HIF-α signaling regulates the macrophage inflammatory response during Leishmania major infection.

Cutaneous leishmaniasis (CL) contributes significantly to the global burden of neglected tropical diseases, with 12 million people currently infected with Leishmania parasites. CL encompasses a range of disease manifestations, from self-healing skin lesions to permanent disfigurations. Currently there is no vaccine available, and many patients are refractory to treatment, emphasizing the need for new therapeutic targets. Previous work demonstrated macrophage HIF-α-mediated lymphangiogenesis is necessary to achieve efficient wound resolution during murine L. major infection. Here, we investigate the role of macrophage HIF-α signaling independent of lymphangiogenesis. We sought to determine the relative contributions of the parasite and the host-mediated inflammation in the lesional microenvironment to myeloid HIF-α signaling. Because HIF-α activation can be detected in infected and bystander macrophages in leishmanial lesions, we hypothesize it is the host's inflammatory response and microenvironment, rather than the parasite, that triggers HIF-α activation. To address this, macrophages from mice with intact HIF-α signaling (LysM Cre ARNT f/+ ) or mice with deleted HIF-α signaling (LysM Cre ARNT f/f ) were subjected to RNASequencing after L. major infection and under pro-inflammatory stimulus. We report that L. major infection alone is enough to induce some minor HIF-α-dependent transcriptomic changes, while infection with L. major in combincation with pro-inflammatory stimuli induces numerous transcriptomic changes that are both dependent and independent of HIF-α signaling. Additionally, by coupling transcriptomic analysis with several pathway analyses, we found HIF-α suppresses pathways involved in protein translation during L. major infection in a pro-inflammatory environment. Together these findings show L. major induces a HIF-α-dependent transcriptomic program, but HIF-α only suppresses protein translation in a pro-inflammatory environment. Thus, this work indicates the host inflammatory response, rather than the parasite, largely contributes to myeloid HIF-α signaling during Leishmania infection.

Mitochondrial protein deacetylation by SIRT3 in osteoclasts promotes bone resorption with aging in female mice.

OBJECTIVES: The mitochondrial deacetylase sirtuin-3 (SIRT3) is necessary for the increased bone resorption and enhanced function of mitochondria in osteoclasts that occur with advancing age; how SIRT3 drives bone resorption remains elusive. METHODS: To determine the role of SIRT3 in osteoclast mitochondria, we used mice with conditional loss of Sirt3 in osteoclast lineage and mice with germline deletion of either Sirt3 or its known target Pink1. RESULTS: SIRT3 stimulates mitochondrial quality in osteoclasts in a PINK1-independent manner, promoting mitochondrial activity and osteoclast maturation and function, thereby contributing to bone loss in female but not male mice. Quantitative analyses of global proteomes and acetylomes revealed that deletion of Sirt3 dramatically increased acetylation of osteoclast mitochondrial proteins, particularly ATPase inhibitory factor 1 (ATPIF1), an essential protein for mitophagy. Inhibition of mitophagy via mdivi-1 recapitulated the effect of deletion of Sirt3 or Atpif1 in osteoclast formation and mitochondrial function. CONCLUSIONS: Decreasing mitophagic flux in osteoclasts may be a promising pharmacotherapeutic approach to treat osteoporosis in older adults.

Quantitative plasma proteomic analysis in children after superior cavopulmonary anastomosis with pulmonary arteriovenous malformations.

Approximately 1000 children are born every year in the United States with one effective cardiac pumping chamber, or single ventricle heart disease. One of the early causes of mortality in this population is pulmonary arteriovenous malformations (PAVMs), which allow blood to bypass gas exchange in the lungs. PAVMs most frequently occur in children after superior cavopulmonary anastomosis (SCPA), a procedure that redirects venous blood from the upper body to the lungs. Because plasma proteins are in part responsible for directing angiogenesis, we hypothesized that differential protein concentrations would be observed in superior caval blood among children after SCPA according to PAVM status. We performed quantitative plasma proteomics from 11 children with PAVMs and in seven children without PAVMs; an additional 11 children with Fontan circulation were included as a reference. Among children with SCPA, there were no significant differences in the plasma proteomes for those with and without PAVMs. When comparing children with Fontan circulation to those with SCPA and PAVMs, 18 proteins exhibited differential expression (10 downregulated and eight upregulated) in superior caval plasma. These results suggest that factors other than, or in addition to, plasma proteins may be responsible for single ventricle patients' susceptibility to PAVMs after SCPA. IMPACT: What is the key message of your article? We did not identify significant differences in plasma proteins when comparing those children with and without pulmonary arteriovenous malformations (PAVMs) after superior cavopulmonary anastomosis (SCPA). What does it add to the existing literature? The etiology of PAVMs in this population is likely due to factors other than, or in addition to, differences in plasma proteins. What is the impact? Further studies are needed to identify causes of PAVMs among children after SCPA.

Enhanced translational activity is linked to lymphatic endothelial cell activation in cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a significant public health problem leading to permanently disfiguring skin lesions caused by Leishmania parasites. Lesion severity stems from an excessive host inflammatory response that prevents healing. Here, we characterized the transcriptional and translational responses of lymphatic endothelial cells (LECs) during murine CL using historical single-cell RNA sequencing data combined with flow cytometry and in vivo puromycin incorporation to assess translational activity. We identified upregulation of antigen presentation pathways including MHC-I, MHC-II, and immunoproteasome transcripts in dermal LECs from Leishmania major -infected mice compared to naive controls. LECs also exhibited increased expression of guanylate binding proteins and interferon-inducible genes, indicative of immune activation. Moreover, our findings demonstrate that LECs in leishmanial lesions displayed heightened translational activity relative to LECs from uninflamed ears, and LEC translational activity was highest in activated LECs. Furthermore, LEC translational activity exceeded that of other cell types within the lesion microenvironment. Validating the transcriptomic data, LECs in lesions expressed elevated MHC-II and programmed death-ligand 1 (PDL-1), supporting their potential role in antigen presentation. Functional assays using DQ-OVA confirmed that LECs from leishmanial lesions efficiently uptake and process antigens, highlighting their capability as antigen presenting cells in the inflamed dermal microenvironment. Overall, our study reveals the activation status of LECs in leishmanial lesions, shedding light on their potential role in shaping local immunity and inflammation in a variety of skin diseases.

A three-dimensional valve-on-chip microphysiological system implicates cell cycle progression, cholesterol metabolism and protein homeostasis in early calcific aortic valve disease progression.

BACKGROUND: Calcific aortic valve disease (CAVD) is one of the most common forms of valvulopathy, with a 50 % elevated risk of a fatal cardiovascular event, and greater than 15,000 annual deaths in North America alone. The treatment standard is valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. The development of early diagnostic and therapeutic strategies requires the fabrication of effective in vitro valve mimetic models to elucidate early CAVD mechanisms. METHODS: In this study, we developed a multilayered physiologically relevant 3D valve-on-chip (VOC) system that incorporated aortic valve mimetic extracellular matrix (ECM), porcine aortic valve interstitial cell (VIC) and endothelial cell (VEC) co-culture and dynamic mechanical stimuli. Collagen and glycosaminoglycan (GAG) based hydrogels were assembled in a bilayer to mimic healthy or diseased compositions of the native fibrosa and spongiosa. Multiphoton imaging and proteomic analysis of healthy and diseased VOCs were performed. RESULTS: Collagen-based bilayered hydrogel maintained the phenotype of the VICs. Proteins related to cellular processes like cell cycle progression, cholesterol biosynthesis, and protein homeostasis were found to be significantly altered and correlated with changes in cell metabolism in diseased VOCs. This study suggested that diseased VOCs may represent an early, adaptive disease initiation stage, which was corroborated by human aortic valve proteomic assessment. CONCLUSIONS: In this study, we developed a collagen-based bilayered hydrogel to mimic healthy or diseased compositions of the native fibrosa and spongiosa layers. When the gels were assembled in a VOC with VECs and VICs, the diseased VOCs revealed key insights about the CAVD initiation process. STATEMENT OF SIGNIFICANCE: Calcific aortic valve disease (CAVD) elevates the risk of death due to cardiovascular pathophysiology by 50 %, however, prevention and mitigation strategies are lacking, clinically. Developing tools to assess early disease would significantly aid in the prevention of disease and in the development of therapeutics. Previously, studies have utilized collagen and glycosaminoglycan-based hydrogels for valve cell co-cultures, valve cell co-cultures in dynamic environments, and inorganic polymer-based multilayered hydrogels; however, these approaches have not been combined to make a physiologically relevant model for CAVD studies. We fabricated a bi-layered hydrogel that closely mimics the aortic valve and used it for valve cell co-culture in a dynamic platform to gain mechanistic insights into the CAVD initiation process using proteomic and multiphoton imaging assessment.

Understanding proteome quantification in an interactive learning module on Google Cloud Platform.

This manuscript describes the development of a resource module that is part of a learning platform named 'NIGMS Sandbox for Cloud-based Learning' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module delivers learning materials on protein quantification in an interactive format that uses appropriate cloud resources for data access and analyses. Quantitative proteomics is a rapidly growing discipline due to the cutting-edge technologies of high resolution mass spectrometry. There are many data types to consider for proteome quantification including data dependent acquisition, data independent acquisition, multiplexing with Tandem Mass Tag reporter ions, spectral counts, and more. As part of the NIH NIGMS Sandbox effort, we developed a learning module to introduce students to mass spectrometry terminology, normalization methods, statistical designs, and basics of R programming. By utilizing the Google Cloud environment, the learning module is easily accessible without the need for complex installation procedures. The proteome quantification module demonstrates the analysis using a provided TMT10plex data set using MS3 reporter ion intensity quantitative values in a Jupyter notebook with an R kernel. The learning module begins with the raw intensities, performs normalization, and differential abundance analysis using limma models, and is designed for researchers with a basic understanding of mass spectrometry and R programming language. Learners walk away with a better understanding of how to navigate Google Cloud Platform for proteomic research, and with the basics of mass spectrometry data analysis at the command line. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

Understanding PTM Cross Talk Through a Visualization Tool, PTMViz.

The advancement of sequencing technologies has expanded our understanding of biological complexity through mechanisms such as allelic variations, alternative splicing of RNA, degradation of RNA by microRNAs, and posttranslational modifications (PTMs). In this chapter, we describe a method, PTMViz, for analyzing proteoforms identified by mass spectrometry. This interactive platform provides differential abundance analysis and visualization of protein and posttranslational modifications. We describe the detailed steps to prepare mass spectrometry database search results into the necessary format for PTMViz, how to set up the experimental conditions for differential abundance analysis, and the visualization of the results. The application is freely available at https://github.com/ByrumLab/PTMViz .

An ex vivo human precision-cut lung slice platform provides insight into SARS-CoV-2 pathogenesis and antiviral drug efficacy.

Coronavirus disease 2019 (COVID-19) has claimed millions of lives since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and lung disease appears the primary cause of death in COVID-19 patients. However, the underlying mechanisms of COVID-19 pathogenesis remain elusive, and there is no existing model where human disease can be faithfully recapitulated and conditions for the infection process can be experimentally controlled. Herein we report the establishment of an ex vivo human precision-cut lung slice (hPCLS) platform for studying SARS-CoV-2 pathogenicity and innate immune responses, and for evaluating the efficacy of antiviral drugs against SARS-CoV-2. We show that while SARS-CoV-2 continued to replicate during the course of infection of hPCLS, infectious virus production peaked within 2 days, and rapidly declined thereafter. Although most proinflammatory cytokines examined were induced by SARS-CoV-2 infection, the degree of induction and types of cytokines varied significantly among hPCLS from individual donors. Two cytokines in particular, IP-10 and IL-8, were highly and consistently induced, suggesting a role in the pathogenesis of COVID-19. Histopathological examination revealed focal cytopathic effects late in the infection. Transcriptomic and proteomic analyses identified molecular signatures and cellular pathways that are largely consistent with the progression of COVID-19 in patients. Furthermore, we show that homoharringtonine, a natural plant alkaloid derived from Cephalotoxus fortunei, not only inhibited virus replication but also production of pro-inflammatory cytokines, and thus ameliorated the histopathological changes caused by SARS-CoV-2 infection, demonstrating the usefulness of the hPCLS platform for evaluating antiviral drugs. IMPORTANCE: Here, established an ex vivo human precision-cut lung slice platform for assessing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, viral replication kinetics, innate immune response, disease progression, and antiviral drugs. Using this platform, we identified early induction of specific cytokines, especially IP-10 and IL-8, as potential predictors for severe coronavirus disease 2019 (COVID-19), and uncovered a hitherto unrecognized phenomenon that while infectious virus disappears at late times of infection, viral RNA persists and lung histopathology commences. This finding may have important clinical implications for both acute and post-acute sequelae of COVID-19. This platform recapitulates some of the characteristics of lung disease observed in severe COVID-19 patients and is therefore a useful platform for understanding mechanisms of SARS-CoV-2 pathogenesis and for evaluating the efficacy of antiviral drugs.

One-pot method for preparing DNA, RNA, and protein for multiomics analysis.

Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver, and lung tissue.

Trichloroethylene metabolite modulates DNA methylation-dependent gene expression in Th1-polarized CD4+ T cells from autoimmune-prone mice.

Trichloroethylene (TCE) is an industrial solvent and widespread environmental contaminant associated with CD4+ T-cell activation and autoimmune disease. Prior studies showed that exposure to TCE in the drinking water of autoimmune-prone mice expanded effector/memory CD4+ T cells with an interferon-γ (IFN-γ)-secreting Th1-like phenotype. However, very little is known how TCE exposure skews CD4+ T cells towards this pro-inflammatory Th1 subset. As observed previously, TCE exposure was associated with hypermethylation of regions of the genome related to transcriptional repression in purified effector/memory CD4 T cells. We hypothesized that TCE modulates transcriptional and/or epigenetic programming of CD4+ T cells as they differentiate from a naive to effector phenotype. In the current study, purified naive CD4 T cells from both male and female autoimmune-prone MRL/MpJ mice were activated ex vivo and polarized towards a Th1 subset for 4 days in the presence or absence of the oxidative metabolite of TCE, trichloroacetaldehyde hydrate (TCAH) in vitro. An RNA-seq assessment and reduced representation bisulfite sequencing for DNA methylation were conducted on Th1 cells or activated, non-polarized cells. The results demonstrated TCAH's ability to regulate key genes involved in the immune response and autoimmunity, including Ifng, by altering the level of DNA methylation at the gene promoter. Intriguing sex differences were observed and for the most part, the effects were more robust in females compared to males. In conclusion, TCE via TCAH epigenetically regulates gene expression in CD4+ T cells. These results may have implications for mechanistic understanding or future therapeutics for autoimmunity.

Characterization of methionine dependence in melanoma cells.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of S-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma.

Expression of the serine/threonine kinase never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is essential for entry into mitosis via its role in facilitating centrosome separation. Its overactivity can lead to tumorigenesis and drug resistance through the activation of several oncogenic pathways, including AKT. Although the cancer-enabling activities of NEK2 are documented in many malignancies, including correlations with poor survival in myeloma, breast, and non-small cell lung cancer, little is known about the role of NEK2 in lymphoma. Here, in tumors from patients with diffuse large B-cell lymphoma (DLBCL), the most common, aggressive non-Hodgkin lymphoma, we found a high abundance of NEK2 mRNA and protein associated with an inferior overall survival. Using our recently developed NEK2 inhibitor, NBI-961, we discovered that DLBCL cell lines and patient-derived cells exhibit a dependency on NEK2 for their viability. This compromised cell fitness was directly attributable to efficient NEK2 inhibition and proteasomal degradation by NBI-961. In a subset of particularly sensitive DLBCL cells, NBI-961 induced G2/mitosis arrest and apoptosis. In contrast, an existing indirect NEK2 inhibitor, INH154, did not prevent NEK2 autophosphorylation, induce NEK2 proteasomal degradation, or affect cell viability. Global proteomics and phospho-proteomics revealed that NEK2 orchestrates cell-cycle and apoptotic pathways through regulation of both known and new signaling molecules. We show the loss of NEK2-sensitized DLBCL to the chemotherapy agents, doxorubicin and vincristine, and effectively suppressed tumor growth in mice. These studies establish the oncogenic activity of NEK2 in DLBCL and set the foundation for development of anti-NEK2 therapeutic strategies in this frequently refractory and relapse-prone cancer.

Anterior Pituitary Transcriptomics Following a High-Fat Diet: Impact of Oxidative Stress on Cell Metabolism.

Anterior pituitary cell function requires a high level of protein synthesis and secretion which depend heavily on mitochondrial adenosine triphosphate production and functional endoplasmic reticula. Obesity adds stress to tissues, requiring them to adapt to inflammation and oxidative stress, and adding to their allostatic load. We hypothesized that pituitary function is vulnerable to the stress of obesity. Here, we utilized a 10- to 15-week high-fat diet (HFD, 60%) in a thermoneutral environment to promote obesity, testing both male and female FVB.129P mice. We quantified serum hormones and cytokines, characterized the metabolic phenotype, and defined changes in the pituitary transcriptome using single-cell RNA-sequencing analysis. Weight gain was significant by 3 weeks in HFD mice, and by 10 weeks all HFD groups had gained 20 g. HFD females (15 weeks) had increased energy expenditure and decreased activity. All HFD groups showed increases in serum leptin and decreases in adiponectin. HFD caused increased inflammatory markers: interleukin-6, resistin, monocyte chemoattractant protein-1, and tumor necrosis factorα. HFD males and females also had increased insulin and increased TSH, and HFD females had decreased serum prolactin and growth hormone pulse amplitude. Pituitary single-cell transcriptomics revealed modest or no changes in pituitary cell gene expression from HFD males after 10 or 15 weeks or from HFD females after 10 weeks. However, HFD females (15 weeks) showed significant numbers of differentially expressed genes in lactotropes and pituitary stem cells. Collectively, these studies reveal that pituitary cells from males appear to be more resilient to the oxidative stress of obesity than females and identify the most vulnerable pituitary cell populations in females.

TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons.

Trimethylation of histone H3 lysine 9 (H3K9me3) is crucial for the regulation of gene repression and heterochromatin formation, cell-fate determination and organismal development1. H3K9me3 also provides an essential mechanism for silencing transposable elements1-4. However, previous studies have shown that canonical H3K9me3 readers (for example, HP1 (refs. 5-9) and MPP8 (refs. 10-12)) have limited roles in silencing endogenous retroviruses (ERVs), one of the main transposable element classes in the mammalian genome13. Here we report that trinucleotide-repeat-containing 18 (TNRC18), a poorly understood chromatin regulator, recognizes H3K9me3 to mediate the silencing of ERV class I (ERV1) elements such as LTR12 (ref. 14). Biochemical, biophysical and structural studies identified the carboxy-terminal bromo-adjacent homology (BAH) domain of TNRC18 (TNRC18(BAH)) as an H3K9me3-specific reader. Moreover, the amino-terminal segment of TNRC18 is a platform for the direct recruitment of co-repressors such as HDAC-Sin3-NCoR complexes, thus enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18(BAH)-mediated H3K9me3 engagement caused neonatal death in mice and, in multiple mammalian cell models, led to derepressed expression of ERVs, which affected the landscape of cis-regulatory elements and, therefore, gene-expression programmes. Collectively, we describe a new H3K9me3-sensing and regulatory pathway that operates to epigenetically silence evolutionarily young ERVs and exert substantial effects on host genome integrity, transcriptomic regulation, immunity and development.

Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

Exosomal MicroRNA and Protein Profiles of Hepatitis B Virus-Related Hepatocellular Carcinoma Cells.

Infection with hepatitis B virus (HBV) is a main risk factor for hepatocellular carcinoma (HCC). Extracellular vesicles, such as exosomes, play an important role in tumor development and metastasis, including regulation of HBV-related HCC. In this study, we have characterized exosome microRNA and proteins released in vitro from hepatitis B virus (HBV)-related HCC cell lines SNU-423 and SNU-182 and immortalized normal hepatocyte cell lines (THLE2 and THLE3) using microRNA sequencing and mass spectrometry. Bioinformatics, including functional enrichment and network analysis, combined with survival analysis using data related to HCC in The Cancer Genome Atlas (TCGA) database, were applied to examine the prognostic significance of the results. More than 40 microRNAs and 200 proteins were significantly dysregulated (p < 0.05) in the exosomes released from HCC cells in comparison with the normal liver cells. The functional analysis of the differentially expressed exosomal miRNAs (i.e., mir-483, mir-133a, mir-34a, mir-155, mir-183, mir-182), their predicted targets, and exosomal differentially expressed proteins (i.e., POSTN, STAM, EXOC8, SNX9, COL1A2, IDH1, FN1) showed correlation with pathways associated with HBV, virus activity and invasion, exosome formation and adhesion, and exogenous protein binding. The results from this study may help in our understanding of the role of HBV infection in the development of HCC and in the development of new targets for treatment or non-invasive predictive biomarkers of HCC.

Cutting-Edge Technologies Driving Quantitative Mass Spectrometry.

ARTICLES DISCUSSED: Correa, C. N., Fiametti, L. O., Esquinca M. E. M., de Castro, L. M. Sample preparation and relative quantitation using reductive methylation of amines for peptidomics studies. Journal of Visualized Experiments. (177), doi:10.3791/62971 (2021). Vanderwall, D. et al. JUMPn: A streamlined application for protein co-expression clustering and network analysis in proteomics. Journal of Visualized Experiments. (176), doi:10.3791/62796 (2021). Qiu, D., Eisenbeis, V. B., Saiardi, A., Jessen, H. J. Absolute quantitation of inositol pyrophosphates by capillary electrophoresis electrospray ionization mass spectrometry. Journal of Visualized Experiments. (174), doi:10.3791/62847 (2021). Smolen, K. A., Kettenbach, A. N. A mass spectrometry-based approach to identify phosphoprotein phosphatases and their interactors. Journal of Visualized Experiments. (182), doi:10.3791/63805 (2022).

Targeting mitochondria in the aged cerebral vasculature with SS-31, a proteomic study of brain microvessels.

Cognitive impairment and dementias during aging such as Alzheimer's disease are linked to functional decline and structural alterations of the brain microvasculature. Although mechanisms leading to microvascular changes during aging are not clear, loss of mitochondria, and reduced efficiency of remaining mitochondria appear to play a major role. Pharmacological agents, such as SS-31, which target mitochondria have been shown to be effective during aging and diseases; however, the benefit to mitochondrial- and non-mitochondrial proteins in the brain microvasculature has not been examined. We tested whether attenuation of aging-associated changes in the brain microvascular proteome via targeting mitochondria represents a therapeutic option for the aging brain. We used aged male (> 18 months) C57Bl6/J mice treated with a mitochondria-targeted tetrapeptide, SS-31, or vehicle saline. Cerebral blood flow (CBF) was determined using laser speckle imaging during a 2-week treatment period. Then, isolated cortical microvessels (MVs) composed of end arterioles, capillaries, and venules were used for Orbitrap Eclipse Tribrid mass spectrometry. CBF was similar among the groups, whereas bioinformatic analysis revealed substantial differences in protein abundance of cortical MVs between SS-31 and vehicle. We identified 6267 proteins, of which 12% were mitochondria-associated. Of this 12%, 107 were significantly differentially expressed and were associated with oxidative phosphorylation, metabolism, the antioxidant defense system, or mitochondrial dynamics. Administration of SS-31 affected many non-mitochondrial proteins. Our findings suggest that mitochondria in the microvasculature represent a therapeutic target in the aging brain, and widespread changes in the proteome may underlie the rejuvenating actions of SS-31 in aging.

Musashi Exerts Control of Gonadotrope Target mRNA Translation During the Mouse Estrous Cycle.

The anterior pituitary controls key biological processes, including growth, metabolism, reproduction, and stress responses through distinct cell types that each secrete specific hormones. The anterior pituitary cells show a remarkable level of cell type plasticity that mediates the shifts in hormone-producing cell populations that are required to meet organismal needs. The molecular mechanisms underlying pituitary cell plasticity are not well understood. Recent work has implicated the pituitary stem cell populations and specifically, the mRNA binding proteins of the Musashi family in control of pituitary cell type identity. In this study we have identified the target mRNAs that mediate Musashi function in the adult mouse pituitary and demonstrate the requirement for Musashi function in vivo. Using Musashi RNA immunoprecipitation, we identify a cohort of 1184 mRNAs that show specific Musashi binding. Identified Musashi targets include the Gnrhr mRNA, which encodes the gonadotropin-releasing hormone receptor (GnRHR), and the Fshb mRNA, encoding follicle-stimulating hormone (FSH). Reporter assays reveal that Musashi functions to exert repression of translation of the Fshb mRNA, in addition to the previously observed repression of the Gnrhr mRNA. Importantly, mice engineered to lack Musashi in gonadotropes demonstrate a failure to repress translation of the endogenous Gnrhr and Fshb mRNAs during the estrous cycle and display a significant heterogeneity in litter sizes. The range of identified target mRNAs suggests that, in addition to these key gonadotrope proteins, Musashi may exert broad regulatory control over the pituitary proteome in a cell type-specific manner.