Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin?

PURPOSE: Two mutations (R555Q and R124L) in the BIGH3 gene have been described in anterior or Bowman's layer dystrophies (CDB). The clinical, molecular, and ultrastructural findings of five families with CDB was reviewed to determine whether there is a consistent genotype:phenotype correlation. METHODS: Keratoplasty tissue from each patient was examined by light and electron microscopy (LM and EM). DNA was obtained, and exons 4 and 12 of BIGH3 were analyzed by polymerase chain reaction and single-stranded conformation polymorphism/heteroduplex analysis. Abnormally migrating products were analyzed by direct sequencing. RESULTS: In two families with type I CDB (CDBI), the R124L mutation was defined. There were light and ultrastructural features of superficial granular dystrophy and atypical banding of the "rod-shaped bodies" ultrastructurally. Patients from three families with "honeycomb" dystrophy were found to carry the R555Q mutation and had characteristic features of Bowman's dystrophy type II (CDBII). CONCLUSIONS: There is a strong genotype:phenotype correlation among CBDI (R124L) and CDBII (R555Q). LM and EM findings suggest that epithelial abnormalities may underlie the pathology of both conditions. The findings clarify the confusion over classification of the Bowman's layer dystrophies.

Intracortical connectivity of layer VI pyramidal neurons in the somatosensory cortex of normal and barrelless mice.

In mice, barrels in layer IV of the somatosensory cortex correspond to the columnar representations of whisker follicles. In barrelless (BRL) mice, barrels are absent, but functionally, a columnar organization persists. Previously we characterized the aberrant geometry of thalamic projection of BRL mice using axonal reconstructions of individual neurons. Here we proceeded with the analysis of the intracortical projections from layer VI pyramidal neurons, to assess their contribution to the columnar organization. From series of tangential sections we reconstructed the axon collaterals of individual layer VI pyramidal neurons in the C2 barrel column that were labelled with biocytin [controls from normal (NOR) strain, 19 cells; BRL strain, nine cells]. Using six morphological parameters in a cluster analysis, we showed that layer VI neurons in NOR mice are distributed into four clusters distinguished by the radial and tangential extent of their intracortical projections. These clusters correlated with the cortical or subcortical projection of the main axon. In BRL mice, neurons were distributed within the same four clusters, but their projections to the granular and supragranular layers were significantly smaller and their tangential projection was less columnar than in NOR mice. However, in both strains the intracortical projections had a preference for the appropriate barrel column (C2), indicating that layer VI pyramidal cells could participate in the functional columnar organization of the barrel cortex. Correlative light and electron microscopy analyses provided morphometric data on the intracortical synaptic boutons and synapses of layer VI pyramidal neurons and revealed that projections to layer IV preferentially target excitatory dendritic spines and shafts.

New perspectives in the use of ink evidence in forensic science: Part II. Development and testing of mathematical algorithms for the automatic comparison of ink samples analysed by HPTLC

In the first part of this research, three stages were stated for a program to increase the information extracted from ink evidence and maximise its usefulness to the criminal and civil justice system. These stages are (a) develop a standard methodology for analysing ink samples by high-performance thin layer chromatography (HPTLC) in reproducible way, when ink samples are analysed at different time, locations and by different examiners; (b) compare automatically and objectively ink samples; and (c) define and evaluate theoretical framework for the use of ink evidence in forensic context. This report focuses on the second of the three stages. Using the calibration and acquisition process described in the previous report, mathematical algorithms are proposed to automatically and objectively compare ink samples. The performances of these algorithms are systematically studied for various chemical and forensic conditions using standard performance tests commonly used in biometrics studies. The results show that different algorithms are best suited for different tasks. Finally, this report demonstrates how modern analytical and computer technology can be used in the field of ink examination and how tools developed and successfully applied in other fields of forensic science can help maximising its impact within the field of questioned documents.

Short- and long-term effects of litter size manipulation in a small wild-derived rodent.

Iteroparous organisms maximize their overall fitness by optimizing their reproductive effort over multiple reproductive events. Hence, changes in reproductive effort are expected to have both short- and long-term consequences on parents and their offspring. In laboratory rodents, manipulation of reproductive efforts during lactation has however revealed few short-term reproductive adjustments, suggesting that female laboratory rodents express maximal rather than optimal levels of reproductive investment as observed in semelparous organisms. Using a litter size manipulation (LSM) experiment in a small wild-derived rodent (the common vole; Microtus arvalis), we show that females altered their reproductive efforts in response to LSM, with females having higher metabolic rates and showing alternative body mass dynamics when rearing an enlarged rather than reduced litter. Those differences in female reproductive effort were nonetheless insufficient to fully match their pups' energy demand, pups being lighter at weaning in enlarged litters. Interestingly, female reproductive effort changes had long-term consequences, with females that had previously reared an enlarged litter being lighter at the birth of their subsequent litter and producing lower quality pups. We discuss the significance of using wild-derived animals in studies of reproductive effort optimization.

Value of PET/CT versus contrast-enhanced CT in identifying chest wall invasion (T3) by NSCLC [B-671]

Purpose: To determine the diagnostic value of 18F-FDG PET/CT versus contrastenhanced CT in identifying chest wall invasion by NSCLC. Methods and Materials: The primary selection criterion was a peripheral tumor of any size with contact to the chest wall. A total of 25 patients with pathologically proven NSCLC satisfied these criteria. Chest wall invasion was interpreted upon PET/CT when a frank costal or intercostal 18F-FDG uptake was identified with or without concomitant morphologic alterations. On the other hand, the existence of periosteal rib reaction/erosion, chest wall thickening or obliteration of the pleural fat layer either separately or combined were considered essential diagnostic criteria for disease extension into the chest wall upon contrast-enhanced CT. The results were correlated with the final histological analysis. Results: Among the studied cohort, 13/25 (52%) patients had chest wall invasion consistent with T3 disease. Both PET/CT and contrast-enhanced CT successfully identified 12/13 (92%) of these patients. The single false-negative result was due to parietal pleural invasion. On the other hand, one false-positive result was encountered by PET/CT in a dyspneic patient; whereas, CT analysis revealed false-positive results in six patients. In these patients, periosteal rib reaction (n = 2) or asymmetric enlargement of adjacent chest wall muscles (n = 1) were identified along with an obliterated pleural fat layer (n = 6). The sensitivity, specificity, and accuracy of PET/CT and contrast-enhanced CT were 92, 91 and 92% versus 92, 50 and 72%. Conclusion: 18F-FDG PET/CT is an accurate diagnostic modality in identifying.

Identification by a monoclonal antibody of a glycolipid highly expressed by cells from the human myeloid lineage.

A monoclonal antibody (MAb) HL-C5, which bound selectively to cells of the myeloid lineage tested, was derived from a fusion between P3/NS2/1-AG8 myeloma cells and splenocytes from a mouse immunized with cells of the promyelocytic leukemia line HL-60. Among a panel of 29 human cell lines derived from either hematopoietic or solid tumors, MAb HL-C5 was found to react exclusively with cells from the five differentiated acute myeloid leukemia lines, HL-60, ML1, ML2, ML3, KG-1B and not with the less differentiated myeloid lines. Fluorescence-activated cell sorter analysis of normal bone marrow samples confirmed that the reactivity of MAb HL-C5 was limited to myeloid cells, from the promyelocytic stage of differentiation to the mature granulocytes. Indirect immunoperoxidase staining of cytocentrifuge preparations of normal bone marrow and peripheral blood leukocytes confirmed these results and showed that MAb HL-C5 stained neutrophils but not eosinophils or basophils. The antigen recognized by HL-C5 was recovered in the upper phase of chloroform-methanol-water lipid extracts prepared from HL-60 cells. By competitive binding experiments, it was found that MAb HL-C5 recognizes the same antigenic determinant as MAb WGHS 29-1, which has been reported to react with glycolipids containing the sugar sequence lacto-N-fucopentaose 111. Autoradiographs of thin layer chromatograms of HL-60 glycolipid extracts which were revealed by incubation with MAb HL-C5 or WGHS 29-1 followed by the addition of 125I-labelled rabbit anti-mouse immunoglobulin antibody confirmed that the two MAbs reacted with the same or structurally very similar glycolipids.

A double layer model of the gas bubble/water interface

Zeta potential is a physico-chemical parameter of particular importance to describe sorption of contaminants at the surface of gas bubbles. Nevertheless, the interpretation of electrophoretic mobilities of gas bubbles is complex. This is due to the specific behavior of the gas at interface and to the excess of electrical charge at interface, which is responsible for surface conductivity. We developed a surface complexation model based on the presence of negative surface sites because the balance of accepting and donating hydrogen bonds is broken at interface. By considering protons adsorbed on these sites followed by a diffuse layer, the electrical potential at the head-end of the diffuse layer is computed and considered to be equal to the zeta potential. The predicted zeta potential values are in very good agreement with the experimental data of H-2 bubbles for a broad range of pH and NaCl concentrations. This implies that the shear plane is located at the head-end of the diffuse layer, contradicting the assumption of the presence of a stagnant diffuse layer at the gas/water interface. Our model also successfully predicts the surface tension of air bubbles in a KCl solution. (c) 2012 Elsevier Inc. All rights reserved.

Cricotracheal resection for laryngeal invasion by thyroid carcinoma: our experience.

Invasion of the laryngeal framework by thyroid carcinoma requires specific surgical techniques and carries a higher rate of complications that deserve to be highlighted. We reviewed our data from 1995 to 2012 and found six patients with laryngotracheal invasion by thyroid carcinoma. All underwent total thyroidectomy and single-stage cricotracheal resection, plus anterolateral neck dissection. Three had airway obstruction that necessitated prior endoscopic debulking. None of the patients needed a tracheotomy. There were four cases of papillary carcinoma, and two cases of undifferentiated carcinoma. One patient died of complications of the procedure (anastomotic dehiscence and tracheo-innominate artery fistula). Another died 2 months after the procedure from local recurrence and aspiration pneumonia. One case presented recurrence at 15 months, which was managed by re-excision and adjuvant radiotherapy; after 26 months of follow-up, he has no evidence of locoregional recurrence. The three other patients are alive without evidence of disease at 6, 18 and 41 months, respectively. Cricotracheal resection for subglottic invasion by thyroid carcinoma is an effective procedure, but carries significant risks of complications. This could be attributed to the devascularisation of the tracheal wall due to the simultaneous neck dissection, sacrifice of the strap muscles or of a patch of oesophageal muscle layer. We advocate a sternocleidomastoid flap to cover the anastomosis. Cricotracheal resection for subglottic invasion can be curative with good functional outcomes, even for the advanced stages of thyroid cancer. Endoscopic debulking of the airway prior to the procedure avoids tracheotomy.

Multi-layer amniotic membrane (MLAM) transplantation for non traumatic corneal perforations or descemetoceles

Purpose:To describe the indications, the surgical procedure and the clinical outcome of MLAM in the treatment of non traumatic corneal perforations and descemetoceles . Methods:A prospective, non comparative, interventional case series of eight consecutive patients (mean age 59 years old, 6 men and 2 women) with non traumatic corneal perforations or descemetoceles.The surgery consisted in a MLAM transplantation of a cryopreservated human amniotic membrane. The series included: three active herpetic keratitis, one rosacea, one perforation of an hydrops, one cicatricial pemphigoid, one perforation after an abcess in a corneal graft and one perforation after protonbeamtherapy. The clinical outcome included: the follow-up, the integrity of the eye, corneal epithelialization, inflammation and neovascularization, and the integration of the MLAM. Stromal thickness was followed precisely with the slit lamp. A corneal graft was performed at one patient after the MLAM, allowing microscopic investigation of the removed MLAM integrated in the cornea. Results:The mean follow-up was 8.78 months (range 3.57 to 30.17). Amniotic membrane transplantation was successful and reduced inflammation in 7 patients out of 8 ,after one procedure.One patient who presented a large herpetic keratitis epithelial defect with corneal anaesthesia had his MLAM dissolved after two weeks with an aqueous leakage. Epithelium healed within 3 weeks above 7 MLAM and remained stable at 3 months in 7 out of 8 patients. MLAM opacification gradually disappeared over a few months, however, stromal layers filling in the corneal perforations or above the descemetoceles remained stable. Conclusions:MLAM transplantation is a safe, effective and useful technique to cure non traumatic corneal perforations and descemetoceles. It can be performed in emergency despite the presence of an active inflammation or infection. By facilitating epithelialization, reducing inflammation and neovascularization, it allows corneal surface reconstruction in patients with persistent epithelial defects and corneal melting that usually ends in a perforation. For full visual rehabilitation, a delayed penetrating keratoplasty is required.

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon.

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.

Validation of methane measurement using headspace-GC-MS and quantification by a stable isotope-labeled internal standard generated in situ.

A previous study has shown the possibility to identify methane (CH4 ) using headspace-GC-MS and quantify it with a stable isotope as internal standard. The main drawback of the GC-MS methods discussed in literature for CH4 measurement is the absence of a specific internal standard necessary to perform quantification. However, it becomes essential to develop a safer method to limit the manipulation of gaseous CH4 and to precisely control the injected amount of gas for spiking and calibration by comparison with external calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate a labeled gas as an internal standard in a vial on the basis of the formation of CH4 by the reaction of Grignard reagent methylmagnesium chloride with deuterated water. This method allows precise measurement of CH4 concentrations in gaseous sample as well as in a solid or a liquid sample after a thermodesorption step in a headspace vial. A full accuracy profile validation of this method is then presented.

Experimental manipulation of colony genetic diversity had no effect on short-term task efficiency in the Argentine ant Linepithema humile

Genetic diversity might increase the performance of social groups by improving task efficiency or disease resistance, but direct experimental tests of these hypotheses are rare. We manipulated the level of genetic diversity in colonies of the Argentine ant Linepithema humile, and then recorded the short-term task efficiency of these experimental colonies. The efficiency of low and high genetic diversity colonies did not differ significantly for any of the following tasks: exploring a new territory, foraging, moving to a new nest site, or removing corpses. The tests were powerful enough to detect large effects, but may have failed to detect small differences. Indeed, observed effect sizes were generally small, except for the time to create a trail during nest emigration. In addition, genetic diversity had no statistically significant impact on the number of workers, males and females produced by the colony, but these tests had low power. Higher genetic diversity also did not result in lower variance in task efficiency and productivity. In contrast to genetic diversity, colony size was positively correlated with the efficiency at performing most tasks and with colony productivity. Altogether, these results suggest that genetic diversity does not strongly improve short-term task efficiency in L. humile, but that worker number is a key factor determining the success of this invasive species.

Experimental conversion of colony social organization in fire ants (Solenopsis invicta): worker genotype manipulation in absence of queen effects

Colony social organization in the fire ant Solenopsis invicta appears to be under strong genetic control. In the invasive USA range, polygyny (multiple queens per colony) is marked by the presence of the Gp-9(b) allele in most of a colony's workers, whereas monogyny (single queen per colony) is associated with the exclusive occurrence of the Gp-9(B) allele. Ross and Keller, Behav Ecol Sociobiol 51:287-295 (2002) experimentally manipulated social organization by cross-fostering queens into colonies of the alternate form, thereby changing adult worker Gp-9 genotype frequencies over time. Although these authors showed that social behavior switched predictably when the frequency of b-bearing adult workers crossed a threshold of 5-10%, the possibility that queen effects caused the conversions could not be excluded entirely. We addressed this problem by fostering polygyne brood into queenright monogyne colonies. All such treatment colonies switched social organization to become polygyne, coincident with their proportions of b-bearing workers exceeding 12%. Our results support the conclusion that polygyny in S. invicta is induced by a minimum frequency of colony workers carrying the b allele, and further confirm that its expression is independent of queen genotype or history, worker genotypes at genes not linked to Gp-9, and colony genetic diversity.

Increasing lactate turnover rate during exercise by means of altitude training and fructose ingestion : effects on substrate metabolism and endurance performance

Summary : With regard to exercise metabolism, lactate was long considered as a dead-end waste product responsible for muscle fatigue and a limiting factor for motor performance. However, a large body of evidence clearly indicates that lactate is an energy efficient metabolite able to link the glycolytic pathway with aerobic metabolism and has endocrine-like actions, rather than to be a dead-end waste product. Lactate metabolism is also known to be quickly upregulated by regular endurance training and is thought to be related to exercise performance. However, to what extent its modulation can increase exercise performance in already endurance-trained subjects is unknown. The general hypothesis of this work was therefore that increasing either lactate metabolic clearance rate or lactate availability could, in turn, increase endurance performance. The first study (Study I) aimed at increasing the lactate clearance rate by means of assumed interaction effects of endurance training and hypoxia on lactate metabolism and endurance performance. Although this study did not demonstrate any interaction of training and hypoxia on both lactate metabolism and endurance performance, a significant deleterious effect of endurance training in hypoxia was shown on glucose homeostasis. The methods used to determine lactate kinetics during exercise exhibited some limitations, and the second study did delineate some of the issues raised (Study 2). The third study (Study 3) investigated the metabolic and performance effects of increasing plasma lactate production and availability during prolonged exercise in the fed state. A nutritional intervention was used for this purpose: part of glucose feedings ingested during the control condition was substituted by fructose. The results of this study showed a significant increase of lactate turnover rate, quantified the metabolic fate of fructose; and demonstrated a significant decrease of lipid oxidation and glycogen breakdown. In contrast, endurance performance appeared to be unmodified by this dietary intervention, being at odds with recent reports. Altogether the results of this thesis suggest that in endurance athletes the relationship between endurance performance and lactate turnover rate remains unclear. Nonetheless, the result of the present study raises questions and opens perspectives on the rationale of using hypoxia as a therapeutic aid for the treatment of insulin resistance. Moreover, the results of the second study open perspectives on the role of lactate as an intermediate metabolite and its modulatory effects on substrate metabolism during exercise. Additionally it is suggested that the simple nutritional intervention used in the third study can be of interest in the investigation on the aforementioned roles of lactate. Résumé : Lorsque le lactate est évoqué en rapport avec l'exercice, il est souvent considéré comme un déchet métabolique responsable de l'acidose métabolique, de la fatigue musculaire ou encore comme un facteur limitant de la performance. Or la littérature montre clairement que le lactate se révèle être plutôt un métabolite utilisé efficacement par de nombreux tissus par les voies oxydatives et, ainsi, il peut être considéré comme un lien entre le métabolisme glycolytique et le métabolisme oxydatif. De plus on lui prête des propriétés endocrines. Il est connu que l'entraînement d'endurance accroît rapidement le métabolisme du lactate, et il est suggéré que la performance d'endurance est liée à son métabolisme. Toutefois la relation entre le taux de renouvellement du lactate et la performance d'endurance est peu claire, et, de même, de quelle manière la modulation de son métabolisme peut influencer cette dernière. Le but de cette thèse était en conséquence d'investiguer de quelle manière et à quel degré l'augmentation du métabolisme du lactate, par l'augmentation de sa clearance et de son turnover, pouvait à son tour améliorer la performance d'endurance de sujets entraînés. L'objectif de la première étude a été d'augmenter la clearance du lactate par le biais d'un entraînement en conditions hypoxiques chez des cyclistes d'endurance. Basé sur la littérature scientifique existante, on a fait l'hypothèse que l'entraînement d'endurance et l'hypoxie exerceraient un effet synergétique sur le métabolisme du lactate et sur la performance, ce qui permettrait de montrer des relations entre performance et métabolisme du lactate. Les résultats de cette étude n'ont montré aucun effet synergique sur la performance ou le métabolisme du lactate. Toutefois, un effet délétère sur le métabolisme du glucose a été démontré. Quelques limitations de la méthode employée pour la mesure du métabolisme du lactate ont été soulevées, et partiellement résolues dans la seconde étude de ce travail, qui avait pour but d'évaluer la sensibilité du modèle pharmacodynamique utilisé pour le calcul du turnover du lactate. La troisième étude a investigué l'effet d'une augmentation de la lactatémie sur le métabolisme des substrats et sur la performance par une intervention nutritionnelle substituant une partie de glucose ingéré pendant l'exercice par du fructose. Les résultats montrent que les composants dynamiques du métabolisme du lactate sont significativement augmentés en présence de fructose, et que les oxydations de graisse et de glycogène sont significativement diminuées. Toutefois aucun effet sur la performance n'a été démontré. Les résultats de ces études montrent que la relation entre le métabolisme du lactate et la performance reste peu claire. Les résultats délétères de la première étude laissent envisager des pistes de travail, étant donné que l'entraînement en hypoxie est considéré comme outil thérapeutique dans le traitement de pathologies liées à la résistance à l'insuline. De plus les résultats de la troisième étude ouvrent des perspectives de travail quant au rôle du lactate comme intermédiaire métabolique durant l'exercice ainsi que sur ses effets directs sur le métabolisme. Ils suggèrent de plus que la manipulation nutritionnelle simple qui a été utilisée se révèle être un outil prometteur dans l'étude des rôles et effets métaboliques que peut revêtir le lactate durant l'exercice.

Localization and characterization of an active fault in an urbanized area in central Guatemala by means of geoelectrical imaging

The Polochic and Motagua faults define the active plate boundary between the North American and Caribbean plates in central Guatemala. A splay of the Polochic Fault traverses the rapidly growing city of San Miguel Uspantan that is periodically affected by destructive earthquakes. This fault splay was located using a 2D electrical resistivity tomography (ERT) survey that also characterized the fault damage zone and evaluated the thickness and nature of recent deposits upon which most of the city is built. ERT images show the fault as a similar to 50 m wide, near-vertical low-resistivity anomaly, bounded within a few meters by high resistivity anomalies. Forward modeling reproduces the key aspects of the observed electrical resistivity data with remarkable fidelity thus defining the overall location, geometry, and internal structure of the fault zone as well as the affected lithologies. Our results indicate that the city is constructed on a similar to 20 m thick surficial layer consisting of poorly consolidated, highly porous, water-logged pumice. This soft layer is likely to amplify seismic waves and to liquefy upon moderate to strong ground shaking. The electrical conductivity as well as the major element chemistry of the groundwater provides evidence to suggest that the local aquifer might, at least in part, be fed by water rising along the fault. Therefore, the potential threat posed by this fault splay may not be limited to its seismic activity per se, but could be compounded its potential propensity to enhance seismic site effects by injecting water into the soft surficial sediments. The results of this study provide the basis for a rigorous analysis of seismic hazard and sustainable development of San Miguel Uspantan and illustrate the potential of ERT surveying for paleoseismic studies.