Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By ...combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in
, we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions.
The vascular endothelium serves as a barrier between the intravascular and extravascular compartments. High-density lipoproteins (HDL) have two kinds of interactions with this barrier. First, ...bloodborne HDL must pass the endothelium to access extravascular tissues, for example the arterial wall or the brain, to mediate cholesterol efflux from macrophages and other cells or exert other functions. To complete reverse cholesterol transport, HDL must even pass the endothelium a second time to re-enter circulation via the lymphatics. Transendothelial HDL transport is a regulated process involving scavenger receptor SR-BI, endothelial lipase, and ATP binding cassette transporters A1 and G1. Second, HDL helps to maintain the integrity of the endothelial barrier by (i) promoting junction closure as well as (ii) repair by stimulating the proliferation and migration of endothelial cells and their progenitor cells, and by preventing (iii) loss of glycocalix, (iv) apoptosis, as well as (v) transmigration of inflammatory cells. Additional vasoprotective functions of HDL include (vi) the induction of nitric oxide (NO) production and (vii) the inhibition of reactive oxygen species (ROS) production. These vasoprotective functions are exerted by the interactions of HDL particles with SR-BI as well as specific agonists carried by HDL, notably sphingosine-1-phophate (S1P), with their specific cellular counterparts, e.g., S1P receptors. Various diseases modify the protein and lipid composition and thereby the endothelial functionality of HDL. Thorough understanding of the structure-function relationships underlying the multiple interactions of HDL with endothelial cells is expected to elucidate new targets and strategies for the treatment or prevention of various diseases.
Callose deposition in Arabidopsis has emerged as a popular model system to quantify activity of plant immunity. However, there has been a noticeable rise in contradicting reports about the regulation ...of pathogen-induced callose. To address this controversy, we have examined the robustness of callose deposition under different growth conditions and in response to two different pathogen-associated molecular patterns, the flagellin epitope Flg22 and the polysaccharide chitosan. Based on a commonly used hydroponic culture system, we found that variations in growth conditions have a major impact on the plant's overall capacity to deposit callose. This environmental variability correlated with levels of hydrogen peroxide (H2O2) production. Depending on the growth conditions, pretreatment with abscissic acid stimulated or repressed callose deposition. Despite a similar effect of growth conditions on Flg22- and chitosan-induced callose, both responses showed differences in timing, tissue responsiveness, and colocalization with H2O2. Furthermore, mutant analysis revealed that Flg22- and chitosan-induced callose differ in the requirement for the NADPH oxidase RBOHD, the glucosinolate regulatory enzymes VTC1 and PEN2, and the callose synthase PMR4. Our study demonstrates that callose is a multifaceted defense response that is controlled by distinct signaling pathways, depending on the environmental conditions and the challenging pathogen-associated molecular pattern.
Surveillance for control of antimicrobial resistance Tacconelli, Evelina; Sifakis, Frangiscos; Harbarth, Stephan ...
Lancet. Infectious diseases/The Lancet. Infectious diseases,
March 2018, 2018-03-00, 20180301, 2018-03, Letnik:
18, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Antimicrobial resistance poses a growing threat to public health and the provision of health care. Its surveillance should provide up-to-date and relevant information to monitor the appropriateness ...of therapy guidelines, antibiotic formulary, antibiotic stewardship programmes, public health interventions, infection control policies, and antimicrobial development. In Europe, although the European Antimicrobial Resistance Surveillance Network provides annual reports on monitored resistant bacteria, national surveillance efforts are still fragmented and heterogeneous, and have substantial structural problems and issues with laboratory data. Most incidence and prevalence data cannot be linked with relevant epidemiological, clinical, or outcome data. Genetic typing, to establish whether trends of antimicrobial resistance are caused by spread of resistant strains or by transfer of resistance determinants among different strains and species, is not routinely done. Furthermore, laboratory-based surveillance using only clinical samples is not likely to be useful as an early warning system for emerging pathogens and resistance mechanisms. Insufficient coordination of surveillance systems of human antimicrobial resistance with animal surveillance systems is even more concerning. Because results from food surveillance are considered commercially sensitive, they are rarely released publicly by regulators. Inaccurate or incomplete surveillance data delay a translational approach to the threat of antimicrobial resistance and inhibit the identification of relevant target microorganisms and populations for research and the revitalisation of dormant drug-discovery programmes. High-quality, comprehensive, and real-time surveillance data are essential to reduce the burden of antimicrobial resistance. Improvement of national antimicrobial resistance surveillance systems and better alignment between human and veterinary surveillance systems in Europe must become a scientific and political priority, coordinated with international stakeholders within a global approach to reduce the burden of antimicrobial resistance.
We built a broadband Electron Paramagnetic Resonance (EPR) spectrometer capable of field- and frequency sweep experiments under field-, microwave amplitude- and microwave frequency-modulation ...detection modes (HM, AM, and FM, respectively). The spectrometer is based on a coplanar waveguide (CPW) architecture, with the sample being deposited on top of the transmission line. We tested the functionality of this spectrometer by measuring a standard 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) sample, and complex (N
n
Bu
4
)
2
Cu
3
(μ
3
-Cl)
2
(μ-pz)
3
Cl
3
(
1
), drop-casted on the CPW. Complex
1
had been previously studied by conventional X-band EPR spectroscopy (
Chem. - Eur. J.
, 2020,
26
, 12769-1784), and comparison with the past studies validated the functionality of the spectrometer and confirmed the stability of the sample upon deposition. Moreover, our results highlighted the importance of surface effects and of the orientation of the microwave magnetic component
B
1
on the lineshapes of the recorded spectra.
A new broadband EPR spectrometer capable of measuring in frequency- and field-sweep modes is described and its functionality is demonstrated on a ferromagnetic Cu
3
II
triangle demonstrating a moderate zero-field splitting of its quartet ground state.
Amyloid plaques, consisting of deposited beta-amyloid (Aβ), are a neuropathological hallmark of Alzheimer's Disease (AD). Cerebral vessels play a major role in AD, as Aβ is cleared from the brain by ...pathways involving the cerebrovasculature, most AD patients have cerebrovascular amyloid (cerebral amyloid angiopathy (CAA), and cardiovascular risk factors increase dementia risk. Here we present a notable advance in vascular tissue engineering by generating the first functional 3-dimensioinal model of CAA in bioengineered human vessels. We show that lipoproteins including brain (apoE) and circulating (high-density lipoprotein, HDL) synergize to facilitate Aβ transport across bioengineered human cerebral vessels. These lipoproteins facilitate Aβ42 transport more efficiently than Aβ40, consistent with Aβ40 being the primary species that accumulates in CAA. Moreover, apoE4 is less effective than apoE2 in promoting Aβ transport, also consistent with the well-established role of apoE4 in Aβ deposition in AD.
A two-step “grafting from” method has been successfully carried out, which is based on the electrografting of polyacrylate chains containing an initiator for the atom transfer radical polymerization ...(ATRP) of 2-(tert-butylamino)ethyl methacrylate (TBAEMA) or copolymerization of TBAEMA with either monomethyl ether of poly(ethylene oxide) methacrylate (PEOMA) or acrylic acid (AA) or styrene. The chemisorption of this type of polymer brushes onto stainless steel surfaces has potential in orthopaedic surgery. These films have been characterized by ATR-FTIR, Raman spectroscopy, atomic force microscopy (AFM), and measurement of contact angles of water. The polymer formed in solution by ATRP and that one detached on purpose from the surface have been analyzed by size exclusion chromathography (SEC) and 1H NMR spectroscopy. The strong adherence of the films onto stainless steel has been assessed by peeling tests. AFM analysis has shown that addition of hydrophilic comonomers to the grafted chains decreases the surface roughness. According to dynamic quartz crystal microbalance experiments, proteins (e.g., fibrinogen) are more effectively repelled whenever copolymer brushes contain neutral hydrophilic (PEOMA) co-units rather than negatively charged groups (PAA salt). Moreover, a 2- to 3-fold decrease in the fibrinogen adsorption is observed when TBAEMA is copolymerized with either PEOMA or AA rather than homopolymerized or copolymerized with styrene. Compared to the bare stainless steel surface, brushes of polyTBAEMA, poly(TBAEMA-co-PEOMA) and poly(TBAEMA-co-AA) decrease the bacteria adhesion by 3 to 4 orders of magnitude as revealed by Gram-positive bacteria S. aureus adhesion tests.
Endothelial cells are important constituents of blood vessels and play a critical role in vascular homeostasis. They do not only control the exchanges between the blood and the surrounding tissues, ...but are also essential in regulating blood flow, modulating immune-cell trafficking and controlling vascular growth and repair. Endothelial dysfunction leads to cardiovascular diseases and is characterized by deficiency in secretion of vasodilator molecules, elevated reactive oxygen species (ROS), expression of adhesion molecules and excretion of proinflammatory cytokines. The sex hormones, estrogens, androgens and progestogens, regulate endothelial functions. Because cardiovascular disease risk increases after menopause, it is believed that female hormones, estrogens and progestogens promote endothelial cell health and function whereas androgens, the male hormones, might be detrimental. However, as illustrated in the present review, the picture might not be that simple. In addition, sex influences endothelial cell physiology independently of sex hormones but at genetic level.
Display omitted
•Endothelial cells are major regulators of vascular physiology.•Endothelial cells are different in males and females.•Sex hormones regulate endothelial health and functions.•Sex regulates endothelial cell physiology. independently of sex hormones.
Many organisms coordinate cell growth and division through size control mechanisms: cells must reach a critical size to trigger a cell cycle event. Bacterial division is often assumed to be ...controlled in this way, but experimental evidence to support this assumption is still lacking. Theoretical arguments show that size control is required to maintain size homeostasis in the case of exponential growth of individual cells. Nevertheless, if the growth law deviates slightly from exponential for very small cells, homeostasis can be maintained with a simple 'timer' triggering division. Therefore, deciding whether division control in bacteria relies on a 'timer' or 'sizer' mechanism requires quantitative comparisons between models and data.
The timer and sizer hypotheses find a natural expression in models based on partial differential equations. Here we test these models with recent data on single-cell growth of Escherichia coli. We demonstrate that a size-independent timer mechanism for division control, though theoretically possible, is quantitatively incompatible with the data and extremely sensitive to slight variations in the growth law. In contrast, a sizer model is robust and fits the data well. In addition, we tested the effect of variability in individual growth rates and noise in septum positioning and found that size control is robust to this phenotypic noise.
Confrontations between cell cycle models and data usually suffer from a lack of high-quality data and suitable statistical estimation techniques. Here we overcome these limitations by using high precision measurements of tens of thousands of single bacterial cells combined with recent statistical inference methods to estimate the division rate within the models. We therefore provide the first precise quantitative assessment of different cell cycle models.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Spectral broadenings due to Dzyaloshinskii-Moriya interactions (DMI) were assessed with respect to the decoherence they induce through increased spin-spin interactions, as the role of DMI in ...developing magnetoelectric spin-chirality qubits is gaining recognition. The structurally related spin triangles Fe
3
O(PhCOO)
6
(py)
3
ClO
4
·py (
Fe
3
) and Cr
3
O(PhCOO)
6
(py)
3
ClO
4
·0.5py (
Cr
3
) were studied as frozen py-
d
5
solutions with various pulsed Electron Paramagnetic Resonance (EPR) spectroscopy experiments, and under identical experimental conditions. Field-swept Hahn echo experiments revealed a match with continuous-wave (CW) spectra, while variable-temperature saturation/inversion recovery and Hahn echo decay experiments were used to extract the thermal evolutions of the spin-lattice relaxation and phase-memory times (
T
1
and
T
m
, respectively). Nutation experiments revealed Rabi oscillations demonstrating that the spins of the complexes could be coherently manipulated. Careful comparisons of
T
m
times confirmed hyperfine interactions with the magnetic nuclei of the metal ions as an intrinsic source of decoherence. Comparisons of Rabi damping times revealed that DMI-induced spectral broadenings play a discernible but moderate role as an extrinsic source of decoherence for the nutation experiments and that they are not particularly detrimental to spin manipulations.
Two related iron(
iii
) and chromium(
iii
) spin-triangle molecular qubits show coherent driving of their spins, and decoherence that is not significantly affected by Dzyaloshikskii-Moriya spectral broadenings.
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