The MreA Metal-Binding Sites C40, H65, and C69 Play a Critical Role in the Metal Tolerance of Pseudomonas putida KT2440.

Metal-binding proteins occur in the cytosol of most eubacteria. The hypothetical metal responsive protein MreA (PP-2969 gene; NreA) seems responsible for zinc, chromium, cadmium accumulation, and metal ion homeostasis. However, there is a lack of definitive evidence regarding the specific metal-binding sites of MreA protein. The present study aimed to identify putative metal-binding regions for MreA. In silico analysis revealed that amino acids C40, H65, and C69 (CHC region) seem critical for metal-protein interactions. We created site-directed mutants (SDM's) of MreA for interacted amino acids to validate in silico results. The differential scanning fluorimetry (DSF) and atomic absorption spectroscopy (AAS) showed that SDM strains of MreA protein curtailed metal accumulation compared to the wild types indicating C40, H65, and C69 amino acids are critical for metal binding. Thus, we report potential implications for MreA-bioengineered strains of Pseudomonas putida KT2440 for metal ion homeostasis by alleviating metal toxicity in the biological environment.

Configurational diffusion transport of water and oil in dual continuum shales.

Understanding fluid flow in shale rocks is critical for the recovery of unconventional energy resources. Despite the extensive research conducted on water and oil flow in shales, significant uncertainties and discrepancies remain in reported experimental data. The most noted being that while oil spreads more than water on shale surfaces in an inviscid medium, its uptake by shale pores is much less than water during capillary flow. This leads to misjudgement of wettability and the underlying physical phenomena. In this study, therefore, we performed a combined experimental and digital rock investigation on an organic-rich shale including contact angle and spontaneous imbibition, X-ray and neutron computed tomography, and small angle X-ray scattering tests to study the potential physical processes. We also used non-equilibrium thermodynamics to theoretically derive constitutive equations to support our experimental observations. The results of this study indicate that the pre-existing fractures (first continuum) imbibe more oil than water consistent with contact angle measurements. The overall imbibition is, however, higher for water than oil due to greater water diffusion into the shale matrix (second continuum). It is shown that more water uptake into shale is controlled by pore size and accessibility in addition to capillary or osmotic forces i.e. configurational diffusion of water versus oil molecules. While the inorganic pores seem more oil-wet in an inviscid medium, they easily allow passage of water molecules compared to oil due to the incredibly small size of water molecules that can pass through such micro-pores. Contrarily, these strongly oil-wet pores possessing strong capillarity are restricted to imbibe oil simply due to its large molecular size and physical inaccessibility to the micro-pores. These results provide new insights into the previously unexplained discrepancy regarding water and oil uptake capacity of shales.

A Critical Role for ISGylation, Ubiquitination and, SUMOylation in Brain Damage: Implications for Neuroprotection.

Post-translational modification (PTMs) of proteins by ubiquitin and ubiquitin-like modifiers such as interferon-stimulated gene 15 (ISG15) and small ubiquitin-related modifier (SUMO) play a critical role in the regulation of brain pathophysiology. Protein ISGylation is a covalent attachment of ISG15 to its target proteins, which is a unique PTM among other ubiquitin-like modifiers. Although, ISG15 shares sequence homology to ubiquitin, yet the functional significance of protein ISGylation is distinct from ubiquitination and SUMOylation. Further, ISG15 highly conserved among vertebrate species, unlike the other ubiquitin-like modifiers. ISGylation modulates various intracellular mechanisms such as Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway, autophagy, DNA repair, etc., indicating its biological significance. ISGylation emerged as one of the important mechanisms in the regulation of various neurological disorders including stroke, traumatic brain injury (TBI), basal ganglia calcification, and ataxia-telangiectasia. It appears that protein ISGylation is an endogenous neuroprotective mechanism. This review discusses the role of ISGylation in various brain pathologies with a particular emphasis on cerebral ischemia/stroke and on structural similarities between ISG15 and ubiquitin. Further, recent advancements on the role of ubiquitination and SUMOylation with relevance to ISGylation will also be discussed. The overall goal is to provide better insights on the mechanistic link between ISGylation and other ubiquitin-like modifiers, which may be helpful to establish novel therapeutic strategies for neuroprotection.