Many scientists learn during training to use analysis of variance (ANOVA) when comparing more than 2 groups, yet the principles and follow-up steps are often not well explained. This overview provides a practical guide for selecting appropriate multiple comparisons tests and for interpreting and presenting data accurately. While not exhaustive, it highlights commonly used approaches, many of which are available in GraphPad Prism, a widely used statistical program among cell and molecular biologists. The authors have no affiliation with Dotmatics, the distributor of GraphPad Prism, and this overview is not intended as an endorsement of the software.
Rubicon (RUN domain Beclin-1-interacting and cysteine-rich-containing protein) is a negative regulator of autophagy in nucleated cells; however, its role in platelets is unstudied. Here, we identify an autophagy-independent role for Rubicon in arterial thrombosis. Mice with a platelet/megakaryocyte-specific deletion of Rubicon (RUBCN-/-) showed normal circulating platelet counts, with a slight increase in platelet size. The basal levels of autophagy-related, vesicle-elongation proteins (i.e., ATG5, ATG7, Syntaxin 17, LC3, and Rab7) were unaffected, and there was no disruption in canonical platelet signaling pathways upon activation. Platelet secretion from all three granule classes remained intact. Aggregation in response to thrombin, convulxin, or botrocetin was unaffected. There was no overt defect in activation-dependent autophagic flux (i.e., loss of LC3 or p62). However, RUBCN-/- mice had a significant thrombosis defect in the FeCl3-induced, carotid artery injury model, but no defect in the tail transection hemostasis model. An intrinsic platelet defect was confirmed using microfluidics, where RUBCN-/- platelets had reduced collagen binding under flow at high shear flow but not low shear. RUBCN-/- platelets also had impaired surface exposure of phosphatidylserine (PS) following thrombin and convulxin activation, though there was no effect on total anoctamin 6 (ANO6), which contributes to PS exposure. There was no effect on platelet endocytosis and only modest reductions in mitochondrial membrane potential with a slight delay in clot contraction, suggesting that Rubicon contributes to late stages of platelet function. Collectively, our findings demonstrate that Rubicon contributes to thrombosis and procoagulant platelet formation in a manner that appears independent of classical autophagy.
Glutathione (GSH) serves as an essential guardian for parasites, protecting them from the onslaught of oxidative stress. As hemoglobin breaks down and energy is harvested through the intricate mitochondrial respiratory pathway, GSH meticulously creates a reducing environment, akin to a fortress against oxidative damage. The metabolic pathways involving GSH in parasites are complex and markedly different from those in human hosts, rendering them a compelling target in the battle against multidrug-resistant parasites. This review illuminates the critical role of GSH in the survival of malaria parasites and the impact of key enzyme inhibitors on the GSH redox pathway. Recent discoveries surrounding significant enzyme inhibitors as GST, GR, GS, and GP, are explored, shedding light on the potential of combination therapies while underscoring the obstacles posed by selective drug targeting and the imperative to navigate drug resistance. Moreover, the review delves into the forefront of research on the metabolic pathways of GSH in parasites, unveiling a trove of promising drug targets for strategies aimed at treating malaria in an era plagued by multidrug resistance and offers hope for innovative therapeutic approaches against one of the world’s most persistent diseases.
INTRODUCTION:Cerebral amyloid angiopathy (CAA), marked by amyloid beta (Aβ) accumulation in perivascular spaces (PVSs), contributes to vascular injury and inflammation in Alzheimer's disease. The mechanisms underlying CAA-related vascular pathology remain unclear. Increasing evidence indicates fibrinogen, the main component in blood clots, interacts with Aβ and exacerbates inflammation and co-deposits in PVS of CAA-positive vessels, yet its role in cerebrovascular dysfunction remains poorly defined. METHODS:Using TgSwDI transgenic mice, which develop robust CAA, we examined fibrin(ogen) deposition, aquaporin-4 (AQP4) polarization, and caveolin-1 (Cav-1) expression. We further assessed the effects of fibrinogen depletion via small interfering RNA. RESULTS:TgSwDI mice showed increased fibrin(ogen) extravasation, colocalization with Aβ in PVS, AQP4 depolarization, and elevated Cav-1 expression. Fibrinogen depletion reduced CAA, restored AQP4 polarization, decreased Cav-1 levels, attenuated microglial activation, and improved spatial memory. DISCUSSION:These findings suggest that modulating fibrinogen-related pathways could be a promising strategy for mitigating CAA pathology and its associated cerebrovascular pathology. HIGHLIGHTS:Fibrin(ogen)-Aβ colocalization aggravates CAA. AQP4 depolarization links fibrinogen-Aβ deposits to impaired clearance in CAA. Cav-1 increase drives fibrinogen leakage and neuroinflammation. Fibrinogen depletion reduces CAA, restores AQP4 polarity, and improves memory.
Classifying heterogeneous human B cells into discrete subsets based on surface phenotype alone remains a major challenge. We discovered a mouse splenic IgM+IgDlow/- B-cell subset (BDL) that induces proliferation of CD4+Foxp3+ T regulatory cells, making them a therapeutic target for autoimmunity. Because they are rare and lack sufficient cell surface markers, a multiomics strategy is required to identify and track them in humans. The lack of correlation between mRNA expression levels and cell surface expression is a drawback to a transcriptome-only single-cell RNA sequencing (scRNA-seq) approach because "novel" cell populations identified by surface protein transcripts may prove impossible to validate. To that end, a method was developed using human peripheral blood (PB) IgM+IgDlow/- B cells as a surrogate. A flow cytometry gating scheme (CD27 vs IgD and CD24 vs CD38) capturing all major human PB B-cell subsets was utilized. CD19+IgM+IgDlow/- PB B cells were tagged with the 4 markers using cellular indexing of transcriptomes and epitopes by sequencing. Biaxial plots similar to flow cytometry were used to identify naïve, immature, and memory B cells, and then confirmed with scRNA-seq. These studies using a multiomic workflow allowed the identification of IgM+IgDlow/- B-cell subsets by cell surface expression that could subsequently be analyzed for unique transcriptional profiles.
