Seeding hESCs to achieve optimal colony clonality

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have promising clinical applications which often rely on clonally-homogeneous cell populations. To achieve this, it is important to ensure that each colony originates from a single founding cell and to avoid subsequent merging of colonies during their growth. Clonal homogeneity can be obtained with low seeding densities; however, this leads to low yield and viability. It is therefore important to quantitatively assess how seeding density affects clonality loss so that experimental protocols can be optimised to meet the required standards. Here we develop a quantitative framework for modelling the growth of hESC colonies from a given seeding density based on stochastic exponential growth. This allows us to identify the timescales for colony merges and over which colony size no longer predicts the number of founding cells. We demonstrate the success of our model by applying it to our own experiments of hESC colony growth; while this is based on a particular experimental set-up, the model can be applied more generally to other cell lines and experimental conditions to predict these important timescales

Correlated random walks of human embryonic stem cells in vitro

We perform a detailed analysis of the migratory motion of human embryonic stem cells in two-dimensions, both when isolated and in close proximity to another cell, recorded with time-lapse microscopic imaging. We show that isolated cells tend to perform an unusual locally anisotropic walk, moving backwards and forwards along a preferred local direction correlated over a timescale of around 50 min and aligned with the axis of the cell elongation. Increasing elongation of the cell shape is associated with increased instantaneous migration speed. We also show that two cells in close proximity tend to move in the same direction, with the average separation of m or less and the correlation length of around 25 μm, a typical cell diameter. These results can be used as a basis for the mathematical modelling of the formation of clonal hESC colonies

Glycoform-selective prion formation in sporadic and familial forms of prion disease

The four glycoforms of the cellular prion protein (PrP(C)) variably glycosylated at the two N-linked glycosylation sites are converted into their pathological forms (PrP(Sc)) in most cases of sporadic prion diseases. However, a prominent molecular characteristic of PrP(Sc) in the recently identified variably protease-sensitive prionopathy (VPSPr) is the absence of a diglycosylated form, also notable in familial Creutzfeldt-Jakob disease (fCJD), which is linked to mutations in PrP either from Val to Ile at residue 180 (fCJD(V180I)) or from Thr to Ala at residue 183 (fCJD(T183A)). Here we report that fCJD(V180I), but not fCJD(T183A), exhibits a proteinase K (PK)-resistant PrP (PrP(res)) that is markedly similar to that observed in VPSPr, which exhibits a five-step ladder-like electrophoretic profile, a molecular hallmark of VPSPr. Remarkably, the absence of the diglycosylated PrP(res) species in both fCJD(V180I) and VPSPr is likewise attributable to the absence of PrP(res) glycosylated at the first N-linked glycosylation site at residue 181, as in fCJD(T183A). In contrast to fCJD(T183A), both VPSPr and fCJD(V180I) exhibit glycosylation at residue 181 on di- and monoglycosylated (mono181) PrP prior to PK-treatment. Furthermore, PrP(V180I) with a typical glycoform profile from cultured cells generates detectable PrP(res) that also contains the diglycosylated PrP in addition to mono- and unglycosylated forms upon PK-treatment. Taken together, our current in vivo and in vitro studies indicate that sporadic VPSPr and familial CJD(V180I) share a unique glycoform-selective prion formation pathway in which the conversion of diglycosylated and mono181 PrP(C) to PrP(Sc) is inhibited, probably by a dominant-negative effect, or by other co-factors

Efficiency of Collisionally-activated dissociation and 193-nm photodissociation of peptide ions in fourier transform mass spectrometry

AbstractFor tandem mass spectrometry, the Fourier transform instrument exhibits advantages for the use of collisionally-activated dissociation (CAD). The CAD energy deposited in larger ions can be greatly increased by extending the collision time to as much as 120 s, and the efficiency of trapping and measuring CAD product ions in many times greater than the found for triple-quadrupole or magnetic sector instruments, although the increased pressure from the collision gas is an offsetting disadvantage. A novel system that uses the same laser for photodesorption of ions and their subsequent photodissociation can produce complete dissociation of larger oligopeptide ions and unusually abundant fragment ions. In comparison to CAD, much more internal energy can be deposited in the primary ions using 193-nm photons, sufficient to dissociate peptide ions of m/z > 2000. Mass spectra closely resembling ion photodissociation spectra can also be obtained by neutral photodissociation (193-nm laser irradiation of the sample) followed by ion photodesorption

Trigonometric Regressive Spectral Analysis Reliably Maps Dynamic Changes in Baroreflex Sensitivity and Autonomic Tone: The Effect of Gender and Age

BACKGROUND: The assessment of baroreflex sensitivity (BRS) has emerged as prognostic tool in cardiology. Although available computer-assisted methods, measuring spontaneous fluctuations of heart rate and blood pressure in the time and frequency domain are easily applicable, they do not allow for quantification of BRS during cardiovascular adaption processes. This, however, seems an essential criterion for clinical application. We evaluated a novel algorithm based on trigonometric regression regarding its ability to map dynamic changes in BRS and autonomic tone during cardiovascular provocation in relation to gender and age. METHODOLOGY/PRINCIPAL FINDINGS: We continuously recorded systemic arterial pressure, electrocardiogram and respiration in 23 young subjects (25+/-2 years) and 22 middle-aged subjects (56+/-4 years) during cardiovascular autonomic testing (metronomic breathing, Valsalva manoeuvre, head-up tilt). Baroreflex- and spectral analysis was performed using the algorithm of trigonometric regressive spectral analysis. There was an age-related decline in spontaneous BRS and high frequency oscillations of RR intervals. Changes in autonomic tone evoked by cardiovascular provocation were observed as shifts in the ratio of low to high frequency oscillations of RR intervals and blood pressure. Respiration at 0.1 Hz elicited an increase in BRS while head-up tilt and Valsalva manoeuvre resulted in a downregulation of BRS. The extent of autonomic adaption was in general more pronounced in young individuals and declined stronger with age in women than in men. CONCLUSIONS/SIGNIFICANCE: The trigonometric regressive spectral analysis reliably maps age- and gender-related differences in baroreflex- and autonomic function and is able to describe adaption processes of baroreceptor circuit during cardiovascular stimulation. Hence, this novel algorithm may be a useful screening tool to detect abnormalities in cardiovascular adaption processes even when resting values appear to be normal

Emerging topics in nanophononics and elastic, acoustic, and mechanical metamaterials:An overview

This broad review summarizes recent advances and “hot” research topics in nanophononics and elastic, acoustic, and mechanical metamaterials based on results presented by the authors at the EUROMECH 610 Colloquium held on April 25–27, 2022 in Benicássim, Spain. The key goal of the colloquium was to highlight important developments in these areas, particularly new results that emerged during the last two years. This work thus presents a “snapshot” of the state-of-the-art of different nanophononics- and metamaterial-related topics rather than a historical view on these subjects, in contrast to a conventional review article. The introduction of basic definitions for each topic is followed by an outline of design strategies for the media under consideration, recently developed analysis and implementation techniques, and discussions of current challenges and promising applications. This review, while not comprehensive, will be helpful especially for early-career researchers, among others, as it offers a broad view of the current state-of-the-art and highlights some unique and flourishing research in the mentioned fields, providing insight into multiple exciting research directions

Clustering of classical swine fever virus isolates by codon pair bias

<p>Abstract</p> <p>Background</p> <p>The genetic code consists of non-random usage of synonymous codons for the same amino acids, termed codon bias or codon usage. Codon juxtaposition is also non-random, referred to as codon context bias or codon pair bias. The codon and codon pair bias vary among different organisms, as well as with viruses. Reasons for these differences are not completely understood. For classical swine fever virus (CSFV), it was suggested that the synonymous codon usage does not significantly influence virulence, but the relationship between variations in codon pair usage and CSFV virulence is unknown. Virulence can be related to the fitness of a virus: Differences in codon pair usage influence genome translation efficiency, which may in turn relate to the fitness of a virus. Accordingly, the potential of the codon pair bias for clustering CSFV isolates into classes of different virulence was investigated.</p> <p>Results</p> <p>The complete genomic sequences encoding the viral polyprotein of 52 different CSFV isolates were analyzed. This included 49 sequences from the GenBank database (NCBI) and three newly sequenced genomes. The codon usage did not differ among isolates of different virulence or genotype. In contrast, a clustering of isolates based on their codon pair bias was observed, clearly discriminating highly virulent isolates and vaccine strains on one side from moderately virulent strains on the other side. However, phylogenetic trees based on the codon pair bias and on the primary nucleotide sequence resulted in a very similar genotype distribution.</p> <p>Conclusion</p> <p>Clustering of CSFV genomes based on their codon pair bias correlate with the genotype rather than with the virulence of the isolates.</p

On the barrier properties of the cornea: a microscopy study of the penetration of fluorescently labeled nanoparticles, polymers, and sodium fluorescein

Overcoming the natural defensive barrier functions of the eye remains one of the greatest challenges of ocular drug delivery. Cornea is a chemical and mechanical barrier preventing the passage of any foreign bodies including drugs into the eye, but the factors limiting penetration of permeants and nanoparticulate drug delivery systems through the cornea are still not fully understood. In this study, we investigate these barrier properties of the cornea using thiolated and PEGylated (750 and 5000 Da) nanoparticles, sodium fluorescein, and two linear polymers (dextran and polyethylene glycol). Experiments used intact bovine cornea in addition to bovine cornea de-epithelialized or tissues pretreated with cyclodextrin. It was shown that corneal epithelium is the major barrier for permeation; pretreatment of the cornea with β-cyclodextrin provides higher permeation of low molecular weight compounds, such as sodium fluorescein, but does not enhance penetration of nanoparticles and larger molecules. Studying penetration of thiolated and PEGylated (750 and 5000 Da) nanoparticles into the de-epithelialized ocular tissue revealed that interactions between corneal surface and thiol groups of nanoparticles were more significant determinants of penetration than particle size (for the sizes used here). PEGylation with polyethylene glycol of a higher molecular weight (5000 Da) allows penetration of nanoparticles into the stroma, which proceeds gradually, after an initial 1 h lag phase

Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca2+-induced conformational changes in the regulatory domain of human cardiac troponin C

AbstractTroponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 ± 86 Å2 for apo-cNTnC to 3108 ± 71 Å2 for Ca2+-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca2+-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis