A new method for the study of velopharyngeal function using gated magnetic resonance imaging

The purpose of this project was to assess the feasibility of imaging the velopharynx of adult volunteers during repetitive speech, using gated magnetic resonance imaging (MRI). Although a number of investigators have used conventional MRI in the study of the human vocal tract, the mismatch between the lengthy time necessary to acquire sufficiently detailed images and the rapidity of movement of the vocal tract during speech has forced investigators to acquire images either while the subject is at rest or during sustained utterances. The technique used here acquired a portion of each image during repetitive utterances, building the full image over multiple utterance cycles. The velopharyngeal portal was imaged on a 1.5-Tesla GE Signa LX 8.2 platform with gated fast spoiled gradient echo protocol. An external 1-Hertz trigger was fed to the cardiac gate. Subjects synchronized utterance of consonant-vowel syllables to a flashing light synchronized with the external trigger. Each acquisition of 30 phases per second at a single-slice location took 22 to 29 seconds. Four consonant-vowel syllables (/pa/, /ma/, /sa/, and /ka/) were evaluated. Subjects vocalized throughout the acquisition, beginning 5 to 6 seconds beforehand to establish a regular rhythm. Imaging of the velopharyngeal portal was performed for sagittal, velopharyngeal axial (aligned perpendicular to the "knee" of the velum), axial, and coronal planes. Volumes were obtained by sequential acquisition of six to 10 slices (each with 30 phases) in the axial or sagittal planes during repetition of the /pa/ syllable. Spatiotemporal volumes of the single-slice data were sectioned to provide time-motion images (analogous to M-mode echocardiograms). Three-dimensional dynamic volume renderings of palate motion were displayed interactively (Vortex; CieMed, Singapore). A method suitable for the collection and visualization of four-dimensional information regarding monosyllabic speech using gated MRI was developed. These techniques were applied to a population of adult volunteer subjects with no history of speech problems and two patients with a history of cleft lip and palate. The techniques allowed good real-time visualization of velopharyngeal anatomy during its entire range of motion and was also able to image pathology-specific anatomic differences in the subjects with cleft lip and cleft palate. These methods may be applicable to a wide spectrum of problems in speech physiology research and for clinical decision-making regarding surgery for speech and outcomes analysis.     

Reverse-flow island sural flap

The reverse flow island sural flap is presented as an alternative to flaps currently used for reconstruction of small and medium substance losses in the distal third of the leg, ankle, and heel. This is a random type of flap, based on the reverse flow of the superficial sural artery, which mainly depends on the anatomy of the perforators of the peroneal artery system.The anatomic structures that constitute the pedicle are the superficial and deep fascias, the sural nerve, the short saphenous vein, and the superficial sural artery. The skin island and the subcutaneous cellular tissue complement the flap proper. This skin island was demarcated at any point of the median or distal thirds of the leg, having the short saphenous vein and the sural nerve on its central axis. The distal dissection limit of the pedicle is located 5 centimeters above the lateral malleolus. This limit is established so as to ensure the integrity of the perforators from the principal arteries of the leg, mainly the peroneal artery, responsible for the reverse flow nourishing the flap. These perforators will affect anastomoses with the superficial sural artery in charge of irrigating the structures compounding the flap.A total of 71 patients were operated on with this technique, some of them with basic pathologic abnormalities limiting the distal blood flow, such as diabetes mellitus, and some others having proven vascular insufficiency or displaying unstable areas attributable to problems such as pseudarthrosis and osteomyelitis, which needed to be covered. Fifteen flaps (21.1 percent) suffered partial necrosis, which did not compromise the final result, and another three (4.2 percent) showed total loss. The flap in question has great mobility and versatility, allowing the treatment of specific areas of the lower limb, without sacrificing important arteries or mobilizing structures that might bring about functional deficits.     

Release of salbutamol sulphate and ketoprofen enantiomers from matrices containing HPMC and cellulose derivatives

We studied the release of salbutamol and ketoprofen enantiomers from HPMC K100M matrices containing two types of cellulose derivatives: cellulose tris (3,5-dimethylphenylcarbamate) and cellulose tris (2,3-dichlorophenylcarbamate), chiral excipients used as stationary phases for liquid chromatography. These matrices provided an extended release of both drugs. Ketoprofen release from formulations elaborated with cellulose tris (2,3-dichlorophenylcarbamate) was by anomalous transport, because the value of n (release exponent of the diffusion equation) ranged between 0.60-0.68, whereas for all other formulations the value of exponent n ranged from 0.50-0.54. The drug thus diffuses through the matrix and is released following a quasi-Fickian diffusion mechanism (stereoselective process). The matrices preferentially retained R-salbutamol and S-ketoprofen and cellulose tris (3,5-dimethylphenylcarbamate) showed more capacity of chiral discrimination for both drugs than cellulose tris (2,3-dichlorophenylcarbamate). Moreover, we observed that stereoselectivity is dependent on the amount of chiral excipient in the formulation. Diffusion tests confirmed the chiral interaction between drugs and cellulose derivatives observed in the dissolution assays except for matrices elaborated with ketoprofen and cellulose tris (2,3-dichlorophenylcarbamate), where the low stereoselectivity observed with the matrices is due to the presence of HPMC K100M. We conclude that the inclusion of these cellulose derivatives in HPMC matrices does not result in a relevant stereoselectivity with respect to the two drugs studied.     

Expression and purification of the recombinant ConBr (Canavalia brasiliensis lectin) produced in Escherichia coli cells

ConBr, a D-glucose/D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a (c)DNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.     

Onset of the segmentation clock in the chick embryo: evidence for oscillations in the somite precursors in the primitive streak

Vertebrate somitogenesis is associated with a molecular oscillator, the segmentation clock, which is defined by the periodic expression of genes related to the Notch pathway such as hairy1 and hairy2 or lunatic fringe (referred to as the cyclic genes) in the presomitic mesoderm (PSM). Whereas earlier studies describing the periodic expression of these genes have essentially focussed on later stages of somitogenesis, we have analysed the onset of the dynamic expression of these genes during chick gastrulation until formation of the first somite. We observed that the onset of the dynamic expression of the cyclic genes in chick correlated with ingression of the paraxial mesoderm territory from the epiblast into the primitive streak. Production of the paraxial mesoderm from the primitive streak is a continuous process starting with head mesoderm formation, while the streak is still extending rostrally, followed by somitic mesoderm production when the streak begins its regression. We show that head mesoderm formation is associated with only two pulses of cyclic gene expression. Because such pulses are associated with segment production at the body level, it suggests the existence of, at most, two segments in the head mesoderm. This is in marked contrast to classical models of head segmentation that propose the existence of more than five segments. Furthermore, oscillations of the cyclic genes are seen in the rostral primitive streak, which contains stem cells from which the entire paraxial mesoderm originates. This indicates that the number of oscillations experienced by somitic cells is correlated with their position along the AP axis.     

Role of the ribosomal stalk components in the resistance of Aspergillus fumigatus to the sordarin antifungals

Aspergillus fumigatus, an important human nosocomial pathogen, is resistant to sordarin derivatives, a new family of antifungals that inhibit protein synthesis by interaction with the EF-2-ribosomal stalk complex. To explore the role of the A. fumigatus ribosome in the resistance mechanism, the fungal stalk proteins were biochemically and genetically characterized and expressed in the sensitive Saccharomyces cerevisiae. Two acidic phosphoproteins homologous to the 12 kDa P1 and P2 proteins described in other organisms were found together with the 34 kDa P0 protein, the third stalk component. The genes encoding each fungal stalk protein were expressed in mutant S. cerevisiae strains lacking the equivalent proteins. Both AfP1 and AfP2 proteins interact with their yeast counterparts of the opposite type and bind to the ribosomal particles in the presence of either the S. cerevisiae or the A. fumigatus P0 protein. The A. fumigatus acidic phosphoproteins did not alter the yeast ribosome sordarin sensitivity. On the contrary, the presence of the fungal P0 induces in vivo and in vitro resistance to sordarin derivatives when present in the yeast ribosome. The mutations A117-->E, P122-->R and G124-->V in A. fumigatus P0 reduce the resistance capacity of the protein. An S. cerevisiae strain with the complete ribosomal stalk of A. fumigatus was obtained, which could be useful for the screening of new antifungals against this pathogenic fungus.     

Evidence for the mechanism of the irreversible inhibition of oestrone sulphatase (ES) by aminosulphonate based compounds

In our search for the mechanism of the enzyme oestrone sulphatase (ES) we have synthesised and evaluated a number of compounds that were predicted to possess some inhibitory activity. Some of these compounds were indeed found to be inhibitors of ES, whilst other compounds were not. From a consideration of the structure–activity relationship (SAR) of the inhibitors and non-inhibitors of this enzyme, we discovered a factor which we now believe is the main inhibitory moiety within the aminosulphonated inhibitors. We therefore report the results of our study into a series of phenyl and alkyl sulphamated compounds as inhibitors of ES. The results of the study show that the substituted phenyl sulphamates are potent inhibitors, whereas the alkyl compounds are, in general, non-inhibitors. Using the results of our SAR study, we postulate the probable mechanism for the irreversible and reversible inhibition of ES, and rationalise the role of the different physicochemical factors in the inhibition of this crucial enzyme.     

Preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) is an evolving technique that provides a practical alternative to prenatal diagnosis and termination of pregnancy for couples who are at substantial risk of transmitting a serious genetic disorder to their offspring. Samples for genetic testing are obtained from oocytes or cleaving embryos after in vitro fertilization. Only embryos that are shown to be free of the genetic disorders are made available for replacement in the uterus, in the hope of establishing a pregnancy. PGD has provided unique insights into aspects of reproductive genetics and early human development, but has also raised important new ethical issues about assisted human reproduction.