The development of exo-erythrocytic schizonts ofPlasmodium berghei in vitrofrom gamma-irradiated and non-irradiated sporozoites: a study using confocal laser scanning microscopy

Parasitology ◽  
1991 ◽  
Vol 103 (1) ◽  
pp. 17-21 ◽  
Author(s):  
R. E. Sinden ◽  
A. Couchman ◽  
A. Suhrbier ◽  
F. Marsh ◽  
L. Winger ◽  
...  
Keyword(s):  
Laser Scanning ◽  
De Novo ◽  
Parasitophorous Vacuole ◽  
Hepatoma Cell ◽  
Specific Antigen ◽  
Antigen Expression ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Laser

Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages ofPlasmodium bergheiin cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in ‘mature’ cultures. These did not represent a distinct population, but the ‘tail’ of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigensde novoin the invaded hepatoma cell. A comparison of the reactivity of text-abstract EE parasites with anti-circumsporozoite antibody and with anti-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate.

scholarly journals Raman Spectroscopic Characterization of Endodontic Biofilm Matrices

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Tatiana Ramirez-Mora ◽  
Claudia Dávila-Pérez ◽  
Fernando Torres-Méndez ◽  
Grettel Valle-Bourrouet
Keyword(s):  
Raman Spectroscopy ◽  
Laser Scanning ◽  
Extracellular Polymeric Substances ◽  
Principal Component ◽  
Spectroscopic Characterization ◽  
Laser Scanning Microscopy ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Laser ◽  
Total Biomass ◽  
Endodontic Infections

Endodontic persistent infections are often mediated by bacterial biofilms. This mode of bacterial growth is characterized by the presence of a matrix mainly composed of extracellular polymeric substances (EPSs) that protect the encased microorganisms. To establish better control and disinfection protocols, elucidation of the main components of biofilm matrices present in endodontic infections is required. The aim of the present study was to characterize the principal components ofE. faecalis,A. naeslundii, and dual-species biofilm matrices by means of Raman spectroscopy and confocal scanning laser microscopy (CSLM) techniques. The total biomass of biofilms was quantified via crystal violet assays, and the monospecies biofilms showed higher biomass than the dual-species biofilms. Raman spectroscopy and confocal laser scanning microscopy were used to identify the biochemical composition and structure of the biofilm matrices. Spectra originating from the biofilms of two endodontic pathogens show the presence of carbohydrates, proteins, fatty acids, and nucleic acids in all samples; however, variation in the levels of expression of these biomolecules allows spectroscopic differentiation of the biofilms using principal component analysis. This study is the first attempt to identify the composition of monospecies and dual-species biofilms of endodontic origin. Our data provides an important approach to the understanding of molecular dynamics of endodontic infections.

scholarly journals CONFOCAL SCANNING LASER MICROSCOPY IN ASSESSING THE EFFICACY OF AUTOLOGOUS PLATELET-ENRICHED PLASMA WHEN CORRECTING AGE-RELATED CHANGES IN FACIAL SKIN

2017 ◽  
Vol 20 (5) ◽  
pp. 311-315
Author(s):  
Olga Yu. Olisova ◽  
A. D Karagadyan ◽  
L. G Garanyan ◽  
A. S Allenova
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Clinical Signs ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Laser ◽  
Facial Skin ◽  
Autologous Platelet

The efficacy of using autologous platelet-enriched plasma (auto-OTP) in women of different age groups with involutionally altered facial skin by the method of confocal laser scanning microscopy was evaluated. Own results of treatment of 50 patients aged from 35 to 55 years with clinical manifestations of involutionally altered facial skin are presented. As a result of the therapy, a decrease in the clinical signs of aging was observed in all women, but the clinical effect was more pronounced in the age group from 35 to 45 years (97%, of which 76% was significant improvement, 21% was improvement). In the second group (46-55 years), the decrease in clinical signs of aging after the course of procedures was observed in 86%, of which a significant improvement was in 39%, improvement in 47%, without effect 14%. With the help of confocal laser scanning microscopy it was established that in all patients the intradermal administration of auto-OTP promoted the improvement of the structural and functional organization of the epidermis and dermis, increasing the thickness of the dermal layer. The received data testify to high efficiency and safety of application of auto-OTP in correction of involutional skin changes.

scholarly journals Real-Time Monitoring of nfxB Mutant Occurrence and Dynamics in Pseudomonas aeruginosa Biofilm Exposed to Subinhibitory Concentrations of Ciprofloxacin

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Greta Zaborskyte ◽  
Jens Bo Andersen ◽  
Kasper Nørskov Kragh ◽  
Oana Ciofu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stress Responses ◽  
Laser Scanning ◽  
De Novo ◽  
Rapid Development ◽  
Flow Cell ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Primary Resistance ◽  
Subinhibitory Concentrations

ABSTRACT Biofilm infections caused by Pseudomonas aeruginosa are frequently treated with ciprofloxacin (CIP); however, resistance rapidly develops. One of the primary resistance mechanisms is the overexpression of the MexCD-OprJ pump due to a mutation in nfxB, encoding the transcriptional repressor of this pump. The aim of this study was to investigate the effect of subinhibitory concentrations of CIP on the occurrence of nfxB mutants in the wild-type PAO1 flow cell biofilm model. For this purpose, we constructed fluorescent reporter strains (PAO1 background) with an mCherry tag for constitutive red fluorescence and chromosomal transcriptional fusion between the P mexCD promoter and gfp leading to green fluorescence upon mutation of nfxB. We observed a rapid development of nfxB mutants by live confocal laser scanning microscopy (CLSM) imaging of the flow cell biofilm (reaching 80 to 90% of the whole population) when treated with 1/10 minimal biofilm inhibitory concentration of CIP for 24 h and 96 h. Based on the observed developmental stages, we propose that nfxB mutants emerged de novo in the biofilm during CIP treatment from filamentous cells, which might have arisen due to the stress responses induced by CIP. Identical nfxB mutations were found in fluorescent colonies from the same flow cell biofilm, especially in 24-h biofilms, suggesting selection and clonal expansion of the mutants during biofilm growth. Our findings point at the significant role of high-enough antibiotic dosages or appropriate combination therapy to avoid the emergence of resistant mutants in biofilms.

scholarly journals Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses

2011 ◽  
Vol 77 (10) ◽  
pp. 3413-3421 ◽  
Author(s):  
Rebecca Peyyala ◽  
Sreenatha S. Kirakodu ◽  
Jeffrey L. Ebersole ◽  
Karen F. Novak
Keyword(s):  
Laser Scanning ◽  
Bacterial Species ◽  
Disease Process ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Content Type ◽  
Oral Biofilms ◽  
Multispecies Biofilms

ABSTRACTOral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novelin vitromodel system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprisingStreptococcus gordonii,Streptococcus oralis, andStreptococcus sanguinis;S. gordonii,Actinomyces naeslundii, andFusobacterium nucleatum; orS. gordonii,F. nucleatum, andPorphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.

scholarly journals Salmonella spvC Gene Inhibits Autophagy of Host Cells and Suppresses NLRP3 as Well as NLRC4

2021 ◽  
Vol 12 ◽  
Author(s):  
Liting Zhou ◽  
Yuanyuan Li ◽  
Song Gao ◽  
Haibo Yuan ◽  
Lingli Zuo ◽  
...  
Keyword(s):  
Hela Cells ◽  
Laser Scanning ◽  
Systemic Infection ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Autophagic Flux ◽  
Virulence Determinants ◽  
Pyrin Domain ◽  
Lyase Activity

Salmonella spvC gene, encoding a phosphothreonine lyase on host mitogen-activated protein kinases, facilitates systemic infection of Salmonella while the precise mechanisms remain elusive. Autophagy and pyroptosis dependent on the activation of inflammasomes, as parts of innate immune response, contribute to host defense against Salmonella infection. Recently, we reported that spvC could inhibit pyroptosis. To explore the effect of spvC on autophagy and the relationship between its function in pyroptosis and autophagy, infection models of macrophages J774A.1 and epithelial HeLa cells co-cultured with Salmonella Typhimurium wild type, spvC deletion, site-directed mutant which lacks phosphothreonine lyase activity, or complemented strain were established. The levels of LC3 turnover and Beclin 1 of J774A.1 cells were determined by western blot. Confocal laser scanning microscopy was used to visualize the autophagic flux after being transfected with mRFP-GFP-LC3 plasmid in HeLa cells. Results showed that SpvC inhibited autophagosome formation through its phosphothreonine lyase activity. Additionally, analysis of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) and NLR with CARD domain-containing 4 (NLRC4) in J774A.1 cells indicated that spvC decreased the protein levels of NLRP3 and NLRC4, which were significantly changed by autophagy inhibitor Bafilomycin A1. Together, our observations reveal a novel mechanism of spvC in Salmonella pathogenesis and host inflammatory response via inhibiting autophagy and NLRP3 as well as NLRC4. These pathways and their subversion by diverse pathogen virulence determinants are expected to throw light on the design of anti-infective agents.

scholarly journals Extracellular excystation and development of Cryptosporidium: tracing the fate of oocysts within Pseudomonas aquatic biofilm systems

2021 ◽  
Author(s):  
Wan Koh ◽  
Andrew Thompson ◽  
Hanna Edwards ◽  
Paul Monis ◽  
Peta L Clode
Keyword(s):  
Laser Scanning ◽  
Distribution Systems ◽  
Water Distribution ◽  
Developmental Stages ◽  
Parasitophorous Vacuole ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Growth Environment ◽  
Flow Cytometric ◽  
Harsh Conditions

Background Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages. Results Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls. Conclusions These observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments.

scholarly journals Nodulation of Mimosa spp. by the β-Proteobacterium Ralstonia taiwanensis

2003 ◽  
Vol 16 (12) ◽  
pp. 1051-1061 ◽  
Author(s):  
Wen-Ming Chen ◽  
Euan K. James ◽  
Alan R. Prescott ◽  
Martin Kierans ◽  
Janet I. Sprent
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Nitrogenase Activity ◽  
Light And Electron Microscopy ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Nodule Formation ◽  
Infection Threads ◽  
Infected Cells

Several β-proteobacteria have been isolated from legume root nodules and some of these are thought to be capable of nodulating and fixing N2. However, in no case has there been detailed studies confirming that they are the active symbionts. Here, Ralstonia taiwanensis LMG19424, which was originally isolated from Mimosa pudica nodules, was transformed to carry the green fluorescent protein (gfp) reporter gene before being used to inoculate axenically-grown seedlings of M. pudica and M. diplotricha. Plants were harvested at various intervals for 56 days after inoculation, then examined for evidence of infection and nodule formation. Nodulation of both Mimosa spp. was abundant, and acetylene reduction assays confirmed that nodules had nitrogenase activity. Confocal laser scanning microscopy (CLSM) showed that fresh M. pudica nodules with nitrogenase activity had infected cells containing bacteroids expressing gfp. In parallel, fixed and embedded nodules from both Mimosa spp. were sectioned for light and electron microscopy, followed by immunogold labeling with antibodies raised against gfp and nitrogenase Fe (nifH) protein. Significant immunolabeling with these antibodies confirmed that R. taiwanensis LMG19424 is an effective N2-fixing symbiont of Mimosa spp. Both species were infected via root hairs and, in all respects, the nodule ontogeny and development was similar to that described for other mimosoid legumes. The nodules were indeterminate with a persistent meristem, an invasion zone containing host cells being invaded via prominent infection threads, and an N2-fixing zone with infected cells containing membrane-bound symbiosomes.

scholarly journals 3D Human Periodontal Stem Cells and Endothelial Cells Promote Bone Development in Bovine Pericardium-Based Tissue Biomaterial

Materials ◽  
2019 ◽  
Vol 12 (13) ◽  
pp. 2157 ◽  
Author(s):  
Jacopo Pizzicannella ◽  
Sante D. Pierdomenico ◽  
Adriano Piattelli ◽  
Giuseppe Varvara ◽  
Luigia Fonticoli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Laser Scanning ◽  
De Novo ◽  
Bone Defects ◽  
Bovine Pericardium ◽  
Laser Scanning Microscopy ◽  
Mitogen Activated Protein Kinase ◽  
Confocal Laser ◽  
Collagen Membrane ◽  
Periodontal Ligament Stem Cells

Bone defects repair represents a public and urgent problem in clinical practice, in fact, every year, more than two million patients required new treatments for bone injuries. Today a complete vascularization is strategic in bone formation, representing a new frontier for clinical application. Aim of this research has been developed a three-dimensional (3D) coculture platform using a bovine pericardium collagen membrane (BioR) loaded with human periodontal ligament stem cells (hPDLSCs) and endothelial differentiated cells from hPDLSCs (E-hPDLSCs) able to undergo toward osteoangiogenesis differentiation process. First, we have characterized at confocal laser scanning microscopy (CLSM) level the E-hPDLSCs phenotype profile, through CD31 and CD34 markers expression and the ability to tube vessel formation. Real Time-Polimerase Chain Reaction (RT-PCR) and western blotting analyses revealed the upregulation of Runt-related transcription factor 2 (RUNX2), Collagen 1A1 (COL1A1), Vascular Endothelial Growth Factor-A (VEGF-A) genes and proteins in the living construct composed by hPDLSCs + E-hPDSCs/BioR. Human PDLSCs + E-hPDLSCs/BioR construct showed also an enhacement of de novo synthesis of osteocalcin. Given that, the extracellular-signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) transduction signaling was involved in the osteogenesis and angiogenesis process, the ERK1/2 protein level at biochemical level, in our experimental model, has been investigated. Our results evidenced an upregulation of ERK1/2 proteins level born in the living construct. In conclusion, we believe that the use of the hPDLSCs and E-hPDLSCs coculture togheter with BioR as substrate, could represent an efficient model able to activate through ERK1/2 signaling pathway the osteoangiogenesis process, and then representing a new potential engineered platform for surgeons during the repair and the healing of bone defects.

scholarly journals Cryptides Identified in Human Apolipoprotein B as New Weapons to Fight Antibiotic Resistance in Cystic Fibrosis Disease

2020 ◽  
Vol 21 (6) ◽  
pp. 2049 ◽  
Author(s):  
Rosa Gaglione ◽  
Angela Cesaro ◽  
Eliana Dell’Olmo ◽  
Rocco Di Girolamo ◽  
Luca Tartaglione ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Apolipoprotein B ◽  
Laser Scanning ◽  
Respiratory Infections ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Bacterial Strains ◽  
Human Apolipoprotein ◽  
Conventional Antibiotics

Chronic respiratory infections are the main cause of morbidity and mortality in cystic fibrosis (CF) patients, and are characterized by the development of multidrug resistance (MDR) phenotype and biofilm formation, generally recalcitrant to treatment with conventional antibiotics. Hence, novel effective strategies are urgently needed. Antimicrobial peptides represent new promising therapeutic agents. Here, we analyze for the first time the efficacy of three versions of a cryptide identified in human apolipoprotein B (ApoB, residues 887-922) towards bacterial strains clinically isolated from CF patients. Antimicrobial and anti-biofilm properties of ApoB-derived cryptides have been analyzed by broth microdilution assays, crystal violet assays, confocal laser scanning microscopy and scanning electron microscopy. Cell proliferation assays have been performed to test cryptide effects on human host cells. ApoB-derived cryptides have been found to be endowed with significant antimicrobial and anti-biofilm properties towards Pseudomonas and Burkholderia strains clinically isolated from CF patients. Peptides have been also found to be able to act in combination with the antibiotic ciprofloxacin, and they are harmless when tested on human bronchial epithelial mesothelial cells. These findings open interesting perspectives to cryptide applicability in the treatment of chronic lung infections associated with CF disease.

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