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Histoplasma capsulatum is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte macrophage colony stimulating factor (GM-CSF) induces metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the zinc transporter, ZRT2 , was important for fungal survival in vivo . Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under control of the ZRT2 zinc-regulated promoter. This reporter accurately responds to a medium devoid of Zn. ZRT2 expression increased in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with a reporter yeast strain and assessed ZRT2 expression at 0, 3, 7, and 14 days post-infection (dpi). ZRT2 expression minimally increased at 3 dpi and peaked at 7 dpi, corresponding with the onset of adaptive immunity. We discovered that the major MΦ populations that restrict Zn from the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted the control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control H. capsulatum replication, M-CSF. These findings illustrate the reporter's ability to sense Zn in vitro and in vivo and correlate ZRT2 expression with GM-CSF and M-CSF activation of MΦ.IMPORTANCEPhagocytes use an arsenal of defenses to control the replication of Histoplasma yeasts, one of which is the limitation of trace metals. On the other hand, H. capsulatum combats metal restriction by upregulating metal importers such as the Zn importer ZRT2 . This transporter contributes to H. capsulatum pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 transcriptional reporter to probe H. capsulatum Zn sensing during infection and exposed the role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to study how intracellular pathogens sense and respond to the changing environments of the host., Competing Interests: The authors declare no conflict of interest.

Three 1 2 mass oriented rare earth element (REE) M 2+ correction approaches (fixed factor, a dual internal standard, and an in-sample) are evaluated for use in an ICP-MS environmental method update. The multi-variant-based evaluation includes analyzing the same 19 REE-fortified matrices on eight different days over a two-month period using two instrument tunes. These REE-fortified matrices were also analyzed using HR-ICP-MS and ICP-MS/MS to estimate the reference value for use in the principal component analysis (PCA) and hierarchical modeling evaluation. A fixed factor is unable to compensate for matrix and mass dependent drift and because of this it generates the largest across matrix, tune, and day 95 th percent confidence bounds for the REE corrections on both As (1.1 ppb) and Se (23 ppb) using samples fortified with 100 ppb Nd, Sm & Gd. The PCA analysis indicated that M 2+ ions cluster together across matrix, tune and day better than M 1+ and these tighter correlations are reflected in reduced 95 th percentile confidence bounds for dual M 2+ internal standards (M 2+ ; As = 0.3 ppb; Se = 5.4 ppb; n = 704) relative to M 1+ internal standards (M 1+ ; As = 0.6 ppb; Se = 12.0 ppb; n = 1056). The use of an in-sample M 2+ correction produced comparable 95 th percent confidence bounds (As = 0.2 ppb; Se = 3.4 ppb; n = 352) relative to the M 2+ internal standard approaches. Finally, the hierarchical modeling indicated M 2+ ions as internal standards tend to minimize the across day variability induced by cone changes and the daily reoccurring matrix shifts in the M 2+ /M 1+ ratio associated with 250 ppm matrices of Na, Ca, and Mg. This internal standard driven reduction in variability can be beneficial in compliance monitoring methods., Competing Interests: Conflicts of interest There are no conflicts to declare.

Dupuytren's disease is a progressive fibrotic condition of the hand that causes contracture of fingers in later stages. Our previous in vitro studies suggest that the transformation of fibroblasts to myofibroblasts induced by transforming growth factor-beta can be inhibited by the addition of the antifibrotic drug, pirfenidone (PFD). We hypothesize that the local delivery of PFD directly to nodules can potentially prevent the progression to cords and, furthermore, that injection of PFD after the resection of cords can limit the recurrence of the disease. The purpose of this research was to develop a PFD injectable solution and to assess its safety in mice. Based on preformulation observations, a sterile solution containing up to 8 mg/0.4 mL of PFD was prepared in a phosphate buffer with and without 15%v/v N-methyl-2-pyrrolidone. Accelerated stability studies suggested that the product should be kept at refrigerated temperature (2-8 °C) for long-term storage. Safety studies involving subcutaneous administration to mice showed that 2-4 mg of PFD in 0.4 mL aqueous buffer did not elicit a significant inflammatory reaction. However, 4 mg PFD in 0.4 mL (F B ) of buffer: NMP cosolvent system led to a significant increase in the influx of inflammatory cells and 8 mg PFD (F A ) in the cosolvent system was lethal to the animals.

Macrophages deploy numerous strategies to combat invasion by microbes. One tactic is to restrict acquisition of diverse nutrients, including trace metals, a process termed nutritional immunity. Intracellular pathogens adapt to a resource-poor environment by marshaling mechanisms to harvest nutrients. Carbon acquisition is crucial for pathogen survival; compounds that reduce availability are a potential strategy to control intracellular replication. Treatment of macrophages with the glucose analog 2-deoxy-D-glucose (2-DG) armed phagocytes to eliminate the intracellular fungal pathogen Histoplasma capsulatum in vitro and in vivo. Killing did not rely on altering access to carbon-containing molecules or changes in ATP, ER stress, or autophagy. Unexpectedly, 2-DG undermined import of exogenous zinc into macrophages, decreasing the quantity of cytosolic and phagosomal zinc. The fungus perished as a result of zinc starvation. This change in metal ingress was not ascribed to a defect in a single importer; rather, there was a collective impairment in transporter activity. This effect promoted the antifungal machinery of macrophages and expanded the complexity of 2-DG activities far beyond manipulating glycolysis. Mechanistic metabolic studies employing 2-DG will have to consider its effect on zinc transport. Our preclinical data support consideration of this agent as a possible adjunctive therapy for histoplasmosis.

BACKGROUNDClear cell renal cell carcinoma (ccRCC) is the most common histologically defined renal cancer. However, it is not a uniform disease and includes several genetic subtypes with different prognoses. ccRCC is also characterized by distinctive metabolic reprogramming. Tobacco smoking (TS) is an established risk factor for ccRCC, with unknown effects on tumor pathobiology.METHODSWe investigated the landscape of ccRCCs and paired normal kidney tissues using integrated transcriptomic, metabolomic, and metallomic approaches in a cohort of white males who were long-term current smokers (LTS) or were never smokers (NS).RESULTSAll 3 Omics domains consistently identified a distinct metabolic subtype of ccRCCs in LTS, characterized by activation of oxidative phosphorylation (OXPHOS) coupled with reprogramming of the malate-aspartate shuttle and metabolism of aspartate, glutamate, glutamine, and histidine. Cadmium, copper, and inorganic arsenic accumulated in LTS tumors, showing redistribution among intracellular pools, including relocation of copper into the cytochrome c oxidase complex. A gene expression signature based on the LTS metabolic subtype provided prognostic stratification of The Cancer Genome Atlas ccRCC tumors that was independent of genomic alterations.CONCLUSIONThe work identified the TS-related metabolic subtype of ccRCC with vulnerabilities that can be exploited for precision medicine approaches targeting metabolic pathways. The results provided rationale for the development of metabolic biomarkers with diagnostic and prognostic applications using evaluation of OXPHOS status. The metallomic analysis revealed the role of disrupted metal homeostasis in ccRCC, highlighting the importance of studying effects of metals from e-cigarettes and environmental exposures.FUNDINGDepartment of Defense, Veteran Administration, NIH, ACS, and University of Cincinnati Cancer Institute.

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca 2+ signalling pathway that depends on intracellular Ca 2+ sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca 2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca 2+ -CaM signalling in aspergillosis. Finally, we demonstrate that Ca 2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca 2+ -CaM signalling to inhibit LAP. These findings reveal the important role of Ca 2+ -CaM signalling in antifungal immunity and identify an immunological function of Ca 2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.