ResearchPad - confocal-laser-microscopy https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Confocal Raman microscopy to identify bacteria in oral subgingival biofilm models]]> https://www.researchpad.co/article/elastic_article_7675 The study of oral disease progression, in relation to the accumulation of subgingival biofilm in gingivitis and periodontitis is limited, due to either the ability to monitor plaque in vitro. When compared, optical spectroscopic techniques offer advantages over traditional destructive or biofilm staining approaches, making it a suitable alternative for the analysis and continued development of three-dimensional structures. In this work, we have developed a confocal Raman spectroscopy analysis approach towards in vitro subgingival plaque models. The main objective of this study was to develop a method for differentiating multiple oral subgingival bacterial species in planktonic and biofilm conditions, using confocal Raman microscopy. Five common subgingival bacteria (Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar, Actinomyces naeslundii and Prevotella nigrescens) were used and differentiated using a 2-way orthogonal Partial Least Square with Discriminant Analysis (O2PLS-DA) for the collected spectral data. In addition to planktonic growth, mono-species biofilms cultured using the ‘Zürich Model’ were also analyzed. The developed method was successfully used to predict planktonic and mono-species biofilm species in a cross validation setup. The results show differences in the presence and absence of chemical bands within the Raman spectra. The O2PLS-DA model was able to successfully predict 100% of all tested planktonic samples and 90% of all mono-species biofilm samples. Using this approach we have shown that Confocal Raman microscopy can analyse and predict the identity of planktonic and mono-species biofilm species, thus enabling its potential as a technique to map oral multi-species biofilm models.

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<![CDATA[A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs]]> https://www.researchpad.co/article/N38f7a2a5-9838-4ae0-b206-f959ee03524f

Land plant shoot structures evolved a diversity of lateral organs as morphological adaptations to the terrestrial environment, with lateral organs arising independently in different lineages. Vascular plants and bryophytes (basally diverging land plants) develop lateral organs from meristems of sporophytes and gametophytes, respectively. Understanding the mechanisms of lateral organ development among divergent plant lineages is crucial for understanding the evolutionary process of morphological diversification of land plants. However, our current knowledge of lateral organ differentiation mechanisms comes almost entirely from studies of seed plants, and thus, it remains unclear how these lateral structures evolved and whether common regulatory mechanisms control the development of analogous lateral organs. Here, we performed a mutant screen in the liverwort Marchantia polymorpha, a bryophyte, which produces gametophyte axes with nonphotosynthetic scalelike lateral organs. We found that an Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1 (ALOG) family protein, named M. polymorpha LATERAL ORGAN SUPRESSOR 1 (MpLOS1), regulates meristem maintenance and lateral organ development in Marchantia. A mutation in MpLOS1, preferentially expressed in lateral organs, induces lateral organs with misspecified identity and increased cell number and, furthermore, causes defects in apical meristem maintenance. Remarkably, MpLOS1 expression rescued the elongated spikelet phenotype of a MpLOS1 homolog in rice. This suggests that ALOG genes regulate the development of lateral organs in both gametophyte and sporophyte shoots by repressing cell divisions. We propose that the recruitment of ALOG-mediated growth repression was in part responsible for the convergent evolution of independently evolved lateral organs among highly divergent plant lineages, contributing to the morphological diversification of land plants.

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<![CDATA[Efficacy of liposomal amphotericin B and anidulafungin using an antifungal lock technique (ALT) for catheter-related Candida albicans and Candida glabrata infections in an experimental model]]> https://www.researchpad.co/article/5c75ac7dd5eed0c484d088b2

Objective

The aims of this study were as follows. First, we sought to compare the in vitro susceptibility of liposomal amphotericin B (LAmB) and anidulafungin on Candida albicans and Candida glabrata biofilms growing on silicone discs. Second, we sought to compare the activity of LAmB versus anidulafungin for the treatment of experimental catheter-related C. albicans and C. glabrata infections with the antifungal lock technique in a rabbit model.

Methods

Two C. albicans and two C. glabrata clinical strains were used. The minimum biofilm eradication concentration for 90% eradication (MBEC90) values were determined after 48h of treatment with LAmB and anidulafungin. Confocal microscopy was used to visualize the morphology and viability of yeasts growing in biofilms. Central venous catheters were inserted into New Zealand rabbits, which were inoculated of each strain of C. albicans and C. glabrata. Then, catheters were treated for 48h with saline or with antifungal lock technique using either LAmB (5mg/mL) or anidulafungin (3.33mg/mL).

Results

In vitro: anidulafungin showed greater activity than LAmB against C. albicans and C. glabrata strains. For C. albicans: MBEC90 of anidulafungin versus LAmB: CA176, 0.03 vs. 128 mg/L; CA180, 0.5 vs. 64 mg/L. For C. glabrata: MBEC90 of anidulafungin versus LAmB: CG171, 0.5 vs. 64 mg/L; CG334, 2 vs. 32 mg/L. In vivo: for C. albicans species, LAmB and anidulafungin achieved significant reductions relative to growth control of log10 cfu recovered from the catheter tips (CA176: 3.6±0.3 log10 CFU, p≤0.0001; CA180: 3.8±0.1 log10 CFU, p≤0.01). For C. glabrata, anidulafungin lock therapy achieved significant reductions relative to the other treatments (CG171: 4.8 log10 CFU, p≤0.0001; CG334: 5.1 log10 CFU, p≤0.0001)

Conclusions

For the C. albicans strains, both LAmB and anidulafungin may be promising antifungal lock technique for long-term catheter-related infections; however, anidulafungin showed significantly higher activity than LAmB against the C. glabrata strains.

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<![CDATA[Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis]]> https://www.researchpad.co/article/5c58d622d5eed0c4840316f5

Biofilm formation in the clinical environment is of increasing concern since a significant part of human infections is associated, and caused by biofilm establishment of (opportunistic) pathogens, for instance Candida albicans and Staphylococcus epidermidis. The rapidly increasing number of antibiotic-resistant biofilms urgently requires the development of novel and effective strategies to prevent biofilm formation ideally targeting a wide range of infectious microorganisms. Both, synthesis of extracellular polymeric substances and quorum sensing are crucial for biofilm formation, and thus potential attractive targets to combat undesirable biofilms.We evaluated the ability of numerous recently identified metagenome-derived bacterial quorum quenching (QQ) proteins to inhibit biofilm formation of C. albicans and S. epidermidis. Here, proteins QQ-5 and QQ-7 interfered with the morphogenesis of C. albicans by inhibiting the yeast-to-hyphae transition, ultimately leading to impaired biofilm formation. Moreover, QQ5 and QQ-7 inhibited biofilm formation of S. epidermidis; in case of QQ7 most likely due to induced expression of the icaR gene encoding the repressor for polysaccharide intercellular adhesin (PIA) synthesis, the main determinant for staphylococcal biofilm formation. Our results indicate that QQ-5 and QQ-7 are attractive potential anti-biofilm agents in the prevention and treatment of C. albicans and S. epidermidis mono-species biofilms, and potentially promising anti-biofilm drugs in also combating multi-species infections.

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<![CDATA[Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus]]> https://www.researchpad.co/article/5c3fa5f5d5eed0c484caa846

Tombusviruses depend on subversions of multiple host factors and retarget cellular pathways to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) recruit the cellular Vps34 phosphatidylinositol 3-kinase (PI3K) into the large viral replication compartment. The kinase function of Vps34 is critical for TBSV replication, suggesting that PI(3)P phosphoinositide is utilized by TBSV for building of the replication compartment. We also observed increased expression of Vps34 and the higher abundance of PI(3)P in the presence of the tombusviral replication proteins, which likely leads to more efficient tombusvirus replication. Accordingly, overexpression of PI(3)P phosphatase in yeast or plants inhibited TBSV replication on the peroxisomal membranes and CIRV replication on the mitochondrial membranes. Moreover, the purified PI(3)P phosphatase reduced TBSV replicase assembly in a cell-free system. Detection of PI(3)P with antibody or a bioprobe revealed the enrichment of PI(3)P in the replication compartment. Vps34 is directly recruited into the replication compartment through interaction with p33 replication protein. Gene deletion analysis in surrogate yeast host unraveled that TBSV replication requires the vesicle transport function of Vps34. In the absence of Vps34, TBSV cannot efficiently recruit the Rab5-positive early endosomes, which provide PE-rich membranes for membrane biogenesis of the TBSV replication compartment. We found that Vps34 and PI(3)P needed for the stability of the p33 replication protein, which is degraded by the 26S proteasome when PI(3)P abundance was decreased by an inhibitor of Vps34. In summary, Vps34 and PI(3)P are needed for providing the optimal microenvironment for the replication of the peroxisomal TBSV and the mitochondrial CIRV.

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<![CDATA[Danger signals activate a putative innate immune system during regeneration in a filamentous fungus]]> https://www.researchpad.co/article/5c0ae430d5eed0c4845891aa

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

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<![CDATA[Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus]]> https://www.researchpad.co/article/5989dad2ab0ee8fa60bb67bf

Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

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<![CDATA[Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilms states]]> https://www.researchpad.co/article/5989db51ab0ee8fa60bdc2eb

Background and objective

Porphyromonas gingivalis is a keystone pathogen in the onset and progression of periodontitis. Its pathogenicity has been related to its presence and survival within the subgingival biofilm. The aim of the present study was to compare the genome-wide transcription activities of P. gingivalis in biofilm and in planktonic growth, using microarray technology.

Material and methods

P. gingivalis ATCC 33277 was incubated in multi-well culture plates at 37°C for 96 hours under anaerobic conditions using an in vitro static model to develop both the planktonic and biofilm states (the latter over sterile ceramic calcium hydroxyapatite discs). The biofilm development was monitored by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). After incubation, the bacterial cells were harvested and total RNA was extracted and purified. Three biological replicates for each cell state were independently hybridized for transcriptomic comparisons. A linear model was used for determining differentially expressed genes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to confirm differential expression. The filtering criteria of ±2 change in gene expression and significance p-values of <0.05 were selected.

Results

A total of 92 out of 1,909 genes (4.8%) were differentially expressed by P. gingivalis growing in biofilm compared to planktonic. The 54 up-regulated genes in biofilm growth were mainly related to cell envelope, transport, and binding or outer membranes proteins. Thirty-eight showed decreased expression, mainly genes related to transposases or oxidative stress.

Conclusion

The adaptive response of P. gingivalis in biofilm growth demonstrated a differential gene expression.

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<![CDATA[Growth and adherence of Staphylococcus aureus were enhanced through the PGE2 produced by the activated COX-2/PGE2 pathway of infected oral epithelial cells]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf5d3

Staphylococcus aureus is a major pathogen of varieties of oral mucous infection. Prostaglandin E2 (PGE2) is a pro-inflammatory factor and Cyclooxygenase 2 (COX-2) is a critical enzyme of PGE2 biosynthesis. The purpose of this study is to investigate whether Staphylococcus aureus can increase PGE2 production of oral epithelial cells and how PGE2 functions in the growth and adherence of Staphylococcus aureus. mRNA levels of COX-2, fnbpA and fnbpB were estimated by quantitative PCR. PGE2 production was measured by Enzyme Linked Immunosorbent Assay (ELISA). The binding biomass of Staphylococcus aureus to human fibronectin was investigated by crystal violet staining and confocal laser scanning microscopy and the adherent force was measured by atomic force microscope (AFM). The COX-2 mRNA level and PGE2 production were increased by Staphylococcus aureus. PGE2 promoted the growth and biofilm formation of Staphylococcus aureus, enhanced the attachment of Staphylococcus aureus to the human fibronectin as well as to the HOK cells. The transcription of fnbpB was up-regulated by PGE2 in both early and middle exponential phase but not fnbpA. These results suggest that the activation of COX-2/PGE2 pathway in oral epithelial cell by Staphylococcus aureus can in turn facilitate the growth and the ability to adhere of the pathogen. These findings uncover a new function of PGE2 and may lead to the potential of COX-2/PGE2 targeting in the therapy of inflammation and cancer in both which the COX-2/PGE2 pathway were observed activated.

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<![CDATA[Discovery and Characterization of Novel Anti-schistosomal Properties of the Anti-anginal Drug, Perhexiline and Its Impact on Schistosoma mansoni Male and Female Reproductive Systems]]> https://www.researchpad.co/article/5989dad8ab0ee8fa60bb8ad5

Background

Schistosomiasis, one of the world’s greatest human neglected tropical diseases, is caused by parasitic trematodes of the genus Schistosoma. A unique feature of schistosome biology is that the induction of sexual maturation as well as the maintenance of the differentiation status of female reproductive organs and egg production, necessary for both disease transmission and pathogenesis, are strictly dependent on the male. The treatment and most control initiatives of schistosomiasis rely today on the long-term application of a single drug, praziquantel (PZQ), mostly by campaigns of mass drug administration. PZQ, while very active on adult parasites, has much lower activity against juvenile worms. Monotherapy also favors the selection of drug resistance and, therefore, new drugs are urgently needed.

Methods and Findings

Following the screening of a small compound library with an ATP-based luminescent assay on Schistosoma mansoni schistosomula, we here report the identification and characterization of novel antischistosomal properties of the anti-anginal drug perhexiline maleate (PHX). By phenotypic worm survival assays and confocal microscopy studies we show that PHX, in vitro, has a marked lethal effect on all S. mansoni parasite life stages (newly transformed schistosomula, juvenile and adult worms) of the definitive host. We further demonstrate that sub-lethal doses of PHX significantly impair egg production and lipid depletion within the vitellarium of adult female worms. Moreover, we highlighted tegumental damage in adult male worms and remarkable reproductive system alterations in both female and male adult parasites. The in vivo study in S. mansoni-patent mice showed a notable variability of worm burdens in the individual experiments, with an overall minimal schistosomicidal effect upon PHX treatment. The short PHX half-life in mice, together with its very high rodent plasma proteins binding could be the cause of the modest efficacy of PHX in the schistosomiasis murine model.

Conclusions/Significance

Overall, our data indicate that PHX could represent a promising starting point for novel schistosomicidal drug discovery programmes.

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<![CDATA[A Novel Computerized Cell Count Algorithm for Biofilm Analysis]]> https://www.researchpad.co/article/5989da08ab0ee8fa60b76adf

Biofilms are the preferred sessile and matrix-embedded life form of most microorganisms on surfaces. In the medical field, biofilms are a frequent cause of treatment failure because they protect the bacteria from antibiotics and immune cells. Antibiotics are selected according to the minimal inhibitory concentration (MIC) based on the planktonic form of bacteria. Determination of the minimal biofilm eradicating concentration (MBEC), which can be up to 1,000-fold greater than the MIC, is not currently conducted as routine diagnostic testing, primarily because of the methodical hurdles of available biofilm assessing protocols that are time- and cost-consuming. Comparative analysis of biofilms is also limited as most quantitative methods such as crystal violet staining are indirect and highly imprecise. In this paper, we present a novel algorithm for assessing biofilm resistance to antibiotics that overcomes several of the limitations of alternative methods. This algorithm aims for a computer-based analysis of confocal microscope 3D images of biofilms after live/dead stains providing various biofilm parameters such as numbers of viable and dead cells and their vertical distributions within the biofilm, or biofilm thickness. The performance of this algorithm was evaluated using computer-simulated 2D and 3D images of coccal and rodent cells varying different parameters such as cell density, shading or cell size. Finally, genuine biofilms that were untreated or treated with nitroxoline or colistin were analyzed and the results were compared with quantitative microbiological standard methods. This novel algorithm allows a direct, fast and reproducible analysis of biofilms after live/dead staining. It performed well in biofilms of moderate cell densities in a 2D set-up however the 3D analysis remains still imperfect and difficult to evaluate. Nevertheless, this is a first try to develop an easy but conclusive tool that eventually might be implemented into routine diagnostics to determine the MBEC and to improve outcomes of patients with biofilm-associated infections.

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<![CDATA[Dual-targeting of Arabidopsis DMP1 isoforms to the tonoplast and the plasma membrane]]> https://www.researchpad.co/article/5989db51ab0ee8fa60bdc2ba

The reports of dual-targeted proteins in plants have steadily increased over the past years. The vast majority of these proteins are soluble proteins distributed between compartments of the non-secretory pathway, predominantly chloroplasts and mitochondria. In contrast, dual-targeted transmembrane proteins, especially of the secretory pathway, are rare and the mechanisms leading to their differential targeting remain largely unknown. Here, we report dual-targeting of the Arabidopsis DUF679 Membrane Protein 1 (DMP1) to the tonoplast (TP) and the plasma membrane (PM). In Arabidopsis and tobacco two equally abundant DMP1 isoforms are synthesized by alternative translation initiation: a full length protein, DMP1.1, and a truncated one, DMP1.2, which lacks the N-terminal 19 amino acids including a TP-targeting dileucine motif. Accumulation of DMP1.1 and DMP1.2 in the TP and the PM, respectively, is Brefeldin A-sensitive, indicating transit via the Golgi. However, DMP1.2 interacts with DMP1.1, leading to extensive rerouting of DMP1.2 to the TP and “eclipsed” localization of DMP1.2 in the PM where it is barely visible by confocal laser scanning microscopy but clearly detectable by membrane fractionation. It is demonstrated that eGFP fusion to either DMP1 terminus can cause mistargeting artifacts: C-terminal fusion to DMP1.1 or DMP1.2 results in altered ER export and N-terminal fusion to DMP1.1 causes mistargeting to the PM, presumably by masking of the TP targeting signal. These results illustrate how the interplay of alternative translation initiation, presence or absence of targeting information and rerouting due to protein-protein interaction determines the ultimate distribution of a transmembrane protein between two membranes.

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<![CDATA[Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei]]> https://www.researchpad.co/article/5989da8aab0ee8fa60b9d995

Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0–6 h using the plate count technique on Ashdown’s agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist.

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<![CDATA[Optochemokine Tandem for Light-Control of Intracellular Ca2+]]> https://www.researchpad.co/article/5989d9e8ab0ee8fa60b6c0cb

An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.

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<![CDATA[A Unidirectional Cell Switching Gate by Engineering Grating Length and Bending Angle]]> https://www.researchpad.co/article/5989d9d1ab0ee8fa60b64460

On a microgrooved substrate, cells migrate along the pattern, and at random positions, reverse their directions. Here, we demonstrate that these reversals can be controlled by introducing discontinuities to the pattern. On “V-shaped grating patterns”, mouse osteogenic progenitor MC3T3-E1 cells reversed predominately at the bends and the ends. The patterns were engineered in a way that the combined effects of angle- and length-dependence could be examined in addition to their individual effects. Results show that when the bend was placed closer to one end, migration behaviour of cells depends on their direction of approach. At an obtuse bend (135°), more cells reversed when approaching from the long segment than from the short segment. But at an acute bend (45°), this relationship was reversed. Based on this anisotropic behaviour, the designed patterns effectively allowed cells to move in one direction but blocked migrations in the opposing direction. This study demonstrates that by the strategic placement of bends and ends on grating patterns, we can engineer effective unidirectional switching gates that can control the movement of adherent cells. The knowledge developed in this study could be utilised in future cell sorting or filtering platforms without the need for chemotaxis or microfluidic control.

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<![CDATA[Colonization of Wheat, Maize and Cucumber by Paenibacillus polymyxa WLY78]]> https://www.researchpad.co/article/5989db22ab0ee8fa60bcf9f1

Paenibacillus polymyxa WLY78 is a nitrogen fixer and it can be potentially applied to biofertilizer in agriculture. In this study, P. polymyxa WLY78 is labelled with gfp gene. The GFP-labelled P. polymyxa WLY78 is used to inoculate wheat, maize and cucumber seedlings grown in the gnotobiotic system and in soil, respectively. Observation by confocal laser scanning microscope reveals that the GFP-labeled bacterial cells are mainly located on the root surface and epidermis of wheat, and only a few cells are present within cortical cells. In maize and cucumber seedlings, bacterial cells were colonized in epidermal and cortical cells, intercellular spaces and vascular system of root, stem and leaf tissue interiors besides on root surfaces. Higher densities of the bacterial cells in roots, stems and leaves indicated that P. polymyxa WLY78 cells could migrate from roots to stems and leaves of maize and cucumber. This study will provide insight into interaction between P. polymyxa WLY78 and host cells.

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<![CDATA[Classification and Segmentation of Nanoparticle Diffusion Trajectories in Cellular Micro Environments]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdcfb9

Darkfield and confocal laser scanning microscopy both allow for a simultaneous observation of live cells and single nanoparticles. Accordingly, a characterization of nanoparticle uptake and intracellular mobility appears possible within living cells. Single particle tracking allows to measure the size of a diffusing particle close to a cell. However, within the more complex system of a cell’s cytoplasm normal, confined or anomalous diffusion together with directed motion may occur. In this work we present a method to automatically classify and segment single trajectories into their respective motion types. Single trajectories were found to contain more than one motion type. We have trained a random forest with 9 different features. The average error over all motion types for synthetic trajectories was 7.2%. The software was successfully applied to trajectories of positive controls for normal- and constrained diffusion. Trajectories captured by nanoparticle tracking analysis served as positive control for normal diffusion. Nanoparticles inserted into a diblock copolymer membrane was used to generate constrained diffusion. Finally we segmented trajectories of diffusing (nano-)particles in V79 cells captured with both darkfield- and confocal laser scanning microscopy. The software called “TraJClassifier” is freely available as ImageJ/Fiji plugin via https://git.io/v6uz2.

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<![CDATA[Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbd1e

Leukocyte trafficking is crucial to facilitate efficient immune responses. Here, we report that the large GTPase dynamin2, which is generally considered to have a key role in endocytosis and membrane remodeling, is an essential regulator of integrin-dependent human T lymphocyte adhesion and migration. Chemical inhibition or knockdown of dynamin2 expression significantly reduced integrin-dependent T cell adhesion in vitro. This phenotype was not observed when T cells were treated with various chemical inhibitors which abrogate endocytosis or actin polymerization. We furthermore detected dynamin2 in signaling complexes and propose that it controls T cell adhesion via FAK/Pyk2- and RapGEF1-mediated Rap1 activation. In addition, the dynamin2 inhibitor-induced reduction of lymphocyte adhesion can be rescued by Rap1a overexpression. We demonstrate that the dynamin2 effect on T cell adhesion does not involve integrin affinity regulation but instead relies on its ability to modulate integrin valency. Taken together, we suggest a previously unidentified role of dynamin2 in the regulation of integrin-mediated lymphocyte adhesion via a Rap1 signaling pathway.

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<![CDATA[The Effects of Swiprosin-1 on the Formation of Pseudopodia-Like Structures and β-Adrenoceptor Coupling in Cultured Adult Rat Ventricular Cardiomyocytes]]> https://www.researchpad.co/article/5989db33ab0ee8fa60bd25bc

Background

Recent findings suggest that adult terminally differentiated cardiomyocytes adapt to stress by cellular de- and redifferentiation. In the present study we tested the hypothesis that swiprosin-1 is a key player in this process. Furthermore, the relationship between swiprosin-1 and β-adrenoceptor coupling was analyzed.

Methods

In order to study the function of swiprosin-1 in adult rat ventricular cardiomyocytes (ARVC) they were isolated and cultured in a medium containing 20% fetal calf serum (FCS). Changes in cell morphology of ARVC during cultivation were quantified by light and confocal laser scan microscopy. Small interfering RNA (siRNA) was used to reduce the expression of swiprosin-1. The impact of calcium on swiprosin-1 dependent processes was investigated with Bapta-AM. Immunoblot techniques and qRT-PCR were performed to measure mRNA and protein expression.

Results

In culture, ARVC first lost their contractile elements, which was followed by a formation of pseudopodia-like structures (spreading). Swiprosin-1 was detected in ARVC at all time points. However, swiprosin-1 expression was increased when ARVC started to spread. Reduction of swiprosin-1 expression with siRNA delayed ARVC spreading. Similarly, Bapta-AM attenuated swiprosin-1 expression and spreading of ARVC. Furthermore, swiprosin-1 expression correlated with the expression of G protein-coupled receptor kinase 2 (GRK2). Moreover, silencing of swiprosin-1 was associated with a down regulation of GRK2 and caused a sensitization of β-adrenergic receptors.

Conclusion

Swiprosin-1 is required for ARVC to adapt to culture conditions. Additionally, it seems to be involved in the desensitization of β-adrenergic receptors. Assuming that ARVC adapt to cardiac stress in a similar way, swiprosin-1 may play a key role in cardiac remodeling.

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<![CDATA[An oral multispecies biofilm model for high content screening applications]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc661

Peri-implantitis caused by multispecies biofilms is a major complication in dental implant treatment. The bacterial infection surrounding dental implants can lead to bone loss and, in turn, to implant failure. A promising strategy to prevent these common complications is the development of implant surfaces that inhibit biofilm development. A reproducible and easy-to-use biofilm model as a test system for large scale screening of new implant surfaces with putative antibacterial potency is therefore of major importance. In the present study, we developed a highly reproducible in vitro four-species biofilm model consisting of the highly relevant oral bacterial species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. The application of live/dead staining, quantitative real time PCR (qRT-PCR), scanning electron microscopy (SEM) and urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH) revealed that the four-species biofilm community is robust in terms of biovolume, live/dead distribution and individual species distribution over time. The biofilm community is dominated by S. oralis, followed by V. dispar, A. naeslundii and P. gingivalis. The percentage distribution in this model closely reflects the situation in early native plaques and is therefore well suited as an in vitro model test system. Furthermore, despite its nearly native composition, the multispecies model does not depend on nutrient additives, such as native human saliva or serum, and is an inexpensive, easy to handle and highly reproducible alternative to the available model systems. The 96-well plate format enables high content screening for optimized implant surfaces impeding biofilm formation or the testing of multiple antimicrobial treatment strategies to fight multispecies biofilm infections, both exemplary proven in the manuscript.

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