Characterization of novel human intragenic antimicrobial peptides, incorporation and release studies from ureasil-polyether hybrid matrix.

Intragenic antimicrobial peptides (IAPs) are internal sequences of proteins with physicochemical similarities to Antimicrobial Peptides (AMPs) that, once identified and synthesized as individual entities, present antimicrobial activity. Many mature proteins encoded by the genomes of virtually any organism may be regarded as inner reservoirs of IAPs, conferring them ample biotechnological potential. However, IAPs may also share shortcomings with AMPs, such as low half-life in biological media and non-specific adsorption in eukaryotic cells. The present manuscript reports a translational approach that encompasses the uncovering of two novel IAPs from human proteins as well as the first results concerning the incorporation and sustained release of one of these peptides from ureasil-polyether hybrid polymeric films. For such, the software Kamal was used to scan putative IAPs in the human proteome, and two peptides, named Hs05 and Hs06, were identified, synthesized, and tested as antimicrobials. Biophysical assays were conducted using model phospholipid vesicles and 1H NMR solution structures in phospholipid micelles were obtained for the IAP Hs05. This peptide was incorporated in a polymeric matrix composed of the ureasil/PPO-PEO-PPO triblock copolymer, and the resulting films were evaluated by atomic force microscopy and imaging mass spectrometry. The release rate of Hs05 from the polymeric matrix was assessed and the antimicrobial activity of Hs05-loaded hybrid polymeric films was evaluated against the bacterium Escherichia coli. This study represents the first steps towards the development of polymeric films enriched with IAPs obtained from the human proteome as sustained release devices for topical application.

Relative quantification of plasma N-glycans in type II congenital disorder of glycosylation patients by mass spectrometry.

BACKGROUND: Type II Congenital Disorders of Glycosylation (CDG-II) are a group of diseases with challenging diagnostics characterized by defects in the processing of glycans in the Golgi apparatus. Mass Spectrometry (MS) has been a valuable tool in the definition of CDG-II subtypes. While some CDG-II subtypes are associated with specific N-glycan structures, others only produce changes in relative levels, reinforcing the demand for quantification methods. METHODS: Plasma samples from control individuals were pooled, derivatized with deuterated iodomethane (I-CD3), and used as internal standards for controls and patients whose glycans were derivatized with iodomethane (I-CH3), followed by MALDI MS, LC-MS and -MS/MS analyses. RESULTS: Total N-glycans from fifteen CDG-II patients were evaluated, and 4 cases with molecular diagnosis were considered in detail: 2ATP6V0A2-CDG siblings, and 2 MAN1B1-CDG patients, one of them carrying a previously undescribed p.Gly536Val mutation. CONCLUSIONS: Our methodology offers a feasible alternative to the current methods for CDG-II diagnosis by MS, which quantify glycan structures as fractions of the total summed signal across a mass spectrum, a strategy that lowers the variability of minor components. Moreover, given its sensitivity for less concentrated yet biologically relevant structures, it might assist the uncovering of novel diagnostic glycans in other CDG-II subtypes.

Towards an experimental classification system for membrane active peptides.

Mature proteins can act as potential sources of encrypted bioactive peptides that, once released from their parent proteins, might interact with diverse biomolecular targets. In recent work we introduced a systematic methodology to uncover encrypted intragenic antimicrobial peptides (IAPs) within large protein sequence libraries. Given that such peptides may interact with membranes in different ways, resulting in distinct observable outcomes, it is desirable to develop a predictive methodology to categorize membrane active peptides and establish a link to their physicochemical properties. Building upon previous work, we explored the interaction of a range of IAPs with model membranes probed by differential scanning calorimetry (DSC) and circular dichroism (CD) techniques. The biophysical data were submitted to multivariate statistical methods and resulting peptide clusters were correlated to peptide structure and to their antimicrobial activity. A re-evaluation of the physicochemical properties of the peptides was conducted based on peptide cluster memberships. Our data indicate that membranolytic peptides produce characteristic thermal transition (DSC) profiles in model vesicles and that this can be used to categorize novel molecules with unknown biological activity. Incremental expansion of the model presented here might result in a unified experimental framework for the prediction of novel classes of membrane active peptides.

Encrypted Antimicrobial Peptides from Plant Proteins.

Examples of bioactive peptides derived from internal sequences of proteins are known for decades. The great majority of these findings appear to be fortuitous rather than the result of a deliberate and methodological-based enterprise. In the present work, we describe the identification and the biological activities of novel antimicrobial peptides unveiled as internal fragments of various plant proteins founded on our hypothesis-driven search strategy. All putative encrypted antimicrobial peptides were selected based upon their physicochemical properties that were iteratively selected by an in-house computer program named Kamal. The selected peptides were chemically synthesized and evaluated for their interaction with model membranes. Sixteen of these peptides showed antimicrobial activity against human and/or plant pathogens, some with a wide spectrum of activity presenting similar or superior inhibition efficacy when compared to classical antimicrobial peptides (AMPs). These original and previously unforeseen molecules constitute a broader and undisputable set of evidences produced by our group that illustrate how the intragenic concept is a workable reality and should be carefully explored not only for microbicidal agents but also for many other biological functions.

Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases.

The black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) forms concentration dependent homomultimers, as previously demonstrated by Light scattering and Atomic Force Microscopy. Considering that these self-aggregates might influence their binding to cognate enzymes, we investigated the interaction of BTCI at picomolar concentrations using surface immobilized Chymotrypsin (α-CT) and Trypsin (T) by Surface Plasmon Resonance. Our results indicate that BTCI has subnanomolar affinity to both immobilized enzymes, which is approximately two orders of magnitude higher than previously reported. Moreover, we probed the influence of temperature on protein binding equilibria in order to investigate their interaction energetics. While the BTCI/T interaction concurs with the canonical entropy-driven mechanism described for BBI interactions with serine proteinases, the BTCI/α-CT interaction does not. Our measurements indicate that bimolecular BTCI/α-CT complexes form with a negative enthalpy change and a moderate entropic increase. Direct calorimetric evaluation is in accord with the van't Hoff approximation obtained by SPR. We demonstrate that as protein concentrations increase to the micromolar range, secondary endothermic events become prevalent and affect both the kinetics and thermodynamics of protein associations. Our study reinforces that BBI interactions with serine proteinases should be studied in dilute solutions to abridge often neglected secondary interactions.

Cognitive and outcome measures seem suboptimal in children with cochlear implants - a cross-sectional study.

OBJECTIVE: This study investigates whether cognition as a single variable significantly influences the hearing and speech outcomes of children with cochlear implants (CIs). More specifically, it identifies various types of cognitive disorders among children with CI, comparing these children's cognitive abilities to the outcomes of CI in terms of hearing and speech performance. STUDY DESIGN: Cross-sectional cognitive testing of children with CIs. SETTING: West Danish CI Centre, Aarhus University Hospital, Denmark. MATERIALS AND METHODS: Fifty-eight CI children underwent postoperative cognitive evaluation. Cognitive tests included Bayley Scales of Infant Development (Bayley-III), Snijders-Oomen Nonverbal Intelligence Test Revised (SON-R) and Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV), depending on the age of the child. Outcome measures of hearing and speech capacity after CI consisted of Categories of Auditory Performance (CAP), Speech Intelligibility Rating (SIR) and Peabody Picture Vocabulary Test-4 (PPVT-4). RESULTS: Only PPVT-4 correlated significantly with cognition scores. Over time, the value of CAP and SIR seems limited by a pronounced ceiling effect. CONCLUSION: The most widely used outcome tests after CI, that is CAP and SIR, may not reflect cognitive disabilities sufficiently due to pronounced ceiling effects. International guidelines suggesting a panel of robust age-matched tests are warranted to optimise rehabilitation programmes as well as the counselling of parents.

Only a Touch of the Flu? The Simultaneous Manifestation of Acute Necrotizing Encephalopathy in Two Consanguineous Patients.

This case report describes the simultaneous manifestation of acute necrotizing encephalopathy in 2 consanguineous patients after infection with influenza B based on the autosomal dominant missense mutation of the RANBP2-gene. Differential diagnosis of acute encephalopathy, clinical and radiological clues, and treatment strategies are outlined.

[Fever and lymphadenopathy. Report of 4 cases of tularemia]. / Fieber und Lymphadenopathie. Bericht über 4 Tularämiefälle.

We report on 4 patients with different types of tularemia acquired in Switzerland or nearby countries. All patients presented with fever, moderate to highly elevated signs of inflammation, and local lymphadenopathy. Additionally, 3 patients did not respond to empirical antimicrobial therapy with aminopenicillins. A tick bite was identified as mode of transmission in 2 patients, while 1 patient showed a possible connection to a tick bite. The route of transmission for the fourth patient remained unknown. The diagnosis of tularemia was either based on positive serology, on a positive polymerase chain reaction (PCR) from the lymph node samples or on positive blood cultures. The treatment in adult patients was ciprofloxacin 500-750 mg twice daily orally for 3 weeks. The pediatric patient was treated with gentamicin 4 mg/kg i.v. once daily for 1 week and ciprofloxacin 15 mg/kg twice daily orally for another 2 weeks. All patients recovered completely. Due to the increasing incidence of tularemia in Switzerland, this infection should be considered in patients with fever and lymph node enlargement particularly after tick bite. We recommend treatment with ciprofloxacin orally for 14-12 days.

Bioacumulation of cyanotoxins in Hypophthalmichthys molitrix (silver carp) in Paranoá Lake, Brasilia-DF, Brazil.

Three individuals of silver carp (Hypophthalmichthys molitrix) were collected biweekly from Paranoá Lake (DF, Brazil) for analysis of microcystin (MC) concentrations in their muscle and liver tissue. Analysis by high performance liquid chromatography and mass spectrometry revealed MC masses and fragmentation patterns that were identified as MC-LR (995.04 m/z), MC-LA (909.01 m/z) and an unknown MC (987.07 m/z). Concentrations were calculated as MC-LR equivalents using a calibration curve prepared with a standard of MC-LR. May/06 was the month with the highest MC-LR equivalent concentrations in muscle and liver (3.83 ± 2.78, and 12.94 ± 10.51 µg g(-1), respectively). Our results show that during the drought months (April-September), consumption of fish with these MC concentrations would result in exposure to MCs that greatly exceed the World Health Organization's recommended tolerable daily intake limit of 0.04 µg MC kg(-1) body weight.

Comparison of a new hydro-surgical technique to traditional methods for the preparation of full-thickness skin grafts from canine cadaveric skin and report of a single clinical case.

OBJECTIVE: To compare the hydro-surgical technique to traditional techniques for removal of subcutaneous tissue in the preparation of full-thickness skin grafts. STUDY DESIGN: Ex vivo experimental study and a single clinical case report. SAMPLE POPULATION: Four canine cadavers and a single clinical case. METHODS: Four sections of skin were harvested from the lateral flank of recently euthanatized dogs. Traditional preparation methods used included both a blade or scissors technique, each of which were compared to the hydro-surgical technique individually. Preparation methods were compared based on length of time for removal of the subcutaneous tissue from the graft, histologic grading, and measurable thickness as compared to an untreated sample. RESULTS: The hydro-surgical technique had the shortest skin graft preparation time as compared to traditional techniques (p = 0.002). There was no significant difference in the histological grading or measurable subcutaneous thickness between skin specimens. CLINICAL SIGNIFICANCE: The hydro-surgical technique provides a rapid, effective debridement of subcutaneous tissue in the preparation of full-thickness skin grafts. There were not any significant changes in histological grade and subcutaneous tissue remaining among all treatment types. Additionally the hydro-surgical technique was successfully used to prepare a full-thickness meshed free skin graft in the reconstruction of a traumatic medial tarsal wound in a dog.

SU-E-T-471: Beam Properties of an In-Room Proton Therapy Accelerator.

PURPOSE: To quantify basic beam properties of the first gantry-mounted proton therapy accelerator, situated in the treatment room and delivering beam to the patient without any intermediate bending magnets. METHODS: Monte Carlo simulations for prototype beamlines of the Mevion S250TM proton therapy system were performed using MCNPX. For each configuration, open profiles in air were determined at 3 different positions for 3 range settings (216 profiles). Similarly, half-beam-blocked profiles were determined for the same settings (216 profiles). Additionally, beam axis fluence profiles in air were determined for 3 range settings (72 profiles). Finally, Bragg peak depth-dose curves in water were determined for 3 range settings (72 profiles). Using these 576 profiles, several beamline parameters were determined for each proton range and configuration, including virtual SAD, effective source size, and effective SAD. Additionally, Bortfeld's analytic approximation of the Bragg curve was fit to each depth-dose curve to determine R0, σ and ε. The 72 values determined for each parameter were compared within each configuration and across all configurations. RESULTS: Within any configuration, the change in range was too small (=2.5 cm) to have a large effect on any of the extracted parameters. Comparing parameters among configurations revealed smooth trends. These parameters were then fit as a function of range. CONCLUSIONS: The simplicity of this system results in beams with greater similarity among configurations as compared to other proton therapy systems. Because a beam with fixed initial energy is degraded only by tissue-equivalent materials, range straggling and beam divergence are nearly constant across all beamline configurations. Beam parameters change smoothly as functions of proton range, with no major discontinuities between configurations. This suggests that the entire machine may be fully and accurately characterized in a treatment planning system by fewer measurements than would normally be required.

MO-F-213AB-01: Improving Dose Uniformity in Patch-Field Proton Therapy Using Beam Current Modulation.

PURPOSE: To improve the dose homogeneity in patch-field proton therapy using beam current modulation. METHODS: MCNPX simulations were performed for various configurations of the Mevion S250 beamline. Spread-out Bragg peak (SOBP) dose distributions were calculated, and lateral and distal dose profiles were extracted for through- and patch-field targets at depths ranging from 5.0 to 27.5 cm. We define 'dose non-uniformity' (DNU) as the difference between the maximum and minimum dose relative to the prescription observed in a patched dose distribution. DNU was first calculated for dose distributions combining through-field lateral dose profiles and with patch-field distal dose profiles, assuming a 'standard' delivery with constant beam current. Distal dose profiles were then optimized using beam current modulation to better complement the lateral dose profiles when combined into a patched dose distribution. The DNU for optimized dose distributions was then calculated and compared to the DNU found for standard delivery. RESULTS: Using standard deliveries, DNU was 10% or less when patching lateral profiles 12.5-17.5 cm deep. Significantly greater DNU was observed for patches outside of this range, sometimes exceeding 35%. Using optimized distal profiles, DNU was reduced to 10% or less for all lateral profiles deeper than 12.5 cm. Patches with lateral profiles shallower than 12.5 cm were not improved, as distal dose dropoff could not be improved to better match the sharp lateral dose dropoff. CONCLUSIONS: Optimizing beam current modulation can create distal profiles with more gradual dose falloff than found in a standard delivery, allowing optimized distal dose distributions to sum more homogeneously with lateral dose distributions. Thus, the hot or cold spots that often appear in patched dose distributions from standard deliveries can be mitigated by optimizing beam current. This method may also be applied to feathering methods or scanned beam deliveries to further improve patch-field dose homogeneity. Partially supported by MEVION Medical Systems, Inc.

MO-F-213AB-02: Correcting Spread-Out Bragg Peak Slope Using Time-Resolved Monte Carlo Simulations and Beam Current Modulation.

PURPOSE: To fully characterize the spread-out Bragg peak from the Mevion S250 Proton Therapy System using Monte Carlo simulations, evaluate the slope of the spread-out Bragg peak in the treatment field, and correct this slope by virtually applying beam current modulation. METHODS: MCNPX simulations were performed on the Mevion S250 for each of the 24 beamline configurations, with the modulator wheel rotated in 1 degree increments. Energy deposition was tallied in a water phantom, resulting in 8,640 central axis depth-dose calculations. These data were imported into MATLAB and the slope of the spread-out Bragg peak (SOBP) was evaluated for a constant beam current. An iterative algorithm was developed to determine the optimal beam current modulation (BCM) profile as a function of modulator wheel position for each configuration. These BCM profiles were then applied virtually to the MCNPX SOBP data and slope values were recalculated for the optimized SOBP profiles. RESULTS: Ideally SOBP slope would measure 0.0 %/cm in the treatment field, corresponding to a uniform dose delivery. Prior to BCM correction, slopes between -1.0 and +2.0 %/cm were observed across all beamline configurations. These were reduced to ±0.2 %/cm by applying optimized BCM profiles. The algorithm converged quickly, validating its underlying assumption that the optimal SOBP profile can be determined by considering only a single dose values at the depths of maximum individual Bragg peak dose. CONCLUSIONS: Optimized BCM profiles adequately reduce SOBP slope in the treatment field, and may be further studied using time-resolved MCNPX simulations. The algorithm presented efficiently calculates BCM profiles, fully accounting for beamline scatter and partial-shining effects across adjacent steps on the range modulator wheels, and without requiring direct measurements. Future work includes applying this same algorithm to optimize the SOBP distal dose profile. Partially supported by MEVION Medical Systems, Inc.

MRI for the size assessment of pure ductal carcinoma in situ (DCIS): a prospective study of 33 patients.

OBJECTIVE: To determine whether MRI assesses the size of ductal carcinomas in situ (DCIS) more accurately than mammography, using the histopathological dimension of the surgical specimen as the reference measurement. MATERIALS AND METHODS: This single-center prospective study conducted from March 2007 to July 2008 at the Antoine-Lacassagne Cancer Treatment Center (Nice, France) included 33 patients with a histologically proven DCIS by needle biopsy, who all underwent clinical examination, mammography, and MRI interpreted by an experienced radiologist. All patients underwent surgery at our institution. The greatest dimensions of the DCIS determined by the two imaging modalities were compared with the histopathological dimension ascertained on the surgical specimen. The study was approved by the local Ethical Research Committee and was authorized by the French National Health Agency (AFSSAPS). RESULTS: The mean age of the 33 patients was 59.7 years (± 10.3). Three patients had a palpable mass at clinical breast examination; 82% underwent conservative surgical therapy rather than radical breast surgery (mastectomy); 6% required repeat surgery. MRI detected 97% of the lesions. Non-mass-like enhancement was noted for 78% of the patients. In over 50% of the cases, distribution of the DCIS was ductal or segmental and the kinetic enhancement curve was persistent. Lesion size was correctly estimated (± 5 mm), under-estimated (<5mm), or over-estimated (>5mm), respectively, by MRI in 60%, 19% and 21% of cases and by mammography in 38%, 31% and 31% (p = 0.05). Mean lesion size was 25.6mm at histopathology, 28.1mm at MRI, and 27.2mm on mammography (nonsignificant difference). The correlation coefficient between histopathological measurement and MRI was 0.831 versus 0.674 between histopathology and mammography. The correlation coefficient increased with the nuclear grade of the DCIS on mammography; this coefficient also increased as the mammographic breast density decreased. CONCLUSION: MRI appears to assess the size of DCIS better than mammography by limiting the number of under- and over-estimations compared to histopathology findings.

Efficient implementation of the rank correlation merit function for 2D/3D registration.

A growing number of clinical applications using 2D/3D registration have been presented recently. Usually, a digitally reconstructed radiograph is compared iteratively to an x-ray image of the known projection geometry until a match is achieved, thus providing six degrees of freedom of rigid motion which can be used for patient setup in image-guided radiation therapy or computer-assisted interventions. Recently, stochastic rank correlation, a merit function based on Spearman's rank correlation coefficient, was presented as a merit function especially suitable for 2D/3D registration. The advantage of this measure is its robustness against variations in image histogram content and its wide convergence range. The considerable computational expense of computing an ordered rank list is avoided here by comparing randomly chosen subsets of the DRR and reference x-ray. In this work, we show that it is possible to omit the sorting step and to compute the rank correlation coefficient of the full image content as fast as conventional merit functions. Our evaluation of a well-calibrated cadaver phantom also confirms that rank correlation-type merit functions give the most accurate results if large differences in the histogram content for the DRR and the x-ray image are present.

Frontoxins, three-finger toxins from Micrurus frontalis venom, decrease miniature endplate potential amplitude at frog neuromuscular junction.

Neurotoxicity is a major symptom of envenomation caused by Brazilian coral snake Micrurus frontalis. Due to the small amount of material that can be collected, no neurotoxin has been fully sequenced from this venom. In this work we report six new three-finger like toxins isolated from the venom of the coral snake M. frontalis which we named Frontoxin (FTx) I-VI. Toxins were purified using multiple steps of RP-HPLC. Molecular masses were determined by MALDI-TOF and ESI ion-trap mass spectrometry. The complete amino acid sequence of FTx II, III, IV and V were determined by sequencing of overlapping proteolytic fragments by Edman degradation and by de novo sequencing. The amino acid sequences of FTx I, II, III and VI predict 4 conserved disulphide bonds and structural similarity to previously reported short-chain alpha-neurotoxins. FTx IV and V each contained 10 conserved cysteines and share high similarity with long-chain alpha-neurotoxins. At the frog neuromuscular junction FTx II, III and IV reduced miniature endplate potential amplitudes in a time-and concentration-dependent manner suggesting Frontoxins block nicotinic acetylcholine receptors.

[Breast cancer screening imaging: what do we do]. / Dépistage du cancer du sein : quelle imagerie pour quelles femmes?

The purpose of this review is to evaluate the value of different breast imaging technics and their place for individual and mass screening of breast cancer according to the randomized studies on digital mammography and ultrasound screening. Analogic and numerical mammograms are validated for screening of women aged from 50 and 74 years. The additional value of ultrasound is therefore proven when the increased risk is moderate. When risk is higher (genetic or familial), MRI is the method of choice associated with conventional imaging. Individual screening is recommended before 50 for women aged from 45 and 50 and for those over 74 using the same procedures as organized screening.

[Anatomy of the female pelvis on MRI: value of intravaginal contrast]. / Anatomie pelvienne en IRM: apport du contraste vaginal.

PURPOSE: Prospective monocentric study to determine the feasibility, tolerability and diagnostic value of intravaginal contrast to assess female pelvic anatomy on MRI. MATERIALS AND METHODS: Forty-nine consecutive women referred for MRI evaluation of the pelvis, irrespective of the indication, were included in this study. The MR imaging protocol consisted of axial and sagittal T2W images before and after intravaginal instillation of sterile US gel. Eight anatomical regions were analyzed and their visibility graded from 1 to 4 (1=excellent; 4=non-visualized) by 3 radiologists without and with intravaginal gel. The value of intravaginal gel was determined by calculating the difference in the visibility index for each anatomical region by the Wilcoxon and khi2 tests. Inter-observer agreement was also determined using the kappa test. RESULTS: Two women declined vaginal opacification resulting in an acceptance rate of 96%. The gel instillation procedure had a duration of less than 3 minutes on average and was well tolerated by all patients. Intravaginal gel allowed significantly improved visualization of all anatomical regions (p<0.001); improvement between 0.5 and 2.5 points on average per anatomical region. Inter-observer agreement significantly improved after gel instillation increasing from 72% to 92%. CONCLUSION: Intravaginal instillation of US gel is simple, noninvasive, well-accepted and well-tolerated by patients. It increases visibility of pelvic anatomical structures with improved inter-observer agreement.