scholarly journals Between Amyloids and Aggregation Lies a Connection with Strength and Adhesion

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Peter N. Lipke ◽  
Caleen Ramsook ◽  
Melissa C. Garcia-Sherman ◽  
Desmond N. Jackson ◽  
Cho X. J. Chan ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Human Infection ◽  
Fiber Formation ◽  
Host Responses ◽  
Infection Model ◽  
High Avidity ◽  
Force Microscopy ◽  
Atomic Force ◽  
Fungal Adhesins

We tell of a journey that led to discovery of amyloids formed by yeast cell adhesins and their importance in biofilms and host immunity. We begin with the identification of the adhesin functional amyloid-forming sequences that mediate fiber formation in vitro. Atomic force microscopy and confocal microscopy show 2-dimensional amyloid “nanodomains” on the surface of cells that are activated for adhesion. These nanodomains are arrays of adhesin molecules that bind multivalent ligands with high avidity. Nanodomains form when adhesin molecules are stretched in the AFM or under laminar flow. Treatment with anti-amyloid perturbants or mutation of the amyloid sequence prevents adhesion nanodomain formation and activation. We are now discovering biological consequences. Adhesin nanodomains promote formation and maintenance of biofilms, which are microbial communities. Also, in abscesses within candidiasis patients, we find adhesin amyloids on the surface of the fungi. In both human infection and a Caenorhabditis elegans infection model, the presence of fungal surface amyloids elicits anti-inflammatory responses. Thus, this is a story of how fungal adhesins respond to extension forces through formation of cell surface amyloid nanodomains, with key consequences for biofilm formation and host responses.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Targeting Breast Cancer Cells with G4 PAMAM Dendrimers and Valproic Acid Derivative Complexes

2020 ◽  
Vol 20 (15) ◽  
pp. 1857-1872
Author(s):  
Alberto M. Muñoz ◽  
Manuel J. Fragoso-Vázquez ◽  
Berenice P. Martel ◽  
Alma Chávez-Blanco ◽  
Alfonso Dueñas-González ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Cell Lines ◽  
Water Solubility ◽  
Md Simulations ◽  
Pamam Dendrimer ◽  
Pamam Dendrimers ◽  
Force Microscopy ◽  
Micromolar Concentration ◽  
Atomic Force

Background: Our research group has developed some Valproic Acid (VPA) derivatives employed as anti-proliferative compounds targeting the HDAC8 enzyme. However, some of these compounds are poorly soluble in water. Objective: Employed the four generations of Polyamidoamine (G4 PAMAM) dendrimers as drug carriers of these compounds to increase their water solubility for further in vitro evaluation. Methods: VPA derivatives were subjected to Docking and Molecular Dynamics (MD) simulations to evaluate their affinity on G4 PAMAM. Then, HPLC-UV/VIS, 1H NMR, MALDI-TOF and atomic force microscopy were employed to establish the formation of the drug-G4 PAMAM complexes. Results: The docking results showed that the amide groups of VPA derivatives make polar interactions with G4 PAMAM, whereas MD simulations corroborated the stability of the complexes. HPLC UV/VIS experiments showed an increase in the drug water solubility which was found to be directly proportional to the amount of G4 PAMAM. 1H NMR showed a disappearance of the proton amine group signals, correlating with docking results. MALDI-TOF and atomic force microscopy suggested the drug-G4 PAMAM dendrimer complexes formation. Discussion: In vitro studies showed that G4 PAMAM has toxicity in the micromolar concentration in MDAMB- 231, MCF7, and 3T3-L1 cell lines. VPA CF-G4 PAMAM dendrimer complex showed anti-proliferative properties in the micromolar concentration in MCF-7 and 3T3-L1, and in the milimolar concentration in MDAMB- 231, whereas VPA MF-G4 PAMAM dendrimer complex didn’t show effects on the three cell lines employed. Conclusion: These results demonstrate that G4 PAMAM dendrimers are capableof transporting poorly watersoluble aryl-VPA derivate compounds to increase its cytotoxic activity against neoplastic cell lines.

scholarly journals Rhinovirus induces MUC5AC in a human infection model and in vitro via NF- B and EGFR pathways

2010 ◽  
Vol 36 (6) ◽  
pp. 1425-1435 ◽  
Author(s):  
C. A. Hewson ◽  
J. J. Haas ◽  
N. W. Bartlett ◽  
S. D. Message ◽  
V. Laza-Stanca ◽  
...  
Keyword(s):  
Human Infection ◽  
Infection Model

scholarly journals Osteoblast-Like Cell Behavior on Porous Scaffolds Based on Poly(styrene) Fibers

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Andrada Serafim ◽  
Romain Mallet ◽  
Florence Pascaretti-Grizon ◽  
Izabela-Cristina Stancu ◽  
Daniel Chappard
Keyword(s):  
Interference Microscopy ◽  
Porous Scaffolds ◽  
Allogenic Bone ◽  
Force Microscopy ◽  
Atomic Force ◽  
Dry Spinning ◽  
Bone Trabeculae ◽  
Large Bone

Scaffolds of nonresorbable biomaterials can represent an interesting alternative for replacing large bone defects in some particular clinical cases with massive bone loss. Poly(styrene) microfibers were prepared by a dry spinning method. They were partially melted to provide 3D porous scaffolds. The quality of the material was assessed by Raman spectroscopy. Surface roughness was determined by atomic force microscopy and vertical interference microscopy. Saos-2 osteoblast-like cells were seeded on the surface of the fibers and left to proliferate. Cell morphology, evaluated by scanning electron microscopy, revealed that they can spread and elongate on the rough microfiber surface. Porous 3D scaffolds made of nonresorbable poly(styrene) fibers are cytocompatible biomaterials mimicking allogenic bone trabeculae and allowing the growth and development of osteoblast-like cellsin vitro.

scholarly journals Nanostructural and mechanical changes in the sclera following proteoglycan depletion

2018 ◽  
Vol 2 (2) ◽  
pp. 14-17
Author(s):  
Zhuola Zhuola ◽  
Steve Barrett ◽  
Yalda Ashraf Kharaz ◽  
Riaz Akhtar
Keyword(s):  
Mechanical Properties ◽  
Collagen Fibril ◽  
Enzymatic Degradation ◽  
Ocular Tissues ◽  
Force Microscopy ◽  
Nanomechanical Properties ◽  
Atomic Force ◽  
Fibril Morphology

The mechanical properties of ocular tissues, such as the sclera, have a major impact on healthy eye function, and are governed by the properties and composition of the microstructural components. For example, biomechanical degradation associated with myopia occurs alongside a reduction of proteoglycans (PGs). In this study, the role of PG degradation in the nanomechanical properties of the porcine sclera is explored. In-vitro enzymatic degradation of PGs was conducted with α-amylase and chondroitinase ABC enzymes. Collagen fibril morphology and nanomechanical stiffness were measured with atomic force microscopy (AFM). The elastic modulus of the tissue was reduced in all enzyme-treated samples relative to controls. In addition, collagen fibril organization was disrupted by PG depletion. Our data demonstrate that PGs play an important role in determining not only the mechanical properties at these length scales, but also collagen fibril arrangement.

scholarly journals Visualization by atomic force microscopy of tobacco mosaic virus movement protein–RNA complexes formed in vitro

2001 ◽  
Vol 82 (6) ◽  
pp. 1503-1508 ◽  
Author(s):  
O. I. Kiselyova ◽  
I. V. Yaminsky ◽  
E. M. Karger ◽  
O. Yu. Frolova ◽  
Y. L. Dorokhov ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Structural Conversion ◽  
Force Microscopy ◽  
Rna Transcripts ◽  
Atomic Force ◽  
Rna Complexes

The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP–RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60–100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive ‘beads-on-a-string’ into a ‘thick string’ form that was partly resistant to RNase. The ‘thick string’-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The ‘thick string’ complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.

scholarly journals Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

2021 ◽  
Author(s):  
Kazuto Yoshimi ◽  
Kohei TAKESHITA ◽  
Noriyuki Kodera ◽  
Satomi Shibumura ◽  
Yuko Yamauchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Speed ◽  
Dna Helicase ◽  
Type I ◽  
Loop Formation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Target Strand ◽  
Target Dna

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

scholarly journals Effect of Gold Nanoparticles on the TLR2-Mediated Inflammatory Responses Induced by Leptospira in TLR2-Overexpressed HEK293 Cells

Nanomaterials ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2522
Author(s):  
Kanidta Sooklert ◽  
Chawikan Boonwong ◽  
Pattama Ekpo ◽  
Rojrit Rojanathanes ◽  
Kanitha Patarakul ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Proinflammatory Cytokines ◽  
Inflammatory Responses ◽  
Hek293 Cells ◽  
Infection Model ◽  
Tlr2 Expression ◽  
Inflammatory Parameters ◽  
Factor Α ◽  
20 Nm

Leptospira infection can cause potential hazards to human health by stimulating inflammation, which is mediated mainly through the Toll-like receptor 2 (TLR2) pathway. Gold nanoparticles (AuNPs) are promising for medical applications, as they display both bioinert and noncytotoxic characteristics. AuNPs have been shown to have the ability to modify immune responses. To understand the in vitro immunomodulatory effect of AuNPs in a Leptospira infection model, the activation of TLR2 expression was examined in HEK-Blue-hTLR2 cells treated with Leptospira serovars and/or AuNPs (10 and 20 nm). The ability of AuNPs to modulate an inflammatory response induced by Leptospira was examined in terms of transcript expression level modulation of three proinflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1β and IL-6) using two-stage quantitative real-time reverse transcriptase PCR. The results revealed that the administration of 10 nm AuNPs could augment the Leptospira-induced TLR2 signaling response and upregulate the expression of all three cytokine gene transcripts, whereas the 20 nm AuNPs attenuated the TLR2 activation and expression of proinflammatory cytokines. This indicates that AuNPs can modulate inflammatory parameters in Leptospira infection and different-sized AuNPs had different immunomodulatory functions in this model.

scholarly journals In vitro evaluation of microbial adhesion on the different surface roughness of acrylic resin specific for ocular prosthesis

2018 ◽  
Vol 12 (02) ◽  
pp. 176-183 ◽  
Author(s):  
Agda Marobo Andreotti ◽  
Cecília Alves De Sousa ◽  
Marcelo Coelho Goiato ◽  
Emily Vivianne Freitas da Silva ◽  
Cristiane Duque ◽  
...  
Keyword(s):  
Surface Roughness ◽  
Staphylococcus Epidermidis ◽  
Microbial Growth ◽  
Acrylic Resin ◽  
Microbial Adhesion ◽  
Force Microscopy ◽  
Atomic Force ◽  
Colony Forming Units ◽  
Ocular Prostheses

ABSTRACT Objective: The purpose of this study was to evaluate the influence of surface roughness in biofilm formation of four microorganisms (Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans) on acrylic resin surface of ocular prostheses. Materials and Methods: Acrylic resin samples were divided into six groups according to polishing: Group 1200S (1200 grit + silica solution); Group 1200; Group 800; Group 400; Group 120 and Group unpolished. Surface roughness was measured using a profilometer and surface images obtained with atomic force microscopy. Microbial growth was evaluated after 4, 24, and 48 hours of incubation by counting colony-forming units. Statistical Analysis Used: For roughness, it was performed 1-way ANOVA and parametric Tukey test α5% (P ≤ 0.05). For CFU data found, it was applied Kruskal-Wallis and Mann-Whitney tests. Results: Group 120 and 400 presented the highest roughness values. For S. epidermidis and S. aureus, Group 1200S presented the lowest values of microbial growth. For E. faecalis at 4 hour, microbial growth was not observed. C. albicans did not adhere to the acrylic resin. Except for Group 1200S, different surface roughnesses did not statistically interfere with microbial adhesion and growth on acrylic surfaces of ocular prostheses. Conclusions: The roughness did not interfere with the microbial adhesion of the microorganisms evaluated. The use of silica decreases significantly microbial growth.

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