We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log 10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV—CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences—is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence.
BackgroundDefensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis.ResultsMD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein.ConclusionThe study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions.
BackgroundGlioblastoma multiforme (GBM) is a highly aggressive form of brain cancer with marginal survival rates. GBM extracellular acidosis can profoundly impact its cell fate heterogeneities and progression. However, the molecules and mechanisms that enable GBM tumor cells acid adaptation and consequent cell fate competencies are weakly understood. Since extracellular proton concentrations (pHe) directly intercept the tumor cell plasma membrane, surface lipids must play a crucial role in pHe-dependent tumor cell fate dynamics. Hence, a more detailed insight into the finely tuned pH-dependent modulation of surface lipids is required to generate strategies that can inhibit or surpass tumor cell acid adaptation, thereby forcing the eradication of heterogeneous oncogenic niches, without affecting the normal cells.ResultsBy using image-based single cell analysis and physicochemical techniques, we made a small-scale survey of the effects of pH ranges (physiological: pHe 7.4, low: 6.2, and very low: 3.4) on LN229 glioblastoma cell line surface remodeling and analyzed the consequent cell fate heterogeneities with relevant molecular targets and behavioral assays. Through this basic study, we uncovered that the extracellular proton concentration (1) modulates surface cholesterol-driven cell fate dynamics and (2) induces ‘differential clustering’ of surface resident GM3 glycosphingolipid which together coordinates the proliferation, migration, survival, and death reprogramming via distinct effects on the tumor cell biomechanical homeostasis. A novel synergy of anti-GM3 antibody and cyclophilin A inhibitor was found to mimic the very low pHe-mediated GM3 supraclustered conformation that elevated the surface rigidity and mechano-remodeled the tumor cell into a differentiated phenotype which eventually succumbed to the anoikis type of cell death, thereby eradicating the tumorigenic niches.Conclusion and significanceThis work presents an initial insight into the physicochemical capacities of extracellular protons in the generation of glioblastoma tumor cell heterogeneities and cell death via the crucial interplay of surface lipids and their conformational changes. Hence, monitoring of proton–cholesterol–GM3 correlations in vivo through diagnostic imaging and in vitro in clinical samples may assist better tumor staging and prognosis. The emerged insights have further led to the translation of a ‘pH-dependent mechanisms of oncogenesis control’ into the surface targeted anti-GBM therapeutics.
Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumor with an extremely poor prognosis. Recent evidences have shown that the “biomechanical imbalances” induced in GBM patient-derived glioblastoma cells (GC) and in vivo via the administration of synthetic small molecules, may effectively inhibit disease progression and prolong survival of GBM animal models. This novel concept associated with de novo anti-GBM drug development has however suffered obstacles in adequate clinical utility due to the appearance of unrelated toxicity in the prolonged therapeutic windows. Here, we took a “drug repurposing approach” to trigger similar physico-chemical disturbances in the GBM tumor cells, wherein, the candidate therapeutic agent has been previously well established for its neuro-protective roles, safety, efficacy, prolonged tolerance and excellent brain bioavailability in human subjects and mouse models. In this study, we show that the extracts of an Indian traditional medicinal plant Bacopa monnieri (BM) and its bioactive component Bacoside A can generate dosage associated tumor specific disturbances in the hydrostatic pressure balance of the cell via a mechanism involving excessive phosphorylation of calcium/calmodulin-dependent protein kinase IIA (CaMKIIA/CaMK2A) enzyme that is further involved in the release of calcium from the smooth endoplasmic reticular networks. High intracellular calcium stimulated massive macropinocytotic extracellular fluid intake causing cell hypertrophy in the initial stages, excessive macropinosome enlargement and fluid accumulation associated organellar congestion, cell swelling, cell rounding and membrane rupture of glioblastoma cells; with all these events culminating into a non-apoptotic, physical non-homeostasis associated glioblastoma tumor cell death. These results identify glioblastoma tumor cells to be a specific target of the tested herbal medicine and therefore can be exploited as a safe anti-GBM therapeutic.
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