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Hakimi, A.

Publications and source records attributed to Hakimi, A..

7 recordsLinked to original sources

Spatiotemporal dynamics of cryptococcal infection reveal novel immune modulatory mechanisms and antifungal targets

The threat and incidence of fungal diseases are increasing, as is the severity and mortality rates associated with these infections. New strategies to combat fungal infections are urgently needed to overcome rising rates of resistance and the emergence of new pathogens. To promote invasion within a host, fungi use highly adapted and regulated virulence factors, and, in turn, the host adopts an active and dynamic immune response to suppress infection. Understanding the interplay between these processes is crucial to move fungal disease management and treatment forward and improve global health outcomes. Within the present study, we tackle these challenges using state-of-the-art mass spectrometry instrumentation to explore proteome remodeling during active infection of Cryptococcus neoformans at an unprecedented depth with spatiotemporal resolution. Our prioritization of three host organs (i.e., lungs, brain, spleen) critical to initiation, progression, and response of disease discovers tissue-specific remodeling across time. Within the lungs, we revealed early and sustained activation of the host immune response integrated with characterization of a promising new antifungal target, and we propose the discovery of a competitive inhibitor for functional target disruption. Within the brain, proteome remodeling aligns with disease progression, and we define a new mechanistic role for haptoglobin in fungal cell modulation, as well as showcasing an adaptive survival response of C. neoformans within an hypoxic environment. Within the spleen, we reveal new dynamics of immune system activation upon cryptococcal infection. Overall, we provide the deepest integrated view of cryptococcal disease dynamics across temporal and spatial scales, revealing unrecognized mechanisms of host immunity and fungal pathogenesis that offer new avenues for targeted therapeutic intervention and disease management.

microbiology↗

Whole blood proteome dynamics defines predictive diagnostic and prognostic signatures of cryptococcal infection

Across the globe, fungi are impacting the lives of millions of people through the development of infections ranging from superficial to systemic with limited treatment options. To effectively combat fungal disease, rapid and reliable diagnostic methods are required, including current methodologies using antigen detection, culturing, microscopy, and molecular tools. However, the flexibility of these platforms to diagnose infection using non-invasive methods and predict the outcome of disease are limited. In this study, we apply state-of-the-art mass spectrometry-based proteomics to perform dual perspective (i.e., host and pathogen) profiling of cryptococcal infection. Whole blood collected over a temporal scale following murine model challenged with the human fungal pathogen, Cryptococcus neoformans, detected >3,000 host proteins and 160 fungal proteins. From the host perspective, temporal regulation of known immune-associated proteins, including eosinophil peroxidase and lipocalin-2, along with suppression of lipoproteins, demonstrated infection- and time-dependent host remodeling. Conversely, from the pathogen perspective, known and putative virulence-associated proteins were detected, including proteins associated with fungal extracellular vesicles and host immune modulation. We also observed and validated a new mechanism of immune system response to C. neoformans through modulation of haptoglobin. Further, we assessed the predictive power of dual perspective proteome profiling toward prognostics of cryptococcal infection and report a previously undisclosed integration among virulence factor production, immune system modulation, and individual model survival. Together, our findings pose novel biomarkers of cryptococcal infection from whole blood and highlight the potential of personal proteome profiles to determine the prognosis of cryptococcal infection, a new parameter in fungal disease management.

microbiology↗

Protein Coronas on Functionalized Nanoparticles Enable Quantitative and Precise Large-Scale Deep Plasma Proteomics

BackgroundThe wide dynamic range of circulating proteins coupled with the diversity of proteoforms present in plasma has historically impeded comprehensive and quantitative characterization of the plasma proteome at scale. Automated nanoparticle (NP) protein corona-based proteomics workflows can efficiently compress the dynamic range of protein abundances into a mass spectrometry (MS)-accessible detection range. This enhances the depth and scalability of quantitative MS-based methods, which can elucidate the molecular mechanisms of biological processes, discover new protein biomarkers, and improve comprehensiveness of MS-based diagnostics. MethodsInvestigating multi-species spike-in experiments and a cohort, we investigated fold-change accuracy, linearity, precision, and statistical power for the using the Proteograph Product Suite, a deep plasma proteomics workflow, in conjunction with multiple MS instruments. ResultsWe show that NP-based workflows enable accurate identification (false discovery rate of 1%) of more than 6,000 proteins from plasma (Orbitrap Astral) and, compared to a gold standard neat plasma workflow that is limited to the detection of hundreds of plasma proteins, facilitate quantification of more proteins with accurate fold-changes, high linearity, and precision. Furthermore, we demonstrate high statistical power for the discovery of biomarkers in small- and large-scale cohorts. ConclusionsThe automated NP workflow enables high-throughput, deep, and quantitative plasma proteomics investigation with sufficient power to discover new biomarker signatures with a peptide level resolution.

biochemistry↗

CaBLAM! A high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase

Measuring ongoing cellular activity is essential to understanding the dynamic functions of biological organisms. The most popular current approach is imaging fluorescence-based genetically encoded Ca2+ indicators (GECIs). While fluorescent probes are useful in many contexts, bioluminescence-based GECIs--probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein--have several distinct advantages. Because bioluminescent (BL) GECIs do not use the bright extrinsic excitation light required for fluorescence, BL GECIs do not photobleach, do not suffer from nonspecific autofluorescent background, and do not cause phototoxicity. Further, BL GECIs can be applied in contexts where directly shining photons on an imaging target is not possible. Despite these advantages, the use of BL GECIs has to date been limited by their small changes in bioluminescence intensity, high baseline signal at resting Ca2+ concentrations, and suboptimal Ca2+ affinities. Here, we describe a new BL GECI, CaBLAM (Ca2+ BioLuminescence Activity Monitor), that displays much higher dynamic range than previous BL GECIs and has a Ca2+ affinity suitable for capturing physiological changes in cytosolic Ca2+ concentration. With these improvements, CaBLAM captures single-cell and subcellular resolution activity at high frame rates in cultured neurons and in vivo, and allows multi-hour recordings in mice and behaving zebrafish. This new advance provides a robust alternative to traditional fluorescent GECIs that can enable or enhance imaging across many experimental conditions.

neuroscience↗

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend towards high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time, and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass resolving quadrupole, the Orbitrap and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared to state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with high dynamic range and resolution, synchronized with Astral analyzers acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/543408v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1eedfdeorg.highwire.dtl.DTLVardef@1ab7939org.highwire.dtl.DTLVardef@a3b2f8org.highwire.dtl.DTLVardef@13ac46b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

IRF8 Governs Tumor-Associated Macrophage Control of T Cell Exhaustion

Tumor progression is associated with overstimulation of cytotoxic T lymphocytes (CTLs), resulting in a dysfunctional state of exhaustion. How T cell exhaustion is elicited in the tumor remains poorly understood. Here we show that tumor-associated macrophages (TAMs) present cancer cell antigen and induce CTL exhaustion through a gene expression program dependent on the transcription factor interferon regulatory factor-8 (IRF8). In a transgenic model of murine breast cancer, CTL priming was supported by IRF8-dependent dendritic cells; yet, CTL exhaustion required TAM expression of IRF8, and its ablation suppressed tumor growth. An analysis of the highly immune-infiltrated human renal cell carcinoma tumors revealed abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. The IRF8 signature co-segregated with T cell exhaustion markers and was negatively associated with long-term patient survival. Thus, CTL exhaustion is promoted by TAMs via IRF8, and this crosstalk may be disrupted in TAM-targeted therapies.

immunology↗

TGFβ Suppresses Type 2 Immunity to Cancer

The immune system employs two distinct defense strategies against infections: pathogen elimination typified by type 1 immunity, and pathogen containment exemplified by type 2 immunity in association with tissue repair. Akin to infectious diseases, cancer progresses with cancer cell acquisition of microorganism-like behavior propagating at the expense of the host. While immunological mechanisms of cancer cell elimination are well defined, whether immune-mediated cancer cell containment can be induced is poorly understood. Here we show that ablation of transforming growth factor-{beta} receptor II (TGF{beta}RII) in CD4+ T cells promotes tumor tissue healing and halts cancer progression. Notably, the restorative response is dependent on the T helper 2 cytokine IL-4 fortifying vasculature organization that spares only proximal layers of cancer cells from hypoxia, nutrient starvation and death. Thus, type 2 immunity represents an effective cancer defense mechanism, and TGF{beta} signaling in helper T cells may be targeted for novel cancer immunotherapy.

immunology↗