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Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion
Stephen M. Christensen,
Affiliation Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America
Laura A. L. Dillon,
Affiliations Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America, Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America
Lucas P. Carvalho,
Affiliation Universidade Federal da Bahia Salvador, Bahia, Brazil
Sara Passos,
Fernanda O. Novais,
Affiliation Department of Pathobiology, School of Veterinary Medicine, The University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
V. Keith Hughitt,
Daniel P. Beiting,
Edgar M. Carvalho,
Phillip Scott,
Najib M. El-Sayed,
David M. Mosser
* E-mail: dmosser@umd.edu
Najib M. El-Sayed
Stephen M. Christensen Laura A. L. Dillon ... David M. Mosser
3 May 2017: Christensen SM, Dillon LAL, Carvalho LP, Passos S, Novais FO, et al. (2017) Correction: Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion. PLOS Neglected Tropical Diseases 11(5): e0005588. https://doi.org/10.1371/journal.pntd.0005588 View correction
Host and parasite gene expression in skin biopsies from Leishmania braziliensis-infected patients were simultaneously analyzed using high throughput RNA-sequencing. Biopsies were taken from 8 patients with early cutaneous leishmaniasis and 17 patients with late cutaneous leishmaniasis. Although parasite DNA was found in all patient lesions at the time of biopsy, the patients could be stratified into two groups: one lacking detectable parasite transcripts (PTNeg) in lesions, and another in which parasite transcripts were readily detected (PTPos). These groups exhibited substantial differences in host responses to infection. PTPos biopsies contained an unexpected increase in B lymphocyte-specific and immunoglobulin transcripts in the lesions, and an upregulation of immune inhibitory molecules. Biopsies without detectable parasite transcripts showed decreased evidence for B cell activation, but increased expression of antimicrobial genes and genes encoding skin barrier functions. The composition and abundance of L. braziliensis transcripts in PTPos lesions were surprisingly conserved among all six patients, with minimal meaningful differences between lesions from patients with early and late cutaneous leishmaniasis. The most abundant parasite transcripts expressed in lesions were distinct from transcripts expressed in vitro in human macrophage cultures infected with L. amazonensis or L. major. Therefore in vitro gene expression in macrophage monolayers may not be a strong predictor of gene expression in lesions. Some of the most highly expressed in vivo transcripts encoded amastin-like proteins, hypothetical genes, putative parasite virulence factors, as well as histones and tubulin. In summary, RNA sequencing allowed us to simultaneously analyze human and L. braziliensis transcriptomes in lesions of infected patients, and identify unexpected differences in host immune responses which correlated with active transcription of parasite genes.
Leishmania spp are intracellular protozoan parasites that replicate primarily within host tissue macrophages. In this paper we simultaneously query host and parasite gene expression in human cutaneous L. braziliensis lesions. We observe an unexpectedly prominent role for B cells and immunoglobulins in lesions in which actively transcribing parasites reside. We also observe that parasite gene expression is surprisingly conserved among L. braziliensis lesions, and the genes that are expressed in lesions are not those that have been previously associated with parasite growth in vitro. This analysis of parasite and host gene expression in lesions may lead to the identification of new parasite virulence factors and may identify host responses that promote parasite persistence in lesions.
Citation: Christensen SM, Dillon LAL, Carvalho LP, Passos S, Novais FO, Hughitt VK, et al. (2016) Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion. PLoS Negl Trop Dis 10(9): e0004992. https://doi.org/10.1371/journal.pntd.0004992
Editor: Abhay R. Satoskar, Ohio State University, UNITED STATES
Received: March 28, 2016; Accepted: August 19, 2016; Published: September 15, 2016
Copyright: © 2016 Christensen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Data are available from the Sequence Read Archive (www.ncbi.nlm.nih.gov) under the project accession PRJNA307599.
Funding: This work was supported by R01 grants from the National Institutes of Health NIAID (AI094773) and NIGMS (GM102589), and an International Collaboration for Infectious Disease Research (ICIDR) grant (U01-AUI088650). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Leishmaniasis is characterized as a spectral disease, with clinical presentations ranging from a self-healing cutaneous form to a visceral form associated with high morbidity and mortality. The immune response to the various species is also spectral in nature, with L. donovani inducing minimal immune responses or actually inhibiting inflammation and immunity [1], and L. braziliensis inducing immune activation and immune-mediated pathology [2]. L. braziliensis is the causative agent of tegumentary leishmaniasis in South America. Approximately 3–5% of patients infected with L. braziliensis will eventually develop mucocutaneous disease, a disfiguring manifestation involving the nasal and oropharyngeal mucosa [3]. L. braziliensis infections are typically associated with a strong Th1 response and a positive skin test response to soluble leishmanial antigens characterized by high levels of TNF and IFN-γ [4].
'Early' L. braziliensis lesions frequently present as a small papule that can eventually progress to larger 'late' ulcerative lesions, typically containing small numbers of parasites. The more severe mucocutaneous forms of the disease are associated with increased cytokine responses and increased T cell proliferation responses to parasite antigens [4]. The robust immune response to this organism despite low or undetectable numbers of parasites in lesions, and the increased immune responses in mucocutaneous forms of the disease have led to the suggestion that exaggerated Th1 host immune responses contribute to the pathological tissue damage associated with L. braziliensis infections [5]. Recent studies suggest that a major factor in the development of disease caused by L. braziliensis is the recruitment of cytolytic CD8+ T cells that promote increased inflammation [6,7].
Immune responses in the skin of infected patients may not be accurately reflected by the type and magnitude of host responses in peripheral blood. Previous studies using immunohistochemistry have described the infiltration of T cells, macrophages, B cells, NK cells, and granulocytes into lesions [8–10]. Some research has implicated CCR2-positive monocytes in parasite killing [11,12], while other studies have described the contribution of the skin micro-environment to the local immune response [13–16]. For these reasons, a meta-transcriptomic profiling of lesions may provide a more complete picture of host and parasite responses during infection.
A microarray-based transcriptomic profiling of skin lesions from patients infected with L. braziliensis was recently reported [17]. This work uncovered an association between chemokines and cytotoxic T cell responses and disease-associated pathology. We extended this work using RNA-seq to simultaneously analyze host and parasite transcripts in these lesions. This approach allowed the detection of parasite transcripts expressed in the skin of infected patients, and revealed unexpected differences in the host response associated with the detectability of parasite transcripts in lesions. It also revealed that the parasite transcripts most abundantly produced in human lesions could not be predicted by in vitro cultivation of parasites in human macrophages.
The human host transcriptome in lesions of L. braziliensis infected patients
RNA sequencing analysis was performed on biopsies from 10 healthy and 25 L. braziliensis infected patients. Principal Component Analysis (PCA) (Fig 1A) and hierarchical clustering (S1A Fig) of the human transcriptome data revealed distinct differences between healthy skin and lesions from infected patients, as expected. Lesions are clinically defined as either 'early' (small papule, no ulceration, ≤ 30 days illness duration) or 'late' (ulcerated lesions, ≥ 30 days illness duration) (S1 Table). Within the group of biopsies from the 25 infected individuals, there were minimal appreciable differences in host responses between patients with early and late cutaneous leishmaniasis (Fig 1B, S2 Table), as previously reported [17]. There was also no separation in host responses as a function of sex (S1B Fig), lesion size (S1C Fig), illness duration (S1D Fig), or age (S1E Fig).
Fig 1. Human and parasite transcriptomes in lesions of L. braziliensis infected patients.
(1A) Principal component analysis (PCA) of human transcriptomes from 10 healthy volunteers (light gray) and 25 L. braziliensis-infected patients (dark gray) is shown. The first two principal components (PC) are displayed on each axis along with the variance (56.79% and 7.61%). (1B) Principal component analysis (PCA) plot of human transcriptomes from 8 early-stage (pink), and 17 late-stage (maroon) leishmaniasis patients is shown. The first two principal components (PC) are displayed on each axis along with the variance (24.91% and 12.64%). (1C) The percentage of total reads that mapped to the Leishmania braziliensis genome from each of the 25 infected patient samples are plotted as bars. The samples containing > 0.5% reads are labeled as parasite transcript positive (PTPos, orange), 0.01–0.5% reads as parasite transcript intermediate (PTInt, yellow), and the rest < 0.01% reads as parasite transcript negative (PTNeg, blue). (1D) A PCA plot of patient samples demonstrates grouping according to parasite transcript levels: PTNeg (blue), PTInt (yellow), and PTPos (orange). The first two principal components (PC) are displayed on each axis along with the variance (24.91% and 12.64%).
RNA-seq allows the simultaneous analysis of both host and parasite transcriptomes, and we sought to determine whether the presence or absence of detectable parasite transcripts in lesions could impact host responses. Lesions from six of the patients were considered parasite transcript positive (PTPos) when greater than 0.5% of the total reads mapped to the parasite genome (Fig 1C, orange). Ten lesions were considered parasite transcript negative (PTNeg) because less than 0.01% of the total reads mapped to the parasite (Fig 1C, blue). This is the same proportion of parasite reads that map non-specifically to highly conserved homologous sequences in the human genome. Nine lesions were considered parasite transcript intermediate (PTInt) when percent reads ranged from 0.01–0.2 (Fig 1C, yellow). The number of parasite reads in this group was too low to provide a meaningful analysis of parasite gene expression, and therefore they were excluded from the PTPos group. By PCA, there was a clear separation in host responses of the PTPos (Fig 1D, orange) and PTNeg groups (Fig 1D, blue, note dashed line separating blue and orange), with the PTInt group (Fig 1D, yellow) falling in between these two. This separation between groups becomes even more obvious with the addition of the third principal component, accounting for 11.89% of the variance, where there is a clear separation in host responses between PTPos and PTNeg lesions (S1F Fig). Thus, there is a surprising separation of host responses, depending on the presence or absence of detectable parasite transcripts in lesions, allowing us to correlate host responses with parasite persistence or elimination.
Host transcriptomic responses in PTPos and PTNeg samples
We found large differences in host transcripts between healthy controls and infected patients, as expected, with 4579 host genes being differentially expressed in lesions relative to normal skin using a cutoff fold change > 2 and P value < 0.05 (S3 Table). A total of 3884 differentially expressed genes were common to PTNeg and PTPos, but there was a total of 477 differentially expressed genes unique to PTPos lesions (Fig 2A, orange circle) and 241 differentially expressed genes unique to PTNeg lesions (Fig 2A, blue circle), relative to healthy controls. The intermediate (PTInt) samples shared 3817 differentially expressed genes with PTNeg and PTPos, as well as an additional 987 genes with PTPos and 214 genes with PTNeg. A total of 238 genes showed differential expression unique to the PTInt group (Fig 2A, yellow circle). S4 Table shows direct quantitative comparisons between gene expression in PTPos and PTNeg lesions, totaling 719 differentially expressed genes. Twenty-five of the top 30 upregulated genes in PTPos lesions relative to PTNeg lesions encoded immunoglobulin fragments, including 9 of the top 10 genes (Fig 2B, black bars). This group also included CXCL8 (granulocyte migration), IL-21 (B cell proliferation), and granulysin (cellular cytotoxicity). The genes most highly expressed in PTNeg lesions relative to PTPos lesions included genes involved in skin defenses and epidermal cell development. The top 10 genes in this category included loricrin, filaggrin-1, filaggrin-2, and hornerin, all involved in skin development (Fig 2B, light gray bars).
Fig 2. Comparison of human host transcriptomes in lesions with parasite detectable-positive transcripts (PTPos) versus parasite detectable-negative (PTNeg) transcripts.
(2A) A Venn diagram shows differentially expressed genes in three patient populations compared to healthy controls (HC): PTPos (orange circle), PTNeg (blue circle), PTInt (yellow circle). (2B). Shown are the top ten significantly upregulated (black) and downregulated (light gray) host genes when comparing PTPos to PTNeg (mean plus SEM). (2C) The 25 immunoglobulin genes exhibiting the highest differential expression between PTNeg and PTPos samples are shown. Bars depict the fold change in expression in PTNeg (blue), PTInt (yellow), and PTPos (orange) samples relative to healthy controls (mean plus SEM).
Although immunoglobulin gene expression in PTNeg lesions increased slightly relative to uninfected healthy skin (Fig 2C, blue), immunoglobulin gene expression in PTPos lesions was substantially higher (367 fold ± 261 compared to 56 fold ± 32) (Fig 2C, orange). The level of immunoglobulin transcripts in the PTInt group (Fig 2B, yellow) fell between the PTPos and PTNeg groups (122 fold ± 75). Thus, high immunoglobulin levels in lesions may portend a poor prognosis in this disease, as previously suggested (23).
Immune response signatures of PTPos and PTNeg patients
An assessment of transcripts encoding cell-specific markers pointed to an increase in B cells and their products in the PTPos lesions. B cell transcripts encoding CD79A, CD19, and CD20, were upregulated in PTPos lesions, relative to healthy controls (Fig 3A) and relative to PTNeg lesions (indicated by a # symbol). Transcript levels in the PTInt group fell between PTPos and PTNeg (Fig 3A, yellow). Increases in transcripts encoding B cell markers were not observed when comparing between early and late cutaneous leishmaniasis lesions (S2A Fig). T cell markers (CD3e, CD4, and CD8a) demonstrated no significant differences between PTPos, intermediate and PTNeg lesions (Fig 3A). No significant differences in T cell markers were detected between early and late cutaneous lesions (S2A Fig) as previously reported [17]. Transcripts for inflammatory molecules (IFN-γ, TNF, IL-1β and FASLG), inhibitory molecules (IL-10, CTLA4, PD1, PDL1, and LAG3), and activation markers (CD80 and CD38) were all higher in PTPos lesions compared to PTNeg lesions (Fig 3B), with PTInt lesions exhibiting intermediate transcript levels (Fig 3B). In contrast to the differences observed between PTPos and PTNeg lesions, we saw no significant difference in the levels of transcripts for cytokines or signaling molecules in early versus late lesions (S2B Fig).
Fig 3. Immune response signatures in parasite transcript positive (PTPos) and negative (PTNeg) lesions.
(3A) Bars represent the fold change of RNA expression of cell-specific markers in PTNeg (blue), PTInt (yellow), and PTPos (orange) samples, each compared to healthy controls (mean plus SEM). Statistically significant differentially expressed genes, when comparing PTPos to PTNeg are identified with a # symbol (p<0.05) (3B) Bars depict fold changes of transcripts encoding inflammatory cytokines (ifnγ, tnf, IL12p40, IL-10, faslg), anti-inflammatory and inhibitory signals (il10, ctla4, pdcd1, cd274, lag3), and activation markers (cd80, cd38) in lesions of all three classifications compared to healthy controls (mean plus SEM). Statistically significant differentially expressed genes when comparing PTPos to PTNeg are identified with a # symbol (p<0.05). (3C) Shown is the top module (by p-value, see * in S3A Fig) and representative genes from within the module correlated to percent parasite reads using WGCNA analysis. The left plot demonstrates correlation to the module eigengene versus correlation to parasite percent reads for each gene. On the right, plots demonstrate normalized expression (rpkm) versus parasite percent reads for selected genes from within the module (highlighted in red in the left plot).
We used the weighted gene co-expression network analysis (WGCNA) program to cluster human host gene expression through comparison of gene expression profiles using pair-wise correlations. Cluster profiles were then assessed as a function of parasite transcript abundance. This analysis yielded three modules of genes (among a total of 51 modules in the network) whose expression exhibited a significant correlation with parasite transcript abundance (S3A Fig). The most highly correlated module (see * in S3A Fig) contained 100 genes (Fig 3C, left). Eight of the top 14 genes and their normalized expression versus parasite transcript number are shown in Fig 3C, right. These host genes included IL-10, IFN-γ, TNF, granzyme B and fas ligand. All of the genes in this module exhibited a higher expression in PTpos lesions (Fig 3C, orange dots). The two additional modules that also showed a significant correlation between host gene expression and parasite transcript numbers contained immunoglobulin transcripts, chemokine genes, cytokines and growth factors (S3 Fig).
Functional interactions and distinct clustering among differentially expressed genes
The Reactome FI plugin for Cytoscape was used to observe known interactions between the 719 genes that were differentially expressed between PTNeg and PTPos (S4 Table). An additional 149 linker genes, known to influence or connect multiple genes in the gene set, were included in this analysis. A network of 558 genes generated numerous clusters showing a dense interaction of genes differentially expressed between PTNeg and PTPos. When clustering genes by functional interactions from the Reactome database, the top three clusters highlight specific immune and cellular pathways (Fig 4). The largest cluster (Fig 4, blue nodes) consists of 137 genes associated with immune cell activation, costimulation, and cytokine and chemokine signaling, including TNF, IL-10, and multiple C-C motif chemokines (S4A Fig). Gene regulation in this category is associated with NF-κβ, CREB/STAT3, JUN, and SP1 signaling. The second largest cluster (Fig 4, red nodes) includes 103 genes involved in B and T cell activation and inhibition, regulated by SYK, SRC, FYN, PLCG1 and 2, and PTPN11 (S4B Fig). Cluster 3 (Fig 4, yellow nodes) includes 72 genes associated with cell growth and proliferation signals (S4C Fig). The transcriptomic differences between PTPos and PTNeg patients demonstrate the potential of these clusters to lead to the identification of new drug targets for treating cutaneous leishmaniasis.
Fig 4. Functional interactions among differentially expressed genes.
(4A) A group of 237 of the 719 differentially expressed genes between PTNeg and PTPos (circular nodes) along with 75 associated linker genes (diamond nodes, black border) show numerous functional interactions (edges). Node size is relative to the number of interactions. Gene clusters are depicted by color (blue, red, and yellow) and direction of differential expression of genes within the clusters is depicted by node border, with green borders designating upregulated and red borders designating downregulated genes. Edges represent numerous known (solid line) and predicted (dashed line) functional interactions.
Identification of L. braziliensis transcripts in lesions
We analyzed parasite gene expression in the six PTPos patient samples and despite substantial differences in lesion size and duration (three early and three late cutaneous PTPos lesions), there was a high degree of uniformity in L. braziliensis transcript expression in all six patient lesions. Pearson correlation analysis of the top 50 parasite genes in each sample quantitatively demonstrated the similarity between samples, and ranged from 0.83 to 0.92 (Fig 5A). The 20 most highly-expressed parasite transcripts by average RPKM, listed in Table 1, were fairly randomly dispersed across the parasite genome (S5 Fig). Six of the parasite chromosomes are illustrated in Fig 5B and the parasite gene expression (in RPKM) is shown by vertical lines in the 6 patients (each patient designated by a different color intensity). Again, there is remarkable uniformity in parasite gene expression from patient to patient. Within these six chromosomes, 12 of the top 20 parasite genes (by RPKM) are noted, including cysteine peptidases, cysteine synthase, a proteasome subunit, and various hydrolase-like and hypothetical proteins.
Fig 5. L. braziliensis gene expression in human lesions.
RPKM values of all samples were scaled to the sample with the highest total normalized reads. (5A) A heatmap demonstrates Pearson correlation of parasite gene expression of the top 50 parasite genes in the six PTPos samples. (5B) A representation of six parasite chromosomes (16, 21, 28, 29, 33, 35) shows the RPKM levels for each PTPos patient, early patients depicted in blue hues, late patients in brown hues. RPKM is depicted on the y axis (0 to 50,000), and genes that appear in the 20 most-highly expressed parasite genes are labeled.
Table 1. Top 20 L. braziliensis genes expressed in detectable-positive lesions as ranked by RPKM.
All 20 of the most highly expressed parasite genes were expressed in all six patients, regardless of whether the lesions were early or late (S5 Fig). The top parasite transcripts (Table 1) consisted of cysteine peptidases, a phosphodiesterase, as well as kinases and transport proteins. The presence of hypothetical and putative proteins in this list illustrates our lack of understanding of parasite gene expression during natural infections. Several amastin family genes and known virulence factors including GP63, heat-shock proteins 70 and 83, and cysteine peptidases appear in the top 100 observed transcripts (S5 Table).
Our recent work demonstrated the benefits of dual-transcriptomic analyses, using in vitro infection of macrophages with L. major and L. amazonensis to observe expression pattern changes in the host cell and parasite gene expression, ranging from 4 to 72 hours [18]. To determine whether the L. braziliensis transcripts identified in lesions were the most highly expressed parasite genes expressed in vitro, we compared the top 40 L. braziliensis transcripts identified in lesions (ranked by RPKM), to the homologous genes expressed by L. amazonensis or L. major 72 hours after an in vitro infection of human macrophages, as previously described by us [18] (S6A Fig). A comparison of homologous genes by rank showed that more than 60% of the most highly expressed L. braziliensis genes in vivo (S6A Fig, red) did not match the most highly expressed parasite genes in cultivated macrophages following in vitro infection with L. amazonensis (S6A Fig, green) or L. major (S6A Fig, blue). Only 15 of the top 40 genes expressed by L. braziliensis during in vivo infections appeared in the top 40 L. major genes expressed in vitro, and only 5 of the top 40 genes appeared in the top 500 L. amazonensis genes expressed in vitro. The converse of matching the most highly expressed genes was also true. The most highly expressed L. amazonensis genes expressed in vitro (S6B Fig, green) were not highly expressed by L. braziliensis in vivo or by L. major in vitro (S6B Fig, red and blue). A direct comparison to L. braziliensis genes expressed in vitro was not possible because RNA-seq analysis of L. braziliensis transcripts following in vitro infection of human macrophages has not been performed. This work is underway. Therefore, definitive conclusions regarding differences in gene expression between in vitro and in vivo infections cannot be made. However the results from this inter-species comparison suggests that the transcripts identified in L. braziliensis lesions were not simply the most highly expressed leishmanial genes, but rather might be transcripts involved in promoting parasite persistence in the lesion microenvironment.
By applying RNA sequencing technology to skin biopsy material, we were able to capture in detail the transcriptomes of both the parasite and the human host during L. braziliensis localized cutaneous infection. This analysis allowed us to examine host responses as a function of parasite persistence. L. braziliensis infections have often been described as having low parasite numbers in lesions [19]. This is consistent with our observation that lesions from 10 of the 25 patients had no detectable parasite transcripts in them at the time of biopsy. This lack of detectable transcripts is consistent with an active elimination of parasites by the host. In contrast, six of the 25 patients had ample evidence of high confidence parasite transcripts in lesions (averaging nearly 1M parasite reads/sample; see S1 Table). The presence of these transcripts indicates that viable parasites were persisting in lesions and continuing to produce transcripts that could contribute to their survival. Therefore, we compared host immune responses in lesions where parasites were producing detectable transcripts (PTPos) to those where parasite transcripts were undetectable (PTNeg).
The progression of L. braziliensis disease has been well-studied, and the small nodules of early tegumentary leishmaniasis typically progress to the larger sometimes necrotic lesions associated with late cutaneous leishmaniasis [20]. Studies have been done to examine differences in immune responses between these two groups [21], and comparisons with regard to treatment efficacy between early and late disease have also been made [19,22]. A surprising result from the previous transcriptomic comparison between early and late L. braziliensis lesions was that the host immune response was initiated early during infection and persisted throughout the course of the disease [17]. Our RNA-seq confirms this previous observation. We could not separate the host response during early and late tegumentary leishmaniasis by Principal Component Analysis (Fig 1). However, when these responses were stratified by the presence or absence of parasite transcripts, a clear separation could be achieved between the host responses in lesions where parasite transcripts were detectable (PTpos) versus those where they were not detectable (PTneg). We chose to contrast those two groups with an eye to understanding host responses that may be associated with parasite persistence or parasite elimination.
One of the surprising observations from this work was the degree to which B cell transcripts were associated with lesions containing detectable levels of parasite transcripts. Of the top 100 genes that were differentially upregulated in PTPos lesions relative to healthy controls, 80 encoded immunoglobulin-related transcripts. Lesions in which parasite transcripts were not detectable (PTNeg) also showed some evidence of B cell activation, but not to the same extent. The quantity of immunoglobulin transcripts in PTPos lesions was higher than PTNeg lesions (Fig 2C), as were the number of B cells as judged by transcripts encoding CD79A, CD19, and CD20 (Fig 3A). One interpretation of these results is that the persistence of actively transcribing parasites in the lesion prolongs the host immune response. However, we previously reported that in human visceral leishmaniasis, high levels of IgG were associated with on-going disease, and that IgG levels decreased as cell mediated immunity developed following treatment [23]. Furthermore, addition of parasite-specific IgG to L. major-infected JH mice exacerbated disease, increasing lesion size and inducing IL-10 production [23]. These previously published observations, along with the present association of high immunoglobulin transcripts in PTPos lesions, suggest that B cells and host IgG may be strong contributors to parasite persistence.
We and others previously demonstrated that IL-10 could contribute to parasite survival, and a correlation between IgG levels and IL-10 production was identified in visceral leishmaniasis [23,24]. The present studies extend this correlation to American tegumentary leishmaniasis, and demonstrate that PTpos lesions had higher levels of IL-10 than PTNeg lesions (Fig 3B). The association between parasite survival and cytokine production may be more complex than originally perceived, however, since PTPos lesions also expressed higher levels of IFN-γ and TNF, two cytokines that have well-established roles in classical macrophage activation and parasite elimination.
In addition to an in depth look at the host response, RNA-seq also provides a picture of the parasite transcriptome during infection. Viewing parasite gene expression within the lesion provided some interesting surprises. First and foremost, the uniformity of parasite gene expression across all six patients, given the differences in lesion size, duration, and degree of necrosis among them was unexpected. It is possible that the most highly expressed parasite genes in lesions have the potential to contribute to parasite persistence. The identification of these gene products may lead to new candidates for vaccine development or new targets for diagnosis. In fact, recently published work from Khare et al. [25] targets the conserved kinetoplastid parasite proteasome, of which we observed a subunit highly expressed by L. braziliensis in patient lesions (Table 1). Also of note is that 8 of the 20 most highly expressed parasite genes encode "hypothetical proteins" of unknown function. The identification and characterization of these proteins may shed new light on how this parasite establishes infection, persists within mammalian cells, or escapes these cells to spread disease. We tested whether the parasite transcripts detected in vivo were a reflection of the most abundant transcripts expressed by amastigotes growing in human macrophages in vitro. The top 40 transcripts detected in lesions were different from the transcripts most highly expressed in vitro, and there was less than 40% (L. major) or essentially no (L. amazonensis) overlap between parasite gene expression in cultivated cells and in lesions. Although the parasite species and time post-infection may confound these findings, we believe that a contrast of the Leishmania genes expressed in vivo versus in vitro provides a useful baseline for future comparisons.
We also observed a correlation between the prevalence of parasite transcripts in the lesion and the expression of a subset of host immune response genes. Using the weighted gene co-expression network analysis program (WGCNA), we identified several key host response genes whose expression increased in lesions containing high levels of parasite transcripts (Fig 3C). IL-10 has been previously associated with disease progression, so perhaps its inclusion in this category may not have been unexpected. However the association of TNF, granzyme B, and IFN-γ with parasite persistence was not expected. These observations may indicate that immunopathology is a driving factor in L. braziliensis infections. The second and third most significant modules (S3 Fig) correlate with several genes involved in B cell responses, immunoglobulin production and T cell interactions with parasite transcriptional activity. These observations are consistent with the hypothesis that B cells and IgG production may contribute to parasite survival.
This study was conducted according to the principles specified in the Declaration of Helsinki and under local ethical guidelines and this study was approved by the Ethics Committees of the Federal University of Bahia (Salvador,Bahia, Brazil)(010/10), University of Maryland (College Park)(395840–3) and the University of Pennsylvania IRB (Philadelphia, Pa)(813390). All patients provided written informed consent for the collection of samples and subsequent analysis.
Patients and procedures
All cutaneous leishmaniasis (CL) patients were seen at the health post in Corte de Pedra, Bahia, Brazil, an area endemic to L. braziliensis. Diagnosis consisted of visual confirmation of a lesion characteristic of CL and parasite DNA detection and/or a positive delayed-type hypersensitivity response to Leishmania antigen. Biopsies were collected at the border of the lesions using a 4 mm punch before therapy. Patients consisted of 15 males and 10 females with illness duration ranging from 15 to 90 days and lesion sizes ranging from 4–960 mm2 (S1 Table). Healthy (uninfected) skin samples were taken from volunteers living in a non-endemic area without a history of leishmaniasis, as described [17].
RNA isolation and cDNA library preparation
Samples were placed in RNA later and homogenized using a rotor-stator. Total RNA was isolated using the RNeasy Plus Kit from Qiagen. RNA integrity was assessed using an Agilent 2100 bioanalyzer. Poly(A)+-enriched cDNA libraries were generated using the Illumina TruSeq Sample Preparation kit (San Diego, CA) and checked for quality and quantity using bioanalyzer and qPCR (KAPA Biosystems).
RNA-seq data generation, pre-processing, and quality trimming
Paired end reads (100 bp) were obtained using the Illumina HiSeq 1500 platform. Trimmomatic [26] was used to remove any remaining Illumina adapter sequences from reads and to trim bases off the start or the end of a read when the quality score fell below a threshold of 20. Sequence quality metrics were assessed using FastQC [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/].
Mapping cDNA fragments to the reference genome, abundance estimation, and data normalization
TopHat (v 2.0.13) [27] was used to align reads to the applicable genome(s) with each genome alignment performed independently. Reads from healthy, early infection, and late infection skin samples were aligned to the human genome (v. hg19/GRCh37) obtained from the UCSC genome browser (http://genome.ucsc.edu). Reads from early infection and late infection samples were additionally aligned to the L. braziliensis (v. MHOM/BR/75M2904, Sanger Institute) genome obtained from the TriTrypDB database (www.tritrypdb.org). Two mismatches per read were permitted (default TopHat parameter) and reads were allowed to map only to a single locus (TopHat option –g 1). Additionally, gene model annotations were provided for the mapping (TopHat option –G) with limitations on the identification of novel splice junctions (TopHat option –no-novel-juncs). The abundance of reads mapping to each gene feature in the aligned genome was determined using HTSeq [28]. Each resulting count table was restricted to protein-coding genes (20,956 genes for human and 8,556 genes for L. braziliensis). Non-expressed and weakly expressed genes, defined as having less than 1 read per million in n of the samples, where n is the size of the smallest group of replicates [29] (here n = 8 for both human and parasite), were removed prior to subsequent analyses, resulting in count tables of 15,256 and 8,556 genes for human and L. braziliensis, respectively.
Global data assessment, visualization and differential expression analysis
Samples were classified as PTPos and PTNeg based on the percentage of reads mapping to the L. braziliensis genome. Parasite detectable-positive samples were defined as those with more than 0.5% of reads mapping to the parasite genome (see Fig 1C). In the six PTpos samples, the average proportion of reads that mapped to the parasite genome was 0.98% (see Fig 1C), for an average of 867,489 parasite reads per tissue sample sequenced to an average depth of 88 million reads (S1 Table). The 10 PTNeg samples were defined as those with fewer than 0.01% reads mapping to the parasite; the proportion of reads in the PTNeg samples that mapped to the parasite genome was not different than the 10 healthy controls. We designated nine additional samples as PTInt because they expressed low levels of transcripts (between 0.01–0.2% of total reads) that could only tentatively be assigned to the parasite. The number of parasite reads in this group was too low to provide a meaningful analysis of parasite gene expression and therefore they were excluded from the PTPos group. Quantile normalization was applied to all samples [30] and data were log2-transformed. Multiple approaches were used to evaluate replicates and to visualize the relationships between samples, including Pearson correlation and Principal Component Analysis (PCA). Limma (a Bioconductor package) was used to conduct differential expression analyses [31]. The voom module was used to transform the data based on observational level weights derived from the mean-variance relationship prior to statistical modeling [32]. Pairwise contrasts were done within limma to identify differentially expressed (DE) genes between conditions. Genes with a Benjamini-Hochberg (BH) multiple-testing adjusted P value of < 0.05 were defined as differentially expressed. Components of our statistical pipeline, named cbcbSEQ, can be accessed on GitHub (https://github.com/kokrah/cbcbSEQ/).
(S1A) Panel A shows a heatmap enriched pathways (using GSEA) comparing healthy controls (gray line, top) and leishmaniasis patients (orange line). 680 pathways showed ≥2 fold differences that were clustered hierarchically. (S1B) Principal component analysis (PCA) plot of human transcriptomes from 15 female (light gray), and 10 male (dark gray) leishmaniasis patients. The first two principal components (PC) are displayed on each axis along with the variance (24.91% and 12.64%). (S1C) Principal component analysis (PCA) plot of human transcriptomes from 25 leishmaniasis patients, colored by lesion size. The first two principal components (PC) are displayed on each axis along with the variance (24.91% and 12.64%). (S1D) Principal component analysis (PCA) plot of human transcriptomes from 25 leishmaniasis patients, colored by illness duration. The first two principal components (PC) are displayed on each axis along with the variance (24.91% and 12.64%). (S1E) Principal component analysis (PCA) plot of human transcriptomes from 25 leishmaniasis patients, colored by patient age. The first two principal components (PC) are displayed on each axis along with the variance (24.91% and 12.64%). (S1F) 3-D Principal component analysis (PCA) plot of human transcriptomes from 25 leishmaniasis patients, colored by parasite transcript status. Blue = PTNeg, Yellow = PTInt, Orange = PTPos. The first three principal components (PC) are displayed on each axis (PC1: 24.91%, PC2: 12.64%, PC3: 11.89%).
S2 Fig. RNA expression signatures of immune responses comparing early and late-stage cutaneous lesions.
Bars represent the fold change (mean plus SEM) of RNA expression of cell-specific markers in early (light grey) and late (dark grey) cutaneous samples, each compared to healthy patients, showing (A) infiltration of B cell (cd79a, cd19, cd20) and T cell (cd3e, cd4, cd8a) biomarkers, and (B) increased inflammatory (ifnγ, tnf, IL12p40, IL-10, faslg), anti-inflammatory and inhibitory signals (il10, ctla4, pdcd1, cd274, lag3), and activation markers (cd80, cd38) in lesions with no difference observed between early and late. NS = not significant.
S3 Fig. Module correlation to parasite percent reads using WGCNA analysis.
(A) Heatmap comparing the module eigengene relationship to the trait of percent parasite reads. Heatmap color indicates correlations of -1 (blue) to +1 (red) and p-values are shown in parentheses. Modules are sorted top to bottom by significance and each module is represented by a different color block. The three most significant modules are indicated in red type with the most significant module, indicated by a *, which is explored further in Fig 3C. (B) The second (light yellow, top) and third (cyan, bottom) module (by p-value) and representative genes from within the module correlated to percent parasite reads using WGCNA analysis. The left plots demonstrate module membership versus correlation to parasite percent reads for each gene. On the right, plots demonstrate normalized expression (rpkm) versus parasite percent reads for selected genes from within the modules (highlighted in red in the left plots).
S4 Fig. Functional interactions among differentially expressed genes.
The largest three clusters consisting of 137 (A), 103 (B), and 72 (C) genes show numerous functional interactions (edges). Gene clusters are depicted by color and direction of differential expression is depicted by node border, with green borders designating upregulated and red borders designating downregulated genes. Node size indicates the number of interactions. (A) Network of genes associated with immune cell activation, costimulation, and cytokine and chemokine signaling, including TNF, IL-10, and multiple C-C motif chemokines regulated by NFκβ, CREB/STAT3, JUN, and SP1 signaling. (B) Network of 103 genes involved in B and T cell activation and inhibition, regulated by SYK, SRC, FYN, PLCG1 and 2, and PTPN11. (C) Interaction of 72 genes associated with cell growth and proliferation signals.
S5 Fig. Parasite gene expression.
Representation of the L. braziliensis genome with the outermost ring (labeled 1–35) showing each individual chromosome. The subsequent concentric circles represent each of the six PTPos patient samples (3 early cutaneous shown in blue, 3 late cutaneous in orange). Vertical lines designate the relative expression levels of L. braziliensis gene expression in RPKM with the top 20 most highly-expressed genes marked by a red line, and numbered from 1–20. Chromosomes labeled in bold are viewed closer in Fig 5.
S6 Fig. L. braziliensis gene expression in lesions and homologous gene expression by L. amazonensis and L. major following in vitro infection of human macrophages.
RPKM values of all samples were scaled to the sample with the highest total normalized reads. (A) The top 40 L. braziliensis genes (red) expressed in vivo as ranked by average RPKM (left axis) matched to the ranking of their homologous genes in L. major (blue, left axis, 1–50+) and L. amazonensis (green, right axis, 1–500+) expressed during in vitro infection of human macrophages at 72 hours post-infection. (B) The top 40 L. amazonensis genes (green) expressed during in vitro infection of human macrophages at 72 hours post-infection as ranked by average RPKM (left axis) matched to the ranking (1–500+) of their homologous genes in L. braziliensis (red, right axis) expressed during in vivo and L. major (blue, right axis) expressed during in vitro infection of human macrophages at 72 hours post-infection.
S1 Table. Experimental design, phenotypic data, and accession numbers.
S2 Table. Differentially expressed genes comparing early and late cutaneous lesions (p<0.05, FC>2).
(XLS)
S3 Table. Differentially expressed genes comparing healthy controls and infected patients (p<0.05, FC>2).
S4 Table. Differentially expressed genes comparing PTPos and PTNeg patients (p<0.05, FC>2).
S5 Table. Top 100 L. braziliensis genes by expression level (RPKM).
All RNA sequencing was performed at the University of Maryland Institute for Bioscience and Biotechnology Research sequencing core.
Conceived and designed the experiments: SMC NMES DMM.
Performed the experiments: SMC LALD LPC SP FON.
Analyzed the data: SMC LALD VKH DPB NMES DMM.
Contributed reagents/materials/analysis tools: LPC SP EMC FON DPB PS.
Wrote the paper: SMC DPB PS NMES DMM.
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Related PLOS Articles
has CORRECTION
Correction: Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion
Is the Subject Area "Gene expression" applicable to this article?
Is the Subject Area "Parasitic diseases" applicable to this article?
Is the Subject Area "Lesions" applicable to this article?
Is the Subject Area "Leishmaniasis" applicable to this article?
Is the Subject Area "Principal component analysis" applicable to this article?
Transcriptome analysis
Is the Subject Area "Transcriptome analysis" applicable to this article?
Is the Subject Area "Immune response" applicable to this article?
Is the Subject Area "Macrophages" applicable to this article?


text:
CAS investigating African football's Presidential vote rules
By Emily Goddard
By Andrew Warshaw
November 29 - The power struggle to determine who can and who cannot challenge for the top job in African football is being taken all the way to sport's highest court of law, the Court of Arbitration for Sport (CAS).
Two months ago, the Confederation of African Football (CAF) passed an amendment banning anyone from outside its inner sanctum from challenging Issa Hayatou (pictured top) of Cameroon for the Presidency.
The contentious move put paid to the aspirations of contenders wishing to unseat the long-serving Hayatou at elections in Morocco next March.
But now Hayatou, a member of the International Olympic Committee, is having his authority challenged.
The CAS has confirmed that Liberian officials have appealed to overturn the election rules, which are supposed to take effect from December 3.
The appeal is seeking interim verdicts to block the amendments until a full hearing is held.
"The [CAF] has been formally notified of the appeals and has been invited to file an answer to the requests for provisional measures," the CAS said in a statement.
Hayatou's grip on African football has been watertight ever since he took charge in 1987 but he was forced to steer through the latest controversial rule change after his FIFA Executive Committee colleague Jacques Anouma emerged as a rival.
Anouma, from Ivory Coast, and South Africa's Danny Jordaan - who led the 2010 FIFA World Cup Organising Committee – were seen as potential challengers although neither are voting members of CAF.
As a result Hayatou quickly called an extraordinary meeting of CAF's 54 member nations where an Algerian proposal was passed limiting the Presidential race to candidates who were "current or former members of the Executive Committee".
Jacques Anouma has slammed the voting rules calling them "undemocratic"
The proposal, passed by a huge majority, is understood to have been backed by FIFA President Sepp Blatter who defeated Hayatou in the 2002 FIFA elections.
Anouma has denounced the move to extend Hayatou's command as totally unjustified.
He was backed by Ivory Coast Sports Minister Philippe Legré, who described the amendment as a "political ruse".
"In the 21st century, such an undemocratic act should not take place," said Anouma.
"I still do not understand how we could have passed an amendment that is clearly anti-democratic.
"The Executive Committee does not have the right to make themselves the only people that can contest the Presidency of CAF.
"But the pressure people were under and the other things that I have heard happened may be responsible for this."
Last year FIFA was forced to withdraw its appointment of Hayatou as chairman of the Organising Committee of the London 2012 football tournament because he was being investigated for alleged corruption by an independent Ethics Commission of the IOC.
February 2012: Hayatou remains as CAF President despite IOC warning
December 2011: "There is no action required" over Hayatou claims FIFA President
December 2011: Hayatou is "incorruptible" claims former England 2018 bid adviser
December 2011: IOC have "disrespected" Africa over Hayatou reprimand, claim CAF
December 2011: Hayatou reprimanded, Diack warned for roles in ISL corruption scandal
Emily Goddard Subeditor and reporter
Emily Goddard is a subeditor and reporter for insidethegames.biz. She has broken a number of exclusives including that News International had lost their exclusive deal with London 2012 following the phone-hacking scandal that rocked Britain in 2011, Richard Caborn's future as chairman of the Amateur Boxing Association of England was in danger, and she was also the journalist to report the first comments from the IOC following the Russian anti-gay legislation.
Contact Emily
Read more of Emily's articles

redpajama_set_name: RedPajamaCommonCrawl


Fatma Ergkiounger is an independent artist living and working in Athens, Greece. Studied at The Athens School of Fine Arts majoring in painting. Other subjects include sculpting, photography and multimedia. Currently working on a project with the subject of location and allocation.


the pixel art is probably one of the best i've ever seen, but damn, all your universe is straight up amazing. Remind me of old children books. Very unique, i love it.
Ух, несётся-то, несётся как стремительно! Как всегда, изумительный арт!
Excellent job! Also I've noticed two extra pixels at the bottom close to right "leg"
Funny that there have already been comments about the roof texture. I noticed that immediately as well. Wonderful. I also love the texture of the leaves, and the little details scattered about as well.
Really good, I especially like the wood texture you made for the roof.
Great Stuff as always? Your color choices are fantastic.
@nottogood: Pretty sure the source of inspiration is Baba Yaga's hut ( https://en.wikipedia.org/wiki/Baba_Yaga ).
And now - animate it!
Indeed, the toilet paper reminds me of dramatic robes from late renaissance works of art. This picture is truly amazing. I'm not sure about the anathomy, but judging from joints, the hut seems to have one leg with a knee and an arm with an elbow. Still I'm not a specialist as far as walking huts are concerned.
Amazinge...the weed keeps dragging my attention!
Awesome...the toilet paper keeps dragging my attention!


text:
Samsung HW-S800B S-Series review
The bar is 'Ultra Slim', the wireless sub is a cracker. This is an impressive combo given the style focus. Tested at £649 / $899 / AU$999
By Jez Ford
( Sound+Image )
(Image: © Samsung Electronics Australia)
Early Verdict
An excellent little subwoofer allows this combo to deliver a high quality sound despite the bar's slender dimensions, justifying even its full price – and it is often available for less.
Crisp dynamic sound
Clean and balanced bass
Genuine height delivery
Optional rear expansion
HDMI ARC only, so check compatibility in advance
Mini-HDMI socket
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Samsung has been releasing great-sounding soundbars ever since it set up an audio lab in California, as part of Samsung Research America under Allan Devantier.
The lab includes testing and measuring facilities that follow the tenets of the legendary National Research Council of Canada as developed by the equally legendary Dr Floyd Toole, and this is no coincidence, since Devantier came to Samsung from Harman (before Samsung bought the company). He professed his devotion to Floyd Toole's methods when we spoke to him back in 2017, just as the first fruits of the Californian lab arrived in Australia.
"I've known Floyd since I was about 17 years old, so he's like a father to me," Allan Devantier told us then. "I wouldn't be here today if it wasn't for him and my years at Harman learning from him, and of course other people like Laurie Fincham was there at the time, Don [D.B.] Keale, Richard Small – these are all giants of the industry.
"So the learning experience there, I was soaking it all up in the 1990s, and the idea of really well-controlled blind listening tests, really well-done measurements – all the things that Floyd talked about for 30 years – has been just as important for me to do, and translate that and get that done at headquarters, as much as just bringing new technology like better woofers and tweeters.
"And it's interesting that now, today, my job is to do for Samsung what Floyd did for me, right?"
Also interesting was that from the start the Samsung lab in California didn't use its facilities to design some new esoteric high-end stereo speakers, but instead applied its abilities to the more effective design of soundbars, and their subwoofers.
The results have been wildly successful in both sound and sales; Samsung is now world soundbar leader.
(Image credit: Samsung Electronics Australia)
Build & design
As the California lab enters its tenth year, the S800B represents something new. It slims down the bar quite remarkably, so slim that we could close our forefinger and thumb around it; the cross-section is roughly a 4cm square.
The bar is long, however – 116cm – but since this matches a 55-inch TV nicely, it's unlikely to outscale a modern TV bench, and it certainly looks far less daft when wallmounted than do larger soundbars. A full wall-mount kit and instructions are included with the S800B.
Visually desirable, then, but such an "Ultra Slim" design creates issues, and not just the obvious sonic one of having much reduced cabinet volume.
The drivers are also necessarily smaller: unspecified, but we think three sets of small tweeters and elongated 65×25mm drivers facing forward, plus two oval 25×40mm drivers firing up for height content. There may be something else in the ends; we couldn't tell through the grille.
Indeed the bar is so slim that Samsung hasn't been able to fit in proper a proper HDMI socket. Instead it's got a mini-HDMI connector, with a mini-HDMI-to-HDMI cable provided in the box (see image above).
And there are no other sockets apart from the power connection, so it's HDMI or the highway, no fallback optical or analogue input. You'd better hope your TV's HDMI ARC works with this one. Although there is one alternative if you have a 2022 Samsung TV (BU8000 and above, say the specs): wireless Dolby Atmos transmission by Wi-Fi. Clever!
This bar appears in one product video in white; this seems a finish given only to certain retail outlets, and released as the HW-S801B. It seems otherwise identical.
There are a great many features included, such as Samsung's SpaceFit Sound+ and Q-Symphony, which will work when used with a recent Samsung TV, but the key takeaway for all users is the inclusion of Dolby Atmos and DTS Virtual:X processing. (Why not actual DTS:X, given its upfiring drivers? Presumably because very few TVs will pass DTS:X to their HDMI ARC output from a connected device, while no streaming services use DTS:X sound.)
Once on Wi-Fi, the bar can be connected to and controlled from Google or Alexa voice assistants, in addition to the little physical remote control, and most likely your TV remote too.
Given the slim bar, the subwoofer is crucial to fill out the sound. Samsung's sub is neatly compact, roughly a 24cm cube, with an active driver on one side, passive radiator on the other. You can also add physical rear speakers to this system, via the SWA-9500S/XY (£199 / $299 / A$349); these wireless rears, each of which will require a mains cable, combine rear surrounds with Atmos-enabled height drivers, so adding these would complete a 5.1.4-channel system.
Other top Dolby Atmos soundbars to consider
Sound & performance
Samsung HW-S800B specifications
Inputs: HDMI ARC, Bluetooth (SBC only), Wi-Fi, Chromecast, AirPlay 2
Soundbar dimensions (whd): 116 x 38 x 40cm
Subwoofer dimensions (whd): 23.8 x 24 x 23.8cm
With no inputs other than the mini-HDMI ARC connection, we were pleased to find the bar worked instantly with the Samsung S95B OLED TV into which we first plugged it, and also with a visiting TCL Roku TV. However we never got it to play from 2021 Samsung TV we tried, nor an older Toshiba TV, no matter how we fiddled with their settings. So we'd suggest having your dealer check compatibility in advance for your particular TV, and to allow a return if it doesn't work.
It's certainly worth doing, because Samsung has delivered another dynamite soundbar performance for the price, and it's a success of teamwork, with both the bar and the subwoofer being essential to the sound.
Indeed the sub is something of a star. Its bass descends to real depths, creating output from signal even below 30Hz, and it manages to sound not only resounding but astoundingly tight for a subwoofer doing so much from a cabinet so small.
There's a moment 1:16 into Jumanji: Welcome To The Jungle where a deep chugging shakes a huge shed before a helicopter bursts through its timbers: the chugging not only had huge weight but stopped and started within each chugbeat, where many small subs deliver this as a blur.
The helicopter exits and flies overhead – an Atmos treat with genuine surround, but not possible here with 3.1.2. Nevertheless, the space generated was impressive; the soundstage stretched wide beyond the bounds of the bar despite there being no side-firing speakers, and yes, large in height too, as if a large semi-circle of sound was being created up and around the screen area.
Better still, the orchestral score sailed on unaffected above the action in the best Indiana Jones fashion. It sounded just a little thinned of lower mids, and a frequency sweep revealed a significant dip in response centred on 170Hz, just above the crossover point; this is perhaps a gap between the ability of the sub to go high and the super-thin bar to go low.
It doesn't intrude on listening much. Nor was there much sense of power limitation, so that while of course the mayhem of the final fight in Justice League will sound bigger and excite more air from a full surround system, it's hard to criticise the size and quality of sound here. This UHD Blu-ray also had DTS-MA 5.1 available; we tried this with the DTS:X Virtualiser setting but it sounded a little phasey, perhaps because of the vertical upscaling.
'Standard' mode seemed the clearest rendition of 5.1 material, even though the top drivers are out of action in this mode.
'Standard' is also the only mode which doesn't upscale stereo content to the full driver complement; everything else plays two-channel content in 3.1.2, even, surprisingly, the Game mode, which you might expect to switch off all non-essential processing.
The 'Adaptive Sound' setting is said to "analyse the audio signal to deliver optimised sound based on each scene", which sounds a bit worrying but actually yielded good sound in nearly every scenario we gave it, excepting only our preference for 5.1 as 3.1.
As for music, once we'd connected the bar to Wi-Fi using Samsung's SmartThings app we enjoyed pretty enjoyable tunes sent by AirPlay 2 from an iPad Pro. There's a wide stereo spread, just a little peakiness on female vocals (that thinning again), and this was accentuated when we tried any sound mode other than Standard. A strong pass here.
What didn't we like? As a byproduct of the Ultra Slim design, there's no display, just a few lights, and a lightly thin computery voice, which we couldn't find an option to turn off (it's redundant if you use the SmartThings app to show status). Otherwise nothing else, so long as HDMI-ARC proves compatible.
See the Sound+Image Showcase of Australia's best home theatres & custom installation
We'd rate this slim bar and wireless subwoofer package as performing very well at its price, though its price seems unusually variable, and the 'RRPs' pleasingly favourable to Sound+Image's Australian readers.
So it is currently listed at £649 in the UK, $899 (plus taxes) in the US, and AU$999 in Australia. But as we write this, the actual prices on Samsung's websites are £649 in the UK, down to US$599 in the States, and Aussies offered AU$599 for the black S800B, and a remarkable AU$499 for that white S801B version. Samsung varies such offers frequently and without warning, so choose your time to buy!
Whatever you end up paying, for your money you'll get a brilliantly-engineering superslim bar, not only useful for its size but sophisticated in having Wi-Fi to allow plenty of streaming options. Its excellent little subwoofer allows the combo to deliver a high quality sound which justifies even its full price, especially given the bar's slender dimensions and style advantages.
It's another undeniable triumph for the team at Samsung's audio lab.
Check out all our Australian reviews from Sound+Image & Australian Hi-Fi magazines.
Jez Ford
Editor, Sound+Image magazine
Jez is the Editor of Sound+Image magazine, having inhabited that role since 2006, more or less a lustrum after departing his UK homeland to adopt an additional nationality under the more favourable climes and skies of Australia. Prior to his desertion he was Editor of the UK's Stuff magazine, and before that Editor of What Hi-Fi? magazine, and before that of the erstwhile Audiophile magazine and of Electronics Today International. He makes music as well as enjoying it, is alarmingly wedded to the notion that Led Zeppelin remains the highest point of rock'n'roll yet attained, though remains willing to assess modern pretenders. He lives in a modest shack on Sydney's Northern Beaches with his Canadian wife Deanna, a rescue greyhound called Jewels, and an assortment of changing wildlife under care.


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ART: HAM & others
We <3 HAM
And HAM loves Heino. HAM, Helsinki Art Museum, started off completely renewed last autumn by bringing Chinese Ai Weiwei's show to Helsinki. The museum continues by exhibiting one of the major private collections in Finland, the Heino Art Foundation collection. The museum is situated in Tennispalatasi, a combination of a multiplex cinema and a world-class art museum.
Heino uncovers its comprehensive collection to the public with love. The collection is bewilderingly contemporary and includes important art by key contemporary Finnish artists. Most of all, the exhibition is curated with love for the field. The Heinos are also important gallerists.
The arched halls at HAM Tennispalatsi are an invitation to large-scale works. Nearly a hundred pieces, paintings, photographs, drawings and sculptures inhabit the space. From Heino With Love is a comprehensive overview of the most prestigious Finnish contemporary art. Why not fall in love with Finnish art?
HAM. From Heino with Love until 28.8.
HAM. Tue–Sun 11–19. Tennispalatsi, Eteläinen Rautatiekatu 8.
From Heino with Love
GALLERY PICKS
Galerie Anhava. Tue–Fri 11–17, Sat–Sun 12–16. Fredrikinkatu 43.
Galerie Forsblom. Tue–Fri 11–18, Sat 11–16, Sun 12–16. Lönnrotinkatu 5.
Helsinki Contemporary. Tue–Wed & Fri 11–17, Thu 11–18, Sat–Sun 12–16. Bulevardi 10.
Saariaho Järvenpää. Wed–Fri 14–18, Sat–Sun 12–16. Merimiehenkatu 31.
Big and Bold
It's never just the magnificient Steven Holl building, they have great exhibitions too.
Ernesto Neto is at the cutting edge of international contemporary art. Neto's art offers relief from the hustle of everyday life, soothes the mind and primes the senses. In the Kiasma show the viewer takes of his shoes, walks on crocheted rugs and ends up, perchance, lying inside a hut enjoying the scents of lavender and basil. The central themes for Neto are the oneness of man and nature, sensuality, experience and the creation of positive energy. Now he speaks out for the rights and traditions of indigenous peoples and respect for cultural pluralism.
Choi Jeong Hwa builds his large installations by cleverly combining local and Korean, old and new, unique and mass-produced elements. Historical handmade items meet giant blow-up sculptures. Traditional Korean imagery blends in with the global consumer culture. He creates enclosed spaces out of everyday consumer products, such as colourful plastic crockery and cheap plastic toy bricks.
In Happy Happy, for example, the viewer walks through a colourful plastic jungle. A glowing paradise is created with chains of household items that hang from ceiling to floor. "I believe that everything can be art. All materials that you can find in the kitchen, your room, the street. Therefore, I can't really say what kind of an artist I am," Choi has stated. We can, just as many others can: "You're a wonderful artist, Jeong Hwa."
Kiasma. Ernesto Neto 'Boa' until 4.9. Choi Jeong Hwa 'Happy Happy' until 18.9.
Kiasma. Tue 10–17, Wed–Fri 10–20.30, Sat 10–18, Sun 10–17. Mannerheiminaukio 2.
It's About Time!
Alice Neel is one of the most important figurative painters and one of the greatest 20th century American artists. She is also a pioneer for female painters. Although her influence has been great on both her contemporaries and internationally the Ateneum show is only the second large exhibition in Europe. Perhaps because she was not altogether fashionable nor was she part of the avantgarde movement.
Alice Neel is known for her distinctive portraits, in which she depicts the personality of her models directly and without
flattery. Ateneum is the initiator of the retrospective that gathers around 70 paintings together. The museum is also publishing a book on the exhibition. The long-awaited Alice Neel European tour proceeds from Helsinki to The Hague, Arles and Hamburg.
Alice Neel 10.6–2.10.
Ateneum. Tue & Fri 10–18, Wed–Thu 10–20, Sat–Sun 10–17. Kaivokatu 2.
The Most Influential Finn
Tom of Finland has shaped gay culture around the world with his homo-erotic and masculine drawings. His influence is greater today than ever. As fetishes have become more commonplace, his imagery has become more commonplace.
Muscles. Leather. Sex. Tom's bold and humorous images have aroused talk about sexual diversity, equality and the relationship between pornography and art; and ended up in duvet covers and postage stamps.
The exhibition in Taidehalli is the largest collection of Tom of Finland's art, including drawings, paper dolls, gouaches, strips of images and collages ranging over six decades. The artist's personal life is also exhibited through paintings and photographs.
Tom of Finland until 7.8.
Taidehalli. Nervanderinkatu 3. Tue & Thu–Fri 11–18, Wed 11–20, Sat–Sun 11–17.


text:
The GiBLi G10, Aero testing for the masses?
The GibLi G10, aero testing for the masses? Over the last few years there has been a steady flow of companies releasing aero testing products – or have been rumoured to be – aimed at (eventually) offering real-time detecting of a cyclist's aerodynamic drag or 'CdA'....
Tom Fowler joins Advisory Board
Tom Fowler Joins Advisory Board at GiBLI Tech GiBLI is pleased to announce that Tom Fowler has joined the company's advisory board with a focus on strategy and global marketing activation. He brings an extensive background in the cycling and sports tech industries...
GiBLI Wins Eurobike Start-up Award
Sep 1, 2021 | News
Halifax, NS, Canada – GiBLI is excited to announce that they have been selected as a winner of the prestigious annual EUROBIKE Start-Up Award 2021. GiBLI is honoured to be among the winning companies to receive the EUROBIKE Start-Up Award 2021. As a global leader in...
GiBLI Appoints Bill Barrett, Executive Chair
GiBLI brings on New Brunswick native, Bill Barrett as Executive Chair William Barrett is a shareholder and Co-CEO of Barrett Corporation, a privately held international management company, based in Woodstock, New Brunswick, Canada. Founded in 1976 and held members of...
GiBLI Collaborates With BikeValley
GiBLI Collaborates With BikeValley Halifax, NS, Canada – GiBLI will be working closely with BikeValley to ensure that the GIBLI G10 sensor continues to deliver world class real-time aerodynamic data and that BikeValley can provide real-world aerodynamic testing to its...
GiBLI & Cycling Canada Partnership
Mar 23, 2021 | News
GiBLI Becomes Cycling Canada's Official Partner In Real-time Aerodynamics. Halifax, NS, Canada – GiBLI is excited to announce a 3-year partnership with Cycling Canada, to be their Official Partner in Real-Time Aerodynamics. As an Official Partner in Real-Time...


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Trade spats or not, we must learn to co-exist with China
China relations
This was published 1 year ago
Amanda Vanstone
Former Howard government minister
August 23, 2020 — 11.01pm
What do you think when someone mentions Richard Nixon? The nickname "Tricky Dicky" comes to mind, as does Watergate. Do you baulk at the idea that he changed the world? Like so many things in life it depends on the perspective.
Nixon formally recognised the People's Republic of China. He had said long before his 1972 visit: "We simply cannot afford to leave China forever outside the family of nations, there to nurture its fantasies, cherish its hates, and threaten its neighbours. There is no place on this small planet for a billion of its potentially most able people to live in angry isolation."
Nixon helped China open up to the world. Credit:AP
China joined the United Nations. Taiwan lost out. Some say Mao got everything he wanted and that Nixon shamefully didn't push for China to lessen its support for North Vietnam to save American troops' lives. Less than a decade later China started opening up to the world. Without Nixon's efforts that may have taken many more years.
In China, this opening up to the world created incredible opportunities. Millions have been lifted out of abject poverty. But the story doesn't end in China. China's opening up to the world meant the world had access to much cheaper white goods, airconditioners, clothing, plastics and small electrical goods. All around the globe people with less money could now buy some things that were previously out of their reach. Many everyday items were cheaper for everyone. That means an improved standard of living.
China, the new industrial powerhouse, needed iron, coal and grains. Credit:Bloomberg
From Australia's position the story was even more beneficial. China, the new industrial powerhouse, needed iron, coal and grains. We had them to sell and that made us richer.
Nixon may have richly deserved the nickname "Tricky Dicky" but we do ourselves a disservice if that's all we remember. I think "I did not have sexual relations with that woman" is a bit tricky, as is describing Bill Clinton's womanising as "he's a hard dog to keep on the porch", but one aspect of a person is rarely the whole story. Like him or not, Nixon's decision to recognise the PRC changed our world for the better.
Now we have to deal with that changed world. It's difficult to see in the near future China becoming unproblematic. The recent announcement on looking at Australian wine imports in the context of dumping is just one example of why. Apparently we need to be sent a message, threatened for not kowtowing to the Chinese perspective. It's no way to win friends.
Using trade to threaten is never smart in the long term. But to choose wine was particularly lacking in skill. Obviously such a move, with the recent dramatic rise in sales to China, would be bound to get the government's attention and plenty of media focus. But the wine industry has a special place in the hearts of Australians. Attack it and you're attacking us. That just engenders ill will in the hearts and minds of Australians. Imagine what the Chinese would say if we put a levy on exported minerals and energy. We might call it the "wine industry support levy".
In the context of anti-dumping cases one might imagine China is most at risk. Anti-dumping was my portfolio responsibility for a few years and it is the job from hell.
If the decision is that something is being dumped, the importers hate you because their cheap supply is about to become more expensive.
If the decision is that there is no dumping the Australian producers hate you because the cheap competition will keep coming. Perhaps we should give all new ministers a turn in this job; you pretty soon learn that you can't please everyone so bad luck, just get on with it and make the decision.
What is taken into account to ascertain whether or not a product is being dumped might not make it easy for goods from China, which are either produced directly by state-owned enterprises or by factories supported by them. That means a lot of production. My own nightmare was Chinese clear float glass. In any event the rules are there to protect free trade, which has brought so many people out of starvation and poverty. Playing with them for political messaging seems a bit inept.
The world rightly marvels at how far China has come in the last half-century. There is more opportunity to be seized. It will be many years before China gets the full benefit of Nixon's decision.
Xi Jinping speaks confidently about further reducing poverty. The Premier, Li Keqiang, brought a note of reality into the debate when he said that more than 600 million Chinese lived on less than $200 a month. Out of a population of about 1.4 billion that's an unattractively high proportion living poorly. Add to that just about zip in terms of healthcare or welfare and it's a pretty dark outlook for many.
China is not going away nor one imagines is it going to suddenly become democratic. On the other side, those of us in democratic countries are quite taken with individual freedom, protection from persecution by the state, freedom of speech, good healthcare and decent welfare. We're not going anywhere either.
We will all have to work out how to co-exist. That's possible. Sticking fingers in another's eye doesn't augur well for happy co-existence.
Amanda Vanstone is a former Coalition minister.
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Amanda Vanstone is a former Howard government minister, and regular columnist.Connect via email.


That's a lot of applications vying for network capacity — not to mention a lot of end users IT needs to keep satisfied. But the sheer volume of apps, users and data that IT needs to parse in order to respond to events is just one factor prohibiting enterprise IT from speeding up their mean-time-to-resolution (MTTR).
Across the enterprise landscape, companies are retiring their data-center-centric network architectures for more flexible deployments, forcing teams to adopt new techniques to get to the root cause of performance issues than they had in the past. That's because although IT controlled all of the connections that supported legacy apps in the pre-SaaS and pre-cloud days, they don't, on their own, have visibility into programs and routes managed by third-parties or partners — ie. SaaS and cloud vendor networks or ISPs.
With enterprise networks now defined by a mix of cloud, MPLS, and direct internet access (DIA), it's increasingly difficult for IT to — on their own — even spot where issues occur on the network in the first place, let alone provide a speedy resolution. This gets compounded when a problem isn't actually the fault of the network, but an issue with the SaaS app itself that enterprise IT no longer has control over.
Today, IT has become the one-stop-shop for blame when applications, increasingly served through web browsers over vast networks, aren't performing they way users expect. Due to mobile apps and the prevalence of WiFi users expect instantaneous load times. When that expectation isn't met they end up turning to the people close by with the technical skills to fix what they perceive to be the problem–the network. In this increasingly common scenario, a new acronym comes to define a big piece of the network-diagnosis workflow: MTTI, or mean-time-to-innocence where IT is assumed guilty until proven innocent.
While IT may not always be able to manage or fix SaaS applications that they don't control, they should, at the very least, be able to give end users an explanation as to what went down, where, and who was really to blame. For frustrated users, "I don't know" is not an acceptable answer.
To speed up MTTI (and ideally accelerate MTTR), teams need to look beyond standard network monitoring or management solutions. They need a tool that can see the entire delivery path, end-to-end, of all apps leveraging the network, regardless of where they originate or where they're going.
For instance, if an app appears to be down from the perspective of an end user, teams need to be able to take a "problem domain isolation" approach that moves step-by-step, beginning with ensuring the app was delivered to the end user successfully in the first place. An SD-WAN controller may be able to tell IT that the app made it to the LAN gateway at a remote office, for instance, but that's only the beginning of tracking down what parties are actually to blame — the app provider or the network.
A comprehensive monitoring solution should be able to fill in the important hop-by-hop details that show the actual journey the app took from A to B.
If there was a server error, for instance, that caused an application to be rerouted over a sub-optimal network pathway that delays delivery, teams can quickly point to the network as the culprit for latency. But the situation isn't always so easy to suss out.
The app delivery path may be relatively routine and unchanged from a point in time when app performance is generally meeting end user expectations. But still, users aren't experiencing their apps as expected, and they'll want to know why.
In this scenario, if teams are employing a monitoring solution that can look at the entire network path in detail, they have all of the backup they need to demonstrate that nothing was out of the ordinary on the network level, and that they'll need to put pressure on the SaaS provider or app owner to resolve the issue. While this situation doesn't, on its own, result in faster MTTR, IT can still demonstrate that they're on top of the issue, even if a third-party needs to ultimately resolve it.
While, ideally, IT teams would like to achieve MTTI in lock-step with MTTR, they can buy themselves a bit more credibility when they can get past visibility challenges that otherwise plague network teams when they adopt new workflows without a monitoring solution.


SKU: N/A. Category: Bracelets. Tags: Austrian Crystals, Fan, Necklace, Rose Gold Plated, Silver Plated.
The fan epitomises romance and sophistication, a touch of the oriental and class. Showcasing a grey faceted resin stone accented by a single round Austrian crystal.


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Android / iOS / Microsoft / Mobile / Windows Phone
Microsoft Acquires Cross-Platform Productivity App Wunderlist
By John Vincent
In an open letter posted June 2 on their website, 6Wunderkinder Founder & CEO Christian Reber announced that his company would be "joining Microsoft". Founded in 2010, the software development company is best known for its productivity app, Wunderlist.
In his letter Reber states that nothing will change for now, and they will continue to develop and support Wunderlist.
"Over the next few months as Wunderlist becomes a part of the Microsoft family, we'll introduce a host of new features, continue growing the ecosystem of partner integration and progress in delivering Wunderlist to billions of people," Reber wrote.
Wunderlist is a cross-platform task manager boasting 13+ million users, and is this author's favorite app for keeping track of multiple to-do lists. The Wunderlist app exists for iOS, Android, Windows Phone, Mac, Windows, and the web. This is something I've appreciated about the app since I began using it – the respect the company has for multiple platforms.
Currently, Wunderlist can integrate with several other third-party apps, including Sunrise Calendar, Slack, and Google Calendar (my personal favorite). It will be interesting to see if Microsoft will continue to allow the use of the API's, or if they'll sunset that.
What are my feelings on this acquisition by Microsoft? My feelings are mixed. No one ever likes to see their favorite app get bought up. I went through several "to-do" apps before finding Wunderlist, and none of them worked for me like Wunderlist does. I don't think I can explain why, but there was just something about the interface and cross-platform syncing that just really clicked for me.
I do fear for what might happen to Wunderlist if it gets sucked into the void of Microsoft. If they decide to take the technology and coding and strip it out of Wunderlist for other purposes, leaving behind a shell of a great app, it will be a sad day.
However, on the flipside, I am somewhat excited for what this could mean for Office integration. For my personal things I tend to use Google Drive, but like many, at work I'm locked into MS Office. If Wunderlist gets integrated into apps like OneNote and Outlook while remaining a standalone app, it could really be the best of both worlds.
Only time will tell. For now, I guess I need to start looking at task manager apps again just in case.
What do you think about Microsoft's acquisition of Wunderlist? Let us know in the comments below, or on Google+, Twitter, or Facebook.
Source: Wunderlist
Related Items:Android apps, Business, IOS, Microsoft, technology, Windows Phone
Xiaomi's Mi Store Opens In Four Countries
Divide By Sheep Coming To iOS And Steam


text:
function eureka-list() {
  # curl -s silent
  # jq -r raw output
  curl -H Accept:application/json -s \
  "http://eureka.apps.${2:-test}.cirrostratus.org/eureka/v2/apps/${1:-mylists-service}" | \
   jq -r '.application|(if ([.instance|arrays]|length>0) then .instance|map(select(.status=="UP")|.hostName) else [.instance|select(.status=="UP")|.hostName]  end)[]'
}

function get_kafka_brokers() {
    SGK_ENVIRONMENT=${1:-test};curl -s -H 'Accept: application/json' http://eureka.${SGK_ENVIRONMENT}.cirrostratus.org/eureka/v2/apps/kafka |    jq -r         '[.application.instance |
        if (. | arrays | length) // (-1) >= 0 then .[]
        else .
        end |
        select(.hostName != null) |
        .hostName + ":9092"] | join(",")'
}

function get_zookeeper_nodes() {
    SGK_ENVIRONMENT=${1:-test};curl -s -H 'Accept: application/json' http://eureka.${SGK_ENVIRONMENT}.cirrostratus.org/eureka/v2/apps/zookeeper |    jq -r         '[.application.instance |
        if (. | arrays | length) // (-1) >= 0 then .[]
        else .
        end |
        select(.hostName != null) |
        .hostName + ":2181"] | join(",")'
}

function list_eureka_instances(){
# Script to discover and list instances based on a eureka service name
# Use this script to discover the IP to SSH to for a given deployment
# Usage example:   list_eureka_instances.sh mylists-service test

    if [[ -z $1 ]]
    then
        echo "Please specify an app service name as an argument"
        echo
        return 1
    fi
    APP=$(echo $1 | tr '[:upper:]' '[:lower:]')
    ENVIRONMENT=${2:-test}

    #Verify we have installed dependencies
    hash jq 2> /dev/null || {
        echo "Please make sure jq is installed and in your path"
        return 1
    }

    hash curl 2> /dev/null || {
        echo "Please make sure curl is installed and in your path"
        return 1
    }

    echo "Looking up eureka registry for ${APP} in ${ENVIRONMENT}"
    echo "HostName Port Status Instance-id"
    curl -s -H 'Accept: application/json' "http://eureka.${ENVIRONMENT}.cirrostratus.org/eureka/v2/apps/${APP}" |\
    jq -r \
        '.application.instance |
        if (. | arrays | length) // (-1) >= 0 then .[]
        else .
        end |
        select(.hostName != null) |
        .hostName+ " " + (.port["$"] | tostring) + " " + .status + " " + .dataCenterInfo.metadata["instance-id"]'

}

redpajama_set_name: RedPajamaGithub


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Pioneers Adapt Their Work to Help Out During COVID-19y
You can always count on the SaskTel Pioneers to be there for Saskatchewan!
These days, as the world finds its way through the challenges of COVID-19, the Pioneers continue to work diligently for people and organizations in the communities we serve.
"We're working as hard as ever but have adjusted and adapted what we're doing to benefit people during this pandemic," said Pioneer Manager Darrell Liebrecht. "The creation of masks is an example of this."
The use of masks has been found to be an important way of limiting the spread of COVID-19. To date, almost 3,000 masks have been created by Pioneers and donated to places like the Grace Within Care Community and the Pasqua Hospital Day Surgery Unit in Regina and various care homes and transition houses across the province.
Besides being available for the community, SaskTel employees could also pick a mask up for a donation of their choice, with the proceeds going to a provincial charity. The sale of masks to employees has raised almost $2000.
The Pioneers have also made and distributed 895 mask extenders to go along with the masks they donated. Mask extenders attach to mask straps to provide a more custom and comfortable fit while alleviating painful rubbing and pressure around the ears.
Through the Computers for Schools program, the Pioneers have also donated 606 laptops, iPads and mobile devices to long term care facilities and schools. This allows residents to reach out to people who are important to them, while giving peace of mind to those who are not able to visit the residents but can still contact them.
The program has also provided desktop computers to schools for distribution to students who do not have any other device available that can be used for distance learning during the pandemic. The Pioneers have also made cash donations totaling $66,782.00 to a number of Saskatchewan charitable groups. Recipients included the Weyburn and Regina Food Banks, the Prince Albert SPCA and the Saskatoon Interval House. The funds for these donations came directly from money raised by the Pioneers through various fundraisers.
With all this activity, the Pioneers have clearly been up to the challenge created by the pandemic. "Through all the projects we've undertaken in this challenging time, our members have continued to give their time selflessly," said Darrell. "We're at 1,084 volunteer hours today (and counting)."
"It speaks to the giving nature of our company and of the Pioneers that our members have reached out to our communities in a time like this," said Darrell. "It's something we can all be proud of!"
Our Impact Areas


Award-winning singer-songwriter Jack Carty and former Papa vs. Pretty bassist Gus Gardiner have been inseparable since meeting in a university elevator in 2009. It's a friendship and creative collaboration that's seen them through shoestring tours, share-houses, breakups and bridal parties. Gardiner has also held bass and 'cello credits on three of Carty's four studio albums.


Description Oskar Schindler uses his connections within the Nazi party to secure factory premises. Aided by Itzhak Stern, Schindler hires Jews in his factory because they cost less. He subsequently witnesses a massacre ordered by SS officer Amon Goth and vows to save his Jewish workforce from Auschwitz.