Semi-Automatic Identification of the Fetal Profile and Nasal Bone Measurement at the Time of the Routine Mid-Trimester Ultrasound Scan.

PURPOSE: This study was designed to compare nasal bone length (NBL) measurements using a manual multiplanar mode with those made using a newer semi-automatic technique (Volume NT™) acquired by an experienced operator as well as measurements done by two independent observers with different levels of ultrasound experience (conventional 2 D vs. Volume NT™). MATERIALS AND METHODS: Ultrasound examination was performed prospectively on 81 pregnant women with a singleton pregnancy at the time of their routine mid-trimester ultrasound scan. RESULTS: The correct mid-sagittal plane of the fetal profile was successfully obtained using the semi-automatic technique in 53 of 81 cases. CONCLUSION: NBL measurements using conventional two-dimensional techniques showed significantly higher inter-observer variability than the semi-automatic program. Our study shows the feasibility of using a semi-automatic technique, especially for less experienced operators. Measurements obtained with the semi-automatic technique produced much less variable results around a mean than those obtained with conventional two-dimensional ultrasound.

Fluorescence in situ hybridization for identification of microorganisms in acute chorioamnionitis.

The relevance of microorganisms in preterm birth is still under discussion. Using a diagnostic fluorescence in situ hybridization probe panel, we visualized Staphylococcus aureus and Streptococcus mitis group in two cases of acute chorioamnionitis. This technique provides spatial resolution and quantity of bacteria, clarifying the epidemiology and pathogenic pathways of acute chorioamnionitis.

Fluorescence in situ hybridization to improve the diagnosis of endocarditis: a pilot study.

Infective endocarditis is a rare but life-threatening disease associated with high mortality. Early diagnosis of the causative microorganism is critical to patient outcome. However, conventional diagnostic methods are often unsatisfactory in achieving this goal. As a proof of concept, we applied fluorescence in situ hybridization (FISH) for detection and identification of bacteria in histological sections of heart valves. Biopsy specimens from 54 suspected endocarditis patients were obtained during valve surgery and analysed via FISH. Specimens were screened with a probe panel that identifies the most common bacteria implicated in endocarditis. Results were compared with those of culture-based diagnostics and clinical data. Discrepant results were subjected to comparative sequence analysis of PCR-amplified 16S rRNA genes. FISH detected bacteria in 26 of the 54 heart valves. FISH allowed successful diagnosis of infective endocarditis in five of 13 blood culture-negative cases and in 11 of 37 valve culture-negative cases, showing the bacteria within their histological context. This technique allows the simultaneous detection and identification of microorganisms at the species or genus level directly from heart valves and might be a valuable tool for diagnosis of endocarditis.

Plasmids for efficient single-copy gene cloning into gdh2 and trpC of Bacillus megaterium DSM319 and QM B1551.

We constructed integrative plasmids to place xylA-lacZ indicator gene fusions into two different loci of the Bacillus megaterium chromosome, gdh2 and trpC, in lac mutants of strains DSM 319 and QM B1551, which differ markedly. Single-crossover integration was achieved in all cases while double crossovers occurred only in gdh2 of DSM 319 and QM B1551 and in trpC of QM B1551. Neither of the loci affected regulation of the xylA-lacZ fusions. These results confirm the suitability of the two gene loci for single-copy cloning.

Regulation of expression, genetic organization and substrate specificity of xylose uptake in Bacillus megaterium.

Xylose uptake in Bacillus megaterium depends on expression of a putative H+/xylose symporter encoded by xylT, the last gene in the xyl operon. Insertional inactivation of xylT leads to an apparent uptake deficiency determined with whole cells and severely slower growth on xylose as sole carbon source. Expression of xylT is xylose inducible and subject to carbon catabolite repression mediated by CcpA and cre. Northern analysis of the xyl mRNA reveals that a potential stem-loop structure located in the non-translated region between xylA and xylB presumably acts as a transcriptional terminator, as it leads to different amounts of the respective mRNA sections: the 5'-xylA portion is very abundant, while the 3'-xylBT portion constitutes only a fraction of it. XylT has an apparent Michaelis constant (KM) of approx. 100 microM and is competitively inhibited by glucose with an inhibitor constant KI of 16 mM.

Contributions of XylR CcpA and cre to diauxic growth of Bacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose.

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion. xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation of xylR yields a two-fold increase in expression of xylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. When xylR and cre are inactivated together a residual two-fold repression of xylA is found. Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40 min. In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.

Glucose and glucose-6-phosphate interaction with Xyl repressor proteins from Bacillus spp. may contribute to regulation of xylose utilization.

The xyl operons of several gram-positive bacteria are regulated at the level of transcription by xylose-responsive repressor proteins (XylR). In addition, they are catabolite repressed. Here, we describe a mechanism by which glucose metabolism can affect both regulatory mechanisms. Glucose-6-phosphate appeared to be an anti-inducer of xyl operon transcription, since it could compete with xylose in interaction in vitro with XylR from Bacillus subtilis, B. megaterium, and B. licheniformis. On the other hand, glucose was a low-efficiency inactivator of XylR from B. subtilis and B. megaterium and a weak anti-inducer of XylR from B. licheniformis. Thus, the chemical nature of the substituent at C-5 of xylose and the primary structure of XylR determine the effect of these compounds on xyl operon transcription.

Cloning, expression and functional analyses of the catabolite control protein CcpA from Bacillus megaterium.

A mutant of Bacillus megaterium relieved from catabolite repression has been used to clone ccpA from B. megaterium by complementation. ccpA is the first gene of a presumed operon, in which it is followed by the motA homologue ORF1 and the motB homologue ORF2. The mutation maps in the 3'-terminal region of ccpA, where an in-frame duplication of 84 nucleotides located between two 9 bp direct repeats leads to an insertion of 28 amino acids near the C-terminus of CcpA. An in-frame deletion of 501 bp in ccpA exhibits the same phenotype as the 84 bp duplication. Deletion of ORF1 and ORF2 does not yield an apparent phenotype. A single-copy ccpA::lacZ transcriptional fusion is constitutively expressed, independent of whether the growth medium triggers catabolite repression or not. The ccpA mutation leads to relief of catabolite repression exerted by glucose, fructose, mannitol, glucitol and glycerol, whereas only smaller effects were found with ribose, citrate and glutamate. The respective growth rates on these carbon sources are uniformly reduced to a generation time of about 90 min in the ccpA mutant. Catabolite repression of a plasmid-encoded xylA::ccpA fusion is less efficient than that of a xylA::lacZ fusion in the same vector. Furthermore, overproduction of CcpA decreases catabolite repression of a single-copy xylA::lacZ fusion approximately twofold. Thus, overexpression of CcpA may be counterproductive for catabolite repression, supporting the hypothesis that CcpA by itself may not bind sufficiently strongly to the cis-active catabolite-responsive element to exert catabolite repression.

An operator binding-negative mutation of Xyl repressor from Bacillus subtilis is trans dominant in Bacillus megaterium.

We have selected a Bacillus subtilis 168-borne xylR Ser to Leu mutation at position 41 of the encoded amino acid sequence showing a constitutive expression phenotype for the xyl operon. When cloned on a multi-copy plasmid in a B. megaterium strain harbouring a single-copy xylA-lacZ fusion it leads to derepression of beta-galactosidase expression. Thus, it is trans dominant over the endogenous xylR, indicating that Xyl repressor functions as a multimer. This result also supports the assumption that the mutation is in a putative alpha-helix-turn-alpha-helix operator binding motif of Xyl repressor.