Impact of Airborne Particle Size, Acoustic Airflow and Breathing Pattern on Delivery of Nebulized Antibiotic into the Maxillary Sinuses Using a Realistic Human Nasal Replica

International audiencePurpose:Improvement of clinical outcome in patients with sinuses disorders involves targeting delivery of nebulized drug into the maxillary sinuses. We investigated the impact of nebulization conditions (with and without 100 Hz acoustic airflow), particle size (9.9 μm, 2.8 μm, 550 nm and 230 nm) and breathing pattern (nasal vs. no nasal breathing) on enhancement of aerosol delivery into the sinuses using a realistic nasal replica developed by our team.Methods:After segmentation of the airways by means of high-resolution computed tomography scans, a well-characterized nasal replica was created using a rapid prototyping technology. A total of 168 intrasinus aerosol depositions were performed with changes of aerosol particle size and breathing patterns under different nebulization conditions using gentamicin as a marker.Results:The results demonstrate that the fraction of aerosol deposited in the maxillary sinuses is enhanced by use of submicrometric aerosols, e.g. 8.155 ± 1.476 mg/L of gentamicin in the left maxillary sinus for the 2.8 μm particles vs. 2.056 ± 0.0474 for the 550 nm particles. Utilization of 100-Hz acoustic airflow nebulization also produced a 2- to 3-fold increase in drug deposition in the maxillary sinuses (e.g. 8.155 ± 1.476 vs. 3.990 ± 1.690 for the 2.8 μm particles).Conclusions:Our study clearly shows that optimum deposition was achieved using submicrometric particles and 100-Hz acoustic airflow nebulization with no nasal breathing. It is hoped that our new respiratory nasal replica will greatly facilitate the development of more effective delivery systems in the future

Ultrastructural detection of nucleic acids on thin sections of tissue embedded in hydrophilic resin.

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Ultrastructural in situ hybridization: a review of technical aspects.

International audienceDetection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters; the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters; the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure

Expression of type I and type V collagen mRNAs in the elasmoid scales of a teleost fish as revealed by in situ hybridization.

International audienceThe ability of scale-forming cells to produce both type I and type V collagens was investigated by in situ hybridization at the light and electron microscope levels. Biochemical analyses reported that type I collagen, the predominant component, was associated with the minor type V collagen in the collagenous matrix of the teleost scales where, thin and thick collagen fibrils formed distinct layers. Thin collagen fibrils of the external layer were produced by the episquamal scleroblasts scattered on the outer scale surface, while thick collagen fibrils forming the compact basal plate were produced by the hyposquamal scleroblasts lining the inner surface of the scale. We demonstrated that episquamal and hyposquamal scleroblasts contained mRNAs for alpha1(I) and alpha1(V) collagens. Quantification by image analysis of the relative amount of alpha1(I) and alpha1(V) mRNAs in episquamal and hyposquamal scleroblasts suggests that the gene expression of type V collagen was proportionally higher in episquamal scleroblasts. These results support our hypothesis that the diameter of the thin fibrils of the external layer is regulated by the significant amount of type V collagen that interacts with type I collagen.The ability of scale-forming cells to produce both type I and type V collagens was investigated by in situ hybridization at the light and electron microscope levels. Biochemical analyses reported that type I collagen, the predominant component, was associated with the minor type V collagen in the collagenous matrix of the teleost scales where, thin and thick collagen fibrils formed distinct layers. Thin collagen fibrils of the external layer were produced by the episquamal scleroblasts scattered on the outer scale surface, while thick collagen fibrils forming the compact basal plate were produced by the hyposquamal scleroblasts lining the inner surface of the scale. We demonstrated that episquamal and hyposquamal scleroblasts contained mRNAs for alpha1(I) and alpha1(V) collagens. Quantification by image analysis of the relative amount of alpha1(I) and alpha1(V) mRNAs in episquamal and hyposquamal scleroblasts suggests that the gene expression of type V collagen was proportionally higher in episquamal scleroblasts. These results support our hypothesis that the diameter of the thin fibrils of the external layer is regulated by the significant amount of type V collagen that interacts with type I collagen

Changes in location of type I collagen synthesis in two stages of fetal calf skin as revealed by in situ hybridization.

International audienceThe distribution of sites of type I collagen gene expression was studied in frozen sections of skin of 4 and 9 month-old calf fetuses by in situ hybridization using a human pro-alpha 1 type I collagen cDNA. The labelling varied with the different layers of the dermis and with the developmental stage considered. In the 4 month old fetus skin, the label appeared concentrated in the upper layer of the dermis at the lewel of the hair follicles. In the 9 month-old fetus skin, the difference of labelling between upper papillary dermis and lower dermis was less marked. Comparatively the distribution of the extracellular type I collagen was determined by indirect immunofluorescence. This collagen appeared present throughout the whole dermis with slight variations at 4 months, where there was less extracellular collagen near the hair bulbs. These results are in agreement with the idea that the collagen synthesis follows cutaneous differentiation. In addition, they support the hypothesis that collagen is deposited once morphogenetic events have occurred and plays thus a stabilizing role in formation of cutaneous appendages.The distribution of sites of type I collagen gene expression was studied in frozen sections of skin of 4 and 9 month-old calf fetuses by in situ hybridization using a human pro-alpha 1 type I collagen cDNA. The labelling varied with the different layers of the dermis and with the developmental stage considered. In the 4 month old fetus skin, the label appeared concentrated in the upper layer of the dermis at the lewel of the hair follicles. In the 9 month-old fetus skin, the difference of labelling between upper papillary dermis and lower dermis was less marked. Comparatively the distribution of the extracellular type I collagen was determined by indirect immunofluorescence. This collagen appeared present throughout the whole dermis with slight variations at 4 months, where there was less extracellular collagen near the hair bulbs. These results are in agreement with the idea that the collagen synthesis follows cutaneous differentiation. In addition, they support the hypothesis that collagen is deposited once morphogenetic events have occurred and plays thus a stabilizing role in formation of cutaneous appendages

Distribution of minor collagens during skin development.

International audienceThe skin is a tissue containing a large number of collagen types. Several collagens are restricted at the dermo-epidermal junction, contrarily to others present throughout the dermis. However, the distribution of the dermal collagen varies during embryonic development. In this contribution, we have been interested in the collagen types associated with the major collagenous components of the dermis, which are the collagen types I and III. Type V collagen, which is mixed with collagen types I and III to form heterotypic fibrils, has been studied during mouse embryo development. Transcripts of the alpha 1 (V) gene have been localized by in situ hybridization, on flattened cells of the stratum germinativum first, and then only on dermal cells. The expression of the gene decreases at birth, while the expression of the alpha 1(I) gene remains constant, with, however, a ring of high intensity around hair follicles. Other collagen types (VI, and the fibril-associated collagens XII and XIV) have been studied during calf embryonic development by immunofluorescence and ultrastructural immunogold detection. Type VI collagen appears homogeneously distributed throughout the dermis. Type XII collagen is first widely distributed and becomes restricted in the upper, papillary dermis after 6 months of gestation. Type XIV collagen, on the contrary, is first located as a delicate framework around hair follicles (at 19 weeks of gestation), and progressively invades the whole dermis where it appears abundant just before birth. The different functions of all these collagens are discussed in terms of dermis architecture, mechanical properties and physiology.The skin is a tissue containing a large number of collagen types. Several collagens are restricted at the dermo-epidermal junction, contrarily to others present throughout the dermis. However, the distribution of the dermal collagen varies during embryonic development. In this contribution, we have been interested in the collagen types associated with the major collagenous components of the dermis, which are the collagen types I and III. Type V collagen, which is mixed with collagen types I and III to form heterotypic fibrils, has been studied during mouse embryo development. Transcripts of the alpha 1 (V) gene have been localized by in situ hybridization, on flattened cells of the stratum germinativum first, and then only on dermal cells. The expression of the gene decreases at birth, while the expression of the alpha 1(I) gene remains constant, with, however, a ring of high intensity around hair follicles. Other collagen types (VI, and the fibril-associated collagens XII and XIV) have been studied during calf embryonic development by immunofluorescence and ultrastructural immunogold detection. Type VI collagen appears homogeneously distributed throughout the dermis. Type XII collagen is first widely distributed and becomes restricted in the upper, papillary dermis after 6 months of gestation. Type XIV collagen, on the contrary, is first located as a delicate framework around hair follicles (at 19 weeks of gestation), and progressively invades the whole dermis where it appears abundant just before birth. The different functions of all these collagens are discussed in terms of dermis architecture, mechanical properties and physiology

Detection of the messenger RNAs coding for the opioid peptide precursors in pituitary and adrenal by "in situ' hybridization: study in several mammal species.

International audienceThe messenger RNAs coding for opioid peptide precursors have been detected and mapped in histological sections by "in situ' hybridization using specific DNA probes labelled with 32P. Using bovine preproenkephalin A (PPA) cDNA, PPA mRNA was detected in adrenal medulla of bull, hamster and guinea pig. No signal was detected in adrenal of man, rat and cat. The pro-opiomelanocortin (POMC) mRNA was detected in pituitary of man, bull, cat, rat and pig, in all cells of the intermediate lobe as well as in scattered cells of the anterior lobe producing POMC. Adequate controls demonstrated the specificity of the labelling. These results provide evidence of the expression of the gene coding for PPA in the adrenal and for POMC in the pituitary. They show cross-hybridization of one DNA probe with mRNAs of various mammals and then provide evidence that one single probe can be used to analyze expression of a given gene in tissues of several animal species by "in situ' hybridization.The messenger RNAs coding for opioid peptide precursors have been detected and mapped in histological sections by "in situ' hybridization using specific DNA probes labelled with 32P. Using bovine preproenkephalin A (PPA) cDNA, PPA mRNA was detected in adrenal medulla of bull, hamster and guinea pig. No signal was detected in adrenal of man, rat and cat. The pro-opiomelanocortin (POMC) mRNA was detected in pituitary of man, bull, cat, rat and pig, in all cells of the intermediate lobe as well as in scattered cells of the anterior lobe producing POMC. Adequate controls demonstrated the specificity of the labelling. These results provide evidence of the expression of the gene coding for PPA in the adrenal and for POMC in the pituitary. They show cross-hybridization of one DNA probe with mRNAs of various mammals and then provide evidence that one single probe can be used to analyze expression of a given gene in tissues of several animal species by "in situ' hybridization

An Air Quality CFD Model Performance in Complex Environment with EMU Observations

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Short chain collagens in sponges are encoded by a family of closely related genes.

International audienceTwo previously described sponge cDNAs, EmC4 and C23, respectively encoding a short chain collagen and a fibrillar collagen, were used to characterize collagen gene families in a freshwater sponge. EmC4 detected several clones when used to screen a cDNA library. Two overlapping clones, EmC13 1 and 2, were sequenced and appeared highly homologous to EmC4. Contrarily to C23, EmC4 hybridized with 10-12 fragments of genomic DNA digested with restriction endonucleases and detected 10 times more positive clones than C23 when used to screen a genomic library. The genomic clone G41 contained two closely related genes, COLNF13, corresponding to EmC13 and COLNF6. Partial characterization of COLNF13 revealed two partial exons and four complete exons of 153, 219, 207, and 144 base pairs, with split glycine codons at their boundaries. The deduced encoded protein is a short chain collagen containing two uninterrupted collagenous domains of 66 and 171 amino acids and non-collagenous domains. A characterized 207-base pair exon of COLNF6 is 77% identical with the comparable COLNF13 exon. In situ hybridization using EmC4 cDNA and electron microscopy suggested that the cells expressing these genes were secreting spongin, a non-fibrillar, surface collagen of these sponges.Two previously described sponge cDNAs, EmC4 and C23, respectively encoding a short chain collagen and a fibrillar collagen, were used to characterize collagen gene families in a freshwater sponge. EmC4 detected several clones when used to screen a cDNA library. Two overlapping clones, EmC13 1 and 2, were sequenced and appeared highly homologous to EmC4. Contrarily to C23, EmC4 hybridized with 10-12 fragments of genomic DNA digested with restriction endonucleases and detected 10 times more positive clones than C23 when used to screen a genomic library. The genomic clone G41 contained two closely related genes, COLNF13, corresponding to EmC13 and COLNF6. Partial characterization of COLNF13 revealed two partial exons and four complete exons of 153, 219, 207, and 144 base pairs, with split glycine codons at their boundaries. The deduced encoded protein is a short chain collagen containing two uninterrupted collagenous domains of 66 and 171 amino acids and non-collagenous domains. A characterized 207-base pair exon of COLNF6 is 77% identical with the comparable COLNF13 exon. In situ hybridization using EmC4 cDNA and electron microscopy suggested that the cells expressing these genes were secreting spongin, a non-fibrillar, surface collagen of these sponges

Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio).

International audienceThe first part of this article is a review of the current status of knowledge of the fish skin, with particular attention to its development. In the second part we present original results obtained in zebrafish (Danio rerio), with particular emphasis on the deposition and organisation of the dermal collagenous stroma. Using a series of zebrafish specimens aged between 15 hours postfertilization (hpf) and 4.5 years old, we have combined Transmission Electron Microscopy (TEM) observations and in situ hybridisation using type I collagen a2 chain (Col1a2) probe. Collagen fibrils, with a diameter of 22 nm, appear first in an acellular subepidermal space at 24 hpf, are first all oriented in the same direction, and form the primary dermal stroma. Subsequently, three events occur. (1) From 5-7 days pf (dpf) onwards the collagen fibrils self-organise into several lamellae arranged in a plywood-like structure, starting in the upper layers and progressing throughout the entire thickness of the dermis. (2) At 20-26 dpf, fibroblasts of unknown origin progressively invade the acellular collagenous stroma, some of them accumulating below the epidermis. (3) Concomitant with the invasion of fibroblasts, the collagen fibrils increase progressively in diameter to reach 160 nm towards the end of the fish life. In situ hybridisation experiments reveal that, between 24 and 48 hpf, the collagen matrix is produced by the epidermis only. From 72 hpf to 20-26 dpf, both the basal epidermal cells and the dermal cells bordering the deep region of the dermis are involved in the production of collagen. When the fibroblasts invade the plywood-like structure, the epidermal cells progressively cease to synthesise collagen, which from this point is produced only by the fibroblasts. This suggests that the fibroblasts secrete a still unidentified signalling molecule that downregulates collagen production by the epidermis.The first part of this article is a review of the current status of knowledge of the fish skin, with particular attention to its development. In the second part we present original results obtained in zebrafish (Danio rerio), with particular emphasis on the deposition and organisation of the dermal collagenous stroma. Using a series of zebrafish specimens aged between 15 hours postfertilization (hpf) and 4.5 years old, we have combined Transmission Electron Microscopy (TEM) observations and in situ hybridisation using type I collagen a2 chain (Col1a2) probe. Collagen fibrils, with a diameter of 22 nm, appear first in an acellular subepidermal space at 24 hpf, are first all oriented in the same direction, and form the primary dermal stroma. Subsequently, three events occur. (1) From 5-7 days pf (dpf) onwards the collagen fibrils self-organise into several lamellae arranged in a plywood-like structure, starting in the upper layers and progressing throughout the entire thickness of the dermis. (2) At 20-26 dpf, fibroblasts of unknown origin progressively invade the acellular collagenous stroma, some of them accumulating below the epidermis. (3) Concomitant with the invasion of fibroblasts, the collagen fibrils increase progressively in diameter to reach 160 nm towards the end of the fish life. In situ hybridisation experiments reveal that, between 24 and 48 hpf, the collagen matrix is produced by the epidermis only. From 72 hpf to 20-26 dpf, both the basal epidermal cells and the dermal cells bordering the deep region of the dermis are involved in the production of collagen. When the fibroblasts invade the plywood-like structure, the epidermal cells progressively cease to synthesise collagen, which from this point is produced only by the fibroblasts. This suggests that the fibroblasts secrete a still unidentified signalling molecule that downregulates collagen production by the epidermis