Checkpoint blockade immunotherapy has become a first-line treatment option for cancer patients, with success in increasingly diverse cancer types. Still, many patients do not experience durable responses and the reasons for clinical success versus failure remain largely undefined. Investigation of immune responses within the tumor microenvironment can be highly informative but access to tumor tissue is not always available, highlighting the need to identify biomarkers in the blood that correlate with clinical success. Here, we used single-cell RNA sequencing coupled with T cell receptor sequencing to define CD8 T cell responses in peripheral blood of two patients with melanoma before and after immunotherapy with either anti-PD-1 (nivolumab) alone or the combination of anti-PD-1 and CTLA-4 (ipilimumab). Both treatment regimens increased transcripts associated with cytolytic effector function and decreased transcripts associated with naive T cells. These responses were further evaluated at the protein level and extended to a total of 53 patients with various cancer types. Unexpectedly, the induction of CD8 T cell responses associated with cytolytic function was variable and did not predict therapeutic success in this larger patient cohort. Rather, a decrease in the frequency of T cells with a naive-like phenotype was consistently observed after immunotherapy and correlated with prolonged patient survival. In contrast, a more detailed clonotypic analysis of emerging and expanding CD8 T cells in the blood revealed that a majority of individual T cell clones responding to immunotherapy acquired a transcriptional profile consistent with cytolytic effector function. These results suggest that responses to checkpoint blockade immunotherapy are evident and traceable in patients' blood, with outcomes predicted by the simultaneous loss of naive-like CD8 T cells and the expansion of mostly rare and diverse cytotoxic CD8 T cell clones.

Although fungal diseases are a major and growing public health concern, there are only four major classes of drug to treat primary fungal pathogens. The pipeline of new antifungals in clinical development is relatively thin compared with other disease classes. One approach to rapidly identify and provide novel treatment options is to repurpose existing drugs as antifungals. However, such proposed drug-repurposing candidates often suffer suboptimal efficacy and pharmacokinetics (PK) for fungal diseases. Herein, we briefly review the current antifungal drug pipeline and recent approaches to optimize existing drugs into novel molecules with unique modes of action relative to existing antifungal drug classes.

The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa-fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood.

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.

The fungal pathogen Cryptococcus neoformans causes up to 278 000 infections each year globally, resulting in up to 180,000 deaths annually, mostly impacting immunocompromised people. Therapeutic options for C. neoformans infections are very limited. Caspofungin, a member of the echinocandin class of antifungals, is generally well tolerated but clinically ineffective against C. neoformans. We sought to identify biological processes that can be targeted to render the cell more susceptible to echinocandins by screening the available libraries of gene deletion mutants made in the KN99α background for caspofungin sensitivity. We adapted a Candida albicans fungal biofilm assay for the growth characteristics of C. neoformans and systematically screened 4,030 individual gene deletion mutants in triplicate plate assays. We identified 25 strains that showed caspofungin sensitivity. We followed up with a dose dependence assay, and 17 of the 25 were confirmed sensitive, 5 of which were also sensitive in an agar plate assay. We made new deletion mutant strains for four of these genes: , encoding an iron transporter; , encoding a sterol desaturase; , encoding a myosin heavy chain; and , encoding a sterol transporter. All were more sensitive to membrane stress and showed significantly increased sensitivity to caspofungin at higher temperatures. Surprisingly, none showed any obvious cell wall defects such as would be expected for caspofungin-sensitive strains. Our microscopy analyses suggested that loss of membrane integrity contributed to the caspofungin sensitivity, either by allowing more caspofungin to enter or remain in the cell or by altering the location or orientation of the enzyme target to render it more susceptible to inhibition. The intrinsic resistance of Cryptococcus neoformans to the cell wall inhibitor caspofungin limits the available therapies for treating cryptococcal infections. We screened a collection of more than 4,000 gene deletion strains for altered caspofungin sensitivity to identify biological processes that could be targeted to render the cell more susceptible to caspofungin. We identified multiple genes with an effect on caspofungin susceptibility and found that they were associated with altered membrane permeability rather than the expected cell wall defects. This suggests that targeting these genes or other genes affecting membrane permeability is a viable path for developing novel therapies for treating this global fungal pathogen.

Human protein disulfide isomerase (PDI) is an essential redox-regulated enzyme required for oxidative protein folding. It comprises four thioredoxin domains, two catalytically active (a, a') and two inactive (b, b'), organized to form a flexible abb'a' U-shape. Snapshots of unbound oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDI's structure changes in response to the redox environment and inhibitor binding remains controversial. Here, we used multiparameter confocal single-molecule FRET to track the movements of the two catalytic domains with high temporal resolution. We found that at equilibrium, PDI visits three structurally distinct conformational ensembles, two "open" (O and O) and one "closed" (C). We show that the redox environment dictates the time spent in each ensemble and the rate at which they exchange. While oxidized PDI samples O, O, and C more evenly and in a slower fashion, reduced PDI predominantly populates O and O and exchanges between them more rapidly, on the submillisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the R300-W396 cation-π interaction and active site cysteines dictate, in unexpected ways, how the catalytic domains relocate. Finally, we show that irreversible inhibitors targeting the active sites of reduced PDI did not abolish these protein dynamics but rather shifted the equilibrium toward the closed ensemble. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize its multifaceted role in biology, and should be considered when designing PDI-targeted therapeutics.

Neutrophils are important cellular mediators of injury and repair in diseases including ischemic heart disease, atherosclerosis, and sepsis. Myeloperoxidase-derived (MPO)-oxidants released from neutrophils are potential mediators of endothelial injury in disease. MPO-derived HOCl attacks plasmalogen phospholipid to liberate 2-chlorofatty aldehyde (2-ClFALD). Both 2-ClFALD and its oxidation product, 2-chlorofatty acid (2-ClFA), are electrophilic lipids, and both probably react with proteins through several mechanisms. In the present study, we investigate protein modification specifically by 2-ClFALD under non-reducing conditions (e.g., without stabilizing Schiff base bonds), which likely reflects nucleophilic targeting of the electrophilic chlorinated carbon. Protein modification by the ω-alkyne analog of 2-chlorohexadecanal (2-ClHDA), 2-ClHDyA, was compared to that with the ω-alkyne analog of 2-chlorohexadecanoic acid (2-ClHA), 2-ClHyA, in multiple cell lines, which demonstrated 2-ClFALD preferentially modifies proteins compared to 2-ClFA. The 2-ClHDyA modified proteins from EA.hy926 cells and human lung microvascular endothelial cells analyzed by shotgun proteomics and over-representation analysis included adherens junction, cell adhesion molecule binding, and cell substrate junction enrichment categories. It is possible that proteins in these groups may have roles in previously described 2-ClFALD-elicited endothelial barrier dysfunction.

Loss of TDP-43 protein homeostasis and dysfunction, in particular TDP-43 aggregation, are tied to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is an RNA binding protein tightly controlling its own expression levels through a negative feedback loop, involving TDP-43 recruitment to the 3' untranslated region of its own transcript. Aberrant TDP-43 expression caused by autoregulation defects are linked to TDP-43 pathology. Therefore, interactions between TDP-43 and its own transcript are crucial to prevent TDP-43 aggregation and loss of function. However, the mechanisms that mediate this interaction remain ill-defined. We find that a central RNA sequence in the 3' UTR, which mediates TDP-43 autoregulation, increases the liquid properties of TDP-43 phase separation. Furthermore, binding to this RNA sequence induces TDP-43 condensation in human cell lysates, suggesting that this interaction promotes TDP-43 self-assembly into dynamic ribonucleoprotein granules. In agreement with these findings, our experiments show that TDP-43 oligomerization and phase separation, mediated by the amino and carboxy-terminal domains, respectively, are essential for TDP-43 autoregulation. According to our additional observations, CLIP34-associated phase separation and autoregulation may be efficiently controlled by phosphorylation of the N-terminal domain. Importantly, we find that specific ALS-associated TDP-43 mutations, mainly M337V, and a shortened TDP-43 isoform recently tied to motor neuron toxicity in ALS, disrupt the liquid properties of TDP-43-RNA condensates as well as autoregulatory function. In addition, we find that M337V decreases the cellular clearance of TDP-43 and other RNA binding proteins associated with ALS/FTD. These observations suggest that loss of liquid properties in M337V condensates strongly affects protein homeostasis. Together, this work provides evidence for the central role of TDP-43 oligomerization and liquid-liquid phase separation linked to RNA binding in autoregulation. These mechanisms may be impaired by TDP-43 disease variants and controlled by specific cellular signaling.

Thrombotic microangiopathy (TMA) is characterized by microangiopathic hemolytic anemia, thrombocytopenia and organ injury occurring due to endothelial cell damage and microthrombi formation in small vessels. TMA is primary when a genetic or acquired defect is identified, as in atypical hemolytic uremic syndrome (aHUS) or secondary when occurring in the context of another disease process such as infection, autoimmune disease, malignancy or drugs. Differentiating between a primary complement-mediated process and one triggered by secondary factors is critical to initiate timely treatment but can be challenging for clinicians, especially after a kidney transplant due to presence of multiple confounding factors. Similarly, primary membranous nephropathy is an immune-mediated glomerular disease associated with circulating autoantibodies (directed against the M-type phospholipase A2 receptor (PLA2R) in 70% cases) while secondary membranous nephropathy is associated with infections, drugs, cancer, or other autoimmune diseases. Complement activation has also been proposed as a possible mechanism in the etiopathogenesis of primary membranous nephropathy; however, despite complement being a potentially common link, aHUS and primary membranous nephropathy have not been reported together. Herein we describe a case of aHUS due to a pathogenic mutation in complement factor I that developed after a kidney transplant in a patient with an underlying diagnosis of PLA2R antibody associated-membranous nephropathy. We highlight how a systematic and comprehensive analysis helped to define the etiology of aHUS, establish mechanism of disease, and facilitated timely treatment with eculizumab that led to recovery of his kidney function. Nonetheless, ongoing anti-complement therapy did not prevent recurrence of membranous nephropathy in the allograft. To our knowledge, this is the first report of a patient with primary membranous nephropathy and aHUS after a kidney transplant.

Plasmalogens are plasma-borne antioxidant phospholipid species that provide protection as cellular lipid components during cellular oxidative stress. In this study we investigated plasma plasmalogen levels in human sepsis as well as in rodent models of infection. In humans, levels of multiple plasmenylethanolamine molecular species were decreased in septic patient plasma compared to control subject plasma as well as an age-aligned control subject cohort. Additionally, lysoplasmenylcholine levels were significantly decreased in septic patients compared to the control cohorts. In contrast, plasma diacyl phosphatidylethanolamine and phosphatidylcholine levels were elevated in septic patients. Lipid changes were also determined in rats subjected to cecal slurry sepsis. Plasma plasmenylcholine, plasmenylethanolamine, and lysoplasmenylcholine levels were decreased while diacyl phosphatidylethanolamine levels were increased in septic rats compared to control treated rats. Kidney levels of lysoplasmenylcholine as well as plasmenylethanolamine molecular species were decreased in septic rats. Interestingly, liver plasmenylcholine and plasmenylethanolamine levels were increased in septic rats. Since COVID-19 is associated with sepsis-like acute respiratory distress syndrome and oxidative stress, plasmalogen levels were also determined in a mouse model of COVID-19 (intranasal inoculation of K18 mice with SARS-CoV-2). 3 days following infection, lung infection was confirmed as well as cytokine expression in the lung. Multiple molecular species of lung plasmenylcholine and plasmenylethanolamine were decreased in infected mice. In contrast, the predominant lung phospholipid, dipalmitoyl phosphatidylcholine, was not decreased following SARS-CoV-2 infection. Additionally total plasmenylcholine levels were decreased in the plasma of SARS-CoV-2 infected mice. Collectively, these data demonstrate the loss of plasmalogens during both sepsis and SARS-CoV-2 infection. This study also indicates plasma plasmalogens should be considered in future studies as biomarkers of infection and as prognostic indicators for sepsis and COVID-19 outcomes.