A simple undergraduate laboratory exercise for teaching the role of sentinel-level clinical microbiology testing in biological threat identification.

Undergraduate students in the biomedical sciences are mostly unaware of how clinical microbiology laboratories handle suspected agents of bioterrorism or emerging infectious diseases. The Public Health Security Bioterrorism Preparedness and Response Act of 2002 requires the US Department of Health and Human Services (HHS) to maintain a list of microbes that pose serious biological threats to human health and safety, including Tier 1 agents with the potential for use in bioterrorism. The Laboratory Response Network (LRN), founded by the Centers for Disease Control and Prevention, the Federal Bureau of Investigation, and the Association of Public Health Laboratories, coordinates the response of sentinel, reference, and national laboratories to these biothreats. The sentinel laboratories, which comprise most hospital-based and commercial laboratories, are the first to encounter a suspicious agent. For this reason, the LRN has published a series of testing guidelines to assist the sentinel laboratories in deciding whether a microbial isolate should be considered potentially hazardous and thus sent to a reference or national laboratory for further characterization. Here, we describe a simple laboratory exercise that teaches sentinel-level testing requirements in the context of an applied setting of a potential outbreak of anthrax that would require a sentinel laboratory to recognize a potential threat, attempt to rule it out, and refer to a national laboratory for identification.

Cell death induction in Aspergillus fumigatus: accentuating drug toxicity through inhibition of the unfolded protein response (UPR).

One of the most potent opportunistic fungal pathogens of humans is Aspergillus fumigatus, an environmental mold that causes a life-threatening pneumonia with a high rate of morbidity and mortality. Despite advances in therapy, issues of drug toxicity and antifungal resistance remain an obstacle to effective therapy. This underscores the need for more information on fungal pathways that could be pharmacologically manipulated to either reduce the viability of the fungus during infection, or to unleash the fungicidal potential of current antifungal drugs. In this review, we summarize the emerging evidence that the ability of A. fumigatus to sustain viability during stress relies heavily on an adaptive signaling pathway known as the unfolded protein response (UPR), thereby exposing a vulnerability in this fungus that has strong potential for future therapeutic intervention.

Pleiotropic Effects of the P5-Type ATPase SpfA on Stress Response Networks Contribute to Virulence in the Pathogenic Mold Aspergillus fumigatus.

Aspergillus fumigatus is a human-pathogenic mold that extracts nutrients from the environment or from host tissues by secreting hydrolytic enzymes. The ability of A. fumigatus to adjust secretion levels in proportion to demand relies on the assistance of the unfolded protein response (UPR), an adaptive stress response pathway that regulates the unique protein-folding environment of the endoplasmic reticulum (ER). The P5-type ATPase Spf1 has recently been implicated in a novel mechanism of ER homeostasis that involves correcting errors in ER-membrane protein targeting. However, the contribution of this protein to the biology of A. fumigatus is unknown. Here, we employed a gene knockout and RNA sequencing strategy to determine the functional role of the A. fumigatus gene coding for the orthologous P5 ATPase SpfA. The data reveal that the spfA gene is induced by ER stress in a UPR-dependent manner. In the absence of spfA, the A. fumigatus transcriptome shifts toward a profile of altered redox and lipid balance, in addition to a signature of ER stress that includes srcA, encoding a second P-type ATPase in the ER. A ΔspfA deletion mutant showed increased sensitivity to ER stress, oxidative stress, and antifungal drugs that target the cell wall or plasma membrane. The combined loss of spfA and srcA exacerbated these phenotypes and attenuated virulence in two animal infection models. These findings demonstrate that the ER-resident ATPases SpfA and SrcA act jointly to support diverse adaptive functions of the ER that are necessary for fitness in the host environment. IMPORTANCE The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus.

Mesenchymal stromal cell mitochondrial transfer to human induced T-regulatory cells mediates FOXP3 stability.

The key obstacle to clinical application of human inducible regulatory T cells (iTreg) as an adoptive cell therapy in autoimmune disorders is loss of FOXP3 expression in an inflammatory milieu. Here we report human iTreg co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) during short-term ex vivo expansion enhances the stability of iTreg FOXP3 expression and suppressive function in vitro and in vivo, and further that a key mechanism of action is MSC mitochondrial (mt) transfer via tunneling nanotubules (TNT). MSC mt transfer is driven by mitochondrial metabolic function (CD39/CD73 signaling) in proliferating iTreg and promotes iTreg expression of FOXP3 stabilizing factors BACH2 and SENP3. These results elucidate cellular and molecular mechanisms underlying human MSC mt transfer to proliferating cells. MSC mt transfer stabilizes FOXP3 expression in iTregs, thereby enhancing and sustaining their suppressive function in inflammatory conditions in vitro and in vivo.

Notch-Regulated Dendritic Cells Restrain Inflammation-Associated Colorectal Carcinogenesis.

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.

Effect of Asian dust on respiratory symptoms among children with and without asthma, and their sensitivity.

There has been little study on the effect of Asian dust exposure on respiratory symptoms among children who are vulnerable to environmental factors. In this panel study, we investigated the effect of Asian dust on respiratory symptoms among children with and without asthma, and their sensitivity. Children attending two elementary schools (137 total), and 23 children with asthma from cooperating medical institutions in Fukuoka prefecture were recruited. Subjects measured peak expiratory flow rate (PEF), and recorded asthma-like symptoms, cough, nasal symptoms and use of medication in a diary from April 1, 2013 to June 30, 2013. To assess exposure to Asian dust, we used Light Detection and Ranging (LIDAR) data. For the analysis of the association between Asian dust and respiratory symptoms, the case-crossover design and generalized estimating equation (GEE) models were used. Taking individual sensitivity to respiratory aggravation into consideration, the subjects were classified into three groups: children without asthma, children with asthma who do not use long-term preventive medication (CA) and children with asthma who use long-term preventive medication (CA-LTM). For CA, Asian dust exposure was significantly associated with asthma-like symptoms, with a hazard ratio of 5.17 (95%CI: 1.02=26.12) at Lag0, and the change in %maxPEF, -1.65% (95%CI:-2.82, -0.48) at Lag0. For children without asthma, a statistically significant association was found between Asian dust exposure and the change in %maxPEF, -0.56% (95%CI: -1.31, -0.08) at Lag1. However, no adverse effects were observed in CA-LTM. Temperature had significant effects on %maxPEF for three groups. Asian dust, photochemical oxidant and pollen caused simultaneously additive adverse effects on nasal symptoms for children without asthma. This study suggests the possibility that long-term preventive medication to manage asthma may suppress aggravation of respiratory symptoms due to Asian dust and may be an effective prevention.

A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus Aspergillus fumigatus and Suggests Noncanonical Functions within the Pathway.

The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA, which splices the downstream mRNA hacAu into its active form, hacAi, encoding the master transcriptional regulator of the pathway. Small-molecule inhibitors against IRE1, the human ortholog of IreA, have been developed for anticancer therapy, but their effects on the fungal UPR are unexplored. Here, we demonstrate that the IRE1 RNase inhibitor 4µ8C prevented A. fumigatus from increasing the levels of hacAi mRNA, thereby blocking induction of downstream UPR target gene expression. Treatment with 4µ8C had minimal effects on growth in minimal medium but severely impaired growth on a collagen substrate that requires high levels of hydrolytic enzyme secretion, mirroring the phenotype of other fungal UPR mutants. 4µ8C also increased sensitivity to carvacrol, a natural compound that disrupts ER integrity in fungi, and hygromycin B, which correlated with reduced expression of glycosylation-related genes. Interestingly, treatment with 4µ8C was unable to induce all of the phenotypes attributed to the loss of the canonical UPR in a ΔhacA mutant but showed remarkable similarity to the phenotype of an RNase-deficient IreA mutant that is also unable to generate the hacAi mRNA. These results establish proof of principle that pharmacological inhibition of the canonical UPR pathway is feasible in A. fumigatus and support a noncanonical role for the hacAu mRNA in ER stress response.IMPORTANCE The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response.

Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus.

Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity.IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.

Impact of sleep duration during pregnancy on the risk of gestational diabetes in the Japan environmental and Children's study (JECS).

BACKGROUND: Gestational diabetes mellitus (GDM) has serious effects on both mother and child. Like Type 2 Diabetes Mellitus, it is increasing in prevalence world-wide. In addition to obesity, sleep duration has been named an important risk factor. Using a large cohort study, including data from 48,787 participants of the Japan Environment and Children's Study (JECS), we examined the association between sleep duration and both random blood glucose levels and GDM rates during pregnancy. METHODS: Random blood glucose levels were measured during pregnancy. GDM diagnosis was based on the results of 75 g oral glucose tolerance test. Additional anthropometric data was collected from questionnaires for statistical analysis. RESULTS: Compared to mothers averaging 7 to < 10 h sleep (reference group), women receiving < 5 h or ≥ 10 h sleep exhibited significantly elevated random blood glucose levels. This was associated with an elevated risk for positive GDM screening (< 5 h sleep: OR 1.17 (0.96-1.44) p = 0.126; ≥10 h sleep: OR 1.13 (1.03-1.25) p = 0.006). Calculating the risk for GDM, women sleeping < 5 h or ≥ 10 h exhibited elevated risks of 1.31-fold and 1.21 respectively. However, this trend was not found to be significant. CONCLUSIONS: Sleep is a critical factor in glucose metabolism, with both abnormally long and short sleep duration increasing random blood glucose levels in pregnant women. Moreover, the risk for positive GDM screening increases significantly with elevated sleep, ≥10 h per night. These findings are promising because they support the idea that sleep duration is a modifiable risk factor, and can be focused upon to improve health and pregnancy outcome.

Associations between metal concentrations in whole blood and placenta previa and placenta accreta: the Japan Environment and Children's Study (JECS).

BACKGROUND: Placenta previa and placenta accreta associate with high morbidity and mortality for both mothers and fetus. Metal exposure may have relationships with placenta previa and placenta accreta. This study analyzed the associations between maternal metal (cadmium [Cd], lead [Pb], mercury [Hg], selenium [Se], and manganese [Mn]) concentrations and placenta previa and placenta accreta. METHODS: We recruited 17,414 women with singleton pregnancies. Data from a self-administered questionnaire regarding the first trimester and medical records after delivery were analyzed. Maternal blood samples were collected to measure metal concentrations. The subjects were classified into four quartiles (Q1, Q2, Q3, and Q4) according to metal concentrations. RESULTS: The odds ratio for placenta previa was significantly higher among subjects with Q4 Cd than those with Q1 Cd. The odds ratio for placenta previa was significantly higher for subjects with Q2 Pb than those with Q1 Pb. CONCLUSION: Participants with placenta previa had higher Cd concentrations. However, this study was cross-sectional and lacked important information related to Cd concentration, such as detailed smoking habits and sources of Cd intake. In addition, the subjects in this study comprised ordinary pregnant Japanese women, and it was impossible to observe the relationship between a wide range of Cd exposure and placenta previa. Therefore, epidemiological and experimental studies are warranted to verify the relationship between Cd exposure and pregnancy abnormalities.

Inhibiting Notch1 enhances immunotherapy efficacy in melanoma by preventing Notch1 dependent immune suppressive properties.

We have previously shown that Notch1 plays a critical role in modulating melanoma tumor cell growth and survival. Here we show that Notch1 also contributes to an immune-suppressive tumor microenvironment (TME). Notch1 inhibition reduces immune suppressive cells (i.e. MDSCs and Tregs) while allowing the recruitment of functional CD8(+) T cells, leading to a decrease in the Tregs/CD8(+) ratio, a key parameter in assessing positive responses to immune-checkpoint inhibitors. Inhibition of Notch1 improves the antitumor activity of nivolumab and ipilimumab, particularly when given in combination. Mechanistically, tumor-associated Notch1 regulates the expression of several chemokines involved in MDSCs and Tregs recruitment. Among them, CCL5, IL6 and IL8, or MIP2 in mouse, were consistently reduced by Notch1 depletion in several human and mouse melanoma cell lines. Notch1 controls the transcription of IL8 and IL6; and the secretion of CCL5 likely by inhibiting the expression of SNAP23, a member of the SNAREs family of proteins involved in cell exocytosis. Inhibition of SNAP23 decreases CCL5 secretion similarly to Notch1 inhibition. Hence, targeting Notch1 would affect both melanoma intrinsic growth/survival properties, and provide an immune-responsive TME, thus improving immune therapy efficacy.

Association between daily ambient air pollution and respiratory symptoms in children with asthma and healthy children in western Japan.

OBJECTIVE: In recent years, air pollutant concentrations in Japan have decreased slightly; however, there are growing concerns about the influences of transnational air pollution on respiratory illness. We aimed to clarify the short-term association between the ambient air pollution and respiratory symptoms among children without asthma, children with asthma not using long-term medications (CA-nonLTM), and those using them (CA-LTM). METHODS: A total of 138 children attending 2 primary schools and 71 children with asthma regularly visiting cooperating medical institutions were recruited. Study participants measured peak expiratory flow (PEF) twice a day and recorded coughing, nasal symptoms, and medication use in a diary. Predicted associations between daily air pollutant concentrations and respiratory symptoms, and PEF were evaluated using case-crossover and generalized estimate equation models. RESULTS: Changes in %maxPEF per 10 ppb oxidant (Ox) increase in children without asthma, CA-nonLTM, and CA-LTM were -0.26% (95% CI: -0.49, -0.03), -0.51% (95% CI: -0.89, -0.12), and -0.20% (95% CI: -0.42, 0.01), respectively. The odds ratios for coughing per 10 ppb Ox increase in the Lag0 model were 1.34 (95% CI: 1.11, 1.60), 1.52 (95% CI: 1.12, 2.07), and 1.06 (95% CI: 0.93, 1.20), respectively. These suggested that the Ox concentration has graded effects on %maxPEF and coughing, in the following descending order, CA-nonLTM, children without asthma, and CA-LTM. The Ox concentration was also positively associated with nasal symptoms in children without asthma and CA-LTM. CONCLUSION: Our results suggest that using long-term medications to manage asthma may play an important role in preventing exacerbation of respiratory symptoms due to air pollution.

CCL3 Enhances Antitumor Immune Priming in the Lymph Node via IFNγ with Dependency on Natural Killer Cells.

Lymph node (LN) plays a critical role in tumor cell survival outside of the primary tumor sites and dictates overall clinical response in many tumor types (1, 2). Previously, we and others have demonstrated that CCL3 plays an essential role in orchestrating T cell-antigen-presenting cell (APC) encounters in the draining LN following vaccination, and such interactions enhance the magnitude of the memory T cell pool (3-5). In the current study, we investigate the cellular responses in the tumor-draining lymph nodes (TDLNs) of a CCL3-secreting CT26 colon tumor (L3TU) as compared to wild-type tumor (WTTU) during the priming phase of an antitumor response (≤10 days). In comparison to WTTU, inoculation of L3TU resulted in suppressed tumor growth, a phenomenon that is accompanied by altered in vivo inflammatory responses on several fronts. Autologous tumor-derived CCL3 (aCCL3) secretion by L3TU bolstered the recruitment of T- and B-lymphocytes, tissue-migratory CD103+ dendritic cells (DCs), and CD49b+ natural killer (NK) cells, resulting in significant increases in the differentiation and activation of multiple Interferon-gamma (IFNγ)-producing leukocytes in the TDLN. During this early phase of immune priming, NK cells constitute the major producers of IFNγ in the TDLN. CCL3 also enhances CD8+ T cell proliferation and differentiation by augmenting DC capacity to drive T cell activation in the TDLN. Our results revealed that CCL3-dependent IFNγ production and CCL3-induced DC maturation drive the priming of effective antitumor immunity in the TDLN.

Cyclin-dependent kinase 5 activity is required for allogeneic T-cell responses after hematopoietic cell transplantation in mice.

Molecular intermediates in T-cell activation pathways are crucial targets for the therapy and prevention of graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT). We recently identified an essential role for cyclin-dependent kinase 5 (Cdk5) in T-cell activation and effector function, but the contribution of Cdk5 activity to the development of GVHD has not been explored. Using an established, preclinical, murine, GVHD model, we reveal that Cdk5 activity is increased in key target organs early after allo-HCT. We then generated chimeric mice (Cdk5+/+C or Cdk5-/-C) using hematopoietic progenitors from either embryonic day 16.5 Cdk5+/+ or Cdk5-/- embryos to enable analyses of the role of Cdk5 in GVHD, as germ line Cdk5 gene deletion is embryonically lethal. The immunophenotype of adult Cdk5-/-C mice is identical to control Cdk5+/+C mice. However, transplantation of donor Cdk5-/-C bone marrow and T cells dramatically reduced the severity of systemic and target organ GVHD. This phenotype is attributed to decreased T-cell migration to secondary lymphoid organs (SLOs), reduced in vivo proliferation within these organs, and fewer cytokine-producing donor T cells during GVHD development. Moreover, these defects in Cdk5-/- T-cell function are associated with altered CCR7 signaling following ligation by CCL19, a receptor:ligand interaction critical for T-cell migration into SLOs. Although Cdk5 activity in donor T cells contributed to graft-versus-tumor effects, pharmacologic inhibition of Cdk5 preserved leukemia-free survival. Collectively, our data implicate Cdk5 in allogeneic T-cell responses after HCT and as an important new target for therapeutic intervention.

Transient Surface CCR5 Expression by Naive CD8+ T Cells within Inflamed Lymph Nodes Is Dependent on High Endothelial Venule Interaction and Augments Th Cell-Dependent Memory Response.

In inflamed lymph nodes, Ag-specific CD4(+) and CD8(+) T cells encounter Ag-bearing dendritic cells and, together, this complex enhances the release of CCL3 and CCL4, which facilitate additional interaction with naive CD8(+) T cells. Although blocking CCL3 and CCL4 has no effect on primary CD8(+) T cell responses, it dramatically impairs the development of memory CD8(+) T cells upon Ag rechallenge. Despite the absence of detectable surface CCR5 expression on circulating native CD8(+) T cells, these data imply that naive CD8(+) T cells are capable of expressing surface CCR5 prior to cognate Ag-induced TCR signaling in inflamed lymph nodes; however, the molecular mechanisms have not been characterized to date. In this study, we show that CCR5, the receptor for CCL3 and CCL4, can be transiently upregulated on a subset of naive CD8(+) T cells and that this upregulation is dependent on direct contact with the high endothelial venule in inflamed lymph node. Binding of CD62L and CD11a on T cells to their ligands CD34 and CD54 on the high endothelial venule can be enhanced during inflammation. This enhanced binding and subsequent signaling promote the translocation of CCR5 molecules from intracellular vesicles to the surface of the CD8(+) T cell. The upregulation of CCR5 on the surface of the CD8(+) T cells increases the number of contacts with Ag-bearing dendritic cells, which ultimately results in increased CD8(+) T cell response to Ag rechallenge.

The Toxicity of a Novel Antifungal Compound Is Modulated by Endoplasmic Reticulum-Associated Protein Degradation Components.

In a search for new antifungal compounds, we screened a library of 4,454 chemicals for toxicity against the human fungal pathogen Aspergillus fumigatus. We identified sr7575, a molecule that inhibits growth of the evolutionary distant fungi A. fumigatus, Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae but lacks acute toxicity for mammalian cells. To gain insight into the mode of inhibition, sr7575 was screened against 4,885 S. cerevisiae mutants from the systematic collection of haploid deletion strains and 977 barcoded haploid DAmP (decreased abundance by mRNA perturbation) strains in which the function of essential genes was perturbed by the introduction of a drug resistance cassette downstream of the coding sequence region. Comparisons with previously published chemogenomic screens revealed that the set of mutants conferring sensitivity to sr7575 was strikingly narrow, affecting components of the endoplasmic reticulum-associated protein degradation (ERAD) stress response and the ER membrane protein complex (EMC). ERAD-deficient mutants were hypersensitive to sr7575 in both S. cerevisiae and A. fumigatus, indicating a conserved mechanism of growth inhibition between yeast and filamentous fungi. Although the unfolded protein response (UPR) is linked to ERAD regulation, sr7575 did not trigger the UPR in A. fumigatus and UPR mutants showed no enhanced sensitivity to the compound. The data from this chemogenomic analysis demonstrate that sr7575 exerts its antifungal activity by disrupting ER protein quality control in a manner that requires ERAD intervention but bypasses the need for the canonical UPR. ER protein quality control is thus a specific vulnerability of fungal organisms that might be exploited for antifungal drug development.

Focal transient CNS vessel leak provides a tissue niche for sequential immune cell accumulation during the asymptomatic phase of EAE induction.

Peripheral immune cells are critical to the pathogenesis of neurodegenerative diseases including multiple sclerosis (MS) (Hendriks et al., 2005; Kasper and Shoemaker, 2010). However, the precise sequence of tissue events during the early asymptomatic induction phase of experimental autoimmune encephalomyelitis (EAE) pathogenesis remains poorly defined. Due to the spatial-temporal constrains of traditional methods used to study this disease, most studies had been performed in the spine during peak clinical disease; thus the debate continues as to whether tissue changes such as vessel disruption represent a cause or a byproduct of EAE pathophysiology in the cortex. Here, we provide dynamic, high-resolution information on the evolving structural and cellular processes within the gray matter of the mouse cortex during the first 12 asymptomatic days of EAE induction. We observed that transient focal vessel disruptions precede microglia activation, followed by infiltration of and directed interaction between circulating dendritic cells and T cells. Histamine antagonist minimizes but not completely ameliorates blood vessel leaks. Histamine H1 receptor blockade prevents early microglia function, resulting in subsequent reduction in immune cell accumulation, disease incidence and clinical severity.

Endoplasmic reticulum stress and fungal pathogenesis.

The gateway to the secretory pathway is the endoplasmic reticulum (ER), an organelle that is responsible for the accurate folding, post-translational modification and final assembly of up to a third of the cellular proteome. When secretion levels are high, errors in protein biogenesis can lead to the accumulation of abnormally folded proteins, which threaten ER homeostasis. The unfolded protein response (UPR) is an adaptive signaling pathway that counters a buildup in misfolded and unfolded proteins by increasing the expression of genes that support ER protein folding capacity. Fungi, like other eukaryotic cells that are specialized for secretion, rely upon the UPR to buffer ER stress caused by fluctuations in secretory demand. However, emerging evidence is also implicating the UPR as a central regulator of fungal pathogenesis. In this review, we discuss how diverse fungal pathogens have adapted ER stress response pathways to support the expression of virulence-related traits that are necessary in the host environment.

Differential levels of amino acid transporters System L and ASCT2, and the mTOR protein in placenta of preeclampsia and IUGR.

BACKGROUND: Sufficient amino acid transport activity (AAT) is indispensable for appropriate fetal growth. Studies suggest that placental nutrient uptake activity is responsive to both maternal and fetal nutrient demands. We hypothesize that under conditions of limited nutrient availability to the fetus, as often present in preeclampsia, intrauterine growth restriction (IUGR), and insufficient weight-gain during pregnancy, a general adaptive response aimed to increase amino acid transport activity may be observed in the placenta. METHOD: A total of 40 placentas from full-term (n = 10) and pre-term (average gestational period = 34.8 weeks, n = 10) normal pregnancies, IUGR (n = 10), and preeclampsia (n = 10) associated pregnancies were looked at by immunohistochemistry followed by relative qualitative scoring to compare expression levels and localization of System L, ASCT2, and mTOR proteins. RESULT: Microvillous syncytiotrophoblast (ST) in placenta of pregnancies complicated by IUGR or preeclampsia (PE) showed significant increases in the levels of System L amino acid transport proteins 4F2hc and LAT1 compared to both full-term control and pre-term (early gestation control) pregnancies seperately (p < 0.05). Elevated mTOR protein was uniquely higher in IUGR placentas compared to full-term controls (P = 0.0026). Total cellular ASCT2 transporter protein levels were similar in all groups, however, levels of ASCT2 protein localized to the ST microvillous membrane (MVM) were significantly lower in IUGR compared to both full-term and pre-term pregnancies (P = 0.0006, 0.03, respectively). Additionally, ASCT2 and mTOR protein levels were positively associated with maternal pre-pregnancy BMI (P = 0.046, 0.048, respectively). CONCLUSION: There are three important findings based upon the present study. First, in conditions of limited nutrient availability, such as PE or IUGR, there is an overall increase in the level of System L and mTOR protein expression in the ST, suggestive of an adaptive response. Second, a decrease in ASCT2 protein at the ST MVM suggests a post-translational event that may decrease AAT activity in IUGR placentas. Third, a physiological link between transporter expression and pre-pregnancy BMI is suggested based upon a positive association observed with ASCT2 and mTOR expression values.