The Development of Xenopus tropicalis Transgenic Lines and their Use in Studying Lens Developmental Timing in Living Embryos

The generation of reporter lines for observing lens differentiation in vivo demonstrates a new strategy for embryological manipulation and allows us to address a long-standing question concerning the timing of the onset of differentiation. Xenopus tropicalis was used to make GFP reporter lines with &#;1-crystallin promoter elements directing GFP expression within the early lens. X. tropicalis is a close relative of X. laevis that shares the same ease of tissue manipulation with the added benefits of a diploid genome and faster life cycle. The efficiency of the Xenopus transgenic technique was improved in order to generate greater numbers of normal, adult transgenic animals and to facilitate in vivo analysis of the crystallin promoter. This transgene is transmitted through the germline, providing an accurate and consistent way to monitor lens differentiation. This line permitted us to distinguish models for how the onset of differentiation is controlled: by a process intrinsic to differentiating tissue or one dependent on external cues. This experiment would not have been feasible without the sensitivity and accuracy provided by the in vivo reporter. We find that, in specified lens ectoderm transplanted from neural tube stage donors to younger neural-plate-stage hosts, the onset of differentiation, as measured by expression of the crystallin/GFP transgene, is delayed by an average of 4.4 hours. When specified lens ectoderm is explanted into culture, the delay was an average of 16.3 hours relative to control embryos. These data suggest that the onset of differentiation in specified ectoderm can be altered by the environment and imply that this onset is normally controlled by external cues rather than by an intrinsic mechanism

Wound Complications Following Resection of Adductor Compartment Tumours

Purpose Limb salvage surgery of soft tissue sarcomas is associated with both a risk of local recurrence and wound complications. Although the lower limb appears to be at greater risk of wound-related morbidity, few studies separate anatomical compartments. We believe that the adductor compartment of the thigh has a particularly high rate of complications and so performed a retrospective analysis of all soft tissue sarcomas arising in this region undergoing limb salvage

The Characterization of Ribosomal RNA Gene Chromatin from Physarum Polycephalum

We have isolated ribosomal RNA gene (rDNA) chromatin from Physarum polycephalum using a nucleolar isolation procedure that minimizes protein loss from chromatin and, subsequently, either agarose gel electrophoresis or metrizamide gradient centrifugation to purify this chromatin fraction (Amero, S. A., Ogle, R. C., Keating, J. L., Montoya, V. L., Murdoch, W. L., and Grainger, R. M. (1988) J. Biol. Chem. 263, 10725-10733). Metrizamide-purified rDNA chromatin obtained from nucleoli isolated according to the new procedure has a core histone/DNA ratio of 0.77:1. The major core histone classes comigrate electrophoretically with their nuclear counterparts on Triton-acid-urea/sodium dodecyl sulfate two-dimensional gels, although they may not possess the extent of secondary modification evident with the nuclear histones. This purified rDNA chromatin also possesses RNA polymerase I activity, and many other nonhistone proteins, including two very abundant proteins (26 and 38 kDa) that may be either ribonucleoproteins or nucleolar matrix proteins. Micrococcal nuclease digestion of the metrizamide-purified rDNA chromatin produces particles containing 145-base pair DNA fragments identical in length to those in total chromatin and which contain both transcribed and nontranscribed rDNA sequences. Some smaller fragments (30, 70, and 110 base pairs) are also seen, but their sequence content is not known. These particles sediment uniformly at 11 S in sucrose gradients containing 15 mM NaCl, and at 4-11 S in gradients containing 0.35 M NaCl. Particles enriched in gene or nontranscribed spacer sequences are not resolved in these sucrose gradients or in metrizamide gradients. Our findings suggest that the rDNA chromatin fraction we have identified contains transcriptionally active genes and that an organized, particle-containing structure exists in active rDNA chromatin

The Purification of Ribosomal RNA Gene Chromatin from Physarum Polycephalum

We have undertaken the purification of ribosomal RNA gene (rDNA) chromatin from the slime mold Physarum polycephalum, in order to study its chromatin structure. In this organism rDNA exists in nucleoli as highly repeated minichromosomes, and one can obtain crude chromatin fractions highly enriched in rDNA from isolated nucleoli. We first developed a nucleolar isolation method utilizing polyamines as stabilization agents that results in a chromatin fraction containing far more protein than is obtained by the more commonly used divalent cation isolation methods. The latter method appears to result in extensive histone loss during chromatin isolations. Two methods were then used for purifying rDNA chromatin from nucleoli isolated by the polyamine procedure. We found that rDNA chromatin migrates as a single band in agarose gels, well separated from other components in the chromatin preparation. Although the utility of this technique is somewhat limited by low yields and by progressive stripping of protein from rDNA chromatin, it can provide useful information about rDNA chromatin protein composition. The application of this technique to the fractionation of gene and spacer chromatin fragments produced by restriction enzyme digestion is discussed. We also found that rDNA chromatin, if RNase-treated, bands discretely in metrizamide equilibrium density gradients with a density lighter than that of non-nucleolar chromatin. These characteristics suggest that we have identified a transcriptionally active rDNA chromatin fraction which possesses a lower protein to DNA ratio than does non-nucleolar chromatin. This technique yields sufficient purified rDNA chromatin for further biochemical studies and does not cause extensive protein stripping. The procedures developed here should be applicable to the analysis of a variety of chromatin fractions in other systems

Prospective Study of Antibiotic Prophylaxis for Prostate Biopsy Involving >1100 Men

We aimed to compare infection rates for two 3-day antibiotic prophylaxis regimens for transrectal ultrasound-guided prostate biopsy (TRUSgbp) and demonstrate local microbiological trends. In 2008, 558 men and, in 2009, 625 men had TRUSgpb. Regimen 1 (2008) comprised 400 mg Ofloxacin immediately before biopsy and 200 mg 12-hourly for 3 days. Regimen 2 (2009) comprised Ofloxacin 200 mg 12-hourly for 3 days commencing 24 hours before biopsy. 20/558 (3.6%) men had febrile episodes with regimen 1 and 10/625 (1.6%) men with regimen 2 (P = 0.03). E. coli was the most frequently isolated organism. Overall, 7/13 (54%) of positive urine cultures were quinolone resistant and (5/13) 40% were multidrug resistant. Overall, 5/9 (56%) patients with septicaemia were quinolone resistant. All patients were sensitive to Meropenem. There was 1 (0.2%) death with regimen 1. Commencing Ofloxacin 24 hours before TRUSgpb reduced the incidence of febrile episodes significantly. We observed the emergence of quinolone and multidrug-resistant E. coli. Meropenem should be considered for unresolving sepsis

Bowing fracture of the inferior angle of the scapula, a difficult diagnosis

A 4-year-old boy presented with swelling over the inferior tip of the scapula and an unclear history. Initial radiographic findings were concerning for an aggressive lesion. This case highlights how a multimodality imaging approach was used to relieve uncertainty by diagnosing a paediatric bowing type fracture of the scapular tip

Breaking Up the C Complex Spliceosome Shows Stable Association of Proteins with the Lariat Intron Intermediate

Spliceosome assembly requires several structural rearrangements to position the components of the catalytic core. Many of these rearrangements involve successive strengthening and weakening of different RNA∶RNA and RNA∶proteins interactions within the complex. To gain insight into the organization of the catalytic core of the spliceosome arrested between the two steps of splicing chemistry (C complex), we investigated the effects of exposing C complex to low concentrations of urea. We find that in the presence of 3M urea C complex separates into at least three sub-complexes. One sub-complex contains the 5′exon, another contains the intron-lariat intermediate, and U2/U5/U6 snRNAs likely comprise a third sub-complex. We purified the intron-lariat intermediate sub-complex and identified several proteins, including U2 snRNP and PRP19 complex (NTC) components. The data from our study indicate that U2 snRNP proteins in C complex are more stably associated with the lariat-intron intermediate than the U2 snRNA. The results also suggest a set of candidate proteins that hold the lariat-intron intermediate together in C complex. This information is critical for further interpreting the complex architecture of the mammalian spliceosome

Modelling distributions of Aedes aegypti and Aedes albopictus using climate, host density and interspecies competition.

Florida faces the challenge of repeated introduction and autochthonous transmission of arboviruses transmitted by Aedes aegypti and Aedes albopictus. Empirically-based predictive models of the spatial distribution of these species would aid surveillance and vector control efforts. To predict the occurrence and abundance of these species, we fit a mixed-effects zero-inflated negative binomial regression to a mosquito surveillance dataset with records from more than 200,000 trap days, representative of 53% of the land area and ranging from 2004 to 2018 in Florida. We found an asymmetrical competitive interaction between adult populations of Aedes aegypti and Aedes albopictus for the sampled sites. Wind speed was negatively associated with the occurrence and abundance of both vectors. Our model predictions show high accuracy (72.9% to 94.5%) in validation tests leaving out a random 10% subset of sites and data since 2017, suggesting a potential for predicting the distribution of the two Aedes vectors

Three allele combinations associated with Multiple Sclerosis

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated disease of polygenic etiology. Dissection of its genetic background is a complex problem, because of the combinatorial possibilities of gene-gene interactions. As genotyping methods improve throughput, approaches that can explore multigene interactions appropriately should lead to improved understanding of MS. METHODS: 286 unrelated patients with definite MS and 362 unrelated healthy controls of Russian descent were genotyped at polymorphic loci (including SNPs, repeat polymorphisms, and an insertion/deletion) of the DRB1, TNF, LT, TGFβ1, CCR5 and CTLA4 genes and TNFa and TNFb microsatellites. Each allele carriership in patients and controls was compared by Fisher's exact test, and disease-associated combinations of alleles in the data set were sought using a Bayesian Markov chain Monte Carlo-based method recently developed by our group. RESULTS: We identified two previously unknown MS-associated tri-allelic combinations: -509TGFβ1*C, DRB1*18(3), CTLA4*G and -238TNF*B1,-308TNF*A2, CTLA4*G, which perfectly separate MS cases from controls, at least in the present sample. The previously described DRB1*15(2) allele, the microsatellite TNFa9 allele and the biallelic combination CCR5Δ32, DRB1*04 were also reidentified as MS-associated. CONCLUSION: These results represent an independent validation of MS association with DRB1*15(2) and TNFa9 in Russians and are the first to find the interplay of three loci in conferring susceptibility to MS. They demonstrate the efficacy of our approach for the identification of complex-disease-associated combinations of alleles