Plasmalogens represent ether lipids that play a role in diabesity and previous data show diminished plasmalogen levels in obese subjects. We therefore analyzed transcriptomic and lipidomic changes during monocyte-macrophage differentiation in vitro to macrophages using rhM-CSF under serum-free conditions. Samples were taken on days 0, 1, 4 and 5 and analyzed for their lipidomic and transcriptomic profiles.Elutriated monocytes from 13 healthy donors were differentiated in silico transcription analysis of lipid related regulation revealed known motifs such as PPAR-gamma and KLF4 as well as novel candidates such as NFY, RNF96 and Zinc-finger proteins.Gene expression analysis showed strong regulation of lipidome-related transcripts. Enzymes involved in fatty acid desaturation and elongation were increasingly expressed, peroxisomal and ER stress related genes were induced. Total plasmalogen levels remained unchanged, while the PE plasmalogen species pattern became more similar to circulating granulocytes, showing decreases in PUFA and increases in MUFA. A partial least squares discriminant analysis (PLS/DA) revealed that PE plasmalogens discriminate the stage of monocyte-derived macrophage differentiation. Partial correlation analysis could predict novel potential key nodes including DOCK1, PDK4, GNPTAB and FAM126A that might be involved in regulating lipid and especially plasmalogen homeostasis during differentiation. An Monocyte to macrophage differentiation goes along with profound changes in the lipid-related transcriptome. This leads to an induction of fatty-acid desaturation and elongation. In their PE-plasmalogen profile macrophages become more similar to granulocytes than monocytes, indicating terminal phagocytic differentiation. Therefore PE plasmalogens may represent potential biomarkers for cell activation. For the underlying transcriptional network we were able to predict a range of novel central key nodes and underlying transcription factors using a bioinformatic approach. Macrophages are key players in innate immunity and play an important role in the development of atherosclerosis and insulin resistance in diabesity Lipids regulate biological processes either locally as membrane components or remotely as signaling molecules. The lipid composition of the plasma membrane determines membrane fluidity but direct lipid-protein interactions also play a role in cellular signaling In a previous study we could demonstrate that a SREBP1 dependent induction of monocyte fatty acid synthesis is vital for monocyte-macrophage differentiation 3 (VitD3), all−trans−retinoic acid (ATRA) and ethanol were obtained from Sigma (Germany). Carrier-free macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) were both from R&D systems. HPLC grade methanol and chloroform were from Merck (Germany), plasmalogen standards from Avanti Polar Lipids .If not otherwise stated all materials including 1α,25-dihydroxyvitamin D6 cells/ml in serum-free macrophage medium (Invitrogen) that was supplemented with 50 ng/ml of recombinant human macrophage colony-stimulating factor . 1×107 cells per dish were cultured in 10 cm Cell+ plastic petri dishes (Sarstedt) for RNA isolation and 2×106 cells per well in Cell+ 6-well plates  for triplicates in mass spectrometry. Incubation was performed at 37°C in a humidified atmosphere containing 5% CO2 in an incubator. After washing with PBS, the cell pellets were stored at −80°C. Protein concentrations were measured according to Smith et al. Blood samples were obtained from thirteen healthy normolipidemic volunteers recruited from blood donors with apoE3/E3 genotype. Informed consent and approval of the Hospital Ethics Committee were obtained . Donors were fully informed of the possible complications and gave their written consent for the procedure. Blood cells were collected by leukapheresis in a Spectra cell separator  followed by subsequent counterflow elutriation as described elsewhere 2) in Iscove's Modified Dulbecco's Medium (IMDM) (PAN-Biotech) containing 20% FBS (Biochrom) in polystyrene tissue culture flasks (Sarstedt). Cells were seeded at a density of 0.3×106 cells per ml and 50% of the medium was replaced once per week. Confluent cultures were split 1∶3 to 1∶4 once per week.HL-60 cells  were grown in an incubator at 37°C . 1 μM working solutions of Vitamin D3 (VitD3)and all-trans retinoic acid (ATRA) were prepared in ethanol. Cells were seeded at 0.3×106 cells per ml. Monocytic differentiation was achieved in macrophage serum-free medium (SFM) supplemented with M-CSF (50 ng/ml) and 0.5 μM VitD3. Granulocytic differentiation was carried out in macrophage SFM with GM-CSF (10 ng/ml) and 0.5 μM ATRA. Cells grown in the respective medium containing 0,05% ethanol were used as controls. Cells were harvested by scraping with a rubber policeman and centrifugation for 5 min at 500 g at room temperature. They were finally washed once with PBS. Cell number was determined by counting in a Casy automated cell counter (Roche). Differentiation phenotype and viability were monitored by flow cytometry and Pappenheim staining. Experiments were repeated in two biological replicates.Differentiation experiments were performed in CellCells from nine donors were harvested, washed in PBS, resuspended in buffer RLT and RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Purity and integrity of the RNA were determined on the Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip reagent set (Agilent Technologies). RNA was quantified using a Nanodrop ND-1000 - UV/Vis spectrophotometer (PeqLab).www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2298.For gene array analysis we used a modified Agilent 4×44K microarray (014850) containing 205 free positions (Agilent Technologies). To these positions we added 201 probes, corresponding to 119 genes of the lipidome (LipidomicNet). 300 ng of total RNA were labeled with Cy3 using the Agilent Quick-Amp Labeling Kit - 1 color according to the manufacturer's instructions. cRNA was purified with the RNeasy Mini Kit (Qiagen). The amount and labeling efficiency of cRNA was measured with the NanoDrop ND-1000. For hybridization the Agilent Gene Expression Hybridisation Kit was used and arrays were incubated in Agilent SureHyb chambers for 17 hours in a hybridization oven at 65°C while rotating. Washing was performed according to the manufacturer's instructions. Scanning was done with the Agilent G2565CA Microarray Scanner System. The resulting TIFF files were processed with Agilent Feature Extraction software (10.7). Microarray data are available in the ArrayExpress database  using Pearson's product moment correlational analysis.Microarray experiments were analyzed according to the method described by Kondrakhin et al. In silico promoter analysis was carried out using the TRANSFAC database and the ExPlain software system . For in silico analysis we focused on regions of 1000 bp upstream from the transcription start site, and used a threshold in order to minimize false positive errors.Significantly expressed and regulated lipid related genes were subjected to transcription factor and virtual promoter analysis, predicting potential transcriptional regulators. q-order partial correlation graphs (qpgraphs) were used to reverse engineer the molecular regulatory network from microarray and lipid data. All data was Z-transformed prior to analysis and average non-rejection rates (NRR) were calculated in R 2.15.3 in vitro differentiation. In total 4728 transcripts  showed significant changes as indicated by a hypergeometrical score of <−4 or >+4 between days 1 and 4 of differentiation. Gene Ontology  analysis of the genearray data showed that lipid metabolism is among the most strongly positively enriched categories  until the 16-carbon product palmitic acid (C16:0) is produced. The main function of FAS is therefore to catalyze the synthesis of palmitate from acetyl-CoA and malonyl-CoA, in the presence of NADPH. The resulting saturated fatty acids are consecutively desaturated by desaturases or elongated by elongases at the endoplasmic-reticulum (ER). An overview over this process is given in Desaturases introduce double bonds between defined carbon atoms in fatty acyl chains. In humans four types of fatty acid desaturases could be identified, according to the position of the double bond introduced. In our experiments all analyzed fatty acid desaturases were upregulated with the notable exception of FADS3, which is interesting since the gene encoding FADS3 is clustered with FADS1 and FADS2 The mitochondrial very long-chain specific acyl-CoA dehydrogenase (ACADVL) was strongly up-regulated. Its function is the degradation of very long fatty acids by catalyzing the first step of the mitochondrial fatty acid beta-oxidation pathway. It can accommodate substrates with chain lengths as long as 24 C-atoms Lipoproteins are aggregates of apo-lipoprotein (apo) and lipid that allow the transport of lipids in the aqueous blood stream. Especially plasmalogens are known to correlate positively to serum HDL levels and to decrease with aging Accumulation of excess free cholesterol in macrophages contributes to the development of atherosclerosis by stimulating tumor necrosis factor-α (TNF-α) and interleukin-6 production Plasmalogens are ether glycerol-phospholipids that are integral parts of the plasma membrane as well as storage lipids for acyl residues and cellular anti-oxidants. Their synthesis takes place in a non-redundant pathway in peroxisomes and at the ER-membrane. Interestingly, although the macrophage is prepared for an immune reaction, transcripts for enzymes in plasmalogen synthesis remained mostly unchanged. The rate limiting enzyme for this process, glyceronephosphate O-acyltransferase , was not regulated on the transcriptomic level. The acyl residues in phosphatidyl-ethanolamine (PE) plasmalogens are taken from the cellular free fatty acid pool. For this purpose the enzyme fatty acyl-CoA reductase 1 (FAR1) provides the long chain fatty alcohol by reduction of saturated fatty acyl-CoAs in the peroxisomal membrane. During monocyte-macrophage differentiation its mRNA was downregulated. The resulting alcohols are then further processed by alkyldihydroxyacetonephosphate synthase (AGPS/ADHAP-S) in the peroxisome, an enzyme whose transcript was downregulated on day 4 and increased on day 5. These findings are in agreement with constant levels of total plasmalogens that were detected in lipid mass spectrometry .2 isoforms constitue a large family of enzymes specifically produced for this purpose. With the exception of PLA2G4A all PLA2 and PLD isoforms that were analyzed on the microarray showed either stable or increasing expression levels for their mRNA, while the endoperoxide synthases were downregulated. This points towards a tight regulation of eicosanoid production and processing. PLA2 is required for releasing precursor molecules for eicosanoid biosynthesis from either phosphatidylcholine  or plasmalogens (vinyl-ethers). Although plasmalogen specific PLA2 activity has been decribed in the literature, this enzymatic activity could not be associated with a specific isoform or transcript in the phospholipase family Eicosanoids are inflammatory mediators that are synthesized in macrophages from eicosapentaenoic acid , arachidonic acid , and dihomo-gamma-linolenic acid  Eicosanoid production is subsequently mediated by the PG-endoperoxide synthases PTGS1 and PTGS2, also known as cyclooxigenases COX1 and COX2 In summary these changes in the gene expression profile reflect the major alterations in lipid metabolism: increases in fatty acid synthesis, elongation and desaturation as well as increased cholesterol production, utilization and export from phagocytic macrophages in comparison to blood monocytes. Since the modulated release of inflammatory mediators is a key contribution of macrophages in the development and sustenance of atherosclerotic plaques we next focused on an in depth examination of plasmalogen lipidomics.in vitro differentiation protocol. In general plasmalogen species containing AA in sn-2 position were most abundant. During the course of monocyte to macrophage differentiation, total plasmalogen levels did not change significantly  producing an environment with high levels of oxidative stress, that also affects the deposited LDL  as well as granulocytic  phenotype. We therefore analyzed the plasmalogen species pattern of differentiating HL-60 cells . Furthermore we found a significant correlation between the changes in monocyte to macrophage differentiation and the regulation of plasmalogen species during monocytic differentiation of HL-60 cells . In contrast the changes found in HL-60 derived granulocytic cells were statistically different from monocyte-macrophage differentiation .Since the plasmalogen pattern undergoes very specific changes during phagocytic differentiation we hypothesized that it could serve as a biomarker for predicting the monocyte to macrophage differentiation state. To this end a PLS/DA analysis was performed on the data obtained from primary human monocytes and macrophages. PLS is a mathematical method that allows to analyze the influence of a matrix of predictors on an outcome variable.In this approach 4 different latent variables were computed and plotted graphically to evaluate their power to differentiate the differentiation stage. As shown in We also determined the most influential lipid species by calculating so called Variable Importance for the Projection (VIP) values as shown in Chronic, systemic low-grade inflammatory diseases such as diabesity and the metabolic syndrome are characterized by a strong activation of tissue macrophages that chemotactically also attract additional monocytes from the circulating blood stream In order to identify novel players influencing the regulation of cellular plasmalogens we performed partial correlation analysis of plasmalogen species with gene array data . This biFurthermore a range of proteins that have not yet been implicated in monocyte-macrophage differentiation were identified. DOCK1 is a Rac activator that regulates myoblast fusion Further transcripts that were identified include ARHGAP26, a Rho GTPase involved in the formation of endocytotic vesicles, cell spreding and adhesion Further studies are needed to clarify the role of these potential lipid related modulators of the differentiation process.Major regulatory changes in the cellular transcriptional profile are mediated by transcription factors. We used a bioinformatics approach therefore affecting all target genes that contain a specific binding motif in their promoter region at the same time. This search was based on transcripts listed in the Gene Ontology category lipid metabolism. Transcription factors that are predicted to bind to promoters of these genes are shown in During the differentiation process to mature macrophages monocytes undergo profound changes in their lipidomic and transcriptomic profile to prepare them for their phagocytic and inflammatory function.Fatty acid synthesis pathways show a strong induction of transcripts involved in fatty acid desaturation and elongation. Lipid transport and lipoprotein related transcripts are differentially modulated partly in a pro-inflammatory way partly anti-inflammatory. On the one hand cholesterol biosynthesis is downregulated and export is strongly promoted. At the same time the cell prepares to facilitate the hydrolysis of triglycerides. In depth analysis was perfomed for plasmalogens. Plasmalogen biosynthesis pathways remained mostly unchanged on the mRNA level. This was reflected in lipidomic measurements showing constant total plasmalogen levels. Plasmalogens represent storage lipids for precursor acyl-residues in eicosanoid synthesis. Transcripts for the iso-enzymes involved in eicosanoid production were tightly regulated.in vitro differentiation. The PE plasmalogen species pattern could be shown to be specific for monocytes and monocyte derived macrophages permitting a purely lipidomic discrimination of the differention stage. It might therefore also represent a potential biomarker indicating immune system activation, e.g. in patients with metabolic syndrome that suffer from chronic, low-grade inflammation.Lipidomic analysis of PE plasmalogens showed decreases in polyunsaturated species (PUFA) and increases in monounsaturated species (MUFA). These adaptions rendered monocyte derived macrophages more similar to mature granulocytes in their plasmalogen profile. This observation could be confirmed in an HL-60 model system for granulocytic and monocytic A bioinformatic approach using partial correlation and molecular network reconstruction was able to identify novel potential hubs in the plasmalogen related transcriptional network, such as 70 kDa heatshock proteins, DOCK1, PDK4, GNPTAB, FAM126A, MNS1 and C12orf48. Further studies are currently underway to clarify the potential modulatory role of these proteins during differentiation. Transcription factor motif analysis revealed the involvement of several binding motifs with the remarkable involvement of motifs for NFY, P53, SP1 and EGR in up- and down- regulated genes. Therefore probably the interplay of multiple additional factors such as SREBP, determines the amount of transcription of lipid regulatory transcripts.In summary we could for the first time show the specificity of the changes in the plasmalogen pattern during monocyte to macrophage differentiation. Furthermore using a bioinformatics approach we were able to predict novel regulatory hubs and transcription factors that might play central roles in the complex interplay of transcription-factors, transcripts and lipids during monocyte-macrophage differentiation.Figure S1Variable importance in projection for PE plasmalogens during differentiation. The VIP value summarizes the contribution a variable makes to the model. Therefore higher values  signify a more significant contribution of the respective lipid species to the estimation of the differentiation stage.(TIF)Click here for additional data file.Table S1GO classification of enriched transcripts during differentiation. Normalized enrichment scores (NES) were calculated to reflect the distribution of Gene Ontology categories across a list of genes ranked by hypergeometrical score (HGS). A higher enrichment score indicates a shift of genes belonging to certain GO categories towards either end of the ranked list, representing up or down regulation .(XLSX)Click here for additional data file.Table S2Hypergeometrical score of lipid related transcripts during differentiation. Agilent microarray data shows regulatory activity in fatty acid desaturation and elongation as well as changes in plasmalogen synthesis and degradation. Comparisons were made for day 4 versus day 1 (d4) and for day 5 versus day 1 (d5). Blue color indicates a significantly lower value following differentiation while red indicates significantly higher values.(DOCX)Click here for additional data file.


Drosophila melanogaster were subjected to ‘high’ and ‘low’ mating regimes, resulting in highly significant differences in lifespan. We sampled females for transcriptomic analysis at day 10 of life, before the visible onset of ageing, and used Tiling expression arrays to detect differential gene expression in two body parts (abdomen versus head+thorax). The divergent mating regimes were associated with significant differential expression in a network of genes showing evidence for interactions with ecdysone receptor. Preliminary experimental manipulation of two genes in that network with roles in post-transcriptional modification  tended to enhance sensitivity to mating costs. However, the subtle nature of those effects suggests substantial functional redundancy or parallelism in this gene network, which could buffer females against excessive responses. There was also evidence for differential expression in genes involved in germline maintenance, cell proliferation and in gustation / odorant reception. Interestingly, we detected differential expression in three specific genes  and one class of genes (gustation / odorant receptors) with previously reported roles in determining lifespan. Our results suggest that high and low mating regimes that lead to divergence in lifespan are associated with changes in the expression of genes such as reproductive hormones, that influence resource allocation to the germ line, and that may modify post-translational gene expression. This predicts that the correct signalling of nutrient levels to the reproductive system is important for maintaining organismal integrity.Elevated rates of mating and reproduction cause decreased female survival and lifetime reproductive success across a wide range of taxa from flies to humans. These costs are fundamentally important to the evolution of life histories. Here we investigate the potential mechanistic basis of this classic life history component. We conducted 4 independent replicated experiments in which female  It has long been observed that there are many instances in which elevated rates of mating are costly to females, causing a decrease in female survival and lifetime reproductive success e.g. –7). This. This7])Trade-offs can occur because of the underlying genetic correlations between the traits involved –7. For eD. melanogaster females have been performed .We examined the potential interactions between the candidate differentially expressed genes, using data from the BIOGRID database  and the e (DroID ), which  BIOGRID . We filthttps://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp] . We chose alpha-tubulin as our control after searching multiple datasets for ‘candidate’ control genes (> 30 were surveyed). We investigated both the integrated breadth of tissue expression  using cDNA derived from the same RNA samples as the microarray hybridisation. cDNA was made from 1µg of RNA using Superscript II reverse transcriptase . Primers were designed using the Assay Design Centre (Roche) [dex.jsp] . qPCR waFlyatlas ) and conFlyatlas . The latEcR network that are associated with post-transcriptional gene expression, namely CG11486 (PAN3) and eyegone. We used an EP mis-expression system for CG11486  x wDah female controls were also relatively short lived in comparison to their low mating counterparts . While there was an internal control as there was for each test (high mating versus low mating treatments of the same genotype) we cannot rule out off-target effects of other unknown loci. Future work should therefore focus on tests with lines that have undergone further backcrossing, so that effects of the focal loci targeted can unequivocally be observed.In the second replicate set of experiments we repeated the tests of overexpression of  mutants  and also mutants . In addiEcR network could be due to several reasons. First, effect sizes may be relatively small, making differences statistically difficult to detect. Note for example that even for moderate effect sizes the Cox regression method and other similar survival analyses are relatively insensitive to interaction effects between survival curves. Second, there could be functional redundancy in this EcR network such that the manipulation of individual component genes in it results in less of an overt phenotype than might be expected. Such redundancy in gene networks may provide an effective way in which to buffer the organism against social, sexual and environmental variation. This buffering could therefore protect females against high mating costs. Finally, it is also possible that there are parallel, as opposed to redundant pathways operating, such as is reminiscent of the effects on ageing and survival of many components of dietary restriction  x Act5C Gal4 (CG11486 overexpressing), (b) Act5C Gal4 x wDah (control), (c) EP[CG11486] x wDah (control), and (d) Dahomey (control). Experiment 2 (e)-(i): survivorship of high and low mating females of the following genotypes: (e) EP[CG11486] x Act5C Gal4 (CG11486 overexpressing), (f) Act5C Gal4 x wDah (control), (g) EP[CG11486] x wDah (control), (h) Dahomey (control), (i) eyegone1 (eyegone knockout).Experiment 1 (a)-(d): survivorship of high and low mating females of the following genotypes: (a) (TIF)Click here for additional data file.Table S1CG11486 and eyegone manipulated females.Sample sizes for the initial tests of mating costs in (PDF)Click here for additional data file.Table S2(a) Number of females alive per day and timing of sampling for gene expression analysis. (b) Cumulative number of deaths per day. (c) Number of female dead since last censoring. (d) Survivorship . (e) Survivorship differences between treatments (low minus high treatments).(PDF)Click here for additional data file.Table S3Results are from the RankProd analysis using a pfp<0.05 (see main text for more details). Genes in bold appear in more than one list.(PDF)Click here for additional data file.Table S4Genes and isoforms surveyed by qPCR. Given are PCR primers used for each of the genes tested and the Universal Probe Library probe used in the qPCR analysis.(PDF)Click here for additional data file.Table S5Shown are (a) the fold changes and significance values from the microrarray analysis, and (b) the isoforms tested by qPCR, the fold difference , the Universal Probe Library (UPL) probe used and finally significance values from 1 tailed paired t-tests . See also (PDF)Click here for additional data file.Table S6Enriched functional categories resulting from DAVID/EASE analysis of all differentially expressed genes in the ABD and HT body parts of females subjected to high and low mating treatments (see main text for more details).(PDF)Click here for additional data file.Table S7CG11486 and eyegone manipulated females.Mean ± 95% confidence intervals of female survival in days in the initial tests of mating costs in (PDF)Click here for additional data file.


To compare myeloid-lineage cell infection in monkeys infected with SIVΔvpx compared to SIVmac239, we analyzed lymphoid and gastrointestinal tissues from SIVΔvpx-infected rhesus (n = 5), SIVmac239-infected rhesus with SIV encephalitis (7 SIV239E), those without encephalitis (4 SIV239noE), and other SIV mutant viruses with low viral loads . SIV+ macrophages and the percentage of total SIV+ cells that were macrophages in spleen and lymph nodes were significantly lower in rhesus infected with SIVΔvpx (2.2%) compared to those infected with SIV239E (22.7%), SIV239noE (8.2%), and SIV mutant viruses (10.1%). In colon, SIVΔvpx monkeys had fewer SIV+ cells, no SIV+ macrophages, and lower percentage of SIV+ cells that were macrophages than the other 3 groups. Only 2 SIVΔvpx monkeys exhibited detectable virus in the colon. We demonstrate that Vpx is essential for efficient macrophage infection in vivo and that simian AIDS and death can occur in the absence of detectable macrophage infection.Analysis of rhesus macaques infected with a  SM, and SIVMAC lineages, encode viral protein X (Vpx) vpr gene, it has been theorized that vpx arose as a duplication of an ancestral vprWhile all lineages of HIV and SIV encode an accessory protein termed viral protein R (Vpr), only some, particularly the HIV-2, SIVin vitro, there is limited information defining the role and impact of Vpx in vivo. Two early studies were conducted with Vpx mutant viruses, one study with mucosal inoculation of pigtail macaques (Macaca nemestrina) using a SIVSMPBj6.6 vpx-I activated mutant virus Macaca mulatta) infected intravenously with a vpx deletion mutant of SIVmac239 (SIVΔvpx) vpx-mutant SIVSM during acute infection were significantly hindered vpx replication was diminished relative to the parental wild type virus and chronic infection and survival was prolonged Although recent studies have elucidated Vpx function vpx after chronic infection at terminal AIDS. The SIVΔvpx mutant virus is identical to SIVmac239 wild-type cloned virus except for the 101 base deletion of the vpx gene from the virus. In previous studies, we demonstrated that SIVΔvpx replicates in rhesus PBMC with only slightly reduced kinetics, but its replicates in rhesus macrophages was markedly impaired with no significant replication noted over 30 days In this study, we analyzed the cellular and tissue targets of viral replication in adult rhesus monkeys infected with SIVΔvpx-infected macaques were compared to those of rhesus infected with SIVmac239 with (SIV239E) or without encephalitis (SIV239noE), SIVΔnef, or SIVΔ3. SIVΔ3 is missing nef, vpr, and upstream sequences in U3vpx function results in the near complete absence of infection in myeloid-lineage cells in vivo even after several years. Additionally, the colon is particularly devoid of virus despite the presence of remaining lymphoid cells. Nevertheless, all 5 SIVΔvpx-inoculated rhesus developed AIDS with opportunistic infections and AIDS-defining lesions in vivo and that the development of AIDS can occur in the absence of detectable virus replication in myeloid-lineage cells, including macrophages and dendritic cells.Cellular and tissue tropism of SIVΔvpx was described previously by Gibbs et al. vpx-infected rhesus ranged from 659 to 1241 days post-inoculation (dpi) with median survival of 1036 dpi  compared to both SIV239E and SIV239noE monkeys  , which are 0.5-2.5 logs lower than usually observed in SIVmac239-infected rhesus vpx case 4 had the highest near-terminal plasma viral load (5.1×105), but still survived 1068 dpi  before succumbing to AIDS. SIVΔnef and SIVΔ3 infected rhesus had chronic and near-terminal plasma viral loads comparable to SIVΔvpx cases, as previously reported The original analysis of these SIVΔvpx-vpr region was amplified from frozen plasma samples available from the SIVΔvpx-infected rhesus macaques. Alignments of the predicted amino acid sequences for Vpx, Vpr, and overlapping Vif and Tat segments were constructed. The original 101 base pair deletion in vpx eliminated Vpx function measured in vitro and was carefully constructed so as not to affect the overlapping Vif sequences at its amino terminus or the splice acceptor for Vpr near the C terminus vpx sequences downstream of the deletion would be out-of-frame with stop codons immediately flanking the deletion in vitro . Sequences from 2 of the five animals (cases 4 and 5) revealed additional stop codons in the remaining vpx sequences  did not differ between SIVΔvpx on tissue virus burden and tissue tropism, we compared the numbers of SIV+ cells in available spleen (SPL), peripheral lymph node (LN), and colon tissues  among the 4 groups of monkeys. Photomicrographs of representative sections of in situ labeled SIV+ cells in spleen and lymph node depict high levels of infection in the SIV239E and SIV239noE monkeys compared to the SIVΔvpx cases (nef/SIVΔ3 group (mean 42.5 SIV+ cells), with the least in the SIVΔvpx group (mean 23.5 SIV+ cells)  and the SIVΔnef/SIVΔ3 group (mean 6.5) compared to both the SIV239E (mean 105.3) and the SIV239noE monkeys (mean 33.5) (p<0.05)  had detectable SIV+ cells in the colon, whereas 4/5 SIVΔnef/SIVΔ3 infected rhesus had detectable virus in the colon. In addition, virus was abundant in SIV239E cases and frequent in SIV239noE cases  (in situ hybridization and Ham-56 immunohistochemistry. Images of lymphoid follicles (top row) depict Ham-56+ cells morphologically consistent with follicular DCs, while images in the second row depict HAM56+ macrophage/dendritic cells in the red pulp of the spleen (left and center) or paracortex in lymph node (right) ( A). Although the total number of SIV+ cells in SPL and LN did not differ significantly among the 4 groups, the total number of infected myeloid-lineage cells, including macrophages and dendritic cells, did differ among the groups (ANOVA p = 0.0011) with significantly higher SIV+ macrophage mean in the SIV239E group (mean 13.6 cells) and SIV239noE (mean 4.5 cells) compared to SIVΔvpx monkeys (mean 0.5 cells) (P<0.05 for both) (nef/SIVΔ3 group was significantly higher (p<0.05) than in the SIVΔvpx monkeys while there was no difference in the overall number of infected cells in these tissues . SIV infected macrophages made up a much higher percentage of all SIV+ cells in SPL and LN of SIV239E (22.7%), SIV239noE (8.3%), and SIVΔnef/SIVΔ3 monkeys (10.1%) compared to SIVΔvpx (2.2%) (p<0.05)  that had 2 detectable SIV+ macrophages in LN out of 55 SIV+ cells also had the highest plasma viral load . Of interest, is the observation that Ham-56+ myeloid cells with morphologic features of myeloid dendritic cells in the germinal centers were also negative for virus. Computer spectral imaging was used in order to discern spectrally distinct chromagens and to compare the frequencies of macrophage infection assessed by counting colabeled (yellow) cells on spectrally unmixed images  had higher numbers of SIV+ macrophages in the colon compared to the SIVΔvpx group, in which there were no SIV infected macrophages detected  (vpx rhesus (0%) compared to SIV239E (63.3%), SIV239noE (22.4%), and SIVΔnef/SIVΔ3 groups (25.6%) .We performed the same double labeling on colon sections from the 4 groups of animals ( (25.6%) Table 4.vpx monkeys highlights a potential role of Vpx and macrophage tropism in vivo, reflected in a difference in overall tissue tropism of SIVΔvpx with an apparent reduction of virus trafficking to the colon. The one SIVΔvpx-infected monkey (case 1) with more frequent SIV+ cells in the colon had severe bacterial enterocolitis, which likely contributed to enhanced immune activation and immune cell trafficking facilitating viral transport to the intestinal tract.The surprising scarcity of SIV-infected cells and complete absence of SIV+ macrophages (0/32 SIV+ cells) in the colon of the SIVΔvpx is unable to efficiently infect tissue macrophages in vivo. Although SIVΔvpx-infected animals had prolonged survival, they did eventually succumb to AIDS with CD4 decline despite the fact that there was little to no infection of macrophages Vpx enables infection of myeloid cells, including macrophage and dendritic cells, with HIV-2 and SIVmac viruses by inhibiting the recently identified macrophage restriction factor SAM domain HD domain-containing protein 1 (SAMHD1) DCAF1 E3 ubiquitin ligase so that SAMHD1 undergoes proteaosome degradation allowing the virus to replicate vpx in SIV, only 3 of the 5 SIVΔvpx-infected macaques exhibited rare SIV+ macrophages in lymphoid tissues while the other 2 animals exhibited no macrophage infection. We also observed that SIVΔvpx-infected animals had little to no virus in the colon despite the fact that there were target lymphocytes and macrophages present, and there was a complete absence of detectable SIV+ macrophages in the colon of any of the SIVΔvpx-infected animals. In contrast, macrophage infection in cloned SIVmac239-infected rhesus occurred at significantly higher rates, especially in those cases that developed encephalitis. As an additional comparison group we analyzed tissues from rhesus infected with SIVΔnef or SIVΔ3. Although tissue virus burden in SPL/LN and colon from the SIVΔnef or SIVΔ3 animals were comparable to that of the SIVΔvpx-infected animals, the number of SIV+ tissue macrophages and percentage of SIV+ cells that were macrophages were significantly lower in the SIVΔvpx group. Nevertheless, all of the SIVΔvpx-infected animals developed AIDS. It has long been postulated that HIV evolves broader cellular tropism from CD4+ T lymphocytes to macrophages and that this viral adaptation is necessary for progression to AIDS In this study, we have demonstrated that SIVΔvpx-deleted SIV to replicate in macrophages and dendritic cells in monkeys is consistent with the in vitro data on Vpx function but was not necessarily an obvious outcome. Wide ranges of culture conditions have been used for measuring the replication of SIV and HIV in macrophages in vitro. Both monocyte-derived macrophages obtained from peripheral blood and bronchoalveolar macrophages obtained by lung lavage have been used. Stimulating agents such as granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) are used by some investigators but not others. These stimulants can have a dramatic effect on the ability of virus to replicate in macrophage cultures in vivo setting. Serra-Moreno, et al. have recently demonstrated that a nef-deleted strain of SIV adapted in monkeys to the loss of anti-tetherin activity by establishing such activity through mutation of a completely different region of the viral genome, the cytoplasmic domain of the gp41 transmembrane envelope glycoprotein vpx replication in macrophages in monkeys, even after two to three years of ongoing viral replication to provide an opportunity to adapt, even with the possibility that select pressure to restore this function may be limited, suggests that it is not easy for the virus to re-acquire the macrophage-specific vpx functions lost by its deletion.The profound and prolonged deficit in the ability of the vpx had lower viral loads and a slower progression to AIDS than what is typically observed with the parental SIVmac239, but nonetheless progressed to simian AIDS with low CD4 counts and AIDS-defining lesions. Two of the five SIVΔvpx-infected monkeys that died with AIDS had no detectable infection of macrophages in spleen, lymph node or colon despite the presence of infected T cells in these animals at this terminal stage. Analysis of the other three monkeys revealed a total of four infected macrophages that were found. This dearth of infected macrophages contrasts markedly with all 11 control monkeys infected with parental SIVmac239. The frequency with which SIV encephalitis occurs with SIVmac239 in monkeys (about 30%) and the frequency of infected cells that are macrophages at a terminal stage appear to be similar to what is seen in HIV-1-infected people Monkeys infected with SIVΔvpx-deleted SIV. Alternatively, SAMHD1's ability to restrict infection may be down regulated in some cells. As with monkeys infected with nef-deleted SIV, in which another SIV gene (env) evolved the capacity for anti-tetherin activity in vivo the vpx-deleted SIV managed to evolve some anti-SAMHD1 activity in another SIV gene. In our limited sequence analysis there were no consistent or highly unusual, focused changes in the vpr or tat genes at the terminal stages in these animals to suggest what such change might be, although there were two novel changes in vif.What might be the explanation for the few infected macrophages that were observed? As with any post-entry restriction factor, it is likely that there is a significant cell-to-cell variation in the amounts of SAMHD1 present in any individual macrophage or in different monocyte-macrophage subtypes. Some rare macrophages may end up with particularly low levels of SAMHD1 based on stage of differentiation, source, history, and activation state and these may be weakly susceptible to infection by vpx-infected monkeys. Not only were macrophages not infected, but also T cells were rarely infected. In fact, there was a near complete absence of detectable virus in the colon of the SIVΔvpx-infected monkeys. Perhaps infection of macrophages or dendritic cells is important for promulgating the infection of the small numbers of CD4+ CCR5+ lymphocytes remaining in the gut. This supports the recent in vitro findings that Vpx, by inducing SAMHD1 degradation, is important for HIV-1 transmission between T cells and dendritic cells, which is important for overall viral dissemination and persistence Findings in the colon are interesting and potentially informative. It is now clear that both SIV and HIV-1 cause extremely severe, sustained depletion of CD4+ CCR5+ T lymphocytes in gut-associated lymphoid tissue (GALT), including the colon. Severe depletion of this population of cells has been suggested to drive the evolution to CXCR4-using HIV-1 late in the course of disease vpx is required for efficient myeloid cell infection in experimentally infected rhesus monkeys and that progression to AIDS and death can occur in the absence of Vpx and macrophage infection. This is interesting in light of the recent revelation that Vpx targets SAMHD1 for degradation vpr, the paralog of vpx, does not inhibit or antagonize SAMHD1 while other primate Vpr proteins do In summary, we have shown that All animal studies were performed in accordance with federal laws and regulations, international accreditation standards, and institutional policies, including approval by the Institutional Animal Care and Use Committee of Harvard Medical School. All animals received two nutritional meals a day and fresh water ad libitum. All animals received required and approved environmental enrichment and were monitored daily for evidence of disease and changes in attitude, appetite, or behavior suggestive of illness. The enrichment program includes weekly rotation of enrichment devices including a wide variety of manipulanda, foraging feeders, sensory enrichment, grooming devices, destructible enrichment, and enrichment food items. Animals are socially housed except when this interferes with a study. Additional enrichment is targeted to animals that are assessed as having abnormal behavior or alopecia. Animals housed in the biocontainment facilities received a daily health check by both animal care technicians and veterinary professional staff. Appropriate clinical support was administered under the direction of the attending veterinarian and included analgesics, antibiotics, intravenous fluids, and other supportive care as needed. To ameliorate pain and suffering, all procedures were performed under ketamine, Telazol, or an inhalant anesthesia. Analgesics, such as buprenex, were used preemptively and following each potentially painful procedure. Animals were humanely euthanized when they presented with advanced stages of AIDS; criteria for euthanasia included 15% weight loss in two weeks, unresponsive opportunistic infection, persistent anorexia, intractable diarrhea, progressive neurologic signs, significant cardiac or pulmonary signs or other serious illness. The Harvard Medical School animal management program is accredited by the Association For Assessment and Accreditation of Laboratory Animal Care (AAALAC) and meets National Institutes of Health standards as set forth in “The Guide for the Care and Use of Laboratory Animals” . The institution also accepts as mandatory the PHS “Policy on Humane care and use of Laboratory Animals by Awardee Institutions” and the NIH “Principles for the Utilization and Care of Vertebrate Animals used in Testing, Research and Training.”vpx-deleted derivative of it called SIVΔvpxnefvpx deletion mutant virus based on parental cloned SIVmac239 (SIVΔvpx) as previously described vpx-infected monkeys (nef (n = 4), or SIVΔ3 (n = 2) were also evaluated for comparison  using techniques as previously described with some modifications Plasma viral RNA from SIVΔ5′-AAAAGGGTGGCTCAGTACTTATGCAGT-3′; positions 5829–5857 in SIVmac239 sequence) and S-VVout.as  for 40 cycles with primer annealing at 58oC for 30″ and primer extension at 68oC for 1′20″. PCR fragments were cloned directly into pCR2.1 vector using TOPO TA Cloning kit (Invitrogen) and inserts sequenced on both strands with S-VxVp.s  and S-VxVp.as  primers at CSHL shared DNA sequencing resource. The vpx-vpr region was amplified and sequenced successfully from plasma samples available from the SIVΔvpx -infected rhesus macaques. Viral vpx gene sequences were analyzed and compared to parental viral clone SIVmac239. Predicted amino acid sequences for Vpx, Vpr, and overlapping Vif and Tat segments were constructed, aligned, and compared to parental viral clone SIVmac239. Alterations in amino acid sequences were searched against existing NCBI databases to determine novel mutations.Viral RNA was isolated from the plasma samples collected from the infected animal using High Pure Viral RNA Kit (Roche), reverse transcribed with SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen). SIV sequences were amplified by polymerase chain reaction using Superscript III kit (Invitrogen) with S-VVout.s  from each infected macaque in FFPE sections by in situ hybridization for SIVmac and immunohistochemistry with either anti-CD3 for T lymphocytes or anti-Ham56 for monocyte/macrophage lineage cells. In situ hybridization was performed as detailed above followed by routine immunoperoxidase staining on 5-µm formalin-fixed paraffin-embedded sections using standard avidin-biotin peroxidase complex techniques  as previously described Infected cells were characterized by double-label analysis vpx, SIVΔnef, and SIVΔ3 cases, the entire slide was examined and all positive cells were counted for each case. All counting tasks were performed by two individuals blinded to the original of the slides. For double-label immunohistochemistry and in situ hybridization, sections were evaluated to assess percentage of infected cells that were macrophages per slide using a Nuance™ liquid crystal tunable filter based camera and multispectral imaging system  interfaced with a Leica DMRE microscope. Images of tissue sections were captured without manipulation and analyzed using Nuance software. When there were fewer than 100 SIV+ cells the entire section was screened. Control slides of the following chromogens were used to make spectral libraries at each relevant magnification: diaminobenzidine (DAB) for immunohistochemistry, BCIP/NBT (in situ), and Vector Nuclear Fast Red (counterstain). The spectral absorbance of each chromogen was established from single stain controls and the contribution of each stain calculated from this reference to determine the relative contribution of the stain at each pixel. Images were automatically acquired from 420 nm to 720 nm at 20 nm steps and spectral components unmixed. Brightfield images were unmixed into a composite image and individual component images for each spectrum analyzed. With different chromogens unmixed, the numbers of SIV/Ham56 double-positive macrophages and SIV/CD3 double-positive T-lymphocytes were quantified in tissue sections and reported as percent of infected cells.Sections were evaluated from each animal to derive a mean number of CD3+ lymphocytes and Ham56+ macrophages and SIV+ cells per 20× microscopic field as previously described Generation of colocalization images was performed by overlaying masks of positive staining cells from the component images. A standardized threshold of minimum number of pixels determined each component mask. Simulated fluorescence images were converted from unmixed brightfield composite images by the Nuance™ software and then combined into a single image with colocalized signals represented in yellow.in situ hybridization assays was performed with the GraphPad Prism program . Kruskal-Wallis nonparametric analysis of variance (ANOVA) followed by Dunn's multicomparison test were used to assess statistical differences between groups for each test with statistical differences assumed for probability values of p<0.05. For significant ANOVA, nonparametric Mann-Whitney t tests were performed for comparisons of mean number of infected cells and percent of SIV+ cells that are macrophages between groups with statistical differences assumed for p<0.05.Statistical evaluation and generation of graphs of counted cells from immunohistochemistry and Figure S1Complete predicted amino acid sequences for Vpx (S1a) and Vpr (S1b) derived from viral RNA sequences from plasma. Cullin 4 E3 binding region, required for counteraction of SAMHD1, is specified in sequence alignment of Vpx.(DOCX)Click here for additional data file.


It is not possible to identify all pregnancies at risk of neonatal hypoxic-ischemic encephalopathy (HIE). Many women use some form of analgesia during childbirth and some anesthetic agents have been shown to be neuroprotective when used as analgesics at subanesthetic concentrations. In this study we sought to understand the effects of two anesthetic agents with presumptive analgesic activity and known preconditioning-neuroprotective properties (sevoflurane or xenon), in reducing hypoxia-induced brain damage in a model of intrauterine perinatal asphyxia. The analgesic and neuroprotective effects at subanesthetic levels of sevoflurane (0.35%) or xenon (35%) were tested in a rat model of intrauterine perinatal asphyxia. Analgesic effects were measured by assessing maternal behavior and spinal cord dorsal horn neuronal activation using c-Fos. In separate experiments, intrauterine fetal asphyxia was induced four hours after gas exposure; on post-insult day 3 apoptotic cell death was measured by caspase-3 immunostaining in hippocampal neurons and correlated with the number of viable neurons on postnatal day (PND) 7. A separate cohort of pups was nurtured by a surrogate mother for 50 days when cognitive testing with Morris water maze was performed. Both anesthetic agents provided analgesia as reflected by a reduction in the number of stretching movements and decreased c-Fos expression in the dorsal horn of the spinal cord. Both agents also reduced the number of caspase-3 positive (apoptotic) neurons and increased cell viability in the hippocampus at PND7. These acute histological changes were mirrored by improved cognitive function measured remotely after birth on PND 50 compared to control group. Subanesthetic doses of sevoflurane or xenon provided both analgesia and neuroprotection in this model of intrauterine perinatal asphyxia. These data suggest that anesthetic agents with neuroprotective properties may be effective in preventing HIE and should be tested in clinical trials in the future. Neonatal hypoxic ischemic encephalopathy (HIE) affects 1–3 per 1000 term births in the developed world in vivo study assessed 7 day old rats but could not effectively reproduce the intra-uterine environment in which HIE normally occurs. In the present study we have used a validated model of nociceptive parturition We previously reported on the neuroprotective effects of xenon and sevoflurane preconditioning in a model of neonatal asphyxia The study was approved by the Home Office (UK) and conforms to the United Kingdom Animals (Scientific Procedures) Act of 1986. All efforts were made to minimize animal suffering and the number of animals used. Pregnant Sprague-Dawley (SD) rats (300–350 g) were housed in individual cages in a 12-h-light/12-h-dark cycle in temperature (22–23°C) and humidity-controlled environment with free access to standard laboratory chow.We used a validated parturition nociceptive model Behavioral analysis: The numbers of stretching behaviors, either inward turning of one hind paw or straining and squashing of the lower abdomen to the floor Immunohistochemistry: One hour after giving birth to the first pup, the dams were deeply anesthetized with sodium pentobarbital  and perfused transcardially with ice-cold heparinized 0.1 M phosphate buffer solution (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M PBS at pH 7.4 , the whole spinal cord was removed. After 24 hours fixation in 4% PFA, the lumbosacral enlargement of the spinal cord was embedded in optimal cutting temperature compound  and sectioned transversely at 30 µm intervals and stained for c-Fos as previously described Separate cohorts of dams (without mifepristone treatment) received 0.35% sevoflurane or 35% xenon in 30% oxygen balanced by nitrogen for 4 hours on gestation day 22 from 8am. Animals from naïve or control group received 30% oxygen plus 70% nitrogen. Dams were sacrificed by cervical dislocation and uterine horns were removed and placed in a water-bath at 37°C for 10 min to induce hypoxic-ischemic insult 4 hours after gas exposure. The dams giving birth before the surgery and the pups they delivered were discarded from the study. Fetal asphyxia was performed as previously described Immunohistochemistry and histology: On Postnatal Day (PND) 3 and 7 Neurocognitive Testing with Morris Water Maze (WM): Neurocognitive outcome was evaluated daily (starting on PND 50) using Morris water maze (WM) post hoc Newman-Keuls test wherever appropriate or unpaired t-test using SPSSv13.0 . A p value<0.05 was considered to be a statistically significant.The results are expressed as mean ± SEM. Data was analyzed with one-way analyses of variance followed by Nociceptive behavior-labor pain: In order to assess the effects of anesthetic exposure on pain-related behavior we measured stretches. Following exposure to either xenon or sevoflurane, uterine contraction-related pain (stretching behavior) in the 90 min period prior delivery was lower in the xenon (46±6) and sevoflurane (31±2) groups as compared to controls (oxygen enriched air exposure only) (185±47) prior to delivery of the first pup   .Neuronal activation during labor pain: Next we measured the effects of xenon or sevoflurane as labor analgesics on neuronal activity using c-Fos expression in the spinal cord. One hour after delivery of the first pup, the number of c-Fos positive neurons in the spinal cord layer I–II was significantly higher in the control group (22±5) as compared to those in the naïve group (2±0.2), xenon group (5±2) and sevoflurane group  is illustrated in Postnatal memory deficit is improved following treatment with xenon and sevoflurane: In order to assess neuropathological amelioration afforded by xenon or sevofluane exposure prior to injury leading to cognitive improvement, we used WM to assess learning and memory functions at the juvenile stage on PND 50. Latency to locate the hidden platform was significantly decreased in animals exposed to sevoflurane or xenon as compared to pups exposed to hypoxia alone  peri-labor administration of the anesthetic agents sevoflurane or xenon provide effective alleviation of nociception induced by labor in rats, and (ii) preconditioning with either agent for four hours prior to fetal asphyxia offered neuroprotection and ameliorated cognitive dysfunction.The visceral component of labor pain, from the uterus to the lower thoracic and upper lumbar spinal cord segments, relays on c-fiber afferent signaling The analgesic effects of xenon and sevoflurane have been proven pre-clinically and clinically Our previous studies demonstrated that xenon and sevoflurane protected the neonatal neuronal system from brain injury in vivo and in vitro studies N-methyl-D-aspartate (NMDA) receptor antagonist at the glycine site In vitro work showed that the adenosine triphosphate-sensitive potassium (KATP) channel can also be activated by xenon ATPThe neuroprotective properties of xenon and sevoflurane has been evaluated in both Xenon does not appear to be teratogenic nor has been associated with neurodegeneration and has been used in an apparently safe manner in neonates for many years for neuroimaging. However there are some reports about sevoflurane which are less reassuring although it is much less toxic than isoflurane at the equipotent concentration Our data indicate that both xenon and sevoflurane administered for 4 h before asphyxia at concentrations that can provide antinociception during labor and also protect against intrauterine fetal hypoxic-ischemic brain injury in an experimental rat model of hypoxia/ischemia. These data suggest that in the clinical setting, xenon or sevoflurane for labor analgesia may not only provide effective analgesia but also prevent brain injury in the compromised fetus that extends into neuroprotection of the newborn following birth.


Anopheles gambiae would be greatly improved by the development of a binary expression system, which would allow the more rapid and flexible characterisation of genes influencing disease transmission, including those involved in insecticide resistance, parasite interaction, host and mate seeking behaviour. The Gal4-UAS system, widely used in Drosophila melanogaster functional genetics, has been significantly modified to achieve robust application in several different species. Towards this end, previous work generated a series of modified Gal4 constructs that were up to 20 fold more active than the native gene in An. gambiae cells. To examine the Gal4-UAS system in vivo, transgenic An. gambiae driver lines carrying a modified Gal4 gene under the control of the carboxypeptidase promoter, and responder lines carrying UAS regulated luciferase and eYFP reporter genes have been created. Crossing of the Gal4 and UAS lines resulted in progeny that expressed both reporters in the expected midgut specific pattern. Although there was minor variation in reporter gene activity between the different crosses examined, the tissue specific expression pattern was consistent regardless of the genomic location of the transgene cassettes. The results show that the modified Gal4-UAS system can be used to successfully activate expression of transgenes in a robust and tissue specific manner in Anopheles gambiae. The midgut driver and dual reporter responder constructs are the first to be developed and tested successfully in transgenic An. gambiae and provide the basis for further advancement of the system in this and other insect species.Functional genetic analysis in  An. gambiae, has attracted considerable scientific focus in the expectation that increased understanding of fundamental mosquito biology may lead to improved tools for vector control An. gambiae in some cases An. gambiae we are interested in the development of a suitable binary expression system in this species.The major African malaria vector, Drosophila and has proven a powerful functional genomics tool. The system is not only used to directly study phenotypes generated through transgene mis- or over-expression, but has a wide variety of applications including enhancer detection and stable gene knockdown through RNAi and refined mosaic analyses The Gal4-UAS system is used routinely and with great success in The bi-partite Gal4-UAS approach utilizes transgenic “driver” lines carrying the yeast transactivator, Gal4, under the transcriptional control of a specific regulatory region; and transgenic “responder” lines containing a candidate gene under the transcriptional control of Gal4 binding sites (otherwise known as upstream activation sequences or UAS) Drosophila has prompted attempts to transfer it directly to other model organisms. However, despite initial reports utilizing the native form of Gal4 in some species, the system was not widely adopted due to low activity of the transactivator and toxicity when over-produced. More recently, various modified forms of Gal4 have been shown to be more active and robust, and have been successfully employed in zebrafish Tribolium castaneumAedes aegyptiThe value of the Gal4-UAS system in An. gambiae cell culture An. gambiae, a panel of Gal4 variants were developed, including a series carrying short tandemly repeated synthetic activation domains derived from the Herpes simplex virus protein, VP16, which activates transcription of the intermediate early viral genes. These were assayed in conjunction with an array of responder constructs with different numbers of UAS repeats. This work identified several driver elements that were up to 20 fold more active than the native Gal4, and a responder construct, UAS-14i, consisting of 14 tandemly repeated UAS and a small artificial intron, that allowed the greatest range of activation in Anopheles cells Previously it has been demonstrated that the native Gal4 was only minimally active in An. gambiae in vivo, we now report the creation of driver lines containing the Gal4-GFY transactivator An. gambiae carboxypeptidase (CP) promoter. A CP promoter has been used previously to direct transgene expression in the midgut of adult Ae. aegypti, Anopheles stephensi and An. gambiae mosquitoes To examine the Gal4-UAS system in Anopheles by the addition of a second UAS14i controlled reporter gene encoding a nuclear localisation signal (nls) tagged eYFP (UAS-eYFPnls). In addition, gypsy insulator elements were used to flank both reporter genes An. gambiae cells SUA5.1 with the constitutively active LRIM1-Gal4Δ driver plasmid Drosophila Actin5C promoter Anopheles cells, whilst dsRed was observed throughout the cell  were recovered. Surviving adults (G0s) were separated by sex, placed in six pools and out-crossed to wild type mosquitoes. After screening G1 larvae for dsRed expression, 31 isofemale lines were generated. G2 progeny were scored for fluorescent phenotype, and only those lines that yielded approximately 50% dsRed positive larvae were further maintained. A similar strategy was followed to generate the responder lines. In this case, 188 larvae (20%) were recovered from 942 embryos injected with the dual UAS reporter transposon. G1 larvae were screened for eCFP expression and 41 transgenic isofemale lines were established. Only lines that displayed Mendelian inheritance of the marker gene suggestive of single (or tightly linked) transposon insertions were retained. These lines were assigned a unique one or three letter identifier based on the parental ‘cage’ they were derived from.An. gambiae transformation were approximately one line in 46, and one line in 63 embryoes injected for driver and responder lines, respectively. This is a conservative estimate since several lines that potentially carried more than one insertion were discarded without further characterization. Six of the 21 lines had transposon insertions into predicted genes (VectorBase Agam P3.6), of which one (Xnt) was located in an predicted exon of the orthologue of dachs gene, encoding a variant myosin, that is involved in growth of legs and wings Drosophila Furin2, yata, beat IIIC, and Pbp45  were crossed with two Gal4 driver lines (F and Dgl). From all crosses at least 10 female progeny were dissected to examine eYFP expression. In all progeny examined, eYFP was detectable in adult female midguts, when viewed through a stereomicroscope (×40 magnification) and in most cases the signal was sufficiently robust to be visible externally through the abdomen cuticle prior to dissection. As expected, the eYFP reporter gene was effectively targeted to the nuclei of midgut epithelial cells , and wasAll six Gal4 driver lines were then crossed with Mbl and Wnd UAS lines. Again, all the resulting progeny displayed adult female midgut specific eYFP expression in the non-blood fed state, although the intensity and distribution of eYFP appeared to vary to some extent depending on the driver used  progenies than in the respective homozygous UAS lines . Apart fIn agreement with the absence of background expression observed with the fluorescent reporter, the more sensitive luciferase assay detected only a very low level of activity in carcasses taken from all crosses . In addiWhilst eYFP was readily observed in female midguts after dissection, fluorescence in male midguts was apparent, but less intense in progeny from all crosses, except for those derived from driver line F, in which eYFP was not detectable. . SimilarNo obvious variation in eYFP expression in the midgut or other tissues was noted in all transheterozygous individuals examined following ingestion of a bloodmeal. Furthermore, luciferase assays also suggested that activity was not upregulated throughout 24 hours post-bloodfeed (data not shown). This absence of response was then examined at the level of transcription, through semi-quantitative RT-PCR on midgut RNA isolated before and at different time points following a bloodmeal. As is shown in An. gambiae to give robust expression patterns. Crossing of driver and responder lines resulted in successful transactivation of the luciferase and eYFP reporter genes in the midgut epithelia of resultant progeny. This expression was observed in all individuals characterised from all 20 different crosses carried out, involving six different driver lines and six UAS lines. The phenotypes were stable over time and consistent expression was observed in transheterozygous progeny after the driver and responder lines had been maintained for at least 25 and 20 generations respectively.The modified Gal4-UAS system utilized here worked efficiently in An. gambiae, we have utilised a construct carrying both UAS regulated eYFPnls and luciferase. These lines permit fluorescence-based identification of temporal and spatial expression patterns, whilst luciferase activity measurements allow simple and accurate quantification. The nuclear tagged fluorescent protein was chosen to allow differentiation of reporter gene expression from cytoplasmic directed expression of marker genes should future experiments require crossing with lines carrying fluorescent proteins with overlapping emission spectra.As the first responder lines developed for Drosophila, it has been shown that the different sites of transposon integration do not support the expression of transgenes equally well in all tissues, and that this can be largely overcome by the insulation of inserted genes with gypsy sequences Ae. aegypti lines without insulator elements, of which only two robustly expressed the fluorescent marker from an endogenous CP promoter Gypsy insulator sequences were placed around the UAS cassettes to potentially mitigate position effects of transgene insertion Anopheles female and male midguts directed from the CP promoter was in accordance with high throughput transcriptome data which indicates highly enriched CP gene transcription in midguts from both sexes An. stephensiThe specificity of midgut expression in An. gambiae CP gene is induced 4–8 fold within 3 hours of a bloodmeal, and then returns to unfed constitutive levels after 24–48 hours An. gambiae CP promoter have indicated that transgene expression in the midgut followed the endogenous transcription pattern and was induced 3–6 hours after bloodfeeding in vivo.No obvious increase in midgut fluorescence or luciferase activity was observed in all the lines examined following bloodfeeding. Previous research suggests that the The large accumulation of transgene products produced from the CP-Gal4-GFY driver lines will prove useful in studies that examine very early stages of pathogen interaction in the midgut. Currently there are currently no promoters available that direct extensive expression prior to the formation of the peritrophic matrix (a protective chitinous layer produced early after feeding to surround the blood bolus) All driver and responder combinations demonstrated an enrichment of female specific reporter gene expression, as would be expected from recent tissue specific transcriptomic data indicating that male mRNA signal is around 75% lower than in females 24 hours after a blood meal AedesAn. gambiae individuals. However, using the more sensitive luciferase assay Anopheles, a very low level of activity was detected in carcass samples, which appeared to be almost entirely derived from non-Gal4 regulated expression from the UAS cassette insertions, since similar activities were detected in non-crossed responder lines. Similar levels of basal luciferase expression observed in more expansive Drosophila studies support the idea that it is unlikely that any UAS-transgenes are ever completely silent Drosophila would also suggest that this limited basal activity is normally biologically inert and is most likely a consequence of non-specific polymerase II transcription throughout the genome As has been reported recently during Gal4-UAS system development in Tribolium, consistent expression of Gal4 transactivators was only achieved by replacement of the core Drosophila heat shock promoter present on the original UAS constructs with a comparable endogenous sequence An. gambiae and is present on both the UAS regulated reporter genes, as well as the 3×P3 promoter used to express the selectable marker genes. More recent studies in Aedes aegypti have also demonstrated the utility of the core promoter to regulate Gal4-UAS dependant expression Drosophila sequence is sufficiently conserved to allow efficient expression of transgenes in mosquitoes.In related work with An. gambiae, we report the largest generation of independent transgenic An. gambiae lines to date. The work produced 21 transgenic lines, of which six carried intragenic transposon insertions that may have caused hypomorphic alleles. Similar frequencies of intragenic insertions have been achieved in other series of piggyBac mediated transformation of An. gambiaeDrosophila Furin2 and dachs genes have been deposited with the Malaria Research and Reference Reagent Resource Center (MR4) to enable potential phenotypes from gene knock downs or hypomorphic alleles to be examined in the future. In addition, Wlm, displaying bright salivary gland expression, enabling easier dissection of this tissue has also been deposited in MR4.As well as the initial validation of the Gal4 system in An. gambiae to give robust and tissue specific transgene activation. Gal4 transactivation was evident in the progeny of all Gal4-UAS crosses, and appeared not to diminish in intensity over 20 generations. The Gal4-GFY driver used in this study gives intermediate levels of activation when used in conjunction with the UAS14i responder cassette in cell transfections. This Gal4 transactivator clearly works efficiently in vivo in combination with the CP promoter, however it may also be possible to produce different levels of target gene expression from the same promoter by utilizing other Gal4 transactivators described previously An. gambiae is a major step forward for functional genetic characterization in this species. To facilitate distribution to the research community, these constructs and representative driver and responder lines will also be offered for general distribution to the MR4 malaria research facility. The establishment of driver and responder lines that direct expression in other tissues of importance in malaria transmission, insecticide resistance and host and mate seeking behaviour are obvious targets for future development.In conclusion, the variant Gal4-UAS system described here functions well in et alAn. gambiae s.s. DNA as template with NotI and EcoRV tagged primers. Primer sequences given in PCR was used to amplify the 1.7 kb region upstream of the carboxypeptidase gene , described by Edwards attB[3×P3-dsRed2nls-SV40]lox66 The following cloning strategy was taken to construct a piggyBac vector carrying a UAS regulated luciferase and eYFP gene cassette flanked either side by gypsy insulator sequences. The gypsy insulator sequence from pH-Stinger (Drosophila Genomic Resource Centre) An. gambiae cell line Sua5.1, originally generated by Hans-Michael Muller 20. Transfection was carried out using Qiagen Effectene reagent following the manufacturer's protocol with a ratio of 5 µl effectene and 1.6 µl enhancer to 300 ng DNA in 24 well plates. 100 ng of Gal4 driver plasmid  were reared according to the MR4 standard protocol. Early embryos were injected with a solution containing 350 ng/µl of the Gal4 driver plasmid  or the UAS responder plasmid (pB-gUAS-LUC-UAS-eYFPnls-g) and 150 ng/µl of the transposase helper plasmid, phspBac 20. Genomic DNA flanking the piggyBac insertion sites was amplified by the PCR with primer pairs; left arm, ITRL1F and ITRL1R Inverse PCR was carried out on each isofemale transgenic line to establish the location of the insertion event in the genome. DNA from at least 30 male and female adult mosquitoes was extracted using Qiagen GenomicTip 20/G columns as described in manufacturer's standard protocol. 2 µg of genomic DNA was then digested to completion with BfuCI (NEB), purified on diatomaceous earth Crosses were performed on mosquito lines homozygous for the Gal4 (marked by dsRed) and UAS (marked by eCFP) transgenes. At least 10 females were used per cross with at least double the number of males. In total 20 different crosses were carried out with two experimental repeats for each cross. Larval progeny were screened to check expected inheritance of both red and cyan fluorescent eye marker proteins. Adults were examined at 3, 7 and 14 days after emergence. Dissections were carried out using a Leitz MFLZ III microscope using GFP-B, dsRed and CFP filters. Images were taken using a Zeiss Axiovert 200 M confocal microscope, an Olympus BX60 microscope fitted with a Nikon DSU2 camera, or through the dissecting microscope fitted with a Nikon P5100 digital camera.2 and dissections carried out in PBS. Midguts, carcasses, or whole mosquitoes were transferred to 200 µl of luciferase buffer [1× passive lysis buffer (Promega) supplemented with 1 µg/ml aprotinin (Sigma) and 1× EDTA-Free protease inhibitor cocktail (Roche). Samples from three mosquitoes were added per tube and the tube frozen in a dry ice ethanol bath before storage at −70°C. Three replicates for each cross were processed. Samples were later thawed on ice, homogenized for 1 min and then spun at full speed in a microfuge for 10 min at 4°C. The supernatant was kept at 4°C and used directly for luciferase and protein assays. Gal4-UAS midgut and whole mosquito samples were diluted 10 fold in luciferase buffer before luciferase assays were carried out. 1 mg/ml BSA was added to diluted midgut samples to prevent luciferase degradation. A Promega Luciferase Assay Kit (E1500) was used to assay luciferase concentrations in mosquito homogenates using a Lumat LB 9507 tube luminometer. Results were standardized to total protein concentration in samples using BioRad RC DC protein assay kits and measured using a VersaMax microplate reader (Molecular Devices) at 750 nm.Seven day old mosquitoes were anesthetized with COAn. gambiae ribosomal protein gene, rS60 (AGAP002122) Mosquitoes were fed on human blood and midguts dissected at 3, 6, and 24 hours after feeding. Samples  were stored in 500 µl of Tri Reagent (Sigma). Unfed female mosquitoes (at 0 hours) were used as the reference time point. RNA was extracted according to Tri Reagent manufacturer's protocol and treated with Turbo DNase (Ambion). First strand cDNA was prepared from approximately 20 ng total RNA using Superscript III First-strand Synthesis System for RT-PCR (Invitrogen). Transcript abundance of the Gal4, luciferase and carboxypeptidase (AGAP009593) genes were examined by semi-quantitative RT-PCR using two sets of primers for each gene in order to monitor reliability of amplification . cDNA leFigure S1Fluorescent marker protein expression in transheterozygous. Images of three representative mosquitoes, U = UAS responder line, G = Gal4 driver line and GU = progeny of Gal4-UAS cross; (A) Brightfield, (B) DsRed filter, DsRed marker expression from the 3×P3 promoter in the Bolwig organ of the eyes and ganglia indicating G and GU larvae are positive for Gal4 driver. (C) CFP filter, eCFP expression in the same tissues of U and GU larvae. Note discernable lack of bleed-through between filter sets with red and blue markers.(TIF)Click here for additional data file.Figure S2eCFP expression in the salivary glands of responder line Wlm. (A) Ventral view of UAS responder line, Wlm, showing eCFP marker gene expression from the 3×P3 promoter in the Bolwig organ, ganglia, salivary glands and anal papillae. (B) Dorsal view of larvae showing eCFP expression in the Bolwig organ of the eyes, ganglia and salivary glands. (C) eCFP expression in dissected larval salivary glands. (D) eCFP expression in distal lateral lobes of adult salivary glands.(TIF)Click here for additional data file.Figure S3Expression of fluorescent protein in the midgut. Images A to F show a representative image of eYFP expression in the midgut of female progeny of crosses between driver lines Cln, Drt, Dgl, F, G and Ivr respectively with the responder line Wnd, photographed through GFP-B filter. All guts are from sugarfed mosquitoes.(TIF)Click here for additional data file.Figure S4Expression of eYFP in male and female midguts of Gal4-UAS mosquitoes. A representative image of eYFP expression in dissected midguts of a male (top) and female (bottom) heterozygous for the Gal4 and UAS cassettes under a GFP-B filter set for crosses involving the responder line, Mbl, and the driver lines Dgl and F (A and B respectively). All guts are from sugarfed mosquitoes.(TIFF)Click here for additional data file.Table S1Primers used for plasmid construction and inverse PCR.The table describes the region amplified, the primer names and the primer sequences from 5′ to 3′. Bold font shows the non-homologous tag used for restriction enzyme digest and cloning.(DOCX)Click here for additional data file.Table S2Primers used for RT-PCR to determine transgene expression levels. The table lists the target gene for amplification, the primer names, the primer sequences from 5′ to 3′ and the number of PCR cycles used to obtain suitable levels of amplification to enable semi-quantitative PCR.(DOCX)Click here for additional data file.


Syphilis point-of-care tests may reduce morbidity and ongoing transmission by increasing the proportion of people rapidly treated. Syphilis stage and co-infection with HIV may influence test performance. We evaluated four commercially available syphilis point-of-care devices in a head-to-head comparison using sera from laboratories in Australia.Point-of-care tests were evaluated using sera stored at Sydney and Melbourne laboratories. Sensitivity and specificity were calculated by standard methods, comparing point-of-care results to treponemal immunoassay (IA) reference test results. Additional analyses by clinical syphilis stage, HIV status, and non-treponemal antibody titre were performed. Non-overlapping 95% confidence intervals (CI) were considered statistically significant differences in estimates.In total 1203 specimens were tested . Point-of-care test sensitivities were: Determine 97.3%(95%CI:95.8–98.3), Onsite 92.5%(90.3–94.3), DPP 89.8%(87.3–91.9) and Bioline 87.8%(85.1–90.0). Specificities were: Determine 96.4%(94.1–97.8), Onsite 92.5%(90.3–94.3), DPP 98.3%(96.5–99.2), and Bioline 98.5%(96.8–99.3). Sensitivity of the Determine test was 100% for primary and 100% for secondary syphilis. The three other tests had reduced sensitivity among primary (80.4–90.2%) compared to secondary syphilis (94.3–98.6%). No significant differences in sensitivity were observed by HIV status. Test sensitivities were significantly higher among high-RPR titre (RPR≥8) (range: 94.6–99.5%) than RPR non-reactive infections (range: 76.3–92.9%).The Determine test had the highest sensitivity overall. All tests were most sensitive among high-RPR titre infections. Point-of-care tests have a role in syphilis control programs however in developed countries with established laboratory infrastructures, the lower sensitivities of some tests observed in primary syphilis suggest these would need to be supplemented with additional tests among populations where syphilis incidence is high to avoid missing early syphilis cases. Timely diagnosis and treatment of syphilis is crucial to reduce morbidity, and onward transmission of syphilis to sexual partners and newborns, in whom consequences can be devastating Syphilis testing is recommended in clinical guidelines as part of antenatal screening, for people at high risk, for those with symptoms and following sexual contact with confirmed cases This paper presents results of a large dual-site, laboratory-based, head-to-head evaluation of performance of four commercially available rapid POC syphilis tests using archived sera that includes analysis by syphilis stage, HIV status and non-treponemal antibody titre.Ethical approval was granted by the South East Sydney and Illawarra Area Health Service Human Research Ethics Committee (HREC), Melbourne Health HREC and University of New South Wales HREC. Consent was not required from individuals as it was a retrospective study of de-identified stored samples that has previously been tested with the goal standard syphilis assays. Linked clinical data were anonymized and de-identified prior to analysis.Rates of syphilis in Australia are highest among two distinct populations - gay men living in urban centres and Aboriginal and Torres Strait Islander people living in remote regions across Australia We assessed the performance of four syphilis POC tests by comparing results to routinely performed reference treponemal tests (immunoassays [IA]) conducted at the laboratories. Linked clinical data for a subset of specimens were extracted from patient medical records. Inter-technician variability and operational characteristics of the tests were also assessed.POC tests were selected to include commercially available tests with traditional as well as novel test platforms: (i) Determine Syphilis TP® ; (ii) Onsite Syphilis Ab Combo Rapid Test® ; (iii) SD Bioline Syphilis 3.0® ; and (iv) DPP Syphilis Screen and Confirm Assay® . All four tests use an immunochromatographic strip design with the appearance of a visible coloured line if treponemal antibodies are detected in the specimen. The DPP Syphilis test, in addition, simultaneously detects non-treponemal antibodies by a separate coloured line on the same test strip.The respective reference treponemal immunoassay (IA) tests used by Victorian Infectious Diseases Reference Laboratory (VIDRL) in Melbourne and South Eastern Area Laboratory Service (SEALS) in Sydney are: (i) Trepanostika TP recombinant enzyme immunoassay®  Reference test results were extracted from the laboratory databases or patient medical records for comparison with POC test results. A specimen from a patient with a previously documented reactive treponemal IA result was not retested and was considered to be IA reactive for the study analyses.Specimens were selected to include approximately equal numbers of IA reactive and IA non-reactive samples, and a range of RPR titres.high RPR titre syphilis ; low RPR titre syphilis ; inactive syphilis ; no syphilis (IA and RPR non-reactive); and biological false positives .Specimens were categorised by treponemal IA and by non-treponemal (RPR) reference test results as Demographic  and clinical data  were extracted from medical records. Specimens were categorised by clinical stage according to the definitions in Technicians performing the POC tests completed training to ensure consistency in the conduct and interpretation of results. Specimens were tested according to the manufacturer's instructions by one study technician blinded to the reference results. The POC test results were interpreted and recorded on a laboratory data record sheet. A second blinded study technician, independently interpreted and recorded the POC test result within 1 minute of the first read. Discrepancies were discussed and a consensus reached and recorded. Reasons for discrepancies were noted. Invalid tests were repeated using a new test and recorded as such.We assessed selected operational characteristics of each test by self-administered questionnaire among the technicians performing the POC tests. Questions focused on ease of test use and interpretation of the results.The sensitivity and specificity for each syphilis POC test compared to the treponemal IA reference test results was calculated by standard methods and a kappa (k) value was calculated for each test as a summation of the overall performance using Stata . Results were stratified by HIV status and RPR titre (among IA reactive specimens). POC test sensitivity was also compared to clinical stage of syphilis. For the analyses presented here, DPP POC test sensitivity refers the treponemal line result compared to reference IA to allow for consistent comparison across all four POC tests.To compare performance between POC tests and between subcategories, 95% confidence intervals (CI) were calculated Inter-observer variability to determine the discrepancy rate between the two technicians recording results was calculated as the number of test results which differ between the two technicians x 100/total number of tests performed using the same serum specimens.3.In total, 1203 specimens (678 from Melbourne and 525 from Sydney) were tested with each of the four syphilis POC tests. The median age of the cases was 35 years (range 18–85) and 83.4% were men. Additional patient demographic and clinical information were available for 878 (73%) specimens. About half (50.9%) of the male cases identified as having a least one same sex partner during the 12 months, prior to specimen collection. One hundred and fifty four (12.8%) cases were HIV positive and 67 (5.6%) had a most recent CD4 count of <500 cells/mmAs presented in Among the 1203 specimens, 736 (61.2%) were reference IA reactive and 467 (38.5%) were non-reactive. RPR results were available for 1,005 (83.5%) specimens and used to further stratify the specimens as described in POC sensitivities and specificities overall, stratified by HIV status, CD4 count are shown in 3 compared to <500 cells/mm3 across all POC tests.By HIV status, there were no statistically significant differences observed in POC test sensitivities. POC test specificities were lower among HIV positive compared to HIV negative specimens; however the difference was only statistically significant for the Determine test (81.5% vs. 97.8%). Though not statistically significant, POC test specificities were lower among those with CD4 count≥500 cells/mmInter-reader variability analysis (discrepancy rate between results recorded by two technicians) revealed a range of discrepancies across the tests: Determine 0.2% (n = 3), Bioline 1.2% (n = 15), DPP 1.5% (n = 18) and Onsite 2.7% (n = 33). Technician's comments indicated that many of these discrepancies occurred when results lines on the tests kits were faint.Technicians (n = 2) found all the four POC tests were “simple” or “very simple” to conduct. Overall interpretation of the POC result was considered to be “relatively easy” for all POC test devices, however technicians raised concerned regarding interpretation of faint or weak bars. Faint bars were reported to have occurred “sometimes” with all the devices except the Determine, leading to discrepancies in initial results between technician 1 and 2. Technicians noted some problems with the nitrocellulose strip in the Determine test and the cassette casing for the DPP and Onsite devices.This paper reports results from a large, dual-site, head-to-head evaluation of performance of four commercially available syphilis POC test devices using stored sera. This is the first such study exploring the impact of syphilis stage and HIV co-infection on POC performance. Our results provide evidence supporting the good performance of treponemal tests currently available, with the Determine test demonstrating overall superior performance and with technicians experiencing the least number of challenges in interpretation of results. Uniquely demonstrated in our study, test sensitivities did not differ by HIV status but did differ by clinical stage and RPR titre.Though much of the current published literature focuses on the use of these POC tests as part of antenatal screening programs in developing countries, there may be a role for these tests, particularly those able to differentiate potentially active from inactive/past infection, in other settings, including developed countries among populations with high rates of syphilis and HIV co-infection. Our results suggest that such a role be considered in the context of ongoing routine serology to identify false negative results, given the possible poorer sensitivity among primary syphilis of some of these tests.Overall, the Determine test had the highest sensitivity (97.3%), while all four had similarly very high specificities (97.2–98.5%). The overall kappa statistic for each test, a summation of the overall performance of each test against the reference standard, was highest for Determine TP suggesting very substantial agreement between the POC test and laboratory reference results. These individual test performances are not dissimilar to others reported in the literature 3. As numbers were small, these results should be interpreted with some caution.We observed no difference in sensitivity by HIV status, however, we did note a trend towards reduced test specificity among HIV positive compared to negative specimens. Determine was the only test showing a statistically significant lower specificity among HIV positive specimens. This phenomenon has been described elsewhere The Determine test was the best performing POC test and was able to identify 100% of specimens in both primary and secondary stage infection. The other three tests appeared to have a lower sensitivity among primary infection, particularly the Bioline test with a sensitivity of only 77.3%. In all of these tests the sensitivity increased in secondary infections but did not reach 100%. These tests would therefore miss a number of primary and secondary infections. In many countries all patients with a genital ulcer are treated for syphilis as identification of early syphilis infections is important to prevent ongoing transmission and is a key STI control strategy.Although the DPP Screen and Confirm syphilis test has a lower sensitivity compared with the Determine test, it does have the ability to detect both treponemal and non-treponemal antibodies, and therefore help distinguish past, treated infection from possible active infection with some encouraging lab and field results The strength of our study was the majority of specimens tested were collected from clients attending large urban sexual health clinics with substantial expertise in syphilis and HIV and providing for a population at highest risk of syphilis (urban gay men) making our study sample well-suited to evaluate potential differences in POC test sensitivity by clinical stage and HIV status. A smaller proportion of our study population were women and although information regarding pregnancy status was not collected, it is likely that fewer than 1% of these women would have been pregnant in this setting .There are also a few limitations to consider. The two laboratories providing reference results each used a different treponemal IA. Both are highly sensitive and specific and as relative ranking of POC test performance did not differ by site, we believe the use of two different reference IAs did not bias our results. Our study was conducted in a laboratory environment and the tests were performed by skilled technicians with experience in POC test devices, therefore likely represent a best performance scenario. Though results may differ in a clinical setting, the relative performance of the tests would be expected to remain similar. As these tests are not quantitative, training regarding the interpretation of results required technicians to record a positive result if any band appeared on the test strip at the test line site. It is possible that some faint lines were interpreted by our technicians as positive that may have been called negative by others. Though some evaluations have demonstrated lower test sensitivities using whole blood in field settings In conclusion, our results support the potential role for these tests to be implemented more broadly supplementing existing antenatal screening strategies in resource limited settings. Their use in developed countries might also be considered in populations with high rates of syphilis and HIV co-infection and where loss to follow-up is a significant risk. This might be either as part of a screening strategy using the Determine test or to distinguish potentially active from past/treated infection using a newer dual platform test. Field evaluations of selected POC tests are needed to determine the local performance in the hands of end users, relevance, acceptability, economic costs and potential impact associated with implementation in each setting.