USP8 inhibition regulates autophagy flux and controls Salmonella infection.

Introduction: Ubiquitination is an important protein modification that regulates various essential cellular processes, including the functions of innate immune cells. Deubiquitinases are enzymes responsible for removing ubiquitin modification from substrates, and the regulation of deubiquitinases in macrophages during infection with Salmonella Typhimurium and Yersinia enterocolitica remains unknown. Methods: To identify deubiquitinases regulated in human macrophages during bacterial infection, an activity-based proteomics screen was conducted. The effects of pharmacological inhibition of the identified deubiquitinase, USP8, were examined, including its impact on bacterial survival within macrophages and its role in autophagy regulation during Salmonella infection. Results: Several deubiquiitnases were differentially regulated in infected macrophages. One of the deubiquitinases identified was USP8, which was downregulated upon Salmonella infection. Inhibition of USP8 was associated with a decrease in bacterial survival within macrophages, and it was found to play a distinct role in regulating autophagy during Salmonella infection. The inhibition of USP8 led to the downregulation of the p62 autophagy adaptor. Discussion: The findings of this study suggest a novel role of USP8 in regulating autophagy flux, which restricts intracellular bacteria, particularly during Salmonella infection.

Murine Norovirus Interaction with Enterobacter cloacae Leads to Changes in Membrane Stability and Packaging of Lipid and Metabolite Vesicle Content.

Outer membrane vesicles (OMVs) are a primary means of communication for Gram-negative bacteria. The specific role of vesicle components in cellular communication and how components are packaged are still under investigation, but a correlation exists between OMV biogenesis and content. The two primary mechanisms of OMV biogenesis are membrane blebbing and explosive cell lysis, and vesicle content is based on the biogenesis mechanism. Hypervesiculation, which can be induced by stress conditions, also influences OMV content. Norovirus interaction with Enterobacter cloacae induces stress responses leading to increased OMV production and changes in DNA content, protein content, and vesicle size. The presence of genomic DNA and cytoplasmic proteins in these OMVs suggests some of the vesicles are formed by explosive cell lysis, so reduction or loss of these components indicates a shift away from this mechanism of biogenesis. Based on this, further investigation into bacterial stability and OMV content was conducted. Results showed that norovirus induced a dramatic shift in OMV lipid content. Specifically, the increased accumulation of phospholipids is associated with increased blebbing, thereby supporting previous observations that noroviruses shift the mechanism of OMV biogenesis. Slight differences in OMV metabolite content were also observed. While norovirus induced changes in OMV content, it did not change the lipid content of the bacterial outer membrane or the metabolite content of the bacterial cell. Overall, these results indicate that norovirus induces significant changes to OMV lipid architecture and cargo, which may be linked to a change in the mechanism of vesicle biogenesis. IMPORTANCE Extracellular vesicles from commensal bacteria are recognized for their importance in modulating host immune responses, and vesicle content is related to their impact on the host. Therefore, understanding how vesicles are formed and how their content shifts in response to stress conditions is necessary for elucidating their downstream functions. Our recent work has demonstrated that interactions between noroviruses and Enterobacter cloacae induce bacterial stress responses leading to hypervesiculation. In this article, we characterize and compare the lipid and metabolomic cargo of E. cloacae vesicles generated in the presence and absence of norovirus and show that viral interactions induce significant changes in vesicle content. Furthermore, we probe how these changes and changes to the bacterial cell may be indicative of a shift in the mechanism of vesicle biogenesis. Importantly, we find that noroviruses induce significant changes in vesicle lipid architecture and cargo that may be responsible for the immunogenic activity of these vesicles.

Using Activity-Based Proteomics for the Quantification of Deubiquitinases in Animal Tissue.

Ubiquitination is a post-translational modification, that regulates essential cellular functions, and the enzymes that control the removal of this modification, deubiquitinases (DUBs), have been well described for the model organisms. However, the information about DUBs is still largely lacking for the non-model organisms, such as agriculturally relevant animals. To understand the expression of these enzymes in animal tissues, we have used chemical proteomics which can be used to identify biologically active DUBs present in tissues based on their reactivity with the activity-based probes (ABPs). Here we describe a sample preparation protocol for ABP-based purification of DUBs from animal tissue using two approaches to homogenize and lyse the animal tissue compatible with ABP labeling of DUBs, including an ultrasonication-based tissue processing method and bead-beating method. Both of these methods retain the enzymatic activity of DUBs. In addition, we describe a protocol for ABP labeling of DUBs in tissue lysates and the immunoprecipitation of the probe-reactive DUBs that can be used along with mass spectrometric identification of proteins and the detection of these DUBs by Western blotting.

Leishmania infection-derived extracellular vesicles drive transcription of genes involved in M2 polarization.

Although it is known that the composition of extracellular vesicles (EVs) is determined by the characteristics of the cell and its environment, the effects of intracellular infection on EV composition and functions are not well understood. We had previously shown that cultured macrophages infected with Leishmania parasites release EVs (LiEVs) containing parasite-derived molecules. In this study we show that LdVash, a molecule previously identified in LiEVs from L. donovani infected RAW264.7 macrophages, is widely distributed in the liver of L. donovani infected mice. This result shows for the first time that parasite molecules are released in EVs and distributed in infected tissues where they can be endocytosed by cells in the liver, including macrophages that significantly increase numbers as the infection progresses. To evaluate the potential impact of LiEVs on macrophage functions, we show that primary peritoneal exudate macrophages (PECs) express transcripts of signature molecules of M2 macrophages such as arginase 1, IL-10, and IL-4R when incubated with LiEVs. In comparative studies that illustrate how intracellular pathogens control the composition and functions of EVs released from macrophages, we show that EVs from RAW264.7 macrophages infected with Salmonella Typhimurium activate PECs to express transcripts of signature molecules of M1 macrophages such as iNOS, TNF alpha, and IFN-gamma and not M2 signature molecules. Finally, in contrast to the polarized responses observed in in vitro studies of macrophages, both M1 and M2 signature molecules are detected in L. donovani infected livers, although they exhibit differences in their spatial distribution in infected tissues. In conclusion, EVs produced by macrophages during Leishmania infection lead to the gene expression consistent with M2 polarization. In contrast, the EVs produced during S. Typhimurium infection stimulated the transcription of genes associated with M1 polarization.

Extracellular vesicles elicit protective immune responses against Salmonella infection.

Small extracellular vesicles (sEVs) produced by antigen-presenting cells represent a novel mechanism of cell-to-cell communication. The sEVs have been shown to drive Th1-type adaptive immune responses against intracellular infections such as Salmonella. In this study, we have demonstrated that an administration of sEVs produced by Salmonella-infected macrophages to BALB/c mice that were then challenged with Salmonella infection decreased bacterial load in infected animals and led to protection against a lethal dose of Salmonella. Second, the same sEVs induced a robust production of IgA anti-Salmonella antibodies (Abs) in BALB/c mice, including IgA anti-OmpD Abs. These results show that the nanoscale sEVs stimulate adaptive immune responses against intracellular pathogens and that these sEVs can be used to provide animals with complete protection against lethal infection, such as the systemic bacterial infection in immunodeficient BALB/c mice.

Erratum: A CTS Team Approach to Wastewater-Based Epidemiology of Non-Typhoidal Salmonella in Gainesville, FL - ERRATUM.

[This corrects the article DOI: 10.1017/cts.2022.23.].

Bacterial extracellular vesicles control murine norovirus infection through modulation of antiviral immune responses.

Human norovirus is the primary cause of non-bacterial gastroenteritis globally and is the second leading cause of diarrheal deaths in children in developing countries. However, effective therapeutics which prevent or clear norovirus infection are not yet available due to a lack of understanding regarding norovirus pathogenesis. Evidence shows that noroviruses can bind to the surface of commensal bacteria, and the presence of these bacteria alters both acute and persistent murine norovirus infection through the modulation of host immune responses. Interestingly, norovirus-bacterial interactions also affect the bacteria by inducing bacterial stress responses and increasing the production of bacterial extracellular vesicles. Given the established ability of these vesicles to easily cross the intestinal barriers, enter the lamina propria, and modulate host responses, we hypothesized that bacterial extracellular vesicles influence murine norovirus infection through modulation of the antiviral immune response. In this study, we show that murine norovirus can attach to purified bacterial vesicles, facilitating co-inoculation of target cells with both virus and vesicle. Furthermore, we have found that when murine noroviruses and vesicles are used to co-inoculate macrophages, viral infection is reduced compared to virus infection alone. Specifically, co-inoculation with bacterial vesicles results in higher production and release of pro-inflammatory cytokines in response to viral infection. Ultimately, given that murine norovirus infection increases bacterial vesicle production in vivo, these data indicate that bacterial vesicles may serve as a mechanism by which murine norovirus infection is ultimately controlled and limited to a short-term disease.

Fluorescent Labeling of Small Extracellular Vesicles (EVs) Isolated from Conditioned Media.

Extracellular vesicles (EVs), such as exosomes, are produced by all known eukaryotic cells, and constitute essential means of intercellular communication. Recent studies have unraveled the important roles of EVs in migrating to specific sites and cells. Functional studies of EVs using in vivo and in vitro systems require tracking these organelles using fluorescent dyes or, alternatively, transfected and fluorescent-tagged proteins, located either intravesicularly or anchored to the EV bilayer membrane. Due to design simplicity, the fluorescent dye might be a preferred method if the cells are difficult to modify by transfection or when the genetic alteration of the mother cells is not desired. This protocol describes techniques to label cultured cell-derived EVs, using lipophilic DiR [DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide)] fluorophore. This technique can be used to study the cellular uptake and intracellular localization of EVs, and their biodistribution in vivo , which are crucial evaluations of any isolated EVs.

Current understanding of extracellular vesicle homing/tropism.

Extracellular vesicles (EVs) are membrane-enclosed packets released from cells that can transfer bioactive molecules from cell to cell without direct contact with the target cells. This transfer of molecules can activate consequential processes in the recipient cells, including cell differentiation and migration that maintain tissue homeostasis or promote tissue pathology. One controversial aspect of the EV's biology that holds therapeutic promise is their capacity to engage defined cells at specific sites. On the one hand, persuasive studies have shown that EVs express surface molecules that ensure their tissue localization and enable cell-specific interactions, as demonstrated using in vitro and in vivo analyses. Therefore, this feature of EV biology is under investigation in translational studies to control malignancies and deliver chemicals and bioactive molecules to combat several diseases. On the other hand, some studies have shown that EVs fail to traffic in hosts in a targeted manner, which questions the potential role of EVs as vehicles for drug delivery and their capacity to serve as cell-free biomodulators. In this review, the biology of EV homing/tropism in mammalian hosts is discussed, and the biological characteristics that may result in their controversial characteristics are brought to the fore.

Interaction with mammalian enteric viruses alters outer membrane vesicle production and content by commensal bacteria.

Intestinal commensal bacteria contribute to maintaining gut homeostasis. Disruptions to the commensal flora are linked to the development and persistence of disease. The importance of these organisms is further demonstrated by the widespread ability of enteric viruses to exploit commensal bacteria to enhance viral infection. These viruses interact directly with commensal bacteria, and while the impact of this interaction on viral infection is well described for several viruses, the impact on the commensal bacteria has yet to be explored. In this article, we demonstrate, for the first time, that enteric viruses alter the gene expression and phenotype of individual commensal bacteria. Human and murine norovirus interaction with bacteria resulted in genome-wide differential gene expression and marked changes in the surface architecture of the bacterial cells. Furthermore, the interaction of the virus with bacteria led to increased production of smaller outer membrane vesicles (OMVs). Enhanced production of smaller vesicles was also observed when noroviruses were incubated with other commensal bacteria, indicating a potentially broad impact of norovirus interaction. The vesicle production observed in the in vivo model followed a similar trend where an increased quantity of smaller bacterial vesicles was observed in stool collected from virus-infected mice compared to mock-infected mice. Furthermore, changes in vesicle size were linked to changes in protein content and abundance, indicating that viral binding induced a shift in the mechanism of the OMV biogenesis. Collectively, these data demonstrate that enteric viruses induce specific changes in bacterial gene expression, leading to changes in bacterial extracellular vesicle production that can potentially impact host responses to infection.

Identification of active deubiquitinases in the chicken tissues.

The existing protein annotation in chicken is mostly limited to computational predictions based on orthology to other proteins, which often leads to a significant underestimation of the function of these proteins. Genome-scale experimental annotation can provide insight into the actual enzymatic activities of chicken proteins. Amongst post-translational modifications, ubiquitination is of interest as anomalies in ubiquitination are implicated in such diseases as inflammatory disorders, infectious diseases, or malignancies. Ubiquitination is controlled by deubiquitinases (DUBs), which remove ubiquitin from protein substrates. However, the DUBs have not been systematically annotated and quantified in chicken tissues. Here we used a chemoproteomics approach, which is based on active-site probes specific to DUBs, and identified 26 active DUBs in the chicken spleen, cecum, and liver.

Yersinia pseudotuberculosis YopJ Limits Macrophage Response by Downregulating COX-2-Mediated Biosynthesis of PGE2 in a MAPK/ERK-Dependent Manner.

Prostaglandin E2 (PGE2) is an essential immunomodulatory lipid released by cells in response to infection with many bacteria, yet its function in macrophage-mediated bacterial clearance is poorly understood. Yersinia overall inhibits the inflammatory circuit, but its effect on PGE2 production is unknown. We hypothesized that one of the Yersinia effector proteins is responsible for the inhibition of PGE2 biosynthesis. We identified that yopB-deficient Y. enterocolitica and Y. pseudotuberculosis deficient in the secretion of virulence proteins via a type 3 secretion system (T3SS) failed to inhibit PGE2 biosynthesis in macrophages. Consistently, COX-2-mediated PGE2 biosynthesis is upregulated in cells treated with heat-killed or T3SS-deficient Y. pseudotuberculosis but diminished in the presence of a MAPK/ERK inhibitor. Mutants expressing catalytically inactive YopJ induce similar levels of PGE2 as heat-killed or ΔyopB Y. pseudotuberculosis, reversed by YopJ complementation. Shotgun proteomics discovered host pathways regulated in a YopJ-mediated manner, including pathways regulating PGE2 synthesis and oxidative phosphorylation. Consequently, this study identified that YopJ-mediated inhibition of MAPK signal transduction serves as a mechanism targeting PGE2, an alternative means of inflammasome inhibition by Yersinia. Finally, we showed that EP4 signaling supports macrophage function in clearing intracellular bacteria. In summary, our unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription, thereby enhancing the intracellular survival of yersiniae. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic molecule to improve the outcomes of specific bacterial infections. Since other pathogens encode YopJ homologs, this mechanism is expected to be present in other infections. IMPORTANCE PGE2 is a critical immunomodulatory lipid, but its role in bacterial infection and pathogen clearance is poorly understood. We previously demonstrated that PGE2 leads to macrophage polarization toward the M1 phenotype and stimulates inflammasome activation in infected macrophages. Finally, we also discovered that PGE2 improved the clearance of Y. enterocolitica. The fact that Y. enterocolitica hampers PGE2 secretion in a type 3 secretion system (T3SS)-dependent manner and because PGE2 appears to assist macrophage in the clearance of this bacterium indicates that targeting of the eicosanoid pathway by Yersinia might be an adaption used to counteract host defenses. Our study identified a mechanism used by Yersinia that obstructs PGE2 biosynthesis in human macrophages. We showed that Y. pseudotuberculosis interferes with PGE2 biosynthesis by using one of its T3SS effectors, YopJ. Specifically, YopJ targets the host COX-2 enzyme responsible for PGE2 biosynthesis, which happens in a MAPK/ER-dependent manner. Moreover, in a shotgun proteomics study, we also discovered other pathways that catalytically active YopJ targets in the infected macrophages. YopJ was revealed to play a role in limiting host LPS responses, including repression of EGR1 and JUN proteins, which control transcriptional activation of proinflammatory cytokine production such as interleukin-1ß. Since YopJ has homologs in other bacterial species, there are likely other pathogens that target and inhibit PGE2 biosynthesis. In summary, our study's unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic target.

Intravacuolar Pathogens Hijack Host Extracellular Vesicle Biogenesis to Secrete Virulence Factors.

Extracellular vesicles (EVs) have garnered significant interest in recent years due to their contributions to cell-to-cell communication and disease processes. EVs are composed of a complex profile of bioactive molecules, which include lipids, nucleic acids, metabolites, and proteins. Although the biogenesis of EVs released by cells under various normal and abnormal conditions has been well-studied, there is incomplete knowledge about how infection influences EV biogenesis. EVs from infected cells contain specific molecules of both host and pathogen origin that may contribute to pathogenesis and the elicitation of the host immune response. Intracellular pathogens exhibit diverse lifestyles that undoubtedly dictate the mechanisms by which their molecules enter the cell's exosome biogenesis schemes. We will discuss the current understanding of the mechanisms used during infection to traffic molecules from their vacuolar niche to host EVs by selected intravacuolar pathogens. We initially review general exosome biogenesis schemes and then discuss what is known about EV biogenesis in Mycobacterium, Plasmodium, Toxoplasma, and Leishmania infections, which are pathogens that reside within membrane delimited compartments in phagocytes at some time in their life cycle within mammalian hosts. The review includes discussion of the need for further studies into the biogenesis of EVs to better understand the contributions of these vesicles to host-pathogen interactions, and to uncover potential therapeutic targets to control these pathogens.

Roles of Eicosanoids in Regulating Inflammation and Neutrophil Migration as an Innate Host Response to Bacterial Infections.

Eicosanoids are lipid-based signaling molecules that play a unique role in innate immune responses. The multiple types of eicosanoids, such as prostaglandins (PGs) and leukotrienes (LTs), allow the innate immune cells to respond rapidly to bacterial invaders. Bacterial pathogens alter cyclooxygenase (COX)-derived prostaglandins (PGs) in macrophages, such as PGE2 15d-PGJ2, and lipoxygenase (LOX)-derived leukotriene LTB4, which has chemotactic functions. The PG synthesis and secretion are regulated by substrate availability of arachidonic acid and by the COX-2 enzyme, and the expression of this protein is regulated at multiple levels, both transcriptionally and posttranscriptionally. Bacterial pathogens use virulence strategies such as type three secretion systems (T3SSs) to deliver virulence factors altering the expression of eicosanoid-specific biosynthetic enzymes, thereby modulating the host response to bacterial lipopolysaccharides (LPS). Recent advances have identified a novel role of eicosanoids in inflammasome activation during intracellular infection with bacterial pathogens. Specifically, PGE2 was found to enhance inflammasome activation, driving the formation of pore-induced intracellular traps (PITs), thus trapping bacteria from escaping the dying cell. Finally, eicosanoids and IL-1ß released from macrophages are implicated in the efferocytosis of neighboring neutrophils. Neutrophils play an essential role in phagocytosing and degrading PITs and associated bacteria to restore homeostasis. This review focuses on the novel functions of host-derived eicosanoids in the host-pathogen interactions.

Characterization and proteomic analysis of outer membrane vesicles from a commensal microbe, Enterobacter cloacae.

Outer membrane vesicles (OMVs) are membrane-enclosed spherical entities released by gram-negative bacteria and are important for bacterial survival under stress conditions. There have been numerous studies on OMVs released by gram-negative pathogenic bacteria, but an understanding of the functions and characteristics of the OMVs produced by commensal microbes is still lacking. Enterobacter cloacae is a gram-negative commensal bacterium present in the human gut microbiome, but this organism can also function as an opportunistic pathogen. Understanding the OMV-mediated communication route between bacteria-bacteria or bacteria-host is essential for the determination of the biological functions of the commensal bacterium in the gut and delineating between benign and virulent characteristics. In this study, we have described a proteome of E. cloacae OMVs, which are membrane vesicles in a size range of 20-300 nm. Proteomic analysis showed the presence of membrane-bound proteins, including transporters, receptors, signaling molecules, and protein channels. The physical and proteomic analyses also indicate this bacterium uses two mechanisms for OMV production. This study is one of the few existing descriptions of the proteomic profile of OMVs generated by a commensal Proteobacteria, and the first report of OMVs produced by E. cloacae. SIGNIFICANCE: This study prioritizes the importance of understanding the vesicular proteome of the human commensal bacterium, Enterobacter cloacae. We demonstrate for the first time that the gram-negative bacterium E. cloacae ATCC 13047 produces outer membrane vesicles (OMVs). The proteomic analysis showed enrichment of membrane-bound proteins in these vesicles. Understanding the cargo proteins of OMVs will help in exploring the physiological and functional role of these vesicles in the human microbiome and how they assist in the conversion of a bacterium from commensal to pathogen under certain conditions. While EM images reveal vesicles budding from the bacterial surface, the presence of cytoplasmic proteins and genomic DNA within the OMVs indicate that explosive cell lysis is an additional mechanism of biogenesis for these OMVs along with outer membrane blebbing. This research encourages future work on characterizing membrane vesicles produced by commensal bacterial and investigating their role in cell to cell communication.

CNS-Targeting Therapies for Lysosomal Storage Diseases: Current Advances and Challenges.

During the past decades, several therapeutic approaches have been developed and made rapidly available for many patients afflicted with lysosomal storage disorders (LSDs), inborn organelle disorders with broad clinical manifestations secondary to the progressive accumulation of undegraded macromolecules within lysosomes. These conditions are individually rare, but, collectively, their incidence ranges from 1 in 2,315 to 7,700 live-births. Most LSDs are manifested by neurological symptoms or signs, including developmental delay, seizures, acroparesthesia, motor weakness, and extrapyramidal signs. The chronic and later-onset clinical forms are at one end of the continuum spectrum and are characterized by a subtle and slow progression of neurological symptoms. Due to its inherent physiological properties, unfortunately, the blood-brain barrier (BBB) constitutes a significant obstacle for current and upcoming therapies to achieve the central nervous system (CNS) and treat neurological problems so prevalent in these conditions. To circumvent this limitation, several strategies have been developed to make the therapeutic agent achieve the CNS. This narrative will provide an overview of current therapeutic strategies under development to permeate the BBB, and address and unmet need for treatment of the progressive neurological manifestations, which are so prevalent in these inherited lysosomal disorders.

An atlas of the catalytically active liver and spleen kinases in chicken identified by chemoproteomics.

Phosphorylation is a post-translational protein modification regulating most known cellular processes. While protein kinases constitute a large family of highly conserved enzymes, identification of active kinases is challenging due to a low abundance of some of these signaling molecules. Although chicken is the first agricultural animal to have a sequenced genome, annotation of the kinome, i.e., a complement of all protein kinases in the genome is limited. We used chemical probes consisting of ATP and ADP derivatives binding to specific lysine (Lys) residues within the ATP-binding pocket of kinases, combined with proteomics, to identify 267 peptides labeled with the ATP and ADP acyl derivatives and 188 corresponding chicken kinases in chicken spleen and liver. Our description of active chicken kinases and ATP binding sites will support future studies focused on identifying the role of this important class of enzymes in chicken health and disease. SIGNIFICANCE: Advances made in understanding chicken enzymes are critical for the improved knowledge of the regulatory pathways controlling physiological processes in chicken. Since protein phosphorylation controls multiple aspects of cell fate, it is often linked to pathological conditions, and understanding of the kinase expression in chicken is essential for future therapeutic approaches. We coupled proteomics and labeling with active-site probes binding to Lys residues within the ATP-binding pocket of kinases to identify 188 kinases and corresponding 267 peptides labeled with the ATP and ADP acyl derivatives in chicken spleen and liver. Results of the present study describing catalytically active kinases is a starting point for chemoproteomic-based interrogation of kinases in chicken exposed to different conditions. Kinases identified in this study are available through the Chickspress genome browser that has previously published mRNA, miRNA, and shotgun proteomics data.

Exosomes released by breast cancer cells under mild hyperthermic stress possess immunogenic potential and modulate polarization in vitro in macrophages.

Macrophages play a dual role in tumor initiation and progression, with both tumor-promoting and tumor-suppressive effects; hence, it is essential to understand the distinct responses of macrophages to tumor progression and therapy. Mild hyperthermia has gained importance as a therapeutic regimen against cancer due to its immunogenic nature, efficacy, and potential synergy with other therapies, yet the response of macrophages to molecular signals from hyperthermic cancer cells has not yet been clearly defined. Due to limited response rate of breast cancer to conventional therapeutics the development, and understanding of alternative therapies like hyperthermia is pertinent. In order to determine conditions corresponding to mild thermal dose, cytotoxicity of different hyperthermic temperatures and treatment durations were tested in normal murine macrophages and breast cancer cell lines. Examination of exosome release in hyperthermia-treated cancer cells revealed enhanced efflux and a larger size of exosomes released under hyperthermic stress. Exposure of naïve murine macrophages to exosomes released from 4T1 and EMT-6 cells posthyperthermia treatment, led to an increased expression of specific macrophage activation markers. Further, exosomes released by hyperthermia-treated cancer cells had increased content of heat shock protein 70 (Hsp70). Together, these results suggest a potential immunogenic role for exosomes released from cancer cells treated with mild hyperthermia.

Long-term Changes in the Central Amygdala Proteome in Rats with a History of Chronic Cocaine Self-administration.

The central nucleus of the amygdala (CeA) is a striatum-like structure that contains mainly inhibitory circuits controlling a repertoire of (mal)adaptive behaviors related to pain, anxiety, motivation, and addiction. Neural activity in the CeA is also necessary for the expression of persistent and robust drug seeking, also termed 'incubation of drug craving.' However, neuroadaptations within this brain region supporting incubated drug craving have not been characterized. Here, we conducted a comprehensive analysis of protein expression in the CeA of male rats after prolonged (45-day) abstinence from extended-access cocaine self-administration using a quantitative proteomic approach. The proteomic analysis identified 228 unique proteins altered in cocaine rats relative to animals that received saline. Out of the identified proteins, 160 were downregulated, while 68 upregulated. Upregulation of tyrosine hydroxylase and downregulation of neural cell-adhesion protein contactin-1 were validated by immunoblotting. Follow-up analysis by the Ingenuity Pathway Analysis tool revealed alterations in protein networks associated with several neurobehavioral disorders, cellular function and morphology, as well as axogenesis, long-term potentiation, and receptor signaling pathways. This study suggests that chronic cocaine self-administration, followed by a prolonged abstinence results in reorganization of specific protein signaling networks within the CeA that may underlie incubated cocaine craving and identifies potential novel 'druggable' targets for the treatment of cocaine use disorder (CUD).

Modification of the host ubiquitome by bacterial enzymes.

Attachment of ubiquitin molecules to protein substrates is a reversible post-translational modification (PTM), which occurs ubiquitously in eukaryotic cells and controls most cellular processes. As a consequence, ubiquitination is an attractive target of pathogen-encoded virulence factors. Pathogenic bacteria have evolved multiple mechanisms to hijack the host's ubiquitin system to their advantage. In this review, we discuss the bacteria-encoded E3 ligases and deubiquitinases translocated to the host for an addition or removal of eukaryotic ubiquitin modification, effectively hijacking the host's ubiquitination processes. We review bacterial enzymes homologous to host proteins in sequence and functions, as well as enzymes with novel mechanisms in ubiquitination, which have significant structural differences in comparison to the mammalian E3 ligases. Finally, we will also discuss examples of molecular "counter-weapons" - eukaryotic proteins, which counteract pathogen-encoded E3 ligases. The many examples of the pathogen effector molecules that catalyze eukaryotic ubiquitin modification bring to light the intricate pathways involved in the pathogenesis of some of the most virulent bacterial infections with human pathogens. The role of these effector molecules remains an essential determinant of bacterial virulence in terms of infection, invasion, and replication. A comprehensive understanding of the mechanisms dictating the mimicry employed by bacterial pathogens is of vital importance in developing new strategies for therapeutic approaches.