Use of Hyaluronic Acid Filler in Patients with Secondary Cleft Lip Deformity.

METHODS: Retrospective case note review was undertaken of all patients treated in the clinic over a two-year period. RESULTS: 20 patients underwent HA filler injections to the upper lip 26 times. Most were female (F:M = 3:1) and patients were aged 18-58 years. Most patients had a unilateral cleft lip +/- palate (n = 13, 65%). The most common indication was to address upper lip volume (n = 13, 65%). Other indications included vermillion notch (n = 5, 25%), cupid bow peak height asymmetry (n = 4, 20%), scar asymmetry (n = 1, 5%) and nasal sill flattening (n = 1, 5%). Small volumes of filler were used with an average of 0.34 ml (range 0.05-1.2 ml). There were no complications and one patient reported pruritis post procedure. CONCLUSIONS: HA filler is a safe and reliable treatment for certain aspects of asymmetry following cleft lip repair. It can be used to address volume deficiency and asymmetry, cupid bow peak height discrepancies and a vermillion notch for patients who do not want surgery. Injection of HA to the lips can be performed easily, with appropriate training, in the outpatient setting.

Socioeconomic influence on orofacial cleft patient care.

BACKGROUND: Cleft lip and palate is the most common craniofacial birth defect in the UK. Orofacial clefts have functional and aesthetic implications requiring intensive multi-disciplinary follow-up to optimise development. Failure to attend follow-up is likely to have a negative impact on patient outcomes. The aim of this retrospective audit is to establish if socioeconomic status influences attendance, DNA and cancellation rates in cleft patients. METHODS AND RESULTS: A retrospective audit of 74 orofacial cleft patients born and operated on at the Royal Hospital for Sick Children Glasgow between 2006 and 2007. There was higher rate of DNA in more deprived social groups-21% (SIMD 1) against 10% (SIMD 5). A higher rate of DNA in cleft lip and palate patients was noted. This group of patients showed a marked difference in attendance between SIMD 1 (38%) and SIMD 5 (78%). CONCLUSION: More deprived areas have a higher outpatient DNA rate for cleft patients resulting in suboptimal follow-up. Ultimately, causation of poorer outcomes in this group is likely to be multi-factorial but the financial implication of travelling to multiple clinics should be considered and it may be that resource reallocation is the answer to address the current inequality of health care provision.

Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities.

Although there is increasing evidence that virus-specific cytotoxic-T-lymphocyte (CTL) responses play an important role in the control of human immunodeficiency virus (HIV) replication in vivo, only scarce CTL data are available for the ethnic populations currently most affected by the epidemic. In this study, we examined the CD8(+)-T-cell responses in African-American, Caucasian, Hispanic, and Caribbean populations in which clade B virus dominates and analyzed the potential factors influencing immune recognition. Total HIV-specific CD8(+)-T-cell responses were determined by enzyme-linked immunospot assays in 150 HIV-infected individuals by using a clade B consensus sequence peptide set spanning all HIV proteins. A total of 88% of the 410 tested peptides were recognized, and Nef- and Gag-specific responses dominated the total response for each ethnicity in terms of both breadth and magnitude. Three dominantly targeted regions within these proteins that were recognized by >90% of individuals in each ethnicity were identified. Overall, the total breadth and magnitude of CD8(+)-T-cell responses correlated with individuals' CD4 counts but not with viral loads. The frequency of recognition for each peptide was highly correlated with the relative conservation of the peptide sequence, the presence of predicted immunoproteasomal cleavage sites within the C-terminal half of the peptide, and a reduced frequency of amino acids that impair binding of optimal epitopes to the restricting class I molecules. The present study thus identifies factors that contribute to the immunogenicity of these highly targeted and relatively conserved sequences in HIV that may represent promising vaccine candidates for ethnically heterogeneous populations.

A longitudinal study of top-level executive performance.

Competency ratings were obtained from a hybrid selection system on 98 top-level executives in a predictive validity design. Hierarchical linear modeling results indicated that "resource problem-solving-oriented" competency ratings predicted initial performance. "People-oriented" competency ratings predicted subsequent performance trends. Utility estimates suggested that the system generated an additional $3 million in annual profit per candidate selected. Groups of executives with similar performance trends were identified who had encountered qualitatively different situational circumstances. Findings imply that a model of executive performance must contain main effects for person (competencies) and situation (economic-industrial) characteristics on both subsequent performance and performance trends. Future research needs to examine which situational circumstances moderate relationships between executive competencies and initial performance or subsequent performance trends.

Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion.

Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.

Fusion protein of the paramyxovirus SV5: destabilizing and stabilizing mutants of fusion activation.

The fusion (F) protein of the paramyxovirus SV5 strain W3A causes syncytium formation without coexpression of the SV5 hemagglutinin-neuraminidase (HN) glycoprotein, whereas the F protein of the SV5 strain WR requires coexpression of HN for fusion activity. SV5 strains W3A and WR differ by three amino acid residues at positions 22, 443, and 516. The W3A F protein residues P22, S443, and V516 were changed to amino acids found in the WR F protein (L22, P443, and A516, respectively). Three single-mutants, three double-mutants, and the triple-mutant were constructed, expressed, and assayed for fusion using three different assays. Mutant P22L did not cause fusion under physiological conditions, but fusion was activated at elevated temperatures. Compared with the W3A F protein, mutant S443P enhanced the fusion kinetics with a faster rate and greater extent, and had a lower activation temperature. Mutant V516A had little effect on F protein-mediated fusion. The double-mutant P22L,S443P was capable of causing fusion, suggesting that the two mutations have opposing effects on fusion activation. The WR F protein requires coexpression of HN to cause fusion at 37 degrees C, and does not cause fusion at 37 degrees C when coexpressed with influenza virus hemagglutinin (HA); however, at elevated temperatures coexpression of WR F protein with HA resulted in fusion activation. In the crystal structure of the core trimer of the SV5 F protein (Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309-319), S443 is the last residue (with interpretable electron density) in an extended chain region and the temperature factor for S443 is high, suggesting conformational flexibility at this point. Thus, the presence of prolines at residues 22 and 443 may destabilize the F protein and thereby decrease the energy required to trigger the presumptive conformational change to the fusion-active state.

The ectodomain of HA2 of influenza virus promotes rapid pH dependent membrane fusion.

To better understand the roles of different regions of influenza hemagglutinin in membrane fusion, we have studied the fusion properties of large unilamellar vesicles in the presence of constructs comprising the 127 amino acid ectodomain of the HA2 fragment (FHA2) as well as mutated forms of FHA2 containing single amino acid substitutions, the 95 amino acid truncated form of FHA2 lacking the N-terminal fusion peptide (SHA2), the 20 amino acid N-terminal fusion peptide and the ten amino acid peptide corresponding to the kinked loop region of FHA2. The 100 nm liposomes were made from dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol in equimolar ratio. At pH 5 a high rate of lipid mixing was observed with FHA2 present, even at very low molar concentrations, whereas much lower rates were observed using the shorter constructs: SHA2, the fusion peptide, and the loop peptide. Concentrations of FHA2 which promoted extensive lipid mixing also induced leakage of aqueous contents. Marked effects of FHA2 were also observed with liposomes of egg phosphatidylcholine. All of the changes observed with the liposomes were highly pH-dependent, with only negligible changes occurring at pH 7. The results demonstrate the potent action of FHA2 in promoting lipid mixing and demonstrate the contribution of other regions of the ectodomain of FHA2, in addition to the fusion peptide, to the mechanism of acceleration of membrane fusion. The results also indicate that the pH dependence of fusion is not due solely to changes in the interactions between the HA1 and HA2 subunits. Thus, the "spring loaded energy" is not required to bring about the apposition of the two membranes, considering that FHA2 is already in its thermostable conformation. The acidic amino acid residues in the kinked loop region appear to play a particularly important role in the pH-dependent fusion process as demonstrated by the marked loss of lipid mixing activity of mutant forms of FHA2.

The membrane affinities of the aliphatic amino acid side chains in an alpha-helical context are independent of membrane immersion depth.

Understanding, predicting, and designing the binding of peptides and proteins to bilayers require quantifying the intrinsic propensities of individual amino acid residues to bind membranes as a function of structural context and bilayer depth. A host-guest study was performed using the peptide host named helix5 in order to determine the membrane affinities of the aliphatic side chains both in an alpha-helical context and as a function of bilayer depth. Use of the alpha-helical host with a constrained geometry allowed the placement of guest sites at three different depths in bilayers and minimized secondary structural changes due to guest substitutions. Circular dichroism and electron paramagnetic resonance (EPR) were used to characterize the aqueous and bilayer-bound structures of the peptide variants. EPR was also used to measure the bilayer-water partition constants of the peptide variants, and the Delta DeltaGtr values (relative to Gly) of the aliphatic amino acid side chains were subsequently calculated. Surprisingly, the DeltaDeltaGtr values did not significantly vary as a function of the guest site depth in bilayers. In addition, the Delta DeltaGtr values determined in an alpha-helical context are reduced to approximately two-thirds of Delta DeltaGtr values determined in other studies for the bilayer-water and octanol-water partitioning of amino acid side chains in extended and unstructured hosts. Both the relative reduction in Delta DeltaGtr values in the context of an alpha-helical host and the invariance of Delta DeltaGtr values with respect to bilayer depth are consistent with the membrane affinities of the aliphatic residues being largely determined by the classical hydrophobic effect.

De novo design of a peptide which partitions between water and phospholipid bilayers as a monomeric alpha-helix.

To dissect the determinants of protein insertion into membranes, we designed a model peptide which partitions between water and phospholipid bilayers as an alpha-helical monomer. We used a simplex method to optimize the 'a, d hydrophobicity' and 'e, g charge' of a series of five peptides, where 'abcdefg' correspond to the positions in two turns of an alpha-helix. Circular dichroism and analytical ultra-centrifugation experiments showed that the final peptide (helix5) is monomeric and has an alpha-helix content of approximately 89% at 0 degrees C in aqueous solution. In the presence of large unilamellar vesicles (LUVs), helix5 partitions between the aqueous and membranous phases with a partition constant well suited for measurements by electron paramagnetic resonance (EPR) spectroscopy. EPR power saturation experiments with a cysteine-scanning strategy showed that the alpha-helicity of helix5 is conserved upon binding to LUVs and that the alpha-helix binds parallel to the membrane surface with the central axis approximately 5 A below the lipid phosphate groups. Helix5 should be a useful model peptide for studies aimed at dissecting the determinants of the membrane binding of alpha-helices. The simplex-based strategy may be useful in the rational design of proteins when desired structural or partitioning properties cannot be selected or screened from libraries.

Temperature dependence of polypeptide partitioning between water and phospholipid bilayers.

Various thermodynamic forces (e.g., the hydrophobic effect, electrostatic interactions, peptide immobilization, peptide conformational changes, "bilayer effects," and van der Waals dispersion forces) can participate in the transfer of polypeptides from aqueous solution into lipid bilayers. To investigate the contributions of these forces to peptide-membrane thermodynamics, we have studied the temperature dependence of the water-bilayer partitioning of 4 polypeptides derived from the first 25 amino acid residues in the presequence of subunit IV of yeast cytochrome c oxidase (Cox IVp) using electron paramagnetic resonance spectroscopy. The partitioning of the Cox IVp peptides into phospholipid bilayers increases as the temperature is increased from 3 to 40 degrees C. The contribution of bilayer surface expansion to the temperature-dependent partitioning is estimated to be relatively small and to contribute minimally to the increased bilayer binding of the peptides with increasing temperature. Thermodynamic analysis of the data shows that the transfer of the peptides from water into bilayers at 298 K is driven by the entropic term (-T delta Str) with values ranging from -6.7 to -10 kcal mol-1, opposed by the enthalpic term (delta Htr) by approximately 4 kcal mol-1, and accompanied by a change in heat capacity (delta Cp) ranging from -117 to -208 cal K-1 mol-1. Our results indicate that while a variety of forces do, in fact, contribute to the transfer free energies (delta Gtr), the major driving force for the water-to-bilayer transfer is the hydrophobic effect.

Direct determination of the membrane affinities of individual amino acids.

Amino acids have distinct lipid bilayer affinities which influence the insertion and topology of membrane-bound polypeptides and proteins. To measure membrane affinities, 14 uncharged amino acids were introduced individually at a guest site in a 25-residue peptide derived from the membrane-binding presequence of yeast cytochrome c oxidase, and the peptides were labeled with a nitroxide spin-label. The free energies of transfer from phospholipid bilayers to water (delta delta Gbilayer) were determined directly by examination of partitioning into phospholipid bilayers using electron paramagnetic resonance. The delta delta Gbilayer values are in agreement with hydrophobicities assessed from 1-octanol-water partitioning of N-acetyl amino acid amides [Fauchere, J.-L., & Pliska, V. (1983) Eur. J. Med. Chem. 18, 369-375; Eisenberg, D., & McLachlan, A. (1986) Nature 319, 199-203] and quantitatively demonstrate the role of the hydrophobic effect in membrane-protein interactions.

Moderated regression analysis and Likert scales: too coarse for comfort.

One of the most commonly accepted models of relationships among three variables in applied industrial and organizational psychology is the simple moderator effect. However, many authors have expressed concern over the general lack of empirical support for interaction effects reported in the literature. We demonstrate in the current sample that use of a continuous, dependent-response scale instead of a discrete, Likert-type scale, causes moderated regression analysis effect sizes to increase an average of 93%. We suggest that use of relatively coarse Likert scales to measure fine dependent responses causes information loss that, although varying widely across subjects, greatly reduces the probability of detecting true interaction effects. Specific recommendations for alternate research strategies are made.

Gene probe analysis in an informative family with multiple endocrine neoplasia syndrome type 2A (MEN 2A). Improvement in carrier risk estimation.

Gene probe analysis of the MEN 2A locus on chromosome 10 has been undertaken using the markers TB10.163, RBP 3 and TB14.34 in a large kindred with familial medullary thyroid carcinomas, with or without phaeochromocytomas or primary hyperparathyroidism. A maximum LOD score of 2.97 gave strong evidence of close linkage with zero recombination. For 12 members of the family so far not known to be affected by any form of the disease the estimated risk of carrying the gene has been considerably decreased in all but one, whose risk has been greatly increased.

MEN 2A: update on the Northern Ireland and Australian family.

The Northern Ireland/Australian family with multiple endocrine neoplasia type 2A (MEN 2A) originally described in 1987 is presented with a revised and enlarged pedigree. Four members of the first generation studied have died. A seventh member of the second generation studied has developed medullary thyroid carcinoma and has progressed to surgery. None of the third generation members studied has shown any conclusive abnormality in metabolic screening tests. Each member of the third and fourth generations has had genetic counseling and (if appropriate) DNA analysis with gene probes close to the MEN 2A gene locus on chromosome 10. All members of this highly penetrant family have remained asymptomatic for their disease.

Automatic postural responses in the cat: responses of hindlimb muscles to horizontal perturbations of stance in multiple directions.

The effect of the direction of unexpected horizontal perturbations of stance on the organization of automatic postural responses was studied in cats. We recorded EMG activity in eight proximal and distal muscles of the hindlimb along with vertical forces imposed by the limbs in awake behaving cats while they stood on an hydraulic platform. Postural responses to motion of the platform in 16 different horizontal directions were recorded. Vertical force changes were consistent with passive shifts of the center of mass and active correction of stance by the animals. When the perturbation was in the sagittal plane, vertical force changes began about 65 ms following initial platform movement. When the perturbation contained a component in the lateral direction, latency for vertical force changes was about 25 ms and an inflection in the vertical force trace was observed at 65 ms. No EMG responses were observed with latencies that were short enough to account for the early force component and it was concluded that this force change was due to passive shifts of the center of mass. The amplitude of the EMG responses of each muscle recorded varied systematically as perturbation direction changed. The directions for which an individual muscle showed measurable EMG activity were termed the muscle's "angular range of activation." No angular range of activation was oriented strictly in the A-P or lateral directions. Most muscles displayed angular ranges of activation that encompassed a range of less than 180 degrees. Onset latencies of EMG responses also varied systematically with perturbation direction. The amplitude and latency relationships between muscles, which made up the organization of postural responses, also varied systematically as perturbation direction was changed. This result suggests that direction of perturbation determines organizational makeup of postural responses, and for displacements in the horizontal plane, is considered a continuous variable by the nervous system.

Automatic postural responses in the cat: responses of proximal and distal hindlimb muscles to drop of support from a single hind- or forelimb.

Cats respond to drop of the support from beneath a single limb with the "diagonal stance response" (Coulmance et al. 1979). They load the limbs on the diagonal opposite to the one containing the dropped limb and unload the third supporting limb in the diagonal containing the dropped limb. Characteristic biomechanical delays in limb motion and in vertical force changes imposed upon the limbs are observed. These delays range from 30 to 45 ms, depending upon the location of the dropped limb. This study describes the kinematics of the "diagonal stance response" and the activation of selected agonist-antagonist muscle pairs acting on the joints of the hindlimb during the response. Proximal and distal hindlimb muscles respond to perturbations in groups that are appropriate to the vertical forces imposed upon the limb. When the hindlimb containing the recording electrodes is loaded by drop of the contralateral hindlimb or the ipsilateral forelimb medium latency (25-45 ms) EMG responses occur in the extensors. This response serves to stiffen the limb against the increased vertical force of loading. A similar response is observed when the hindlimb is reloaded after being dropped. In this case, however, short latency responses precede the medium latency responses in muscles that are passively stretched by the limb drop. When drop of the diagonal forelimb unloads the hindlimb containing the electrodes, medium latency responses are observed in the distal hindlimb flexors, which indicates that the unloading is evoked in part by active lifting of the limb. In most cases, the medium latency responses precede or are coincident with the changes in force imposed on the limb, suggesting that the observed responses are centrally programmed.

Automatic postural responses in the cat: responses of distal hindlimb muscles to paired vertical perturbations of stance.

The active components of the quadrupedal diagonal stance response to rapid removal of the support from beneath a single limb were studied in cats to further define the mechanisms that trigger and generate the response. We recorded EMG activity from lateral gastrocnemius and tibialis anterior muscles in awake, behaving cats while they stood on an hydraulic posture platform. By dropping the support from beneath a single limb, we evoked the diagonal stance response, with its characteristic changes in vertical force and EMG patterns. As the animal responded to this drop, a second perturbation of posture was then presented at intervals of 10 to 100 ms following the first. The second perturbation, which consisted of dropping the support from beneath the two limbs that were loaded as a result of the initial limb drop, made the first response biomechanically inappropriate. The EMG responses observed in both muscles during paired perturbations were triggered by the somatosensory events related to the perturbations. Muscle responses that were appropriate for the first perturbation always occurred with amplitudes and latencies similar to control trials. This was true even when the second perturbation occurred 10-20 ms after the first, that is, when this perturbation either preceded or was coincident with the response to the initial limb drop. The EMG responses that were normally associated with the second perturbation were delayed and/or reduced in amplitude when the time interval between perturbations was short. As the inter-perturbation interval was lengthened beyond 60-100 ms, however, EMG responses to the second perturbation were unaffected by the occurrence of the first perturbation. When the hindlimb containing the recording electrodes was dropped as part of the second perturbation, a myotatic latency response was observed in tibialis anterior. The amplitude of this response to the second perturbation was greater than controls when this displacement was presented during the period between initiation of the first perturbation and execution of the response to it. When the second displacement was presented after execution of the first response began, the amplitude of the myotatic response was reduced below control levels. While the results do not preclude the possibility that these "automatic" postural responses are segmental or suprasegmental reflexes, they support the hypothesis that the active component of the response to drop of the support beneath a single limb is centrally programmed and that the appropriate response can be triggered very rapidly by the somatosensory information signalling the perturbation.