In silico identification and characterization of small-molecule inhibitors specific to RhoG/Rac1 signaling pathway.

Rho family GTPases serve as molecular switches in numerous cellular processes, and their overexpression is involved in disease conditions. RhoG is one of the less explored Rho GTPases with significant sequential and structural homology with Rac1. Experimental mutations in RhoG (i.e., RhoGG12V and RhoGQ61L) are shown to dysregulate cell migration. Thus, targeting upstream activators of RhoG, such as guanine nucleotide exchange factors (GEFs), maybe an important strategy for inhibiting RhoG activation. In the current study, we have modelled the 3D structure of RhoG with greater accuracy as confirmed through PROCHECK, ProSA, and Verify3D. Our results indicate that 90.4% of residues are in the Ramachandran plots favoured region, with the Z-score of -6.46, and 87.96% of residues had an averaged 3D-1D score ≥0.2. Further, we have evaluated and binding dynamics of ten Rac1 inhibitors to investigate their potential to inhibit RhoG by targeting GEFs binding grooves. To this end, the binding energy of the docked complexes of the wild-type (WT) RhoG and its mutant proteins with inhibitor molecules was calculated using the MM/PBSA method. Our results from docking studies showed that macrolide1 binds efficiently with the GEF site of WT RhoG and the mutants mentioned above. However, an extensive analysis using MD simulations (200 ns) showed that the Rac1 based inhibitor, EHop-016, and NSC23766 might bind with greater affinity to GEF sites of mutants and WT RhoG. Thus, the results from the study indicate that Rac1 inhibitors have the potential for use as therapeutics in conditions involving dysregulation of RhoG.Communicated by Ramaswamy H. Sarma.

Functional and Therapeutic Relevance of Rho GTPases in Innate Immune Cell Migration and Function during Inflammation: An In Silico Perspective.

Systematic regulation of leukocyte migration to the site of infection is a vital step during immunological responses. Improper migration and localization of immune cells could be associated with disease pathology as seen in systemic inflammation. Rho GTPases act as molecular switches during inflammatory cell migration by cycling between Rho-GDP (inactive) to Rho-GTP (active) forms and play an essential role in the precise regulation of actin cytoskeletal dynamics as well as other immunological functions of leukocytes. Available reports suggest that the dysregulation of Rho GTPase signaling is associated with various inflammatory diseases ranging from mild to life-threatening conditions. Therefore, it is crucial to understand the step-by-step activation and inactivation of GTPases and the functioning of different Guanine Nucleotide Exchange Factors (GEFs) and GTPase-Activating Proteins (GAPs) that regulate the conversion of GDP to GTP and GTP to GDP exchange reactions, respectively. Here, we describe the molecular organization and activation of various domains of crucial elements associated with the activation of Rho GTPases using solved PDB structures. We will also present the latest evidence available on the relevance of Rho GTPases in the migration and function of innate immune cells during inflammation. This knowledge will help scientists design promising drug candidates against the Rho-GTPase-centric regulatory molecules regulating inflammatory cell migration.

Climate change: how it impacts the emergence, transmission, resistance and consequences of viral infections in animals and plants.

The ongoing COVID-19 pandemic has made us wonder what led to its occurrence and what can be done to avoid such events in the future. As we document, one changing circumstance that is resulting in the emergence and changing the expression of viral diseases in both plants and animals is climate change. Of note, the rapidly changing environment and weather conditions such as excessive flooding, droughts, and forest fires have raised concerns about the global ecosystem's security, sustainability, and balance. In this review, we discuss the main consequences of climate change and link these to how they impact the appearance of new viral pathogens, how they may facilitate transmission between usual and novel hosts, and how they may also affect the host's ability to manage the infection. We emphasize how changes in temperature and humidity and other events associated with climate change influence the reservoirs of viral infections, their transmission by insects and other intermediates, their survival outside the host as well the success of infection in plants and animals. We conclude that climate change has mainly detrimental consequences for the emergence, transmission, and outcome of viral infections and plead the case for halting and hopefully reversing this dangerous event.

Electrostatic Surface Potential of Macrophages Correlates with Their Functional Phenotype.

Macrophages exist in various functional phenotypes, which could be identified by specific surface molecules. Previous studies have shown that modulation of surface charges could alter the phagocytic function of macrophages. In this study, we show that activation of both human peripheral blood monocyte and THP-1-derived macrophages with lipopolysaccharide (LPS) or IL-1ß resulted in a significant decrease in the zeta potential compared to freshly isolated monocytes and unstimulated macrophages. Interestingly, interaction with bacteria significantly increased the zeta potential of such cells irrespective of activation conditions. Similarly, IFNγ-treated pro-inflammatory macrophages showed lesser negative zeta potential compared to untreated control. A moderate reduction was also seen in IL-4-treated anti-inflammatory subtype. Additionally, in an LPS-induced systemic inflammation model, bone marrow cells isolated after 2 h of LPS injection showed significant reduction in zeta potential compared to naïve cells. Furthermore, electrostatic potential measurement of surface proteins associated with pro-inflammatory and anti-inflammatory macrophages, using in silico modeling under physiological and protonation conditions, showed that the average electrostatic potential of pro-inflammatory type surface proteins was less negative than anti-inflammatory subtype. These data suggest that the expression of different protein molecules on macrophages under different environments may contribute to the zeta potential and that this quick and low-cost technique could be used in monitoring macrophage functional phenotypes.

Investigation of Extracellular Matrix Protein Expression Dynamics Using Murine Models of Systemic Inflammation.

Extracellular matrix (ECM) proteins form the structural support for migration of leukocytes and provide multiple signals to assist in their functions during inflammatory conditions. Presence of pro-inflammatory mediators in the tissues results in the remodelling of matrices which could modify the functions of extravasated leukocytes. Previous reports have shown changes in the expression of ECM proteins during local inflammatory responses. In this study, we have investigated the time- and tissue-specific expression profile of key ECM proteins in systemic inflammation using lipopolysaccharide (LPS)-induced endotoxemia and cecal ligation and puncture (CLP) mouse models. The results show that compared to naïve tissues, within 12 h following CLP surgery, a 20-30-fold increase was observed in the expression of collagen-IV (Col-IV) transcripts in the mesentery tissues with a 2.4-fold increase in the protein by 24 h. However, Western blot band intensities indicated that vimentin and fibrinogen were remarkably expressed in more quantity compared to Col-IV. Secondly, in CLP group of mice, fibrinogen showed 6-40-fold increase in mRNA level in various tissues with about 2-fold increase in the protein level compared to respective naïve tissues. Similar studies in the LPS-injected mice showed up to 2-3 fold increase in the expression of Col-IV, fibrinogen and vimentin at protein level in the lungs. In such animals, although similar pattern was observed for fibrinogen in kidney and liver tissues, the mesentery showed prominent changes in Col-IV and vimentin mRNA compared to CLP. Further, bioinformatics analysis showed multiple pathways which could be associated with vimentin, Col-IV and fibrinogen under inflammatory conditions both in human and mouse. The current study will help in better understanding of possible signalling from ECM proteins in inflammatory microenvironment and may contribute in development of cell adhesion-based therapeutics.

Role of cellular events in the pathophysiology of sepsis.

INTRODUCTION: Sepsis is a dysregulated host immune response due to an uncontrolled infection. It is a leading cause of mortality in adult intensive care units globally. When the host immune response induced against a local infection fails to contain it locally, it progresses to sepsis, severe sepsis, septic shock and death. METHOD: Literature survey was performed on the roles of different innate and adaptive immune cells in the development and progression of sepsis. Additionally, the effects of septic changes on reprogramming of different immune cells were also summarized to prepare the manuscript. FINDINGS: Scientific evidences to date suggest that the loss of balance between inflammatory and anti-inflammatory responses results in reprogramming of immune cell activities that lead to irreversible tissue damaging events and multi-organ failure during sepsis. Many surface receptors expressed on immune cells at various stages of sepsis have been suggested as biomarkers for sepsis diagnosis. Various immunomodulatory therapeutics, which could improve the functions of immune cells during sepsis, were shown to restore immunological homeostasis and improve survival in animal models of sepsis. CONCLUSION: In-depth and comprehensive knowledge on the immune cell activities and their correlation with severity of sepsis will help clinicians and scientists to design effective immunomodulatory therapeutics for treating sepsis.