The Mobilome-Enriched Genome of the Competence-Deficient Streptococcus pneumoniae BM6001, the Original Host of Integrative Conjugative Element Tn5253, Is Phylogenetically Distinct from Historical Pneumococcal Genomes

Streptococcus pneumoniae is an important human pathogen causing both mild and severe diseases. In this work, we determined the complete genome sequence of the S. pneumoniae clinical isolate BM6001, which is the original host of the ICE Tn5253. The BM6001 genome is organized in one circular chromosome of 2,293,748 base pairs (bp) in length, with an average GC content of 39.54%; the genome harbors a type 19F capsule locus, two tandem copies of pspC, the comC1-comD1 alleles and the type I restriction modification system SpnIII. The BM6001 mobilome accounts for 15.54% (356,521 bp) of the whole genome and includes (i) the ICE Tn5253 composite; (ii) the novel IME Tn7089; (iii) the novel transposon Tn7090; (iv) 3 prophages and 2 satellite prophages; (v) 5 genomic islands (GIs); (vi) 72 insertion sequences (ISs); (vii) 69 RUPs; (viii) 153 BOX elements; and (ix) 31 SPRITEs. All MGEs, except for the GIs, produce excised circular forms and attB site restoration. Tn7089 is 9089 bp long and contains 11 ORFs, of which 6 were annotated and code for three functions: integration/excision, mobilization and adaptation. Tn7090 is 9053 bp in size, flanked by two copies of ISSpn7, and contains seven ORFs organized as a single transcriptional unit, with genes encoding for proteins likely involved in the uptake and binding of Mg2+ cations in the adhesion to host cells and intracellular survival. BM6001 GIs, except for GI-BM6001.4, are variants of the pneumococcal TIGR4 RD5 region of diversity, pathogenicity island PPI1, R6 Cluster 4 and PTS island. Overall, prophages and satellite prophages contain genes predicted to encode proteins involved in DNA replication and lysogeny, in addition to genes encoding phage structural proteins and lytic enzymes carried only by prophages. & phi;BM6001.3 has a mosaic structure that shares sequences with prophages IPP69 and MM1 and disrupts the competent comGC/cglC gene after chromosomal integration. Treatment with mitomycin C results in a 10-fold increase in the frequency of & phi;BM6001.3 excised forms and comGC/cglC coding sequence restoration but does not restore competence for genetic transformation. In addition, phylogenetic analysis showed that BM6001 clusters in a small lineage with five other historical strains, but it is distantly related to the lineage due to its unique mobilome, suggesting that BM6001 has progressively accumulated many MGEs while losing competence for genetic transformation

Modified toggle pin technique combined with prosthetic capsular reconstruction for surgical stabilization of coxofemoral luxation in a Shetland pony

Objective To describe open reduction and surgical stabilization of a coxofemoral luxation in a pony using a modified toggle pin technique and prosthetic joint capsule reconstruction without osteotomy of the greater trochanter. Animal A 2-year-old Shetland pony with a bodyweight of 167 kg. Study design Case report. Methods Radiographic examination confirmed craniodorsal luxation of the left coxofemoral joint. An open reduction with the aid of a pulley system was performed. A toggle pin was inserted through a bone tunnel extending from the level of the femoral shaft through the femoral head and the center of the acetabulum for the pin to be positioned on the medial wall of the acetabulum. FiberWire was subsequently passed through the cranial and caudal aspects of the acetabulum as well as a transverse tunnel in the femoral neck in a figure of 8 to facilitate capsular reconstruction. The pony was placed in a sling for 8 weeks and gradually returned to normal activity over 2 months. Results Postoperative radiographic examination confirmed the position of the femoral head in the acetabulum with the implants in place. On 2-year follow-up the pony was sound at walk and trot. Conclusion A combined intra- and extra-articular stabilization technique for coxofemoral luxation in a pony resulted in successful long-term reduction and excellent outcome

Ethidium bromide as a marker of mtDNA replication in living cells.

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE)

Full-aperture extended-depth oblique plane microscopy through dynamic remote focusing

Oblique plane microscopy is a method enabling light-sheet fluorescence imaging through a single microscope objective lens by focusing on a tilted plane within the sample. To focus the fluorescence emitted by the oblique plane on a camera, the light is imaged through a pair of remote objective lenses, facing each other at an angle. The aperture mismatch resulting from this configuration limits the effective numerical aperture of the system, reducing image resolution and signal intensity. This manuscript introduces an alternative method to capture the oblique plane on the camera. Instead of relying on angled objective lenses, an electrically tunable lens is employed. This lens adjusts the focal plane of the microscope synchronously with the rolling shutter of a scientific CMOS camera. In this configuration the entire aperture of the objective is effectively employed, increasing the resolution of the system. Moreover, a variety of objective lenses can be employed, enabling the acquisition of wider axial fields of view compared to conventional oblique plane microscopy

Modified toggle pin technique combined with prosthetic capsular reconstruction for surgical stabilization of coxofemoral luxation in a Shetland pony

Objective To describe open reduction and surgical stabilization of a coxofemoral luxation in a pony using a modified toggle pin technique and prosthetic joint capsule reconstruction without osteotomy of the greater trochanter. Animal A 2-year-old Shetland pony with a bodyweight of 167 kg. Study design Case report. Methods Radiographic examination confirmed craniodorsal luxation of the left coxofemoral joint. An open reduction with the aid of a pulley system was performed. A toggle pin was inserted through a bone tunnel extending from the level of the femoral shaft through the femoral head and the center of the acetabulum for the pin to be positioned on the medial wall of the acetabulum. FiberWire was subsequently passed through the cranial and caudal aspects of the acetabulum as well as a transverse tunnel in the femoral neck in a figure of 8 to facilitate capsular reconstruction. The pony was placed in a sling for 8 weeks and gradually returned to normal activity over 2 months. Results Postoperative radiographic examination confirmed the position of the femoral head in the acetabulum with the implants in place. On 2-year follow-up the pony was sound at walk and trot. Conclusion A combined intra- and extra-articular stabilization technique for coxofemoral luxation in a pony resulted in successful long-term reduction and excellent outcome