Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

Plasma membrane Ca²⁺-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca²⁺]_i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca²⁺]_i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.     

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.     

A prospective observational cohort study in primary care practices to identify factors associated with treatment failure in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> skin and soft tissue infections

Background

The incidence of outpatient visits for skin and soft tissue infections (SSTIs) has substantially increased over the last decade. The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has made the management of S. aureus SSTIs complex and challenging. The objective of this study was to identify risk factors contributing to treatment failures associated with community-associated S. aureus skin and soft tissue infections SSTIs.

Methods

This was a prospective, observational study among 14 primary care clinics within the South Texas Ambulatory Research Network. The primary outcome was treatment failure within 90 days of the initial visit. Univariate associations between the explanatory variables and treatment failure were examined. A generalized linear mixed-effect model was developed to identify independent risk factors associated with treatment failure.

Results

Overall, 21% (22/106) patients with S. aureus SSTIs experienced treatment failure. The occurrence of treatment failure was similar among patients with methicillin-resistant S. aureus and those with methicillin-susceptible S. aureus SSTIs (19 vs. 24%; p = 0.70). Independent predictors of treatment failure among cases with S. aureus SSTIs was a duration of infection of ≥7 days prior to initial visit [aOR, 6.02 (95% CI 1.74–19.61)] and a lesion diameter size ≥5 cm [5.25 (1.58–17.20)].

Conclusions

Predictors for treatment failure included a duration of infection for ≥7 days prior to the initial visit and a wound diameter of ≥5 cm. A heightened awareness of these risk factors could help direct targeted interventions in high-risk populations.

     

Systematic Dissection of the Metabolic-Apoptotic Interface in AML Reveals Heme Biosynthesis to Be a Regulator of Drug Sensitivity

1. Download : Download high-res image (240KB)
2. Download : Download full-size image

     

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Extrapolation to the full‐length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine‐phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated‐tyrosine‐mimic Y492E‐FL‐Grb7 protein (Y492E‐FL‐Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild‐type FL Grb7(WT‐FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization K_d of WT‐FL‐Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT‐FL‐Grb7 in comparison the tyrosine‐phosphorylation mimic Y492E‐FL‐Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.     

A Two-Antibody Pan-Ebolavirus Cocktail Confers Broad Therapeutic Protection in Ferrets and Nonhuman Primates

1. Download high-res image (261KB)
2. Download full-size image

     

Invasive Phragmites australis management outcomes and native plant recovery are context dependent

The outcomes of invasive plant removal efforts are influenced by management decisions, but are also contingent on the uncontrolled spatial and temporal context of management areas. Phragmites australis is an aggressive invader that is intensively managed in wetlands across North America. Treatment options have been understudied, and the ecological contingencies of management outcomes are poorly understood. We implemented a 5‐year, multi‐site experiment to evaluate six Phragmites management treatments that varied timing (summer or fall) and types of herbicide (glyphosate or imazapyr) along with mowing, plus a nonherbicide solarization treatment. We evaluated treatments for their influence on Phragmites and native plant cover and Phragmites inflorescence production. We assessed plant community trajectories and outcomes in the context of environmental factors. The summer mow, fall glyphosate spray treatment resulted in low Phragmites cover, high inflorescence reduction, and provided the best conditions for native plant recruitment. However, returning plant communities did not resemble reference sites, which were dominated by ecologically important perennial graminoids. Native plant recovery following initial Phragmites treatments was likely limited by the dense litter that resulted from mowing. After 5 years, Phragmites mortality and native plant recovery were highly variable across sites as driven by hydrology. Plots with higher soil moisture had greater reduction in Phragmites cover and more robust recruitment of natives compared with low moisture plots. This moisture effect may limit management options in semiarid regions vulnerable to water scarcity. We demonstrate the importance of replicating invasive species management experiments across sites so the contingencies of successes and failures can be better understood.     

Conformation-specific binding of alpha-synuclein to novel protein partners detected by phage display and NMR spectroscopy

Alpha-synuclein (AS) is an intrinsically unstructured protein in aqueous solution but is capable of forming beta-sheet-rich fibrils that accumulate as intracytoplasmic inclusions in Parkinson disease and certain other neurological disorders. However, AS binding to phospholipid membranes leads to a distinct change in protein conformation, stabilizing an extended amphipathic alpha-helical domain reminiscent of the exchangeable apolipoproteins. To better understand the significance of this conformational change, we devised a novel bacteriophage display screen to identify protein binding partners of helical AS and have identified 20 proteins with roles in diverse cellular processes related to membrane trafficking, ion channel modulation, redox metabolism, and gene regulation. To verify that the screen identifies proteins with specificity for helical AS, we further characterized one of these candidates, endosulfine alpha (ENSA), a small cAMP-regulated phosphoprotein implicated in the regulation of insulin secretion but also expressed abundantly in the brain. We used solution NMR to probe the interaction between ENSA and AS on the surface of SDS micelles. Chemical shift perturbation mapping experiments indicate that ENSA interacts specifically with residues in the N-terminal helical domain of AS in the presence of SDS but not in aqueous buffer lacking SDS. The ENSA-related protein ARPP-19 (cAMP-regulated phosphoprotein 19) also displays specific interactions with helical AS. These results confirm that the helical N terminus of AS can mediate specific interactions with other proteins and suggest that membrane binding may regulate the physiological activity of AS in vivo.