Escape of a Small Molecule from Inside T4 Lysozyme by Multiple Pathways

The T4 lysozyme L99A mutant is often used as a model system to study small-molecule binding to proteins, but pathways for ligand entry and exit from the buried binding site and the associated protein conformational changes have not been fully resolved. Here, molecular dynamics simulations were employed to model benzene exit from its binding cavity using the weighted ensemble (WE) approach to enhance sampling of low-probability unbinding trajectories. Independent WE simulations revealed four pathways for benzene exit, which correspond to transient tunnels spontaneously formed in previous simulations of apo T4 lysozyme. Thus, benzene unbinding occurs through multiple pathways partially created by intrinsic protein structural fluctuations. Motions of several α-helices and side chains were involved in ligand escape from metastable microstates. WE simulations also provided preliminary estimates of rate constants for each exit pathway. These results complement previous works and provide a semiquantitative characterization of pathway heterogeneity for binding of small molecules to proteins.     

The cytoplasmic domain of MT1-MMP is dispensable for migration augmentation but necessary to mediate viability of MCF-7 breast cancer cells

Membrane-type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease that regulates ECM degradation, proMMP-2 activation, and varied cellular processes including migration and viability. MT1-MMP is believed to be a central mediator of tumourigenesis whose role is dictated by its functionally distinct protein domains. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain, exemplifying diverse regulatory functions. To further our understanding of the multifunctional contributions of MT1-MMP to cellular processes, we overexpressed cytoplasmic domain altered constructs in MCF-7 breast cancer cells and analyzed migration and viability in 2D culture conditions, morphology in 3D Matrigel culture, and tumorigenic ability in vivo. We found that the cytoplasmic domain was not needed for MT1-MMP mediated migration promotion, but was necessary to maintain viability during serum depravation in 2D culture. Similarly, during 3D Matrigel culture the cytoplasmic domain of MT1-MMP was not needed to initiate a protrusive phenotype, but was necessary to prevent colony blebbing when cells were serum deprived. We also tested in vivo tumorigenic potential to show that cells expressing cytoplasmic domain altered constructs demonstrated a reduced ability to vascularize tumours. These results suggest that the cytoplasmic domain regulates MT1-MMP function in a manner required for cell survival, but is dispensable for cell migration.     

The Nature of Urban Soils and Their Role in Ecological Restoration in Cities

Current and predicted trends indicate that an increasing proportion of the world’s population is living in urban and suburban places. The nature of the urban environment becomes an important factor if we are concerned with the restoration and preservation of biodiversity and ecosystems in and around cities. This article highlights the varied impacts of cities on soils and their implications for restoration planning and expectations of restoration “success.” Urban soils exist in different historical and formational trajectories than their local nonurbanized counterparts due to direct anthropogenic disturbance and indirect environmental impacts from urbanization. Therefore, urban soils often exhibit altered physical, chemical, and biological characteristics in comparison to local nonurbanized soils. Several unique features of urban soils and urban ecosystems pose particular issues for ecological restoration or the improvement of degraded soil conditions in cities. The creation of novel soil types, conditions that promote invasion by non‐natives, the strong influence of past land use on soil properties, and the presence of strong interactions and alternative stable states set up unique difficulties for the restoration of urban soils. Soils in urban restorations are a medium that can be deliberately manipulated to improve site conditions or in the monitoring of soil conditions as indices of ecosystem status. Including an explicit role for strong manipulations of soils in urban ecosystems changes how we approach baselines, management, and reference conditions in urban ecological restoration. With an understanding of urban soil ecological knowledge, we can guide aspects of urban ecological restoration toward successful outcomes.     

Hypocitraturia: Pathophysiology and Medical Management

Low urinary citrate excretion is a known risk factor for the development of kidney stones. Citrate inhibits stone formation by complexing with calcium in the urine, inhibiting spontaneous nucleation, and preventing growth and agglomeration of crystals. Hypocitraturia is a common metabolic abnormality found in 20% to 60% of stone formers. It is most commonly idiopathic in origin but may be caused by distal renal tubular acidosis, hypokalemia, bowel dysfunction, and a high-protein, low-alkali diet. Genetic factors, medications, and other comorbid disorders also play a role. Hypocitraturia should be managed through a combination of dietary modifications, oral alkali, and possibly lemonade or other citrus juice-based therapy. This review concerns the pathophysiology of hypocitraturia and the management of stone formers afflicted with this abnormality.Key words: Hypocitraturia, Citrate metabolism, Citrate pathophysiology, Hypocitraturia etiologies, Medical management of stone disease, Alkali citrate, Potassium citrate, Citrus juice, LemonadeLow urinary citrate excretion is a known risk factor for the development of kidney stones.¹ Hypocitraturia, generally defined as urinary citrate excretion less than 320 mg (1.67 mmol) per day for adults,² is a common metabolic abnormality in stone formers, occurring in 20% to 60%.^1,3–6 Citrate is a known inhibitor of stone formation, working through a variety of mechanisms. In the renal tubule citrate complexes with calcium, increasing its solubility and reducing the concentration of free calcium in the urine. This calcium-citrate complex limits calcium supersaturation and prevents nucleation of both calcium oxalate and calcium phosphate, at least partly through interactions with Tamm-Horsfall protein.^7,8 Additionally, citrate prevents crystal agglomeration and growth through its ability to bind to the crystal’s surface and may also prevent adhesion of calcium oxalate to renal epithelial cells.^9–11 Hypocitraturia may be corrected with dietary modifications and the administration of citrate preparations or other forms of alkali therapy. Citrate excretion is linked to urinary pH and thus may influence the generation of a number of types of stones. Herein we review the pathophysiology of hypocitraturia and the management of stone formers with this abnormality.