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A rapidly expanding range of Pectinatella magnifica has been reported globally. Although many studies have been conducted to manage this invasive species, surveying methods have mainly been limited to visual observation of bryozoan colonies. For efficient monitoring and early detection of bryozoan species, we compared three different monitoring methods: visually detecting bryozoan colonies (visual observation of colonies, VOC), floating statoblasts (statoblasts in filtered water, SFW), and attached statoblasts (statoblasts attached to artificial substrate, SAS). Using these sampling methods, we compared the occurrences of P. magnifica in the littoral zones of lotic ecosystems from 117 sites in South Korea from 2015 to 2019. Occurrence frequency of P. magnifica was higher using both SFW (37.3%) and SAS (72.5%) than the traditional VOC (28.0%) method. The agreement of occurrence detection of P. magnifica between VOC, SFW and SAS was more consistent in tributaries than in main channels. Density estimations using colony (VOC) and statoblast detection methods (SFW, SAS) also showed a higher positive correlation in tributaries. To identify early P. magnifica invasion in an unidentified area, collecting statoblasts is a more efficient alternative to determine the occurrence of bryozoan species in field conditions.

Cowpea [Vigna unguiculate (L.) Walp.] is not only a healthy, nutritious, and versatile leguminous crop; it also has a relatively high adaptation to drought. Research has shown that cowpea lines have a high tolerance to drought, and many of them can survive more than 40 days under scorching and dry conditions. The cowpea (Southern pea) breeding program at the University of Arkansas has been active for more than 50 years and has produced more than 1000 advanced breeding lines. The purpose of this study was to evaluate the drought-tolerant ability in Arkansas cowpea lines and use the drought-tolerant lines in cowpea production or as parents in cowpea breeding. A total of 36 University of Arkansas breeding lines were used to screen drought tolerance at the seedling stage in this study. The experiment was conducted in the greenhouse using a randomized complete block design (RCBD) with two replicates, organized in a split-plot manner, where the drought treatment (drought and nondrought stress) as the main plot and the cowpea genotypes as the subplot. Drought stress was applied for 4 weeks, and three drought-tolerant–related traits were collected and analyzed. Results showed that cowpea breeding lines: ‘17-61’, ‘17-86’, ‘Early Scarlet’, and ‘ARBlackeye #1’ were found to be drought tolerant.

Effective wildlife conservation requires consideration of ecological and social factors, including social acceptability of conservation actions. Using the threatened koala (Phascolarctos cinereus) as a case study, we demonstrate a novel, socio-ecological approach for identifying conservation opportunity that spatially connects landscapes with community preferences to prioritize koala recovery strategies at a regional scale. We conceptualize conservation opportunity as the spatial integration of three sustainability criteria—ecological potential, social acceptability, and economic feasibility. The social acceptability criterion was assessed using a crowdsourced spatial survey that identified spatial preferences for koalas and land uses that impact koala conservation. As a novel approach, we addressed important research questions regarding the design, collection, and analysis of crowdsourced mapping data for identifying socially acceptable conservation opportunities. Public preferences for koalas were mapped closer to home, in higher suitable koala habitats than expected, were more pronounced in conservation and natural areas on public lands, and were mapped less frequently in modified agricultural landscapes. When the multiple criteria (ecological, social, and economic) were included in the conservation assessment, we found the social acceptability criterion exerted the greatest influence on spatial conservation priorities. The systematic assessment of social criteria for conservation using spatial surveys provides information that can be integrated with ecological information to prioritize conservation opportunities. Potential enhancements include expanding survey recruitment efforts and using alternative social data collection methods to achieve greater geographic and socio-demographic representation, and augmenting the economic feasibility assessment with private property values and transaction data from voluntary conservation agreements with private landowners.

Lolalove, a B4 subcluster soil bacteriophage of Mycobacterium smegmatis , was isolated in Charleston, South Carolina. It possesses a 71,111-bp linear double-stranded DNA (dsDNA) genome with 99 protein-coding genes and a GC content of 68.9%. Whole-genome BLASTn alignments indicate high sequence identity to the related B4 subcluster M. smegmatis phages BrownCNA, Mithril, and Hangman.

Preparations of plasma-derived small extracellular vesicles (sEVs) were deployed as liquid biopsy to study cytochrome P450 (CYP) 3A4 (CYP3A4) induction following modafinil 400 mg once daily × 14 days (young healthy volunteers, N = 10 subjects). Induction was confirmed using the 4β-hydroxycholesterol-to-cholesterol (4βHC/C) ratio, a plasma CYP3A4/5 biomarker, with a mean 2.1-fold increase (Day 15 vs. Day 1; 90% confidence interval (CI) = 1.8-2.3; P value = 0.0004). Proteomic analysis revealed the induction (mean Day 15 vs. Day 1 fold-increase (90% CI)) of both liver (1.3 (1.1-1.5), P value = 0.014) and nonliver (1.9 (1.6-2.2), P value = 0.04) sEV CYP3A4 protein expression. In CYP3A5 nonexpresser subjects, the baseline (pre-dose) 4βHC/C plasma ratio was more highly correlated with liver sEVs (r = 0.937, P value = 0.001) than nonliver sEVs (r = 0.619, P value = 0.101) CYP3A4 protein expression. When CYP3A5 expressers (CYP3A5*1/*3) were included, the correlation with liver sEVs (r = 0.761, P value = 0.011) and nonliver sEVs (r = 0.391, P value = 0.264) CYP3A4 protein was weaker. Although modafinil-induced changes in plasma 4βHC/C ratio did not correlate with sEVs CYP3A4 protein expression, the individual subject sEVs proteomic data were used successfully to predict victim drug (midazolam, triazolam, dextromethorphan, 17α-ethinylestradiol, and abemaciclib) area under the plasma concentration-time curve (AUC) ratios (AUCRs) following modafinil. Based on the AUCR values, modafinil was classified as a weak to moderate CYP3A4 inducer (vs. rifampicin). For the first time, it was possible to deploy plasma-derived sEVs to study CYP3A4 induction beyond rifampicin, a more potent CYP3A4 inducer.

Ongoing SARS-CoV-2 vaccine development is focused on identifying stable, cost-effective, and accessible candidates for global use, specifically in low and middle-income countries. Here, we report the efficacy of a rapidly scalable, novel yeast expressed SARS-CoV-2 specific receptor-binding domain (RBD) based vaccine in rhesus macaques. We formulated the RBD immunogen in alum, a licensed and an emerging alum adsorbed TLR-7/8 targeted, 3M-052-alum adjuvants. The RBD+3M-052-alum adjuvanted vaccine promoted better RBD binding and effector antibodies, higher CoV-2 neutralizing antibodies, improved Th1 biased CD4+T cell reactions, and increased CD8+ T cell responses when compared to the alum-alone adjuvanted vaccine. RBD+3M-052-alum induced a significant reduction of SARS-CoV-2 virus in respiratory tract upon challenge, accompanied by reduced lung inflammation when compared with unvaccinated controls. Anti-RBD antibody responses in vaccinated animals inversely correlated with viral load in nasal secretions and BAL. RBD+3M-052-alum blocked a post SARS-CoV-2 challenge increase in CD14+CD16++ intermediate blood monocytes, and Fractalkine, MCP-1, and TRAIL in the plasma. Decreased plasma analytes and intermediate monocyte frequencies correlated with reduced nasal and BAL viral loads. Lastly, RBD-specific plasma cells accumulated in the draining lymph nodes and not in the bone marrow, contrary to previous findings. Together, these data show that a yeast expressed, RBD-based vaccine+3M-052-alum provides robust immune responses and protection against SARS-CoV-2, making it a strong and scalable vaccine candidate.

A combination of vaccination approaches will likely be necessary to fully control the SARS-CoV-2 pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane anchored pre-fusion stabilized spike (MVA/S), but not secreted S1, induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and showed protection from SARS-CoV-2 infection and virus replication in the lung as early as day 2 following intranasal or intratracheal challenge. Single-cell RNA sequencing analysis of lung cells at day 4 post-infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities, and lowered induction of interferon stimulated genes. These results demonstrate that MVA/S vaccination induces both neutralizing antibodies and CD8+ T cells in the blood and lung and serves as a potential vaccine candidate against SARS-CoV-2., Graphical Abstract, Modified vaccinia Ankara (MVA) vector-based vaccines are attractive for their excellent safety and ability to induce long-lived humoral and cellular immunity in humans. Routhu et al. show that an MVA-based COVID-19 vaccine encoding prefusion-stabilized spike MVA/S induces strong neutralizing antibody and CD8+ T cell responses and protects macaques from SARS-CoV2 infection, immunopathology and infection-induced B cell abnormalities in the lung.

Background: Primary cytotoxic T cell lymphomas are a heterogenous collection of ill-defined diseases with aggressive disease course and poor response to conventional therapy. Of these, primary cutaneous CD8+ aggressive epidermotropic T cell lymphoma and cutaneous T cell lymphoma, not otherwise specified, have undergone few to no systematic analyses of its genetic landscape. In this study, we classify these lymphomas based on their histological and clinical features and perform systematic genomic analyses to find novel therapeutic targets. Methods: We clinically characterize 35 patients from 10 institutions with cytotoxic T cell lymphoma and performed integrative clinical, pathological, and genetic analyses. We perform Archer™ Heme Fusion on an initial 24 FFPE samples. In addition, we perform DNA and/or RNA sequencing on 11 cases. We functionally validate our genetic findings by transducing alterations into Ba/F3 cells and subsequently performing biochemical assays. In this setting, we assess sensitivity to FDA-approved small molecule inhibitors. We provide orthogonal evidence of transcriptional upregulation by geneset enrichment analysis. Results: We find kinase fusions impacting JAK-STAT signaling in all primary cutaneous CD8+ aggressive epidermotropic T cell lymphomas in our cohort. Three of these fusions have not previously been described in cancer. Known fusions have been shown to upregulate JAK-STAT signaling. Upon each transduction, CAPRIN1-JAK2 and SELENOI-ABL1 cause increased phosphorylation and display IL-3-independent growth compared to control. Upon treatments to small molecule inhibitors, ruxolitinib and imatinib respectively, CAPRIN1-JAK2 and SELENOI-ABL1 expressing cells exhibit sensitivity. Cutaneous T cell lymphoma, not otherwise specified, harbor JAK-STAT hotspot mutations but not kinase fusions. Conclusions: All 9 primary cutaneous CD8+ aggressive epidermotropic T cell lymphomas in our series harbor kinase fusions, while the majority of the cutaneous T cell lymphoma, not otherwise specified, cases in our cohort harbor hotspot JAK/STAT mutations. We provide evidence that the of presence these alterations, which are associated with inhibitor response, depend on cytotoxic T cell lymphoma histology.

Mounded, tallgrass prairie ecosystems are a unique, endangered ecosystem due to severe disturbance for agricultural production and/or urban expansion. Limited research exists on earthworm (Lumbricus terrestris) dynamics in mounded, tallgrass prairies, particularly in the Ozark Highlands region of northwestern Arkansas. The objectives of this study were to evaluate the effects of soil moisture regime (SMR), landscape position (LP), and sample date on earthworm densities in the top 20 cm of soil in two mounded, native prairies in the Ozark Highlands region of northwestern Arkansas. Juvenile and total earthworm densities were five times greater (P

To mitigate the loss of tallgrass prairies through fragmentation and conversion to other land uses, prairie restoration efforts have been undertaken to reclaim areas that have been previously used for agricultural production. Earthworms are a key component of the soil ecology of natural, undisturbed ecosystems. However, limited research exists on earthworm populations in prairie restorations and how earthworm densities may change over time. The objectives of this study were (a) to evaluate earthworm densities in the upper 20 cm of soil over time (i.e., May 2007 and May 2019) in a native prairie and chronosequence of three prairie restorations (i.e., established in 1979, 2000, and 2002), and (b) to identify relationships between earthworm (i.e., juvenile, adult, and total) density and soil physical and chemical properties. Juvenile (P = .03) earthworm density decreased 25 and 35% and total (P = .03) earthworm density decreased by 33 and 42% in the 2000 and 2002 restorations, respectively, from May 2007 to May 2019. Adult earthworm density did not change over time (P > .05) in any prairie ecosystem. Combined across prairie sites, adult earthworm density was positively correlated (P

Effective therapeutics aimed at mitigating COVID-19 symptoms are urgently needed. SARS-CoV-2 induced hypercytokinemia and systemic inflammation are associated with disease severity. Baricitinib, a clinically approved JAK1/2 inhibitor with potent anti-inflammatory properties is currently being investigated in COVID-19 human clinical trials. Recent reports suggest that baricitinib may also have antiviral activity in limiting viral endocytosis. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages and tissues was not reduced with baricitinib. Type I IFN antiviral responses and SARS-CoV-2 specific T cell responses remained similar between the two groups. Importantly, however, animals treated with baricitinib showed reduced immune activation, decreased infiltration of neutrophils into the lung, reduced NETosis activity, and more limited lung pathology. Moreover, baricitinib treated animals had a rapid and remarkably potent suppression of alveolar macrophage derived production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for severe inflammation induced by SARS-CoV-2 infection.

SARS-CoV-2 induced hypercytokinemia and inflammation are critically associated with COVID-19 disease severity. Baricitinib, a clinically approved JAK1/2 inhibitor, is currently being investigated in COVID-19 clinical trials. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages and tissues was not reduced with baricitinib. Type-I IFN antiviral responses and SARS-CoV-2-specific T-cell responses remained similar between the two groups. Animals treated with baricitinib showed reduced inflammation, decreased lung infiltration of inflammatory cells, reduced NETosis activity, and more limited lung pathology. Importantly, baricitinib treated animals had a rapid and remarkably potent suppression of lung macrophages production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for inflammation induced by SARS-CoV-2 infection., Highlights • SARS-CoV-2 infected RMs mimic signatures of inflammation seen in COVID-19 patients • Baricitinib suppresses production of pro-inflammatory cytokines in lung macrophages • Baricitinib limits recruitment of neutrophils to the lung and NETosis • Baricitinib preserves innate antiviral and SARS-CoV-2-specific T-cell responses, Using a rhesus macaque infection model it is shown that baricitinib treatment started early after infection effectively resolves inflammatory signatures in airway macrophages, with decreased lung pathology and neutrophil infiltration.

The Royal Museum of Central Africa at Tervuren, Belgium, includes a small collection of freshwater bryozoans from Congo and Rwanda. Included are: Plumatella philippinensis with both statoblast types, as well as holotypes of Plumatella ruandensis Wiebach, 1964, Plumatella marlieri Wiebach, 1970, and Plumatella pseudostolonata Borg, 1940. There are also two new species which had been previously misidentified: specimens designated as Plumatella javanica are now recognized as P. wiebachi n. sp.; and specimens labeled Stolella indica now recognized as Plumatella kisalensis n. sp. This paper includes full descriptions of the new species as well as fresh descriptions and illustrations of other species in the collection.

Charcoal rot of soybean, caused by Macrophomina phaseolina, is a disease of economic significance in the United States. Although there are soybean cultivars with moderate resistance, identifying and quantifying resistance is challenging. Existing assays are time consuming, and results are often highly variable. The objectives of this research were to (i) create a reproducible seed plate assay (SPA) for charcoal rot resistance and (ii) correlate field-based disease assessments with SPA results on diverse soybean accessions. To develop the SPA, surface-disinfected seeds from eight soybean genotypes (representing three susceptible and five resistant cultivars) were placed on water agar plates inoculated with M. phaseolina. After incubation at room temperature in darkness for 7 days, percent germination was determined for each cultivar relative to the germination on noninoculated plates. Results from SPA were in general agreement with published responses. None of the soybean genotypes showed complete resistance to M. phaseolina. For the second objective, charcoal rot resistance in 18 soybean accessions was assayed with SPA, and results were analyzed for correlation with field disease assessments from Stuttgart, AR, from 2011 to 2014 and from Rohwer, AR, in 2011 and 2012. SPA consistently categorized soybean genotype resistance compared with field disease assessment averages, and results were consistent with previously published resistance determinations. SPA was significantly correlated with percent height of internal stem discoloration (PHSD) at Stuttgart from 2011 to 2013 and in 2012 at Rohwer, with root and stem severity (RSS) at Rohwer in 2012, and with tap root colonization (CFU) at Stuttgart in 2012. SPA was significantly correlated to yield at Stuttgart in 2011, 2013, and 2014, and in 2011 and 2012 at Rohwer. Yield was not correlated to RSS, PHSD, or CFU at either location or in any year. Therefore, SPA is a reproducible and rapid assay for charcoal rot resistance in soybean and is significantly associated to field performance.

The ability of the pigment dinitrosyl ferrohemochrome to mimic the cured meat color function attributed to nitrite, was evaluated in a number of nitrite-free, model meat systems. In addition, compounds with reported antibotulinal properties were compared to the antibotulinal effect of nitrite. Fifteen treatments were evaluated and compared to 50 and 150 ppm nitrite. Two processing conditions (short and extended heating) were also compared for their ability to enhance pigment color and eliminate the natural meat microbial population. Meat slurries varying in cure composition were inoculated with a composite of six different strains of Clostridium botulinum , types A and B. After processing, the packages were incubated at 10 and 27°C, and were analyzed for toxin. The treatment containing 3000 ppm sodium hypophosphite most closely resembled the 150 ppm nitrite control in its ability to prevent spore outgrowth and toxin production. The treatment containing 1250 ppm monomethyl fumerate also scored better than the other treatments including ethylene diamine tetraacetic acid (EDTA), potassium sorbate and tertiary butyl hydroquinome (TBHQ), but was slightly less inhibitory than sodium hypophosphite. The longer heat treatment eliminated all the natural meat flora (lactic acid bacteria) and enhanced the color production of the pigment.

Public health authorities in Oxford, Middlesex and Elgin Counties, Ontario, seized raw milk Cheddar cheese due to presence of Salmonella muenster . Investigations by these units and the University of Guelph traced the source of Salmonella to one particular milk supplier shipping to a cheese factory. Analysis of milk samples from a herd of 35 cattle revealed only one cow shedding S. muenster directly into the milk (ca. 200 CFU/ml). Eleven of 181 vats of cheese, produced at the factory between May and October 1982, were positive for Salmonella at the curd stage. Only 2 vats of the finished raw milk Cheddar, however, were positive. One lot of Salmonella -positive cheese was still positive after the legally required 60-d holding period and remained so for 125 d.

Gram-negative organisms were isolated from vacuum-packed bologna only after 7-8 weeks of storage at 5 C. These isolates were identified as Acinetobacter sp., Enterobacter hafniae , Enterobacter aerogenes , Serratia marcescens and Serratia liquefaciens . Nitrite tolerance tests on these isolates at pH 5, 5.5 and 6 showed that only Acinetobacter sp. may be controlled by nitrite and pH levels associated with bologna during the initial storage period. The nitrite tolerance of these organisms was linked to their ability to deplete nitrite at 5 C in broth cultures under anaerobic conditions. Associated growth experiments were done at 15 C in APT and BHI broths under anaerobic conditions. These studies with E. aerogenes and S. liquefaciens in the presence of Lactobacillus brevis or Lactobacillus buchneri showed varying degrees of inhibition of the gram-negative isolates. The major factor which appeared to influence this inhibition in both broth cultures and bologna was glucose levels.

The treatment of psoriasis remains elusive, underscoring the need for identifying novel disease targets and mechanism-based therapeutic approaches. We recently reported that the PI3K/Akt/mTOR pathway that is frequently deregulated in many malignancies is also clinically relevant for psoriasis. We also provided rationale for developing delphinidin (Del), a dietary antioxidant for the management of psoriasis. This study utilized high-throughput biophysical and biochemical approaches and in vitro and in vivo models to identify molecular targets regulated by Del in psoriasis.A kinome-level screen and Kds analyses against a panel of 102 human kinase targets showed that Del binds to three lipid (PIK3CG, PIK3C2B, and PIK3CA) and six serine/threonine (PIM1, PIM3, mTOR, S6K1, PLK2, and AURKB) kinases, five of which belong to the PI3K/Akt/mTOR pathway. Surface plasmon resonance and in silico molecular modeling corroborated Del's direct interactions with three PI3Ks (α/c2β/γ), mTOR, and p70S6K. Del treatment of interleukin-22 or TPA-stimulated normal human epidermal keratinocytes (NHEKs) significantly inhibited proliferation, activation of PI3K/Akt/mTOR components, and secretion of proinflammatory cytokines and chemokines. To establish the in vivo relevance of these findings, an imiquimod (IMQ)-induced Balb/c mouse psoriasis-like skin model was employed. Topical treatment of Del significantly decreased (i) hyperproliferation and epidermal thickness, (ii) skin infiltration by immune cells, (iii) psoriasis-related cytokines/chemokines, (iv) PI3K/Akt/mTOR pathway activation, and (v) increased differentiation when compared with controls. Innovation and Conclusion: Our observation that Del inhibits key kinases involved in psoriasis pathogenesis and alleviates IMQ-induced murine psoriasis-like disease suggests a novel PI3K/AKT/mTOR pathway modulator that could be developed to treat psoriasis. Antioxid. Redox Signal. 26, 49-69.

SummaryGenerating tier 2 HIV-neutralizing antibody (nAb) responses by immunization remains a challenging problem, and the immunological barriers to induction of such responses with Env immunogens remain unclear. Here, some rhesus monkeys developed autologous tier 2 nAbs upon HIV Env trimer immunization (SOSIP.v5.2) whereas others did not. This was not because HIV Env trimers were immunologically silent because all monkeys made similar ELISA-binding antibody responses; the key difference was nAb versus non-nAb responses. We explored the immunological barriers to HIV nAb responses by combining a suite of techniques, including longitudinal lymph node fine needle aspirates. Unexpectedly, nAb development best correlated with booster immunization GC B cell magnitude and Tfh characteristics of the Env-specific CD4 T cells. Notably, these factors distinguished between successful and unsuccessful antibody responses because GC B cell frequencies and stoichiometry to GC Tfh cells correlated with nAb development, but did not correlate with total Env Ab binding titers.

// Minakshi Nihal 1, 2 , Gary S. Wood 1, 2, 3 1 Department of Dermatology, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin, USA 2 Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA 3 Wm. S. Middleton VA Medical Centre, Madison, Wisconsin, USA Correspondence to: Minakshi Nihal, email: mnihal@dermatology.wisc.edu Keywords: c-CBL, melanoma, proliferation, migration, invasion Received: April 05, 2016 Accepted: July 10, 2016 Published: July 27, 2016 ABSTRACT Melanoma is one of the most aggressive and lethal forms of skin cancer. Despite recent improvements in targeted therapies, many patients with advanced disease fail to achieve lasting tumor regression. Therefore, it is important to develop novel druggable targets that can be exploited to improve clinical outcome. Here, we studied the role of Casitas B-lineage lymphoma (c-CBL), an E3 ubiquitin ligase, in human melanoma. Employing quantitative real-time PCR and Western blot analysis in a panel of human melanoma cell lines (A375, G361, Hs-294T, SK-Mel-2, SK-Mel-28 and 451Lu), we found that c-CBL is strongly expressed in human melanoma cells at the mRNA and protein levels. Further, we determined c-CBL levels in clinical samples of melanomas and benign melanocytic nevi, using quantitative Nuance multispectral imaging. Compared to benign nevi, melanomas showed an overlapping range of c-CBL immunoreactivity. Small interfering RNA (siRNA)-mediated knockdown of c-CBL resulted in decreased proliferation, clonogenic survival and migration of melanoma cells. Furthermore, it also resulted in decreased cellular invasion in a 3D spheroid assay system. C-CBL and FAK are regulated by SRC, and FAK binds SRC and GRB2. C-CBL E3 ligase domain regulates receptor tyrosine kinase internalization through ubiquitination and its ring finger domain stabilizes the FAK-SRC-actin cytoskeleton thereby promoting cellular motility. C-CBL knockdown was associated with decreased protein and/or mRNA levels of SRC, FAK and GRB2. Taken together, we have provided evidence that c-CBL plays a role in melanoma cell proliferation, migration and invasion as well as inhibition of the FAK-GRB2-SRC nexus. Our findings indicate that additional studies are warranted to further dissect the role of c-CBL in melanoma and determine the therapeutic potential of its inhibition.

Overabundant herbivorous mammals can increase the mortality rates of preferred food species, causing long-term changes to plant communities. When lethal control of the herbivore is not possible, fertility control is often advocated as a management solution. There is, however, little empirical evidence that fertility control is a viable option for managing the impacts of open populations of polygynous mammals. We evaluated the effectiveness of two long-term fertility control programs implemented to reduce overabundant koala (Phascolarctos cinereus) populations and their impacts on preferred food trees (manna gum, Eucalyptus viminalis): one in Victoria (10 years) and one in South Australia (17 years). Fertility control reduced koala recruitment such that population densities more than halved at both sites. Mortality rates of manna gum trees were significantly reduced at one site, and trees with light or moderate defoliation recovered at both sites. Relationships between koala population density and canopy defoliation suggest that if management wishes to alleviate moderate browsing pressure then koala density should be maintained below 1.0 koala/ha. We conclude that population-level fertility control can significantly reduce overabundant koala populations and some of their impacts on food trees. Our empirical study demonstrates that long-term fertility control can reduce the undesirable impacts of open populations of polygynous mammals.

Background Individuals with an extremely rare inherited condition, termed Congenital Insensitivity to Pain (CIP), do not feel pain in response to noxious stimuli. Variants in SCN9A, encoding the transmembrane voltage-gated sodium channel Nav1.7, have previously been reported in subjects with CIP accompanied by anosmia, which are typically transmitted in a recessive pattern. Functional characterisations of some of these SCN9A mutations show that they result in complete loss-of-function of Nav1.7. Methods In a consanguineous family we performed whole exome sequencing of three members who have a diagnosis of CIP and one unaffected family member. The functional effects of the segregating variant in SCN9A were determined using patch clamp electrophysiology in human embryonic kidney (HEK) 293 cells transfected with the variant. Results We found that each CIP subject was homozygous for a putatively nonsense variant, R1488*, in SCN9A. This variant was reported elsewhere in a subject with CIP, though the functional effect was not determined. Using electrophysiology, we confirm that this variant results in a complete loss-of-function of Nav1.7. Conclusions We confirm through electrophysiological analysis that this R1488* variant in SCN9A results in complete loss-of-function of Nav1.7, which is consistent with reports on other variants in this gene in subjects with CIP.

AIMS: Demonstrate the presence of cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT) proteins and mRNAs in isolated human plasma exosomes and evaluate the capacity for exosome‐derived biomarkers to characterize variability in CYP3A4 activity. METHODS: The presence of CYP and UGT protein and mRNA in exosomes isolated from human plasma and HepaRG cell culture medium was determined by mass spectrometry and reverse transcription–polymerase chain reaction, respectively. The concordance between exosome‐derived CYP3A4 biomarkers and midazolam apparent oral clearance (CL/F) was evaluated in a small proof‐of‐concept study involving six genotyped (CYP3A4 *1/*1 and CYP3A5 *3/*3) Caucasian males. RESULTS: Exosomes isolated from human plasma contained peptides and mRNA originating from CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2 J2, 3A4 and 3A5, UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15, and NADPH‐cytochrome P450 reductase. Mean (95% confidence interval) exosome‐derived CYP3A4 protein expression pre‐ and post‐rifampicin dosing was 0.24 (0.2–0.28) and 0.42 (0.21–0.65) ng ml(–1) exosome concentrate. Mean (95% confidence interval) exosome CYP3A4 mRNA expression pre‐ and post‐rifampicin dosing was 6.0 (1.1–32.7) and 48.3 (11.3–104) × 10(–11) 2(‐ΔΔCt), respectively. R(2) values for correlations of exosome‐derived CYP3A4 protein expression, CYP3A4 mRNA expression, and ex vivo CYP3A4 activity with midazolam CL/F were 0.905, 0.787 and 0.832, respectively. CONCLUSIONS: Consistent strong concordance was observed between exosome‐derived CYP3A4 biomarkers and midazolam CL/F. The significance of these results is that CYP3A4 is the drug‐metabolizing enzyme of greatest clinical importance and variability in CYP3A4 activity is poorly described by existing precision dosing strategies.

Non-syndromic craniosynostosis (CS) affects 1 in 2350 live births. Recent studies have shown that a significant fraction of cases are caused by de novo or rare transmitted mutations that promote premature osteoblast differentiation in cranial sutures. Rare heterozygous loss-of-function (LOF) mutations in SMAD6 and TCF12 are highly enriched in patients with non-syndromic sagittal and coronal CS, respectively. Interestingly, both mutations show striking incomplete penetrance, suggesting a role for modifying alleles; in the case of SMAD6, a common variant near BMP2 drastically increases penetrance of sagittal CS. Here, we report a proband presenting with both sagittal and coronal craniosynostosis with the highly unusual recurrence of CS within two months of initial surgery, requiring a second operation to re-establish suture patency at six months of age. Exome sequencing revealed a rare transmitted frameshift mutation in SMAD6 (p. 152 fs*27) inherited from an unaffected parent, absence of the common BMP2 risk variant, and a de novo frameshift mutation in TCF12 (p.E548fs*14). SMAD6 and TCF12 independently inhibit transcriptional targets of BMP signaling. The findings are consistent with epistasis of these mutations, increasing penetrance and severity of CS in this proband. They also add to the list of composite phenotypes resulting from two Mendelian mutations, and support the utility of exome sequencing in atypical CS cases.

During the Gulf War, prophylactic treatment with pyridostigmine bromide (PB) along with the stress of deployment may have caused unexpected alterations in neural and immune function, resulting in a host of cognitive deficits which have become clinically termed Gulf War Illness (GWI). In order to test this interaction between PB and stress, the following study used a rodent model of GWI to examine how combinations of repeated restraint stress and PB induced alterations of peripheral cholinesterase (ChE) activity, corticosterone (CORT) levels, and cytokines on the last day of treatment, and then 10 days and three months post-treatment. Results indicate that PB decreases ChE activity acutely but sensitizes it by three months post-treatment selectively in rats subjected to stress. Similarly, while stress increased CORT levels acutely, rats in the PB/stressed condition continued to exhibit elevations in CORT at the delayed time point, indicating that PB and stress interact to progressively disrupt homeostasis in several peripheral measures. Because memory deficits are also common in clinical populations with GWI, we examined the effects of PB and stress on contextual fear conditioning. PB exacerbates stress-induced impairments in contextual fear conditioning ten days post-treatment, but protects against stress-induced augmentation of contextual fear conditioning at three months post-treatment. Collectively, these results provide critical insight as to how PB and stress may interact to contribute to the pathophysiological progression of GWI.

Seeds of Acorus americanus (Raf.) Raf. (Acoraceae), or ratroot as it is commonly known, were harvested from 3 locations in each of 2 y and subjected to combinations of light and dark treatments, 30 d of stratification, 3 germination temperature regimes, and 4 storage durations (zero to 24 mo). When germinated under ambient conditions, we determined that light is required for germination and that stored seeds germinate better after stratification (moist conditions at 2–4 °C). Observations on ideal storage time and germination temperatures, however, were inconclusive. This species is an important plant for Aboriginal peoples of northern Canada, and it is included in oil sands mining reclamation efforts.

Nine species of freshwater bryozoans were recorded in Lithuania in a survey of 18 various types of freshwater bodies. Eight species were assigned to the Class Phylactolaemata and families Plumatellidae and Cristatellidae (Plumatellarepens,Plumatellafungosa,Plumatellafruticosa,Plumatellacasmiana,Plumatellaemarginata,Plumatellageimermassardi,HyalinellapunctataandCristatellamucedo). The ninth species,Paludicellaarticulata, represented the Class Gymnolaemata.PlumatellageimermassardiandP.casmianawere recorded for the first time in Lithuania. For the plumatellids, species identification was achieved partly by analysing statoblasts’ morphological ultrastructures by scanning electron microscopy.

Agronomic management practices that maximize monoculture switchgrass (Panicum virgatum L.) yield are generally well understood; however, little is known about corresponding effects of differing switchgrass management practices on near-surface soil properties and processes. The objective of the study was to evaluate the effects of cultivar (“Alamo” and “Cave-in-Rock”), harvest frequency (1- and 2-cuts per year), fertilizer source (poultry litter and commercial fertilizer), and irrigation management (irrigated and non-irrigated) on near-surface soil properties and surface infiltration in a Leadvale silt loam (fine-silty, siliceous, semiactive, thermic, Typic Fragiudult) after four years (2008 through 2011) of consistent management in west-central Arkansas. Irrigating switchgrass increased (P 0.05) and averaged 0.79 mm∙min−1. Results from this study indicate that management decisions to maximize switchgrass biomass production affect soil properties over relatively short periods of time, and further research is needed to develop local best management practices to maximize yield while maintaining or improving soil quality.

Histopathological data collected from patients with severe malaria have been instrumental for studying malaria pathogenesis. Animal models of malaria are critical to complement such studies. Here, the histopathological changes observed in a rhesus macaque with severe and complicated Plasmodium cynomolgi malaria are reported. The animal presented with thrombocytopenia, severe anemia, and hyperparasitemia during the acute infection. The macaque was given subcurative antimalarial treatment, fluid support, and a blood transfusion to treat the clinical complications, but at the time of transfusion, kidney function was compromised. These interventions did not restore kidney function, and the animal was euthanized due to irreversible renal failure. Gross pathological and histological examinations revealed that the lungs, kidneys, liver, spleen, and bone marrow exhibited abnormalities similar to those described in patients with malaria. Overall, this case report illustrates the similarities in the pathophysiological complications that can occur in human malaria and cynomolgi malaria in rhesus macaques.

Charcoal rot, caused by Macrophomina phaseolina in soybean [Glycine max (L).Merr.] is a disease of economic significance in the United States. Chemical and cultural control are not effective and host resistance could be a potential method to control the disease. In this study we report the use of bulked-segregant analysis (BSA) to map the genetic basis of resistance to charcoal rot in soybean PI 567562A, using a QTL-seq approach. Resistant and susceptible bulks from progenies of a biparental population (PI 567562A and PI 567437) were sequenced, and reads were aligned to the PI 567562A genome and used to calculate the SNP-index at each SNP position for the resistant and susceptible bulks. The average distributions of the SNP-index and Δ SNP-index across the genome were estimated for genomic intervals using a sliding window analysis with 2-Mb window size and a 10-kb step. Three genomic regions on chromosomes 5, 8 and 14 were identified with positive values in Δ SNP-index plots that potentially correspond to QTL governing the difference between the R-bulk and S-bulk sub-populations. The current study is the first attempt employing QTL-seq to identify genomic regions that may contain QTL controlling charcoal rot disease resistance in soybean.

Melanoma, a highly aggressive form of cancer, is notoriously resistant to available therapies. Methotrexate (MTX), an antifolate, competitively inhibits DNA synthesis and is effective for several types of cancer. In cutaneous T-cell lymphoma (CTCL), MTX increases Fas death receptor by decreasing Fas promoter methylation by blocking the synthesis of SAM, the principal methyl donor for DNMTs, resulting in enhanced Fas-mediated apoptosis. The objective of this study was to explore the effects of MTX in human melanoma. MTX variably inhibited the survival of melanoma cells and induced apoptosis as evident by annexin V positivity and senescence associated β-galactosidase activity induction. Furthermore, MTX caused increased transcript and protein levels of extrinsic apoptotic pathway factors Fas and Fas-ligand, albeit at different levels in different cell lines. Our pyrosequencing studies showed that this increased expression of Fas was associated with Fas promoter demethylation. Overall, the ability of MTX to up-regulate Fas/FasL and enhance melanoma apoptosis through extrinsic as well as intrinsic pathways might make it a useful component of novel combination therapies designed to affect multiple melanoma targets simultaneously. In support of this concept, combination therapy with MTX and interferon-alpha (IFNα) induced significantly greater apoptosis in the aggressive A375 cell line than either agent alone.

Ratroot, or Acorus americanus (Raf.) Raf. (Acoraceae), can be propagated from seed harvested from natural sites in northeastern Alberta. Light is required for germination, and germination is most successful at warm temperatures (15/25 °C). Seedlings can be quickly (

Mycosis fungoides (MF) and Sezary syndrome (SS) are two major forms of cutaneous T-cell lymphoma (CTCL) characterized by resistance to apoptosis. A central pathway for T-cell apoptosis is activation-induced cell death (AICD) which is triggered through the T-cell receptor (TCR). This results in upregulation of FAS-ligand (FASL) and subsequent apoptosis through the FAS death receptor pathway. It has been known for more than a decade that TCR signaling is defective in CTCL; however, the underlying mechanism has not been apparent. In this report, we show that the E3 ubiquitin ligase, c-CBL, is over-expressed in CTCL and that its knockdown overcomes defective TCR signaling resulting in phosphorylation of PLCg1, calcium influx, ROS generation, up-regulation of FASL and extrinsic pathway apoptosis in CTCL cells expressing adequate FAS. In CTCL cells with suboptimal FAS expression, FAS can be upregulated epigenetically by derepression of the FAS promoter using methotrexate (MTX) which we showed previously has activity as a DNA methylation inhibitor. Using these combined strategies, FAS-low as well as FAS-high CTCL cells can be killed effectively.

Significance: Skin, a complex organ and the body's first line of defense against environmental insults, plays a critical role in maintaining homeostasis in an organism. This balance is maintained through a complex network of cellular machinery and signaling events, including those regulating oxidative stress and circadian rhythms. These regulatory mechanisms have developed integral systems to protect skin cells and to signal to the rest of the body in the event of internal and environmental stresses. Recent Advances: Interestingly, several signaling pathways and many bioactive molecules have been found to be involved and even important in the regulation of oxidative stress and circadian rhythms, especially in the skin. It is becoming increasingly evident that these two regulatory systems may, in fact, be interconnected in the regulation of homeostasis. Important examples of molecules that connect the two systems include serotonin, melatonin, vitamin D, and vitamin A. Critical Issues: Excessive reactive oxygen species and/or dysregulation of antioxidant system and circadian rhythms can cause critical errors in maintaining proper barrier function and skin health, as well as overall homeostasis. Unfortunately, the modern lifestyle seems to contribute to increasing alterations in redox balance and circadian rhythms, thereby posing a critical problem for normal functioning of the living system. Future Directions: Since the oxidative stress and circadian rhythm systems seem to have areas of overlap, future research needs to be focused on defining the interactions between these two important systems. This may be especially important in the skin where both systems play critical roles in protecting the whole body. Antioxid. Redox Signal. 20, 2982–2996.

Silent information regulator type-1 (SIRT1) is the best-studied member of the Sirtuin (Sir2) family of nicotinamide dinucleotide (NAD)-dependent class III histone deacetylases (HDACs), but has not yet been explored in cutaneous T-cell lymphoma (CTCL). We analyzed five CTCL cell lines and lesional tissues using flow cytometry, immunostaining, immunoblotting, cell death, viability, and apoptosis assays, small-molecule inhibitors, and shRNA knockdown. We found strong SIRT1 expression among CTCL lines relative to normal lymphocytes. CTCL cells in lesional tissues also expressed SIRT1 strongly. SIRT1 knockdown resulted in reduced cellular metabolism and proliferation, increased apoptosis, and PARP cleavage products. Tenovin-1, which reversibly inhibits class III HDACs (SIRT1 and SIRT2), reduced SIRT enzymatic activity and SIRT1 expression and led to increased apoptosis. These alterations were accompanied by increased forkhead box O3 (FoxO3) in several cell lines and increased nuclear p53, as well as acetylated p53 in wtp53 MyLa CTCL line. A combination of class I/II and class III HDACIs (vorinostat and tenovin-1) produced significantly greater growth inhibition, cell death via apoptosis, as well as superior p53 promoter upregulation in wtp53 MyLa cells as compared with either agent alone. This occurred in a partially p53-dependent manner, as these effects were blunted by p53 knockdown. Our results indicate that SIRT1 is strongly expressed in CTCL. Its inhibition results in reduced growth and increased apoptosis of CTCL cells. Furthermore, our findings suggest that some CTCL patients, such as those with wtp53, might benefit more from treatment with a combination of different classes of HDACIs than with a single agent.

1. In early discovery stages, 2-methyl-N-(2'-(pyrrolidinyl-1-ylsulfonyl)-[1,1'-biphenyl]-4-yl)propan-1-amine (PBPA) demonstrated monoamine oxidase A (MAO-A) and cytochrome P450 (CYP)-mediated clearance. While human liver microsomes predicted low CL(b) PBPA demonstrated a moderate CL(p)/F in humans. The plasma pharmacokinetic (PK) of PBPA was characterized by unexpected high inter-individual variability. Hence, a retrospective analysis was undertaken to understand the disposition processes of PBPA, by applying in vitro mechanistic tools. 2. The in vitro-to-in vivo of rat CL(b) of PBPA was calculated as similar to that of human, suggesting rat to be a better predictor of a MAO-A/CYP substrate, but not dog or monkey; this is consistent with differences in expression of MAO-A in rat, dog, monkey and human. Fraction metabolized (f(m)) of human MAO A (hMAO-A) (50%), CYP3A4 (8%), CYP3A5 (16%) and CYP2D6 (29%) was determined, in vitro. 3. While the fm of CYP3A5 was

Summary A 25-year-old, female rhesus macaque (Macaca mulatta) presented with a history of weight loss despite a normal appetite and supportive care. The animal was humanely destroyed due to poor prognosis. Post-mortem examination revealed a focally extensive, firm, white annular constriction at the ileocaecal junction and an incidental finding of a pale white nodule approximately 0.8 cm in diameter in the left renal pelvis. Based on the microscopical findings, ileocaecal adenocarcinoma and renal pelvis transitional cell carcinoma (TCC) were diagnosed. The use of cytokeratin (CK)-7 and -20 and uroplakin III as potential renal TCC markers was evaluated. The neoplastic cells were labelled intensely with antibodies to uroplakin III, but not to CK-7 or -20. Spontaneous intestinal adenocarcinoma has been documented in the rhesus macaque, but concurrent renal pelvis TCC is highly unusual. 2013 Elsevier Ltd. All rights reserved.

Rohwer et al. (2009) present isotopic evidence of migratory double breeding by five species of birds that were breeding in July and August in northwestern Mexico. The presence of old brood patches in these species, combined with their late breeding in Mexico, suggested that these species had bred earlier in the north. Here we present data showing that Orchard Orioles and Yellow-breasted Chats breed as commonly in coastal Sinaloa in late May and early June as they do later in the summer; further, many females of these species examined in early June 2011 had downy ventral apteria, indicating that they were breeding for the first time in their annual cycle in Sinaloa. Thus, the old brood patches observed in these species in July and August, when they are still breeding in northwestern Mexico, may reflect earlier breeding attempts by those same individuals in Mexico. Yellow-billed Cuckoos seldom call and are uncommon in northwestern Mexico until late June and likely do not begin breeding until July. ...

Delphinidin (Del), [3,5,7,3'-,4'-,5'-hexahydroxyflavylium], an anthocyanidin and a potent antioxidant abundantly found in pigmented fruits and vegetables exhibits proapoptotic effects in many cancer cells. Here, we determined the effect of Del on growth, apoptosis and differentiation of normal human epidermal keratinocytes (NHEKs) in vitro in submerged cultures and examined its effects in a three-dimensional (3D) epidermal equivalent (EE) model that permits complete differentiation reminiscent of in vivo skin. Treatment of NHEKs with Del (10-40 μm; 24-48 h) significantly enhanced keratinocyte differentiation. In Del-treated cells, there was marked increase in human involucrin (hINV) promoter activity with simultaneous increase in the mRNA and protein expressions of involucrin and other epidermal differentiation markers including procaspase-14 and transglutaminase-1 (TGM1), but without any effect on TGM2. Del treatment of NHEKs was associated with minimal decrease in cell viability, which was not associated with apoptosis as evident by lack of modulation of caspases, apoptosis-related proteins including Bcl-2 family of proteins and poly(ADP-ribose) polymerase cleavage. To establish the in vivo relevance of our observations in submerged cultures, we then validated these effects in a 3D EE model, where Del was found to significantly enhance cornification and increase the protein expression of cornification markers including caspase-14 and keratin 1. For the first time, we show that Del induces epidermal differentiation using an experimental system that closely mimics in vivo human skin. These observations suggest that Del could be a useful agent for dermatoses associated with epidermal barrier defects including aberrant keratinization, hyperproliferation or inflammation observed in skin diseases like psoriasis and ichthyoses.

Prolonged survival of lesional T cells plays a central role in the pathogenesis of T-cell-mediated dermatoses. We have recently shown that the ubiquitin ligase c-CBL is highly expressed in cutaneous T-cell lymphoma (CTCL) and that its knockdown increases activation-induced cell death, a key pathway for T-cell apoptosis. Here, we extend our work on c-CBL expression in malignant T cells to their non-neoplastic counterparts in benign inflammatory dermatoses. Immunohistochemical staining with anti-c-CBL antibody was performed on lesional biopsies from a total of 65 patients with atopic dermatitis, allergic contact dermatitis, pityriasis rosea, psoriasis vulgaris, lichen planus, mycosis fungoides (MF)/Sézary syndrome (SS) as well as on tonsil tissue from 5 individuals and on 5 human cutaneous T-cell lymphoma cell lines. Protein levels were measured in situ using multispectral image analysis, a quantitative method that is 5x more sensitive than standard immunohistology for antigen detection. There was a significant (p < 0.05) and progressive increase of mean c-CBL expression across the spectrum of inflammatory dermatoses (2-fold), MF/SS (3-fold) and lymphoma cell lines (4-fold) as compared to tonsillar T lymphocytes. A subset of MF/SS cases expressed mean c-CBL levels above the ranges observed in inflammatory dermatoses. Given our prior finding that c-CBL inhibits activation-induced cell death, c-CBL might play a role in the pathogenesis of inflammatory dermatoses and CTCL.

The melanocortin system has been implicated in the regulation of various physiological functions including melanogenesis, steroidogenesis, energy homeostasis, and feeding behavior. Five melanocortin receptors have been identified to date and belong to the family of G protein-coupled receptors (GPCR). Post-translational modification of the proopiomelanocortin (POMC) prohormone leads to the biosynthesis of the endogenous melanocortin agonists, including α-melanocyte stimulating hormone (α-MSH), β-MSH, γ-MSH, and adrenocorticotropic hormone (ACTH). All the melanocortin agonists derived from the POMC prohormone contain a His-Phe-Arg-Trp tetrapeptide sequence that has been implicated in eliciting the pharmacological responses at the melanocortin receptors. Herein, an alanine (Ala) positional scan is reported for the endogenous α-MSH ligand and the synthetic, more potent, NDP-MSH peptide (Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH2) at the cloned mouse melanocortin receptors to test the assumption that the structure-activity relationships of one ligand would apply to the other. Several residues outside of the postulated pharmacophore altered potency at the melanocortin receptors, most notably the 1560-, 37-, and 15-fold potency loss when the Glu(5) position of α-MSH was substituted with Ala at the mMC1R, mMC3R, and mMC4R, respectively. Importantly, the altered potencies due to Ala substitutions in α-MSH did not necessarily correlate with equivalent Ala substitutions in NDP-MSH, indicating that structural modifications and corresponding biological activities in one of these melanocortin ligands may not be predictive for the other agonist.

Signaling by phosphatidylinositol 4-kinases (PI4Ks) in the Golgi apparatus controls lipid homeostasis and protein-sorting pathways. Signaling is shown to be terminated on the medial cisterna by a complex of a PI4K effector, Vps74, and Sac1, the major PtdIns4P phosphatase in the cell., In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (KD = 3.8 μM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents.

OCA2 is used as a model melanosome cargo protein to define primary sequence elements required for acidic dileucine–motif binding to adaptors AP-1 and AP-3. OCA2 must bind to AP-3 for melanosome localization. BLOC-1 is also required and thus can cooperate with either adaptor for cargo delivery to lysosome-related organelles., Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.

FAS (TNF receptor superfamily member 6, also known as CD95) plays a major role in T-cell apoptosis and is often dysregulated in CTCL. We searched for structural alterations of the FAS gene with the potential to affect its function. Although several heterozygous FAS promoter single nucleotide polymorphisms (SNPs) were detected, the only homozygous one was the -671 GG SNP present in 24/80 CTCL cases (30%). This SNP maps to an interferon response element activated by STAT-1. EMSA and supershift EMSA showed decreased CTCL nuclear protein/STAT-1 binding to oligonucleotides bearing this SNP. Luciferase reporters showed significantly less interferon-alfa responsive expression by FAS promoter constructs containing this SNP in multiple CTCL lines. Finally, FAS was upregulated by interferon-alfa in wildtype CTCL cells but not those bearing the -671 GG SNP. These findings indicate that many CTCL patients harbor the homozygous FAS promoter -671 GG SNP capable of blunting its response to interferon. This may have implications for CTCL pathogenesis, racial incidence and the response of patients to interferon-alfa therapy. In contrast, functionally significant mutations in FAS coding sequences were detected uncommonly. Among CTCL lines with the potential to serve as models of FAS regulation, FAS-high MyLa had both FAS alleles, FAS-low HH was FAS-hemizygous and FAS-negative SeAx was FAS-null.

Summary Background Polo-like kinase 1 (Plk1) has multiple functions throughout mitosis. Plk1 levels are high in a number of cancers and haematological malignancies while being low in most differentiated tissues. Objectives To assess the immunoreactivity of Plk1 in cutaneous T-cell lymphoma (CTCL) as a potential therapeutic target, to differentiate Plk1 levels among lesion types and to compare the detection level of Plk1 in fresh frozen (f) vs. paraffin-embedded (p) tissue. Methods Immunohistochemical staining of CTCL skin lesions with anti-Plk1 antibody was performed in a total of 65 biopsies from 49 patients with CTCL. Both f and p tissue was available for comparison in 46 biopsies. Results Tumour-stage CTCL lesions displayed significantly more Plk1 (mean f 7·7%, p 8·8%) than patch (mean f 0·7%, p 2·0%) and plaque-stage lesions (mean f 1·1%, p 2·0%) (P