Biofunctional Surfaces for Smart Entrapment of Polysomes

Protein synthesis is a central process in all cells, crucial for cell development and maintenance. Translational dysregulation, in fact, is associated with cancer or neurodegenerative diseases. Active protein synthesis occurs on a supramolecular complex, named polyribosome or polysome, formed by a mRNA associated with multiple ribosomes. Polysomes therefore can be considered as a privileged molecular platform to obtain information about the physiological or pathological state in cells. The classical methods for purifying the mRNAs associated with polysomes mainly rely on ultracentrifugation in sucrose gradient followed by standard RNA extraction. This method present several drawbacks, among all it is a time-consuming procedure, which requires a fairly large amounts of starting material. New methods offering an efficient, rapid and user-friendly alternative to standard methods are therefore highly desirable. Here, a panel of surfaces and surface functionalizations were screened for their ability to entrap polysomes with the ultimate aim to set up smart biofunctional surfaces for the purification of nonlabelled polysomes and their associated mRNAs. As a proof-of-concept, prepurified ribosomes and polysomes were incubated on multiple functional surfaces and characterized by atomic force microscopy to assess number and morphology of entrapped polysomes. Surfaces able to efficiently capture polysomes were then included in a microdevice with promising results, opening the future perspective of developing protocols and devices based on biofunctional surfaces.

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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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FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

10.21203/rs.3.rs-20919/v2 ◽

2020 ◽

Cited By ~ 1

Author(s):

Senthilvelrajan Kaniyappan ◽

Katharina Tepper ◽

Jacek Biernat ◽

RamReddy Chandupatla ◽

Sabrina Hübschmann ◽

...

Keyword(s):

Recipient Cell ◽

Pathological State ◽

Tau Pathology ◽

Amyloid Fibers ◽

Force Microscopy ◽

Atomic Force ◽

Transmission Electron ◽

Repeat Domain ◽

Scanning Transmission ◽

Tau Aggregation

Abstract Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~3nm x 4nm) is ~7 times larger than the β-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on Tau or TauRD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused a process distinct from assembly of TauRD filaments.

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Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors

Disease Markers ◽

10.1155/2019/6852917 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 15

Author(s):

Vianey Gonzalez-Villasana ◽

Mohammed H. Rashed ◽

Yessica Gonzalez-Cantú ◽

Recep Bayraktar ◽

Jorge Luis Menchaca-Arredondo ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Rna Extraction ◽

Breast Cancer Patients ◽

High Concentration ◽

Force Microscopy ◽

Atomic Force ◽

Healthy Donors ◽

Mean Size ◽

Noninvasive Biomarkers

miR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.

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New technique for direct fluoroimmunomagnetic detection of rotavirus in water samples

Journal of Water and Health ◽

10.2166/wh.2017.028 ◽

2017 ◽

Vol 15 (6) ◽

pp. 932-941 ◽

Cited By ~ 2

Author(s):

Raquel A. Villamizar-Gallardo ◽

Johann F. Osma ◽

Oscar Orlando Ortíz

Keyword(s):

Monoclonal Antibodies ◽

Water Samples ◽

Rna Extraction ◽

Reliable Technique ◽

Selective Method ◽

Magnetic Microparticles ◽

Force Microscopy ◽

Atomic Force ◽

Transmission Electron ◽

Drinking Water Samples

Abstract A new rapid, sensitive and selective method for rotavirus detection in water samples is described in this paper. Amino pink magnetic microparticles were functionalized with monoclonal antibodies and used to capture, concentrate, separate and detect infectious rotavirus particles in distilled and drinking water samples. The fluorescence of the microparticles was used to determine the presumptive presence of rotaviruses by using confocal microscopy. Atomic force microscopy and transmission electron microscopy were used to confirm the presence of the anti-rotavirus antibodies attached to the surface of the magnetic microparticles as well as that of viruses attached through the antibody. In addition, RNA extraction, quantification and amplification were carried out to validate the microscopic observations. The selectivity of the microparticles was tested in a sample containing a mix of enteric viruses. It was concluded that functionalizing fluoromagnetic microparticles with anti-rotavirus monoclonal antibodies constituted a fast, simple and reliable technique for detecting as low as 10 Rotavirus particles in 1 L of artificial or real water in just 2 hours.

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Antibacterial potentiality of antiulcer and antispasmodic drugs with selected antibiotics against methicillin resistant Staphylococcus aureus: In vitro and in silico studies

Bangladesh Journal of Pharmacology ◽

10.3329/bjp.v10i4.25271 ◽

2015 ◽

Vol 10 (4) ◽

pp. 875

Author(s):

Krishnan Akilandeswari ◽

Kandasamy Ruckmani

Keyword(s):

Antibacterial Properties ◽

Docking Studies ◽

Penicillin Binding Protein ◽

Force Microscopy ◽

Atomic Force ◽

Mebeverine Hydrochloride ◽

Resistant Strains ◽

Polymerase Chain ◽

Set Up

In the present study, the antispasmodic drug mebeverine hydrochloride and the antiulcer drug troxipide were tested for their possible antibacterial properties in vitro. The antimicrobial assays of the above drugs were determined with ampicillin, penicillin and ciprofloxacin against sensitive and resistant strains and their resistance were confirmed through Polymerase Chain Reaction by identifying the presence of the mecA gene. A computer-aided method was used for screening the effectiveness of the drug interactions. Mebeverine and troxipide inhibited most of the sensitive and resistant strains tested in vitro from 32.5 to 125 µg/mL. The loss of structural alterations of the cell wall was analyzed by atomic force microscopy. In docking studies, troxipide and mebeverine were found to have substantial inhibition against penicillin binding protein 2a (IVQQ) and UDP-N-acetylglucosamine 1-carboxyvinyltransferase (2YVW) receptor proteins that seem to have interacted with most of the residues. Video Clips<a href="https://www.youtube.com/v/CpfdUxdNvt0">Set up</a> 2 min 27 sec<a href="https://www.youtube.com/v/IJlkGtIViUU">Broth and drug</a> 4 min 40 sec<a href="https://www.youtube.com/v/_jZHZ8zOJlc">Inoculation</a> 2 min 29 sec<a href="https://www.youtube.com/v/VYT5slycgdE">Incubation and result</a> 46 sec

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Calibration of quartz tuning fork spring constants for non-contact atomic force microscopy: direct mechanical measurements and simulations

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.5.59 ◽

2014 ◽

Vol 5 ◽

pp. 507-516 ◽

Cited By ~ 12

Author(s):

Jens Falter ◽

Marvin Stiefermann ◽

Gernot Langewisch ◽

Philipp Schurig ◽

Hendrik Hölscher ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Tuning Fork ◽

Quartz Tuning Fork ◽

Quantitative Agreement ◽

Experimental Calibration ◽

Force Microscopy ◽

Atomic Force ◽

Strain Stress ◽

Set Up ◽

Spring Constants

Quartz tuning forks are being increasingly employed as sensors in non-contact atomic force microscopy especially in the “qPlus” design. In this study a new and easily applicable setup has been used to determine the static spring constant at several positions along the prong of the tuning fork. The results show a significant deviation from values calculated with the beam formula. In order to understand this discrepancy the complete sensor set-up has been digitally rebuilt and analyzed by using finite element method simulations. These simulations provide a detailed view of the strain/stress distribution inside the tuning fork. The simulations show quantitative agreement with the beam formula if the beam origin is shifted to the position of zero stress onset inside the tuning fork base and torsional effects are also included. We further found significant discrepancies between experimental calibration values and predictions from the shifted beam formula, which are related to a large variance in tip misalignment during the tuning fork assembling process.

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Progress in high resolution atomic force microscopy in biology

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500003061 ◽

1995 ◽

Vol 28 (2) ◽

pp. 195-251 ◽

Cited By ~ 97

Author(s):

Zhifeng Shao ◽

Jie Yang

Keyword(s):

Atomic Force Microscopy ◽

High Resolution ◽

Aqueous Solutions ◽

Biological Samples ◽

Gold Foil ◽

Vertical Resolution ◽

Force Microscopy ◽

Atomic Force ◽

Set Up ◽

Better Than

The atomic force microscope (AFM) was invented by Binnig, Quate and Gerber less than 10 years ago (Binniget al. 1986). In their first prototype, a piece of goldfoil was used as the cantilever, with a crushed diamond tip mounted at the end. On the back of the cantilever, a tunnelling junction was used to monitor the deflection of the cantilever (the gold-foil) when the specimen was scanned with the tip in contact with the surface. Thus, the surface topography of the specimen was obtained with a resolution critically dependent on the sharpness of the tip provided the deformation of the specimen was not serious. Even with such a crude set-up, they managed to obtain a lateral resolution of ˜ 30 Å and a vertical resolution of better than 1 Å on an amorphous A12O3surface. The operating principle of such an instrument is deceptively simple. However, such an arrangement was inconvenient for routine operations and unsuitable for imaging hydrated specimens, because the tunnelling junction is easily contaminated in air and works poorly in aqueous solutions (Alexanderet al. 1989). As a result, the application of this type of AFM to biological samples was rare (Engel, 1991).

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Simultaneous 3D super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for cell imaging

10.1101/638262 ◽

2019 ◽

Author(s):

Ana I. Gómez-Varela ◽

Dimitar R. Stamov ◽

Adelaide Miranda ◽

Rosana Alves ◽

Cláudia Barata-Antunes ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Fluorescence Microscopy ◽

System Integration ◽

Super Resolution ◽

Resolution Limit ◽

Force Microscopy ◽

Spatially Correlated ◽

Atomic Force ◽

Set Up ◽

Cell Variation

AbstractCorrelating data from different microscopy techniques holds the potential to discover new facets of signaling events in cellular biology. Here we report for the first time a hardware set-up capable of achieving simultaneous imaging of spatially correlated super-resolution fluorescence microscopy and atomic force microscopy, a feat only obtained until now by fluorescence microscopy set-ups with spatial resolution restricted to the Abbe resolution limit. We hereby remove the need to perform independent measurement and subsequent data averaging required to eliminate cell-to-cell variation in observed signals. We detail system integration, demonstrate system performance and report imaging of sub-resolution fluorescent beads and genome-engineered human bone osteosarcoma epithelial cells.

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iTaxoTools 0.1: Kickstarting a specimen-based software toolkit for taxonomists

10.1101/2021.03.26.435825 ◽

2021 ◽

Author(s):

Miguel Vences ◽

Aurelien Miralles ◽

Sophie Brouillet ◽

Jacques Ducasse ◽

Alexander Fedosov ◽

...

Keyword(s):

User Interfaces ◽

Species Delimitation ◽

Statistical Tests ◽

Future Perspective ◽

User Friendliness ◽

Bioinformatic Pipeline ◽

Morphometric Analyses ◽

Data Files ◽

Set Up ◽

User Friendly

While powerful and user-friendly software suites exist for phylogenetics, and an impressive cybertaxomic infrastructure of online species databases has been set up in the past two decades, software specifically targeted at facilitating alpha-taxonomic work, i.e., delimiting and diagnosing species, is still in its infancy. Here we present a project to develop a bioinformatic toolkit for taxonomy, based on open-source Python code, including tools focusing on species delimitation and diagnosis and centered around specimen identifiers. At the core of iTaxoTools is user-friendliness, with numerous autocorrect options for data files and with intuitive graphical user interfaces. Assembled standalone executables for all tools or a suite of tools with a launcher window will be distributed for Windows, Linux, and Mac OS systems, and in the future also implemented on a web server. The alpha version (iTaxoTools 0.1) distributed with this paper contains GUI versions of six species delimitation programs (ABGD, ASAP, DELINEATE, GMYC, PTP, tr2) and a simple threshold-clustering delimitation tool. There are also new Python implementations of existing algorithms, including tools to compute pairwise DNA distances, ultrametric time trees based on non-parametric rate smoothing, species-diagnostic nucleotide positions, and standard morphometric analyses. Other utilities convert among different formats of molecular sequences, geographical coordinates, and units; merge, split and prune sequence files and tables; and perform simple statistical tests. As a future perspective, we envisage iTaxoTools to become part of a bioinformatic pipeline for next-generation taxonomy that accelerates the inventory of life while maintaining high-quality species hypotheses.

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iTaxoTools 0.1: Kickstarting a specimen-based software toolkit for taxonomists

Megataxa ◽

10.11646/megataxa.6.2.1 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

MIGUEL VENCES ◽

AURÉLIEN MIRALLES ◽

SOPHIE BROUILLET ◽

JACQUES DUCASSE ◽

ALEXANDER FEDOSOV ◽

...

Keyword(s):

Open Source ◽

User Interfaces ◽

Species Delimitation ◽

Statistical Tests ◽

Future Perspective ◽

User Friendliness ◽

Morphometric Analyses ◽

Data Files ◽

Set Up ◽

User Friendly

While powerful and user-friendly software suites exist for phylogenetics, and an impressive cybertaxomic infrastructure of online species databases has been set up in the past two decades, software targeted explicitly at facilitating alpha-taxonomic work, i.e., delimiting and diagnosing species, is still in its infancy. Here we present a project to develop a bioinformatic toolkit for taxonomy, based on open-source Python code, including tools focusing on species delimitation and diagnosis and centered around specimen identifiers. At the core of iTaxoTools is user-friendliness, with numerous autocorrect options for data files and with intuitive graphical user interfaces. Assembled standalone executables for all tools or a suite of tools with a launcher window will be distributed for Windows, Linux, and Mac OS systems, and in the future also implemented on a web server. The initial version (iTaxoTools 0.1) distributed with this paper (https://github.com/iTaxoTools/iTaxoTools-Executables) contains graphical user interface (GUI) versions of six species delimitation programs (ABGD, ASAP, DELINEATE, GMYC, PTP, tr2) and a simple threshold-clustering delimitation tool. There are also new Python implementations of existing algorithms, including tools to compute pairwise DNA distances, ultrametric time trees based on non-parametric rate smoothing, species-diagnostic nucleotide positions, and standard morphometric analyses. Other utilities convert among different formats of molecular sequences, geographical coordinates, and units; merge, split and prune sequence files, tables and species partition files; and perform simple statistical tests. As a future perspective, we envisage iTaxoTools to become part of a bioinformatic pipeline for next-generation taxonomy that accelerates the inventory of life while maintaining high-quality species hypotheses. The open source code and binaries of all tools are available from Github (https://github.com/iTaxoTools) and further information from the website (http://itaxotools.org)

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