The CD4+AT2R+ T cell subpopulation improves post‐infarction remodelling and restores cardiac function

Myocardial infarction (MI) is a major condition causing heart failure (HF). After MI, the renin angiotensin system (RAS) and its signalling octapeptide angiotensin II (Ang II) interferes with cardiac injury/repair via the AT1 and AT2 receptors (AT1R, AT2R). Our study aimed at deciphering the mechanisms underlying the link between RAS and cellular components of the immune response relying on a rodent model of HF as well as HF patients. Flow cytometric analyses showed an increase in the expression of CD4⁺ AT2R⁺ cells in the rat heart and spleen post‐infarction, but a reduction in the peripheral blood. The latter was also observed in HF patients. The frequency of rat CD4⁺ AT2R⁺ T cells in circulating blood, post‐infarcted heart and spleen represented 3.8 ± 0.4%, 23.2 ± 2.7% and 22.6 ± 2.6% of the CD4⁺ cells. CD4⁺ AT2R⁺ T cells within blood CD4⁺ T cells were reduced from 2.6 ± 0.2% in healthy controls to 1.7 ± 0.4% in patients. Moreover, we characterized CD4⁺ AT2R⁺ T cells which expressed regulatory FoxP3, secreted interleukin‐10 and other inflammatory‐related cytokines. Furthermore, intramyocardial injection of MI‐induced splenic CD4⁺ AT2R⁺ T cells into recipient rats with MI led to reduced infarct size and improved cardiac performance. We defined CD4⁺ AT2R⁺ cells as a T cell subset improving heart function post‐MI corresponding with reduced infarction size in a rat MI‐model. Our results indicate CD4⁺ AT2R⁺ cells as a promising population for regenerative therapy, via myocardial transplantation, pharmacological AT2R activation or a combination thereof.     

Hydrophobic potential of mean force as a solvation function for protein structure prediction

We have developed a solvation function that combines a Generalized Born model for polarization of protein charge by the high dielectric solvent, with a hydrophobic potential of mean force (HPMF) as a model for hydrophobic interaction, to aid in the discrimination of native structures from other misfolded states in protein structure prediction. We find that our energy function outperforms other reported scoring functions in terms of correct native ranking for 91% of proteins and low Z scores for a variety of decoy sets, including the challenging Rosetta decoys. This work shows that the stabilizing effect of hydrophobic exposure to aqueous solvent that defines the HPMF hydration physics is an apparent improvement over solvent-accessible surface area models that penalize hydrophobic exposure. Decoys generated by thermal sampling around the native-state basin reveal a potentially important role for side-chain entropy in the future development of even more accurate free energy surfaces.     

Acetyltransferase p300 inhibitor reverses hypertension‐induced cardiac fibrosis

Epigenetic dysregulation plays a crucial role in cardiovascular diseases. Previously, we reported that acetyltransferase p300 (ATp300) inhibitor L002 prevents hypertension‐induced cardiac hypertrophy and fibrosis in a murine model. In this short communication, we show that treatment of hypertensive mice with ATp300‐specific small molecule inhibitor L002 or C646 reverses hypertension‐induced left ventricular hypertrophy, cardiac fibrosis and diastolic dysfunction, without reducing elevated blood pressures. Biochemically, treatment with L002 and C646 also reverse hypertension‐induced histone acetylation and myofibroblast differentiation in murine ventricles. Our results confirm and extend the role of ATp300, a major epigenetic regulator, in the pathobiology of cardiac hypertrophy and fibrosis. Most importantly, we identify the efficacies of ATp300 inhibitors C646 and L002 in reversing hypertension‐induced cardiac hypertrophy and fibrosis, and discover new anti‐hypertrophic and anti‐fibrotic candidates.     

In vitro measurement of cell death with the annexin A5 affinity assay

One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.     

Direct visualization of the interaction between pilin and exopolysaccharides of Myxococcus xanthus with eGFP-fused PilA protein

Type IV pili (TFP) and exopolysaccharides (EPS) are important components for social behaviors in Myxococcus xanthus, including gliding motility and fruiting body formation. Although specific interactions between TFP and EPS have been proposed, there have as yet been no direct observations of these interactions under native conditions. In this study, we found that a truncated PilA protein (PilACt) containing only the C-terminal domain (amino acids 32-208) is sufficient for EPS binding in vitro. Furthermore, an enhanced green fluorescent protein (eGFP) and PilACt fusion protein were constructed and used to label the native EPS in M. xanthus. Under confocal laser scanning microscope, the eGFP-PilACt-bound fruiting bodies, trail structures and biofilms exhibited similar patterns as the wheat germ agglutinin lectin-labeled EPS structures. This study showed that eGFP-PilACt fusion protein was able efficiently to label the EPS of M. xanthus, providing evidence for the first time of the direct interaction between the PilA protein and EPS under native conditions.     

Interleukin 6, soluble tumor necrosis factor receptor I and red blood cell distribution width as biological markers of functional dependence in an elderly population: a translational approach

In the present investigation we have analyzed the association between functional dependence and inflammatory biomarkers using the Barthel Index (BI) and the Katz Index (KI). This analysis may contribute to translational medicine by incorporating the clinical and laboratory data to better understand the relationship between chronic inflammation and functional dependence in the elderly population. The ultimate goal of this study was to identify possible useful biomarkers of functional dependence in the elderly. Participants in this study consisted of 120 older subjects (90 women and 30 men; range 68-105 years) who were selected from the Santa Teresa nursing home (Oviedo, Spain). We studied functional status using the following tools to diagnose the functional dependence by clinicians: BI and KI for activities of daily living. We analyzed morbidity, sociodemographic characteristics and a panel of inflammatory and inflammatory-related markers. In linear regression models adjusted by age, sex, anti-inflammatory drug use and morbid conditions high levels of interleukin 6 (IL-6) and soluble TNF receptor-I (sTNF-RI) were associated with functional dependence as measured using BI and KI. Elevated levels of red blood cell distribution width (RDW) were also associated with functional dependence measured using the KI after adjusting for the same potential confounders. The current results suggest that high IL-6, sTNF-RI and RDW levels are associated with the functional dependence in the elderly population. The results are consistent with the presumed underlying biological mechanism, in which the up-regulation of inflammatory mediators is associated with functional dependence in elderly subjects.     

Protofibril assemblies of the arctic, Dutch, and Flemish mutants of the Alzheimer's Abeta1-40 peptide

Using a coarse-grained model of the Aβ peptide, we analyze the Arctic (E22G), Dutch (E22Q), and Flemish (A21G) familial Alzheimer's disease (FAD) mutants for any changes in the stability of amyloid assemblies with respect to the wild-type (WT) sequence. Based on a structural reference state of two protofilaments aligned to create the “agitated” protofibril as determined by solid-state NMR, we determine free energy trends for Aβ assemblies for the WT and FAD familial sequences. We find that the structural characteristics and oligomer size of the critical nucleus vary dramatically among the hereditary mutants. The Arctic mutant's disorder in the turn region introduces new stabilizing interactions that better align the two protofilaments, yielding a well-defined protofibril axis at relatively small oligomer sizes with respect to WT. By contrast, the critical nucleus for the Flemish mutant is beyond the 20 chains characterized in this study, thereby showing a strong shift in the equilibrium toward monomers with respect to larger protofibril assemblies. The Dutch mutant forms more ordered protofilaments than WT, but exhibits greater disorder in protofibril structure that includes an alternative polymorph of the WT fibril. An important conclusion of this work is that the Dutch mutant does not support the agitated protofibril assembly. We discuss the implications of the structural ensembles and free energy profiles for the FAD mutants in regards to interpretation of the kinetics of fibril assembly using chromatography and dye-binding experiments.     

Plant root research: the past, the present and the future

This special issue is dedicated to root biologists past and present who have been exploring all aspects of root structure and function with an extensive publication record going over 100 years. The content of the Special Issue on Root Biology covers a wide scale of contributions, spanning interactions of roots with microorganisms in the rhizosphere, the anatomy of root cells and tissues, the subcellular components of root cells, and aspects of metal accumulation and stresses on root function and structure. We have organized the papers into three topic categories: (1) root ecology, interactions with microbes, root architecture and the rhizosphere; (2) experimental root biology, root structure and physiology; and (3) applications of new technology to study root biology. Finally, we will speculate on root research for the future.     

VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy

Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin‐directed AAA‐ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo‐Lysosomal Damage Response. Together, they act downstream of K63‐linked ubiquitination and p62 recruitment, and selectively remove K48‐linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation. Lysosomal clearance is also compromised in MEFs harboring a p97 mutation that causes inclusion body myopathy and neurodegeneration, and damaged lysosomes accumulate in affected patient tissue carrying the mutation. Moreover, we show that p97 helps clear late endosomes/lysosomes ruptured by endocytosed tau fibrils. Thus, our data reveal an important mechanism of how p97 maintains lysosomal homeostasis, and implicate the pathway as a modulator of degenerative diseases.