A dual role for Sox10 in the maintenance of the postnatal melanocyte lineage and the differentiation of melanocyte stem cell progenitors

During embryogenesis, the transcription factor, Sox10, drives the survival and differentiation of the melanocyte lineage. However, the role that Sox10 plays in postnatal melanocytes is not established. We show in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Sox10 knockout (Sox10(fl); Tg(Tyr::CreER)) results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10)) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and Sox10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of Tg(DctSox10) hair graying is through increased expression of Mitf, a target of SOX10, by asking if haploinsufficiency for Mitf (Mitf(vga9) ) can rescue hair graying in Tg(DctSox10) animals. Surprisingly, Mitf(vga9) does not mitigate but exacerbates Tg(DctSox10) hair graying suggesting that MITF participates in the negative regulation of Sox10 in McSCs. These observations demonstrate that while SOX10 is necessary to maintain the postnatal melanocyte lineage it is simultaneously prevented from driving differentiation in the McSCs. This data illustrates how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage

A Microfluidic Platform to Monitor Real-Time Effects of Extracellular Vesicle Exchange between Co-Cultured Cells across Selectively Permeable Barriers

Exosomes and other extracellular vesicles (EVs) play a significant yet poorly understood role in cell–cell communication during homeostasis and various pathological conditions. Conventional in vitro and in vivo approaches for studying exosome/EV function depend on time-consuming and expensive vesicle purification methods to obtain sufficient vesicle populations. Moreover, the existence of various EV subtypes with distinct functional characteristics and submicron size makes their analysis challenging. To help address these challenges, we present here a unique chip-based approach for real-time monitoring of cellular EV exchange between physically separated cell populations. The extracellular matrix (ECM)-mimicking Matrigel is used to physically separate cell populations confined within microchannels, and mimics tissue environments to enable direct study of exosome/EV function. The submicron effective pore size of the Matrigel allows for the selective diffusion of only exosomes and other smaller EVs, in addition to soluble factors, between co-cultured cell populations. Furthermore, the use of PEGDA hydrogel with a very small pore size of 1.2 nm in lieu of Matrigel allows us to block EV migration and, therefore, differentiate EV effects from effects that may be mediated by soluble factors. This versatile platform bridges purely in vitro and in vivo assays by enabling studies of EV-mediated cellular crosstalk under physiologically relevant conditions, enabling future exosome/EV investigations across multiple disciplines through real-time monitoring of vesicle exchange

Extracellular vesicles from infected cells: potential for direct pathogenesis

Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain Pathogen-Associated Molecular Patterns (PAMPs), regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical TLR and NFkB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many pathologies seen in infected hosts

Presence of Viral RNA and Proteins in Exosomes from Cellular Clones Resistant to Rift Valley Fever Virus Infection

Rift Valley Fever Virus (RVFV) is a RNA virus that belongs to the genus Phlebovirus, family Bunyaviridae. It infects humans and livestock and causes Rift Valley fever. RVFV is considered an agricultural pathogen by the USDA, as it can cause up to 100% abortion in cattle and extensive death of newborns. In addition, it is designated as Category A pathogen by the CDC and the NIAID. In some human cases of RVFV infection, the virus causes fever, ocular damage, liver damage, hemorrhagic fever, and death. There are currently limited options for vaccine candidates, which include the MP-12 and clone 13 versions of RVFV. Viral infections often deregulate multiple cellular pathways that contribute to replication and host pathology. We have previously shown that latent HIV-1 and HTLV-1 infected cells secrete exosomes that contain short viral RNAs, limited number of genomic RNAs, and viral proteins. These exosomes largely target neighboring cells and activate the NF-кB pathway, leading to cell proliferation and overall better viral replication. In this manuscript, we studied the effects of exosome formation from RVFV infected cells and their function on recipient cells. We initially infected cells, isolated resistant clones, and further purified using dilution cloning. We then characterized these cells as resistant to new RVFV infection, but sensitive to other viral infections, including Venezuelan Equine Encephalitis Virus (VEEV). These clones contained normal markers (i.e. CD63) for exosomes and were able to activate the TLR pathway in recipient reporter cells. Interestingly, the exosome rich preparations, much like their host cell, contained viral RNA (L, M, and S genome). The RNAs were detected using qRT-PCR in both parental and exosomal preparations as well as in CD63 immunoprecipitates. Viral proteins such as N and a modified form of NSs were present in some of these exosomes. Finally, treatment of recipient cells (T- cells and monocytic cells) showed drastic rate of apoptosis through PARP cleavage and caspase 3 activation from some but not all enriched exosomes. Collectively, these data suggest that exosomes from RVFV infected cells alter the dynamics of the immune cells and may contribute to pathology of the viral infection

Protein Phosphatase-1 regulates Rift Valley fever virus replication

Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target

Optimization of Protein Solubilization for the Analysis of the CD14 Human Monocyte Membrane Proteome Using LC-MS/MS

Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), iii) a combination of both agents (methanol+PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was approximately 98% positive for CD14 expression as determined by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46%) were mapped integral membrane proteins. However, employing PPS 216 proteins were identified of which 75 (35%) were mapped as integral membrane proteins. These results indicate that methanol alone or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone

Host Response during Yersinia pestis Infection of Human Bronchial Epithelial Cells Involves Negative Regulation of Autophagy and Suggests a Modulation of Survival-Related and Cellular Growth Pathways

Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of the signaling network changes in human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm- , Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and treatment of plague

Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40—Bacillus Specific Endolysins

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies

<i>Sox10</i> is required by bulb melanocytes postnatally.

<p>(A–B) <i>Sox10^fl/fl</i> (<i>fl/fl; +/+</i>) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (<i>fl/fl; Cre/+</i>) pups treated with TAM by IP injection to the lactating mother on P0–3 display variegated hypopigmentation on the belly and back and exhibit a white head spot upon the emergence of the morphogenetic coat (P10 shown here, n>5). (C) Number of PAX3⁺ melanocytes per hair bulb in skins harvested from these mice at P10 are significantly decreased in <i>Sox10^fl/fl; Tyr::CreERT2</i> animals compared to similarly-treated <i>Sox10^fl/fl</i> animals (*p = 0.002). (D–E) Adult <i>Sox10^fl/fl; Tyr::CreERT2</i> mice treated with TAM by IP injection on 0–3dpp exhibit white hairs within the plucked region upon hair regrowth that is not visible in similarly treated <i>Sox10^fl/fl</i> mice (brackets indicate plucked region, lower image is a magnification of plucked region). (F) Number of PAX3⁺ melanocytes per hair bulb in skins harvested from similarly-treated mice at 7dpp are significantly decreased in <i>Sox10^fl/fl; Tyr::CreERT2</i> animals compared to <i>Sox10^fl/fl</i> animals (*p = 0.001). (G–H) Fluorescent and corresponding brightfield images of hair bulbs from mice described in D–E. Arrows and arrowheads indicate PAX3⁺/SOX10⁺ and PAX3⁺/SOX10⁻ melanocytes, respectively. (I) Distribution of melanocytes double-labeled for PAX3 and SOX10 within pigmented (gray) and non-pigmented (white) hair bulbs in skins from <i>Sox10^fl/fl</i> (n = 3) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (n = 4) harvested on 7dpp from mice treated with TAM on 0–3dpp (*p<0.006).</p

<i>Tg(DctSox10)</i> results in congenital white spotting and premature hair graying.

<p>(A, B) Ventral and dorsal views demonstrating variable hypopigmentation in <i>Tg(DctSox10)/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i> mice during hair morphogenesis and adult hair cycling. (C) Frequency of pigmented (pig+) and non-pigmented (pig−) anagen III/IV (7dpp) hairs that contain (DCT+ LPP cells) or do not contain (no LPP cells) LPP melanocytes within <i>Tg(DctSox10)</i> or <i>+/+</i> mice. The ages of mice analyzed ranged between 9–22 weeks at harvest. Significance determined by chi-square analysis (p<<0.0001) and evaluation of standardized residuals (*, z = −8.84; **, z = 12.24).</p