessel Analysis in the Quantification of 50-70% Carotid Artery Stenosis.

Purpose. To correlate ultrasonographic peak systolic velocity (US-PSV) and 64-row multidetector computed tomography angiography (MDCTA) with advanced vessel analysis (AVA) software in the quantification of 50-70% carotid artery stenosis. Materials and methods. 199 consecutive patients (247 arteries) with internal carotid artery (ICA) or third proximal bifurcation stenosis. Each patient was studied by duplex US (DUS) and 64-row MDCTA with AVA software. Results. DUS showed PSV measurements less than 125 cm/s in 51 carotid stenosis and a value greater than this in 196 arteries. 64-row MDCTA AVA software showed a grade of stenosis less than 50% in 42 carotid arteries while a greater 70% was found in 4 carotid arteries; then, carotid arteries with stenosis percentage between 50% and 70% were 201. Linear regression analysis showed a good linear correlation (r = 0.88) between MDCTA-AVA software percentage stenosis and PSV: between 50% grade of stenosis and PSV value corresponding to 133,6 cm/sec and between 70% stenosis and PSV value corresponding to 268 cm/sec. The sensitivity, specificity, positive predictive value(PPV), negative predictive value(NPV) of this analysis were 93%, 82%, 97%, 75%, respectively. Conclusion. Linear correlation between PSV data and grade of stenosis from 50% to 70% obtained with 64-row MDCTA AVA software. Main PSV value corresponding to 50% and 70% grade of stenosis at AVA analysis.

Halogenated spirotetronates from Actinoallomurus.

Two new members of the spirotetronate class, nai414-A and nai414-B, were discovered and isolated from an Actinoallomurus sp. Their structures were established by 1D and 2D NMR, UV, and MS analyses and by chemical degradation. They showed antimicrobial and antitumor activity against Gram-positive bacteria and against human microvascular endothelial cells, respectively. Substituting bromide for chloride ions in the growth medium afforded mono- and dibrominated derivatives.

Percutaneous local ablation of unifocal subclinical breast cancer: clinical experience and preliminary results of cryotherapy.

OBJECTIVES: To assess the ablative effectiveness, the oncological and cosmetic efficacy of image-guided percutaneous cryoablation in the treatment of single breast nodules with subclinical dimensions after identification with ultrasonography (US), mammography, magnetic resonance (MRI) and characterization by vacuum assisted biopsy. MATERIALS: Fifteen women with a mean age of 73 ± 5 years (range 64-82 years) and lesion diameter of 8 ± 4 mm were undergoing cryotherapy technology with a single probe under US-guidance associated with intra-procedural lymph-node mapping and excision of the sentinel node. All the patients underwent surgical resection (lumpectomy) from 30 to 45 days after the percutaneous ablation. RESULTS: The iceball size generated by the cryoprobe during the procedure at minus 40°C was 16 × 41 mm. In 14 of the 15 patients was observed a complete necrosis of the cryo-ablated lesion both in post-procedural MRI follow-up and anatomo-pathological evaluation after surgical resection. In one case there was a residual disease in post-procedural MRI and postoperative histological examination, probably justified by an incorrect positioning of the probe. CONCLUSION: The percutaneous cryoablation as a "minimally invasive" technique can provide excellent oncological and cosmetic results on selected cases handled by experienced operators by using the tested devices.

Seasonal variability of bacteria in fine and coarse urban air particulate matter.

The current knowledge about the microbial communities associated with airborne particulate matter, particularly in urban areas, is limited. This study aims to fill this gap by describing the microbial community associated with coarse (PM10) and fine (PM2.5) particulate matter using pyrosequencing. Particulate matter was sampled on Teflon filters over 3 months in summer and 3 months in winter in Milan (Italy), and the hypervariable V3 region of the gene 16S rRNA amplified from the DNA extracted from the filters. The results showed large seasonal variations in the microbial communities, with plant-associated bacteria dominating in summer and spore-forming bacteria in winter. Bacterial communities from PM10 and PM2.5 were also found to differ from each other by season. In all samples, a high species richness, comparable with that of soils, but a low evenness was found. The results suggest that not only can the sources of the particulate influence the presence of specific bacterial groups but also that environmental factors and stresses can shape the bacterial community.

Mixed hepatoblastoma in a young male adult: a case report and literature review.

Hepatoblastoma (HB) is a rare malignant tumour of the liver and usually occurs in the first three years of life. Most of these tumours arise in the embryo; hence it seems to be unusual that hepatoblastoma occurs in adults and is an exceptional cause of primary malignant liver tumour in adult patients. The diagnosis is often overlooked, and patients might be diagnosed at late stages of the disease at risk of increased mortality. In this paper we report a case of a 30-year-old man with mixed hepatoblastoma and abdominal pain, hepatomegaly and fever. The patient under went noninvasive diagnostic methods: duplex scanning, Computed Tomography (CT), and Magnetic Resonance imaging (MRI). In our experience, despite the important role of histological sample provided by biopsy in defining the diagnosis, very important was the role of MRI, more than ultrasonography and enhanced CT. The MRI detects tumor features as size, margins, and ratio with neighboring organs in order to get the best surgical approach.

Adult intra-thoracic kidney: a case report of bochdalek hernia.

Introduction. Bochdalek hernia is a congenital posterior lateral diaphragmatic defect that allows abdominal viscera to herniate into the thorax. Intrathoracic kidney is a very rare finding representing less than 5% of all renal ectopias with the least frequency of all renal ectopias. Case Presentation. We report a case of a 62-year-old man who had a left thoracic kidney associated with left Bochdalek hernia. Abdominal X-ray and chest X-ray revealed dilated loops of the colon above left hemidiaphragm. Abdominal ultrasound (US) showed the right kidney with many fluid and esophytic cysts; left kidney was unfeasible to study because of the impossibility to find it. Computed Tomography (CT) basal scan demonstrated a left-sided Bochdalek hernia with dilatated colon loops and the left kidney within the pleural space. Magnetic Resonance (MR) confirmed a defect in left hemidiaphragm with herniation of left kidney, omento, spleen and colon flexure, and intrarotation with posterior hilum on sagittal plane. Conclusion. The association of a Bochdalek hernia and an intrathoracic renal ectopia is very rare, that pose many diagnostic and management dilemmas for clinicians. Our patient has been visualized by CT and MR imaging. A high index of suspicion can result in early diagnosis and prompt intervention with reduced morbidity and mortality.

A rare case of popliteal venous aneurysm.

We report a case of a 21-year-old man with a popliteal venous aneurysm of the left popliteal fossa, with local symptoms and pain during palpation. Early diagnosis is fundamental in order to prevent the thromboembolic events or other major complications. Duplex scanning, Computed Tomography scanning, and Magnetic Resonance imaging are considered to be important non-invasive diagnostic methods for the diagnosis of PVA. The Angio Computed Tomography acquisition confirmed a 36 mm x 17 mm oval mass in the left popliteal fossa continuous with the popliteal veins. This lesion had presented contrast enhancement only in delayed acquisition (180 sec) and so appeared to be a true venous aneurysm and no arterial. The PVA was repaired surgically via a posterior approach to the popliteal fossa. A 4 x 2 aneurysm was identified. In the same time open tangential aneurysmectomy and lateral vein reconstruction were realised. This case is interesting because the Angio Computed Tomography study, in delayed acquisition, has allowed a correct diagnostic assessment of PVA and the surgical treatment.