Tick tock,tick tock: Mouse culture and tissue aging captured by an epigenetic clock

Aging is associated with dramatic changes to DNA methylation (DNAm), although the causes and consequences of such alterations are unknown. Our ability to experimentally uncover mechanisms of epigenetic aging will be greatly enhanced by our ability to study and manipulate these changes using in vitro models. However, it remains unclear whether the changes elicited by cells in culture can serve as a model of what is observed in aging tissues in vivo. To test this, we serially passaged mouse embryonic fibroblasts (MEFs) and assessed changes in DNAm at each time point via reduced representation bisulfite sequencing. By developing a measure that tracked cellular aging in vitro, we tested whether it tracked physiological aging in various mouse tissues and whether anti‐aging interventions modulate this measure. Our measure, termed CultureAGE, was shown to strongly increase with age when examined in multiple tissues (liver, lung, kidney, blood, and adipose). As a control, we confirmed that the measure was not a marker of cellular senescence, suggesting that it reflects a distinct yet progressive cellular aging phenomena that can be induced in vitro. Furthermore, we demonstrated slower epigenetic aging in animals undergoing caloric restriction and a resetting of our measure in lung and kidney fibroblasts when re‐programmed to iPSCs. Enrichment and clustering analysis implicated EED and Polycomb group (PcG) factors as potentially important chromatin regulators in translational culture aging phenotypes. Overall, this study supports the concept that physiologically relevant aging changes can be induced in vitro and used to uncover mechanistic insights into epigenetic aging.     

Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues

Background

Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples.

Methodology/Principal Findings

We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport.

Conclusions

Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach.     

Invasive and adherent bacterial pathogens co-Opt host clathrin for infection

Infection by the bacterium Listeria monocytogenes depends on host cell clathrin. To determine whether this requirement is widespread, we analyzed infection models using diverse bacteria. We demonstrated that bacteria that enter cells following binding to cellular receptors (termed "zippering" bacteria) invade in a clathrin-dependent manner. In contrast, bacteria that inject effector proteins into host cells in order to gain entry (termed "triggering" bacteria) invade in a clathrin-independent manner. Strikingly, enteropathogenic Escherichia coli (EPEC) required clathrin to form actin-rich pedestals in host cells beneath adhering bacteria, even though this pathogen remains extracellular. Furthermore, clathrin accumulation preceded the actin rearrangements necessary for Listeria entry. These data provide evidence for a clathrin-based entry pathway allowing internalization of large objects (bacteria and ligand-coated beads) and used by "zippering" bacteria as part of a general mechanism to invade host mammalian cells. We also revealed a nonendocytic role for clathrin required for extracellular EPEC infections.     

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating succinyl hydroxamate and iminodiacetic acid hydroxamate moieties

A series of succinyl hydroxamates/bishydroxamates as well as a new structural type of matrix metalloproteinase (MMP)/bacterial protease (BP) inhibitors, incorporating iminodiacetic (IDA) hydroxamate/bishydroxamate moieties, has been synthesized and tested for interaction with four vertebrate proteases, MMP-1, MMP-2, MMP-8 and MMP-9, and a BP, the collagenase isolated from Clostridium histolyticum (ChC). The new derivatives generally showed inhibition constants in the range of 8-62 nM against the five proteases mentioned above.     

Extensive activation,tissue trafficking,turnover and functional impairment of NK cells in COVID-19 patients at disease onset associates with subsequent disease severity

The SARS-CoV-2 infection causes severe respiratory involvement (COVID-19) in 5–20% of patients through initial immune derangement, followed by intense cytokine production and vascular leakage. Evidence of immune involvement point to the participation of T, B, and NK cells in the lack of control of virus replication leading to COVID-19. NK cells contribute to early phases of virus control and to the regulation of adaptive responses. The precise mechanism of NK cell dysregulation is poorly understood, with little information on tissue margination or turnover. We investigated these aspects by multiparameter flow cytometry in a cohort of 28 patients hospitalized with early COVID-19.Relevant decreases in CD56^brightCD16+/- NK subsets were detected, with a shift of circulating NK cells toward more mature CD56^dimCD16+KIR+NKG2A+ and “memory” KIR+CD57+CD85j+ cells with increased inhibitory NKG2A and KIR molecules. Impaired cytotoxicity and IFN-γ production were associated with conserved expression of natural cytotoxicity receptors and perforin. Moreover, intense NK cell activation with increased HLA-DR and CD69 expression was associated with the circulation of CD69+CD103+ CXCR6+ tissue-resident NK cells and of CD34+DNAM-1^brightCXCR4+ inflammatory precursors to mature functional NK cells. Severe disease trajectories were directly associated with the proportion of CD34+DNAM-1^brightCXCR4+ precursors and inversely associated with the proportion of NKG2D+ and of CD103+ NK cells.Intense NK cell activation and trafficking to and from tissues occurs early in COVID-19, and is associated with subsequent disease progression, providing an insight into the mechanism of clinical deterioration. Strategies to positively manipulate tissue-resident NK cell responses may provide advantages to future therapeutic and vaccine approaches.     

Vein recovery from embolism occurs under negative pressure in leaves of sunflower (Helianthus annuus)

Leaf veins undergo cavitation at water potentials (Psi(leaf)) commonly experienced by field-growing plants. Theoretically, embolism reversal should not be possible until xylem pressures rise by several kilopascals of atmospheric pressure, but recent evidence suggests that embolized conduits can be refilled even when surrounded by others at substantial tension (novel refilling). The present study reports 'novel refilling' occurring in leaf veins of sunflower (Helianthus annuus L.) while at Psi(leaf) = -0.33 MPa. Sixty per cent loss of vein hydraulic conductance (K(vein)) was recorded at Psi(leaf) < -0.65 MPa, while stem hydraulic conductance (K(stem)) was unaffected even at Psi(leaf) = -1.1 MPa. Loss of K(vein) was accompanied by stomatal closure. Water-stressed plants (Psi(leaf) = -1.1 MPa) were rehydrated overnight to different target water potentials achieved by using PEG at different concentrations as irrigation medium. K(vein) recovered by 50% at Psi(leaf) = -0.47 MPa and vein refilling was complete at Psi(leaf) = -0.33 MPa, i.e. well below the theoretical limit for conduit refilling (-0.05 MPa as calculated for sunflower minor veins). Mercurials supplied to detached leaves had no effect on the refilling process. Upon rehydration, recovery of K(vein) was not paralleled by recovery of whole-plant hydraulic conductance or leaf conductance to water vapour (g(L)), as a likely consequence of hydraulic failure of other components of the water pathway (root system or extravascular leaf compartments) and/or root-to-leaf chemical signalling. This is the first study providing experimental evidence for 'novel refilling' in a herbaceous dicot and highlighting the importance of this process in the leaf.     

Neuronal selectivity and local map structure in visual cortex

The organization of primary visual cortex (V1) into functional maps makes individual cells operate in a variety of contexts. For instance, some neurons lie in regions of fairly homogeneous orientation preference (iso-orientation domains), while others lie in regions with a variety of preferences (e.g., pinwheel centers). We asked whether this diversity in local map structure correlates with the degree of selectivity of spike responses. We used a combination of imaging and electrophysiology to reveal that neurons in regions of homogeneous orientation preference have much sharper tuning. Moreover, in both monkeys and cats, a common principle links the structure of the orientation map, on the spatial scale of dendritic integration, to the degree of selectivity of individual cells. We conclude that neural computation is not invariant across the cortical surface. This finding must factor into future theories of receptive field wiring and map development.     

Computer simulation of uranyl uptake by the rough lipopolysaccharide membrane of Pseudomonas aeruginosa

Heavy metal environmental contaminants cannot be destroyed but require containment, preferably in concentrated form, in a solid or immobile form for recycling or final disposal. Microorganisms are able to take up and deposit high levels of contaminant metals, including radioactive metals such as uranium and plutonium, into their cell wall. Consequently, these microbial systems are of great interest as the basis for potential environmental bioremediation technologies. The outer membranes of Gram-negative microbes are highly nonsymmetric and exhibit a significant electrostatic potential gradient across the membrane. This gradient has a significant effect on the uptake and transport of charged and dipolar compounds. However, the effectiveness of microbial systems for environmental remediation will depend strongly on specific properties that determine the uptake of targeted contaminants by a particular cell wall. To aid in the design of microbial remediation technologies, knowledge of the factors that determine the affinity of a particular bacterial outer membrane for the most common ionic species found in contaminated soils and groundwater is of great importance. Using our previously developed model for the lipopolysaccharide (LPS) membrane of Pseudomonas aeruginosa, this work presents the potentials of mean force as the estimate of the free energy profile for uptake of sodium, calcium, chloride, uranyl ions, and a water molecule by the bacterial LPS membrane. A compatible classical parameter set for uranyl has been developed and validated. Results show that the uptake of uranyl is energetically a favorable process relative to the other ions studied. At neutral pH, this nuclide is shown to be retained on the surface of the LPS membrane through chelation with the carboxyl and hydroxyl groups located in the outer core.     

Mechanism of inhibition of wt-dihydrofolate reductase from E. coli by tea epigallocatechin-gallate

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in major biological process, including DNA synthesis and cancer inhibition, and its modulation is the object of extensive structural, kinetic, and pharmacological studies. In particular, earlier studies showed that green tea catechins are powerful inhibitors of bovine liver and chicken liver DHFR. In this article, we report the results of inhibition kinetics for the enzyme from another source (DHFR from E. coli) exerted by (-)-epigallocatechingallate (EGCG). Using different analytical techniques, we reported that EGCG acts as a bisubstrate inhibitor on the bacterial DHFR. Moreover, the combined approach of biosensor, kinetic, and molecular modelling analysis disclosed the ability of EGCG to bind to the enzyme both on substrate (DHF) and cofactor (NADPH) site. Collectively, our data have confirmed the selectivity of antifolate compounds with respect to the different source of enzyme (bacterial or mammalian DHFR) and the possible role of tea catechins as chemopreventive agents.     

Males with a Faster Courtship Display have More White Spots and Higher Pairing Success in the Diamond Firetail,Stagonopleura guttata

There is growing evidence that female mate choice could be based on a combination of multiple signals that often involve both ornamental colourful traits and behavioural displays. The Diamond Firetail is an Australian finch with a variable number of white spots on their black flank feathers. The number of white spots is a dimorphic characteristic: females have more spots than males, and males prefer females with many spots. Previously, we found assortative pairing for spot number despite the absence of experimental evidence for female preference for male spot number. Here, we test whether the male behavioural courtship display (bobbing while waving a grass stem) correlates with male spot number and pairing success. We also test whether male spot number predicts the outcome (winner or loser) of intrasexual competition over courtship materials (grass stem, perch, nest site). Males with many spots had higher pairing success, and male spot number correlated with the intensity of courtship display. In a multivariate statistical analysis, male courtship display was the stronger predictor of male pairing success. Finally, male spot number predicted the outcome of intrasexual interactions: males with many spots consistently won contests over grass stems, perches and nest sites. We suggest that intrasexual selection could favour male spot number, whereas courtship intensity appears to be under stronger intersexual selection.