Mycobacterium tuberculosis and Paracoccidioides brasiliensis Formation and Treatment of Mixed Biofilm In Vitro

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3’hydroxychalcone (3’chalc) -loaded nanoemulsion (NE) (NE3’chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3’chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3’hidroxychalcone (3’chalc) range from 0.97- 7.8 µg/mL and NE3’chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3’chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3’chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3’chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.

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

10.26434/chemrxiv.7725002.v1 ◽

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.

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Preparation and Characterization of Chitosan-Casein Phosphopeptides Biocomposite Coatings on the Medical Titanium Alloy

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.480-481.1065 ◽

2011 ◽

Vol 480-481 ◽

pp. 1065-1069

Author(s):

Bin Liu ◽

Lin Wang ◽

Yin Zhong Bu ◽

Sheng Rong Yang ◽

Jin Qing Wang

Keyword(s):

Photoelectron Spectroscopy ◽

Attenuated Total Reflectance ◽

Ti Alloy ◽

Force Microscopy ◽

Implant Materials ◽

Atomic Force ◽

Casein Phosphopeptides ◽

Potential Applications ◽

First Time

Titanium (Ti) and its alloys have been applied in orthopedics as one of the most popular biomedical metallic implant materials. In this work, to enhance the bioactivity, the surface of Ti alloy pre-modified by silane coupling agent and glutaraldehyde was covalently grafted with chitosan (CS) via biochemical multistep self-assembled method. Then, for the first time, the achieved surface was further immobilized with casein phosphopeptides (CPP), which are one group of bioactive peptides released from caseins in the digestive tract and can facilitate the calcium adsorption and usage, to form CS-CPP biocomposite coatings. The structure and composition of the fabricated coatings were characterized by X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and atomic force microscopy (AFM). As the experimental results indicated, multi-step assembly was successfully performed, and the CS and CPP were assembled onto the Ti alloy surface orderly. It is anticipated that the Ti alloys modified by CS-CPP biocomposite coatings will find potential applications as implant materials in biomedical fields.

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A New Sugarcane Cystatin Strongly Binds to Dental Enamel and Reduces Erosion

Journal of Dental Research ◽

10.1177/0022034517712981 ◽

2017 ◽

Vol 96 (9) ◽

pp. 1051-1057 ◽

Cited By ~ 14

Author(s):

A.C. Santiago ◽

Z.N. Khan ◽

M.C. Miguel ◽

C.C. Gironda ◽

A. Soares-Costa ◽

...

Keyword(s):

Potent Inhibitor ◽

Surface Hardness ◽

Dental Enamel ◽

Reversible Inhibitors ◽

Force Microscopy ◽

Atomic Force ◽

Enamel Erosion ◽

Acquired Enamel Pellicle

Cystatin B was recently identified as an acid-resistant protein in acquired enamel pellicle; it could therefore be included in oral products to protect against caries and erosion. However, human recombinant cystatin is very expensive, and alternatives to its use are necessary. Phytocystatins are reversible inhibitors of cysteine peptidases that are found naturally in plants. In plants, they have several biological and physiological functions, such as the regulation of endogenous processes, defense against pathogens, and response to abiotic stress. Previous studies performed by our research group have reported high inhibitory activity and potential agricultural and medical applications of several sugarcane cystatins, including CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4. In the present study, we report the characterization of a novel sugarcane cystatin, named CaneCPI-5. This cystatin was efficiently expressed in Escherichia coli, and inhibitory assays demonstrated that it was a potent inhibitor of human cathepsins B, K, and L ( Ki = 6.87, 0.49, and 0.34 nM, respectively). The ability of CaneCPI-5 to bind to dental enamel was evaluated using atomic force microscopy. Its capacity to protect against initial enamel erosion was also tested in vitro via changes in surface hardness. CaneCPI-5 showed a very large force of interaction with enamel (e.g., compared with mucin and casein) and significantly reduced initial enamel erosion. These results suggest that the inclusion of CaneCPIs in dental products might confer protection against enamel erosion.

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Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913018714 ◽

2013 ◽

Vol 69 (10) ◽

pp. 2050-2060 ◽

Cited By ~ 23

Author(s):

Bodo Sander ◽

Giancarlo Tria ◽

Alexander V. Shkumatov ◽

Eun-Young Kim ◽

J. Günter Grossmann ◽

...

Keyword(s):

Conformational Dynamics ◽

Cd Spectroscopy ◽

X Ray ◽

Force Microscopy ◽

X Ray Scattering ◽

Atomic Force ◽

Extended States ◽

High Degree

Gephyrin is a trimeric protein involved in the final steps of molybdenum-cofactor (Moco) biosynthesis and in the clustering of inhibitory glycine and GABAAreceptors at postsynaptic specializations. Each protomer consists of stably folded domains (referred to as the G and E domains) located at either terminus and connected by a proteolytically sensitive linker of ∼150 residues. Both terminal domains can oligomerize in their isolated forms; however, in the context of the full-length protein only the G-domain trimer is permanently present, whereas E-domain dimerization is prevented. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) reveal a high degree of flexibility in the structure of gephyrin. The results imply an equilibrium between compact and extended conformational states in solution, with a preference for compact states. CD spectroscopy suggests that a partial compaction is achieved by interactions of the linker with the G and E domains. Taken together, the data provide a rationale for the role of the linker in the overall structure and the conformational dynamics of gephyrin.

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In vitro Performance of Nano-heterogeneous Dentin Adhesive

Journal of Dental Research ◽

10.1177/154405910808700911 ◽

2008 ◽

Vol 87 (9) ◽

pp. 829-833 ◽

Cited By ~ 55

Author(s):

Q. Ye ◽

J.G. Park ◽

E. Topp ◽

Y. Wang ◽

A. Misra ◽

...

Keyword(s):

Mechanical Properties ◽

Phase Image ◽

Force Microscopy ◽

Dental Tissues ◽

Atomic Force ◽

Dentin Adhesives ◽

Mechanical Durability ◽

Nanophase Separation ◽

High Degree

Water is ubiquitous in the mouths of healthy individuals and routinely interferes with efforts to bond restorations to dental tissues. Our previous studies using tapping-mode atomic force microscopy (TMAFM) have shown that nanophase separation is a general feature of cross-linked polymethacrylates photocured in the presence of water. To explore the relationship between nanophase separation in dentin adhesives and their long-term mechanical properties, we evaluated model adhesives after 3 months of aqueous storage. The degree of contrast in the TMAFM phase image depended on the formulations used, ranging from ‘not observable’ to ‘very strong’. Correspondingly, the mechanical properties of these model adhesives varied from ‘minimal change’ to ‘significant depreciation’. The results support the hypothesis that a high degree of heterogeneity at the nano-scale is associated with poor mechanical durability in these model adhesives.

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Microstructural differences of fibrin and self-assembling peptide hydrogels in dental pulp stem cell behavior: the effect of chlorite-oxidized oxyamylose

10.21203/rs.3.rs-109250/v1 ◽

2020 ◽

Author(s):

Mostafa EzEldeen ◽

Burak Toprakhisar ◽

Denise Murgia ◽

Nick Smisdom ◽

Olivier Deschaume ◽

...

Keyword(s):

Dental Pulp ◽

Microstructural Stability ◽

Microscopy Imaging ◽

Force Microscopy ◽

Regenerative Endodontics ◽

Self Assembling ◽

Atomic Force ◽

Peptide Hydrogels

Abstract Tailored hydrogels mimicking the native extracellular environment could aid in overcoming the high variability in regenerative endodontics outcomes. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of fibrin and self-assembling peptide (SAP) hydrogels. Further, to assess the influence of the microstructural differences between the hydrogels on the in vitro behavior of dental pulp stem cells (DPSCs).Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and fiber length, diameter, straightness and alignment) and by nanoindentation to measure their stiffness (elastic modulus). DPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity was determined using confocal microscopy imaging, and proliferation was determined using DNA quantification. Inclusion of COAM did not alter the microstructure of the fibrin hydrogels at the fiber level, while affecting the SAP hydrogel microstructure (homogeneity) leading to fiber aggregation. The stiffness of the SAP hydrogels was 7-fold higher than the fibrin hydrogels. The viability and attachment of DPSCs and DNA content was significantly higher in fibrin hydrogels than in SAP hydrogels. The microstructural stability after COAM inclusion and the favorable DPSCs’ response observed in fibrin hydrogels suggest this system as a promising carrier for COAM and for application in endodontic regeneration.

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Exploring the action of RGDV-gemcitabine on tumor metastasis, tumor growth and possible action pathway

Scientific Reports ◽

10.1038/s41598-020-72824-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Xiaoyi Zhang ◽

Jinhuan Zhang ◽

Wenchao Liu ◽

Yaonan Wang ◽

Jianhui Wu ◽

...

Keyword(s):

Tumor Metastasis ◽

A549 Cells ◽

Force Microscopy ◽

Atomic Force ◽

Tnf Α ◽

First Time ◽

Inhibit Tumor Metastasis ◽

Necrosis Factor

Abstract The coupling of Arg-Gly-Asp-Val (RGDV) and gemcitabine led to a hypothesis that the conjugate (RGDV-gemcitabine) could inhibit tumor metastasis. To confirm this hypothesis the activities of RGDV-gemcitabine inhibiting tumor metastasis in vitro and in vivo were presented for the first time. AFM (atomic force microscopy) imaged that RGDV-gemcitabine was able to adhere onto the surface of serum-starved A549 cells, to block the extending of the pseudopodia. Thereby RGDV-gemcitabine was able to inhibit the invasion, migration and adhesion of serum-starved A549 cells in vitro. On C57BL/6 mouse model RGDV-gemcitabine dose dependently inhibited the metastasis of planted tumor towards the lung and the minimal dose was 0.084 µmol/kg/3 days. The decrease of serum TNF-α (tumor necrosis factor), IL-8 (interleukin-8), MMP-2 (matrix metalloprotein-2) and MMP-9 (matrix metalloprotein-9) of the treated C57BL/6 mice was correlated with the action pathway of RGDV-gemcitabine inhibiting the metastasis of the planted tumor towards lung.

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Enzymatic Conversion of Oleuropein to Hydroxytyrosol Using Immobilized β-Glucosidase on Porous Carbon Cuboids

Nanomaterials ◽

10.3390/nano9081166 ◽

2019 ◽

Vol 9 (8) ◽

pp. 1166 ◽

Cited By ~ 2

Author(s):

Chatzikonstantinou ◽

Gkantzou ◽

Thomou ◽

Chalmpes ◽

Lyra ◽

...

Keyword(s):

Porous Carbon ◽

Photoelectron Spectroscopy ◽

Thermotoga Maritima ◽

Covalent Immobilization ◽

Force Microscopy ◽

Atomic Force ◽

Repeated Use ◽

First Time ◽

Hydrolysis Of

In the present study, we developed novel β-glucosidase-based nano-biocatalysts for the bioconversion of oleuropein to hydroxytyrosol. Using non-covalent or covalent immobilization approaches, β-glucosidases from almonds and Thermotoga maritima were attached for the first time on oxidized and non-oxidized porous carbon cuboids (PCC). Various methods were used for the characterization of the bio-nanoconjugates, such as Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and fluorescence spectroscopy. The oxidation state of the nanο-support and the immobilization procedure play a key role for the immobilization efficiency or the catalytic activity of the immobilized β-glucosidases. The nano-biocatalysts were successfully used for the hydrolysis of oleuropein, which leads to the formation of its bioactive derivative, hydroxytyrosol (up to 2.4 g L−1), which is a phenolic compound with numerous health benefits. The bio-nanoconjugates exhibited high thermal and operational stability (up to 240 hours of repeated use), which indicated that they are efficient tools for various bio-transformations.

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Chlorite oxidized oxyamylose differentially influences the microstructure of fibrin and self assembling peptide hydrogels as well as dental pulp stem cell behavior

Scientific Reports ◽

10.1038/s41598-021-84405-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mostafa EzEldeen ◽

Burak Toprakhisar ◽

Denise Murgia ◽

Nick Smisdom ◽

Olivier Deschaume ◽

...

Keyword(s):

Dental Pulp ◽

Microstructural Stability ◽

Force Microscopy ◽

Regenerative Endodontics ◽

Self Assembling ◽

Atomic Force ◽

Human Dental Pulp ◽

Peptide Hydrogels

AbstractTailored hydrogels mimicking the native extracellular environment could help overcome the high variability in outcomes within regenerative endodontics. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of fibrin and self-assembling peptide (SAP) hydrogels. A further goal was to assess the influence of the microstructural differences between the hydrogels on the in vitro behavior of human dental pulp stem cells (hDPSCs). Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and fiber length, diameter, straightness, and alignment) and by nanoindentation to measure their stiffness (elastic modulus). Then, hDPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity were determined using confocal microscopy, and proliferation was determined using DNA quantification. Inclusion of COAM did not alter the microstructure of the fibrin hydrogels at the fiber level while affecting the SAP hydrogel microstructure (homogeneity), leading to fiber aggregation. The stiffness of the SAP hydrogels was sevenfold higher than the fibrin hydrogels. The viability and attachment of hDPSCs were significantly higher in fibrin hydrogels than in SAP hydrogels. The DNA content was significantly affected by the hydrogel type and the presence of COAM. The microstructural stability after COAM inclusion and the favorable hDPSCs' response observed in fibrin hydrogels suggest this system as a promising carrier for COAM and application in endodontic regeneration.

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Perspectives on the Use of Hyaluronidase from the Red King Crab Hepatopancreas in Treating Filler Complications: In Vitro Investigations

10.20944/preprints201909.0001.v1 ◽

2019 ◽

Author(s):

Tatyana Ponomareva ◽

Dmitrii Sliadovskii ◽

Maria Timchenko ◽

Alexander Timchenko ◽

Evgeny Sogorin

Keyword(s):

Red King Crab ◽

King Crab ◽

Force Microscopy ◽

Atomic Force ◽

Safe Product ◽

Crab Hepatopancreas ◽

Aggregation Formation ◽

First Time ◽

Filler Complications

The kinetics of the hydrolysis of hyaluronic acid (HA) of cosmetic fillers using thehomogenate of the red king crab hepatopancreas was studied for the first time. Turbidimetricanalysis of the reaction mixture revealed a bell–shaped time dependence of aggregation formation. The HA fillers were examined by atomic force microscopy (AFM) and it was found that they wererepresented by spherical–like structures. These structures were disrupted under the action of thehomogenate of the red king crab hepatopancreas. It was shown that the prepared homogenate hasthe activity which is similar to that observed in the commercially available hyaluronidase products.The preparation with hyaluronidase activity obtained from the red king crab hepatopancreas couldbe used as potentially safe product for treating filler complications.

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