Design of an artificial phage-display library based on a new scaffold improved for average stability of the randomized proteins.

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the µM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.

[Epidemiologic and histopathologic characteristics of 1280 uterine cervical cancers in Kinshasa]. / Caractéristiques épidémiologiques et histopathologiques de 1280 cancers du col utérin à Kinshasa.

OBJECTIVE: Due to the lack of both cancer registry and large scale cervical screening in most african countries, only theoretical studies are available. The objective of our work was to provide epidemiological and histopathological characteristics of cervical cancer based on concrete observations. METHODS: This study was carried out on all cancers (n=5801) collected in the last 10 years from 5 pathology laboratories of Kinshasa; the histologic slides of the cervical cancers (n=1280) were reviewed by at least two pathologists and classified according to the 2014 OMS classification. RESULTS: The cervical cancers accounted for 22% of all cancers and 40,4% of breast and gynecological cancers. The cervical cancer was the most common among women aged 49-58. Squamous cell carcinomas were the most observed type (73,2%) followed by adenocarcinomas (18,4%) and adenosquamous carcinomas (8,4%). Keratinized (47,2%) and non keratinized squamous carcinoma (20,8%) were the most frequent subtypes among squamous carcinomas and the usual adenocarcinoma among adenocarcinomas (9,6%). In the mucinous adenocarcinoma subtype, only the signet ring cells (1,3%) variant was found. Among cervical cancers, 69% were grade I, 20% grade II and 11% grade III. CONCLUSION: Our study provides a concrete database of epidemiological and histopathological cervical cancer particularities in Kinshasa, useful to initiate a cancer register, as well as cervical screening and HPV vaccine campaigns.

Multiple myeloma presenting as hepatic nodular lesion.

The diffuse infiltration by plasma cells in the liver is not uncommon in multiple myeloma (MM). However, a MM with hepatic mass is very unusual. We report a case of a 75-year-old male with hepatomegaly and a lesion occupying a voluminous space in the liver. A lambda light chain multiple myeloma was found in the check-up of this hepatic mass. We also provide a literature review.

All-trans-retinoic acid-induced disturbanceof forelimb digital apoptosis in mouseembryos: a preliminary scanning electronmicroscope (SEM) tudy

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Cross-sectional anatomy and comparative ultrasonography of the equine medial femorotibial joint and its related structures.

REASONS FOR PERFORMING STUDY: Injuries of the equine stifle are frequent causes of hindlimb lameness. Ultrasonography is useful for the diagnosis of many soft tissue lesions but, until recently, its application to the equine stifle has been limited to a description of normal and abnormal structures. A comparative study using gross anatomical sections to identify all structures visible on ultrasonographic images of the equine stifle has not previously been reported. OBJECTIVES: To provide a comprehensive comparative cross-sectional atlas of the normal equine medial femorotibial (MFT) joint and its related structures using ultrasonography. METHODS: The stifle joints of 15 cadaver limbs were examined ultrasonographically using a systematic approach. Ten different scanning planes were defined. The lateral and medial femorotibial and the femoropatellar joints were injected with pigments of different colours. Each leg was frozen (10 in extension and 5 in flexion) and cut into slices corresponding to the different scanning planes. According to the different ultrasonographic reference images, 10 representative anatomical images were selected and digitised. RESULTS: All bony and soft tissue structures were identified on the anatomical sections and subsequently located on the corresponding ultrasonographic images, except the caudal meniscotibial ligaments. Visualisation of the cruciate ligaments was a challenge due to their position and oblique orientation. CONCLUSIONS: Ultrasonography is a very useful technique for imaging both soft tissue and bony structures of the MFT joint and its related structures, although a protocol is required to perform a systematic and standardised ultrasonographic examination. Good anatomical knowledge is mandatory for identification of the different structures. POTENTIAL RELEVANCE: This study should contribute to 1) knowledge of the ultrasonographic anatomy of the equine MFT joint and its related structures, 2) use of a protocol for a standardised ultrasonographic examination and 3) the ability to recognise abnormal structures.

Histological observations of palatal malformations in rat embryos induced by retinoic acid treatment.

Malformations of the palate were induced in white rat embryos following maternal exposure to retinoic acid (tretinoin). Five experimental groups and the controls were treated by the following protocol: Group 1: pregnant rats received 100 mg retinoic acid (RA)/kg b.w. suspended in corn oil on gestational day (GD) 11.5; Group 2: 20 mg RA/kg b.w. from GD 8-12; Group 3: 20 mg RA/kg b.w. from GD 7.5-11.5; Group 4: 100 mg RA/kg b.w. on GD 10-11; Group 5: 100 mg RA/kg b.w. on GD 10 and 12; Group 6 received corn oil vehicle from GD 7-14.5; and Group 6: served as non-injected controls. In all retinoic acid treated groups, varying degrees of clefts with occasional attempts of fusion were noted. The severity and frequency of the malformations were dependent on dosage or gestational day of drug treatment. Our results indicate that RA, even at the lowest dose tested (20 mg/kg b.w.) severely affects the various tissues constituting the embryonic palatal shelves by altering cell interaction and possibly programmed cell death. These events would then result in lack of or inadequate differentiation with subsequent formation of aberrant craniofacial architecture.

Craniofacial abnormalities induced by retinoic acid: a preliminary histological and scanning electron microscopic (SEM) study.

Exogenous retinoic acid has been found to be teratogenic in animals and man. Craniofacial defects induced by retinoic acid have stimulated considerable research interest. The present report deals with scanning electron microscopical observations of the craniofacial region concurrent with histological examination of craniofacial dysmorphism induced in rat embryos following maternal treatment treated with varying dosages of all-trans-retinoic acid (tretinoin). Two groups of pregnant rats were treated with rat embryos exposed to retinoic acid suspended in corn oil (100 mg/kg b.w. on gestational day 11.5 and 50 mg/kg b.w. on gestational day 10, 11 and 12 respectively). A third group was treated with corn oil (vehicle) while a fourth group remained untreated. A wide spectrum of congenital abnormalities, including exophthalmos, microphthalmia and anophthalmia, maxillo-mandibular dysostosis, micrognathia of both maxilla and mandible, cleft palate, subdevelopment of ear lobe, preauricular tags and macroglossia, were observed in the offspring of retinoic acid treated animals. The abnormalities were both time and dosage dependent, and characteristic of Treacher Collins syndrome when retinoic-acid was administered on gestational day 11.5. In contrast, when retinoic acid was administered were on gestational days 10-12, the defects were similar to those seen in the first and second pharyngeal arch syndrome, as well as in the oculo-auriculo-vertebral spectrum. Whereas our data support the hypothesis that all-trans retinoic-acid disturbs growth and differentiation of several embryonic cell types essential for normal craniofacial development, its mechanism of action remains unclear.

Anterior neural tube malformations induced after all-trans retinoic acid administration in white rat embryos. I. Macroscopical observations.

For this study all-trans-retinoic acid was administered in pregnant white rats in their "prima gravida" pregnancy. Rats were divided in five groups. The first three groups were treated with 20 mg R.A./kg b.w. at several gestational days. The fourth group was treated with corn oil, while the fifth group remained untreated. All the animals were sacrificed during the first hours of the 21st gestational day. In the first group, three embryos, five absorptions and six compact embryonic masses were counted in litters. All the embryos presented exencephaly, combined with external anopthalmia. They also presented severe craniofacial malformations. In the second group, nine embryos and five compact embryonic masses were counted in litters. Three of the embryos presented exencephaly combined with external anopthalmia, while the six remaining presented complex craniofacial anomalies. In the third group, exencephaly was present in two embryos combined with anopthalmia, seven embryos had complex anomalies and four compact embryonic masses were counted in litters. Our results indicate the teratogenic involvement of all-trans-retinoic acid in anterior neural tube differentiation.

All trans retinoic acid interfering with palatal development. Scanning electron microscopical and light microscopical observations on embryonic rat palate.

Diverse studies on retinoic acid teratogenesis, during the recent years, indicate that the drug's analogues target on diverse cell population during differentiation in mammals. During an extended teratological protocol concerning retinoic acid influence in diverse embryonic tissue differentiation in experimental animals we studied all-trans-retinoic acid's influence on palatal development in the white rat embryo. For this purpose, six groups of white rat embryos were studied: Group 1 was treated with 100 mg/kilogram of body weight (k.b.w.) on gestational days (g.d.) 10th and 11th, Group 2 was treated with 100 mg all-trans-retinoic acid/k.b.w. on g.d. 11.5, Group 3 was treated with 50 mg all-trans retinoic acid/k.b.w. on g.d. 10th, 11th and 12th, Group 4 was treated with 50 mg all-trans-retinoic acid/k.b.w. on g.d. 11th and 12th, Group 5 was treated with 20 mg all-trans-retinoic acid/k.b.w. on g.d. 7.5, 8.5, 9.5, 10.5 and 11.5, Group 6 remained untreated. Embryonic heads aged 20 days were observed by light microscopy and scanning electron microscopy. In all treated groups clefts and malformations concerning the differentiation of palatal cell populations were observed. All our findings were compared with normal palatal morphology of untreated "control" embryos. Among the malformations, median clefts were observed, extended along only a part of the primary and all the secondary palate for group 2, the primary and secondary palate for groups 1, 3 and 5 while on group 4, an irregularity of the median palatal raphe and rugae were combined with a median incomplete cleft extended between the primary and secondary palate. Our results are discussed in relation with the international literature results.

Microradiographic aspects of the growing mandibular body during permanent premolar eruption in the dog.

In order to explore the bony changes in the mandibular body during prefunctional intraosseous eruption of premolars, 18 dogs aged from 8 to 16 wk at the beginning of experimental period, were given two intraperitoneal injections of oxytetracycline (50 mg/kg and 35 mg/kg 2 wk later) and 2 wk later a final injection of Alizarin red S (70 mg/kg). Microradiographic and fluorescent light microscopy studies showed that changes of the alveolar bony crypt walls were influenced by the growing dental germs which they surrounded. The cervical volumetric reduction, which indicates the end of crown formation, induced the apposition of lamellar and then woven bone on the adjacent alveolar walls. Furthermore, with occlusal displacement of the dental crown, the space below the tooth was immediately filled with woven bone trabeculae and chondroid tissue. The same phenomenon was observed at the level of the alveolar base, when the speed of tooth eruption was greater than that of root growth. During premolar development, the changes in the dental germ produces accommodating changes in the adjacent alveolar bone walls, and mandibular transversal growth has the same characteristics as that of a growing diaphysis.

Oculo-auriculo-vertebral spectrum: cranial and vertebral malformations due to focal disturbed chondrogenesis.

Microradiographic and histological analyses point out a focal disturbed chondrogenesis of both the skull base and the axial skeleton in a case of oculo-auriculo-vertebral spectrum. Cartilage showed disturbed endochondral ossification with defects in calcification, deficient resorption, and abnormal crumpled areas of mineralized cartilage.

Protection by glutathione against the antiproliferative effects of nitric oxide. Dependence on kinetics of no release.

Pretreatment by L-buthionine sulfoximine (BSO), which inactivates gamma-glutamylcysteine synthetase and, therefore, inhibits glutathione (GSH) synthesis, greatly increased the sensitivity of tumor cells to the antiproliferative effects of several NO-donating compounds. The sensitization that resulted from depletion of cellular GSH pools was observed in tumor cells exhibiting different degrees of resistance to NO. In contrast, GSH depletion of tumor target cells did not affect their sensitivity to the cytostatic activity of activated macrophages and other NO-producing cells (EMT6 cells treated by interferon gamma and LPS). The kinetics for NO generation is a parameter that may differentiate NO-producing cells and short-lived NO donors. To study the relationship between the magnitude of NO fluxes and the increased toxicity on BSO-pretreated cells, two NO-releasing zwitterions derived from polyamines (NONOates) with different half-lives were selected. NO fluxes as a function of time were simulated, according to the donor half-life and initial concentration, and antiproliferative effects on control and BSO-treated cells were compared. GSH depletion increased the sensitivity of tumor cells in the case of the less stable NO donor only. We, thus, propose that intracellular GSH is specifically protective against high fluxes of NO.

Cytochemical localization of Ca(2+)-ATPases and demonstration of ATP-dependent calcium sequestration in giant smooth muscle fibres of Beroe.

A cytochemical analysis of the mechanisms underlying cytosolic calcium regulation was undertaken in the giant smooth muscle fibres of the marine invertebrate Beroe. The ability of the sarcoplasmic reticulum to accumulate Ca2+ was demonstrated on living skinned single cells. In the presence of oxalate, and physiological concentrations of Ca2+, calcium oxalate crystals were formed in the lumen of tubules and cisternae of the sarcoplasmic reticulum. The subcellular distribution of Ca(2+)-ATPase was studied with a cytochemical technique; a dense precipitate resulting from Ca(2+)-ATPase activity was found on the plasma membrane, on the membranes of tubules and cisternae of the sarcoplasmic reticulum, and in mitochondria.

Two distinct distribution patterns of sarcoplasmic reticulum in two functionally different giant smooth muscle cells of Beroe ovata.

The sarcoplasmic reticulum has been studied in radial and longitudinal giant smooth muscle fibres of the marine planktonic invertebrate Beroe. Impregnation with heavy metals has revealed that the smooth component is organised in a longitudinally oriented three-dimensional network of tubules running along the myofilaments. An ultrastructural morphometric analysis has shown that the relative volume of the sarcoplasmic reticulum is the same (1% of the myofilament volume) in both fibres but that the size, number and distribution of the sarcoplasmic reticulum tubules differ significantly. The longitudinal fibres are characterised physiologically by an action potential with a short calcium-dependent plateau that can trigger a short contraction; radial fibres produce action potentials without a plateau and their contraction requires a train of spikes. The sarcoplasmic reticulum tubules in longitudinal fibres are thinner (132 nm in diameter) and more numerous than those in radial fibres (160 nm in diameter). Moreover, the tubules are homogeneously distributed among the myofilaments in radial fibres, whereas they are more numerous in the centre of longitudinal muscles.

Comparative SEM and TEM examination of the ultrastructure of the resin-dentin interdiffusion zone.

The resin-dentin interdiffusion zone produced by a dentin-adhesive system that removes the smear layer and concurrently decalcifies superficial dentin was morphologically examined by both scanning and transmission electron microscopy. Cross-sectioned resin-bonded dentin discs were etched with an argon-ion beam to make the resin-dentin interface observable by SEM. For the TEM examination, the sections were partly decalcified by an aqueous EDTA solution to facilitate ultramicrotomy and to disclose the ultrastructure of the interdiffusion zone. Both SEM and TEM confirmed the presence of the resin-dentin interdiffusion zone as the junction between the deep unaltered dentin structure and the restorative resin. Within the interdiffusion zone, three sublayers with characteristic ultrastructure and staining were identified by TEM. An upper diffuse black layer contained few structural features. Underneath, partially-altered collagen fibrils were closely packed, mostly running parallel with the interface and perpendicular to the dentinal tubules. Their outline was electron-dense, forming tunnel-like structures. At the base of the upper layer, several stained projections were found to bulge out into the underlying collagen network and appeared to be confined by obstructive, parallel-running collagen fibrils. Finally, the third dense layer, containing hydroxyapatite crystals, demarcated the superficially demineralized dentin layer from the deeper unaltered dentin. Resin diffusion into the decalcified dentin surface layer was evident, but diminished with depth, presumably reducing deeper resin impregnation into the interfibrillar spaces. The citric acid dentin-pretreatment probably caused denaturation of the superficial collagen fibrils. Its decalcifying effect gradually weakened with depth, leaving behind hydroxyapatite crystals at the base of the interdiffusion zone.(ABSTRACT TRUNCATED AT 250 WORDS)

The abundance of Aplysia gliagrana depends on Ca2+ and/or Na+ concentrations in sea water.

The glial cells surrounding the identified giant nerve cell bodies R2 or LP1 of Aplysia punctata were studied by quantitative electron microscopy. They contain specific, electron-dense but non-osmiophilic membrane-bound granules, approximately 0.3 microns in diameter, called gliagrana. Similar glial granules are more often found in marine than in freshwater molluscs, possibly because they represent a calcium store used to compensate excess Na+ in the extracellular milieu of marine species and to regulate perineuronal calcium concentration. In agreement with this hypothesis, the abundance of gliagrana (= number of glial granules per microns 2) is found to be higher in animals adapted to low Ca2+ artificial sea water than in animals kept in high Ca2+ (or low Na+) conditions. This finding is not observed after 1 week but after 2 weeks of adaptation.

Ca(2+)-ATPase and Mg(2+)-ATPase in Aplysia glial and interstitial cells: an EM cytochemical study.

The localization of Ca(2+)- and Mg(2+)-ATPases was determined in Aplysia central and peripheral nervous system, using an electron microscopic cytochemical method. The enzyme activity appeared localized to the membrane of glial granules (gliagrana), particularly in the peripheral nervous system of the esophagus, and on the plasma membrane of central glial cells adjacent to neuronal cell bodies. No calcium- and/or magnesium-ATPase activity was detectable on the plasma membrane of glial cells surrounding nerve axons in the pleuro-visceral connectives. These findings are discussed along two main lines: (a) the calcium-ATPase of the gliagrana coincides with a high intragranular calcium and/or proton concentration; and (b) the presence of a calcium-ATPase activity at the glio-neuronal interface around the neuronal cell bodies coincides with the use of calcium ions as charge carriers of the action potential, and its absence at the level of the axon with the concurrent functional use of sodium ions.

The Desheathed Periphery of Aplysia Giant Neuron. Fine Structure and Measurement of [Ca2+]o Fluctuations with Calcium-selective Microelectrodes.

The visceral ganglion of Aplysia was mechanically desheathed after protease softening of the connective tissue to permit the positioning of ion-selective electrodes in the vicinity of the neuronal membrane. The effects of this treatment on satellite glia and neuronal cytology were observed by electron microscopy. The intracellular alterations were not suggestive of serious membrane damage but the cohesion between glial and neuronal membranes was affected-the glial processes appeared to retract from the trophospongium and in some cases the neuronal membrane was completely naked. The external calcium activity [Ca2+]o at the surface of identified giant neuron, R2, was measured using double-barrelled calcium-selective microelectrodes. A decrease of approximately 1 mM in [Ca2+]o could be recorded only during trains of action potentials induced by intracellular depolarizing current injection, and when the electrode was pushed firmly against the neuron surface. A recovery from this decrease in [Ca2+]o could sometimes, but not always, be observed during the phase of induced neuronal activity.

The lacunar glial zone at the periphery of Aplysia giant neuron: volume of extracellular space and total calcium content of gliagrana.

The relative volume of perineuronal extracellular space, the number of gliagrana and their total calcium content have been measured in Aplysia punctata and A. californica, at the periphery of giant neurons R2 and LP1. After chemical fixation, the extracellular space amounts to 26% of the periganglionic glial zone, but this increases to 36% after quick freezing and freeze-substitution. The glial cytoplasm contains gliagrana, membrane-bound granules approximately 0.3 micron in diameter. The number of gliagrana per micron 2 of section, defined as "abundance", was counted in electron micrographs of chemically fixed tissues. The abundance of gliagrana appears to be directly proportional to the volume of the extracellular space when the values are averaged per individual Aplysia. The total calcium concentration of the gliagrana is measured by X-ray microanalysis on sections of ganglia processed by rapid freezing and freeze-substitution in the presence of oxalic acid: it was found to be very high. An individual granule may contain 100 mM Ca in A. californica and 50 mM in A. punctata but in both species the calcium concentration varies along a wide range as if there were different functional states of the granules with respect to this concentration. The total calcium stored in the specific granules of the glial zone was estimated. It was calculated that should the glial calcium store be entirely diluted in the extracellular space of the glial zone, it would raise the calcium concentration of this space by approximately 1 mM (0.1-2.7 mM). These findings are discussed with regard to the hypothesis of glial cells regulating the perineuronal calcium concentration.