Molecular cloning and characterization of NcROP2Fam-1, a member of the ROP2 family of rhoptry proteins in Neospora caninum that is targeted by antibodies neutralizing host cell invasion in vitro.

Recent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

Loss of Raf-1 kinase inhibitor protein (RKIP) is strongly associated with high-grade tumor budding and correlates with an aggressive phenotype in pancreatic ductal adenocarcinoma (PDAC)

BACKGROUND Raf-1 kinase inhibitor protein (RKIP) has emerged as a significant metastatic suppressor in a variety of human cancers and is known to inhibit Ras/Raf/MEK/ERK signaling. By suppressing the activation of the NFkB/SNAIL circuit, RKIP can regulate the induction of epithelial-mesenchymal transition (EMT). The aim of this study was to evaluate RKIP expression and to determine its association with clinicopathological features, including EMT in form of tumor budding in pancreatic ductal adenocarcinoma (PDAC). METHODS Staining for RKIP was performed on a multipunch Tissue Microarray (TMA) of 114 well-characterized PDACs with clinico-pathological, follow-up and adjuvant therapy information. RKIP-expression was assessed separately in the main tumor body and in the tumor buds. Another 3 TMAs containing normal pancreatic tissue, precursor lesions (Pancreatic Intraepithelial Neoplasia, PanINs) and matched lymph node metastases were stained in parallel. Cut-off values were calculated by receiver operating characteristic (ROC) curve analysis. RESULTS We found a significant progressive loss of RKIP expression between normal pancreatic ductal epithelia (average: 74%), precursor lesions (PanINs; average: 37%), PDAC (average 20%) and lymph node metastases (average 8%, p<0.0001). RKIP expression was significantly lower in tumor buds (average: 6%) compared to the main tumor body (average 20%; p<0.005). RKIP loss in the tumor body was marginally associated with advanced T-stage (p=0.0599) as well as high-grade peritumoral (p=0.0048) and intratumoral budding (p=0.0373). RKIP loss in the buds showed a clear association with advanced T stage (p=0.0089). CONCLUSIONS The progressive loss of RKIP seems to play a major role in the neoplastic transformation of pancreas, correlates with aggressive features in PDAC and is associated with the presence of EMT in form of tumor budding.

Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination.

Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04-0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9-2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.

PCR identification of HIV-1 DNA sequences in brain tissue of patients with AIDS encephalopathy.

We analyzed brain tissue from 39 patients for the presence of proviral HIV-1 sequences, using the polymerase chain reaction (PCR) for the amplification of segments of the viral LTR and gag genes. A novel primer extension procedure allowed the detection of a single HIV-1 copy in 1 micrograms DNA. We detected proviral HIV-1 DNA in 16 of 25 brain samples from AIDS patients. Semiquantitative evaluation of the amplified DNAs indicated considerable variation in viral load. Highest levels of proviral DNA were present in brain samples from six patients with clinical evidence of HIV-associated cognitive/motor complex and the histopathologic correlate of HIV leukoencephalopathy or HIV encephalitis. An additional 11 brain samples contained smaller amounts of proviral DNA. In these patients, clinical data were inconclusive regarding the diagnosis of HIV-1 encephalopathy and histopathologically there was no evidence of HIV-1-induced tissue lesions. In nine of 25 seropositive patients with AIDS (36%), brain samples scored negative or did not contain an unequivocal signal indicating the presence of proviral DNA. HIV-1 sequences were not detected in any of 14 control brain samples from HIV-1 seronegative patients. Our data indicate that HIV-1 is present in the central nervous system of the majority (two thirds) of AIDS patients and that the highest levels of proviral DNA in brain tissue are associated with HIV encephalopathy.

The effects of endothelin-1 on coronary perfusion pressure during cardiopulmonary resuscitation in a canine model

OBJECTIVE To study the hemodynamic effects of exogenously administered endothelin-1 (ET-1), a peptide produced by endothelial cells with potent non-adrenergically mediated vasoconstrictor properties. METHODS A prospective drug intervention study was carried out in a resuscitation research laboratory. Fifteen mixed-breed dogs were anesthetized and instrumented for hemodynamic monitoring. Asphyxia arrest was produced by clamping the endotracheal tube. Hemodynamic data were collected continuously. Following loss of aortic fluctuations monitored by thoracic aortic catheter, the animals remained in pulseless electrical activity (PEA) for 10 minutes. After 10 minutes of no-flow PEA, closed-chest CPR was begun and the animals were randomized to one of three treatment groups (EPI, 0.02 mg/kg epinephrine IV every 3 minutes; ENDO, 100 micrograms ET-1 IV at 0 minutes; and EPI/ENDO, a combination of the EPI and ENDO treatments). RESULTS ENDO and EPI alone produced similar coronary perfusion pressures (CPPs). The EPI/ENDO combination produced significantly improved CPP compared with that of either EPI or ENDO alone. In the EPI group, the best mean CPP was 16 +/- 14 mm Hg and occurred 7 minutes after drug administration. In the ENDO group, the best mean CPP was 28 +/- 7 mm Hg and occurred 13 minutes after drug administration. In the EPI/ENDO combination group, the best mean CPP was 61 +/- 37 mm Hg and occurred 7 minutes after drug administration (p < 0.05 compared with the EPI and ENDO groups alone). CONCLUSION ET-1 is a potent vasoconstrictor. The combination of EPI and ENDO significantly improved CPP compared with that for either agent alone. ET-1 should be investigated further as a vasoconstrictor in cardiac arrest.

Role of MicroRNA Modulation in the Interferon-α/Ribavirin Suppression of HIV-1 In Vivo.

BACKGROUND Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo. METHODS Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors. RESULTS miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo. CONCLUSIONS The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.

Quantifying the turnover of transcriptional subclasses of HIV-1-infected cells

HIV-1-infected cells in peripheral blood can be grouped into different transcriptional subclasses. Quantifying the turnover of these cellular subclasses can provide important insights into the viral life cycle and the generation and maintenance of latently infected cells. We used previously published data from five patients chronically infected with HIV-1 that initiated combination antiretroviral therapy (cART). Patient-matched PCR for unspliced and multiply spliced viral RNAs combined with limiting dilution analysis provided measurements of transcriptional profiles at the single cell level. Furthermore, measurement of intracellular transcripts and extracellular virion-enclosed HIV-1 RNA allowed us to distinguish productive from non-productive cells. We developed a mathematical model describing the dynamics of plasma virus and the transcriptional subclasses of HIV-1-infected cells. Fitting the model to the data allowed us to better understand the phenotype of different transcriptional subclasses and their contribution to the overall turnover of HIV-1 before and during cART. The average number of virus-producing cells in peripheral blood is small during chronic infection. We find that a substantial fraction of cells can become defectively infected. Assuming that the infection is homogenous throughout the body, we estimate an average in vivo viral burst size on the order of 104 virions per cell. Our study provides novel quantitative insights into the turnover and development of different subclasses of HIV-1-infected cells, and indicates that cells containing solely unspliced viral RNA are a good marker for viral latency. The model illustrates how the pool of latently infected cells becomes rapidly established during the first months of acute infection and continues to increase slowly during the first years of chronic infection. Having a detailed understanding of this process will be useful for the evaluation of viral eradication strategies that aim to deplete the latent reservoir of HIV-1.

Fibrinogen association with human monocytes: evidence for constitutive expression of fibrinogen receptors and for involvement of Mac-1 (CD18, CR3) in the binding

Radiolabeled fibrinogen (Fg) specifically binds to mononuclear leukocytes (MNL) and to purified monocytes, but not to nylon-nonadherent lymphocytes. The association is rapid, Ca++-dependent and reversible. MNL containing Fg-binding monocytes had not been exposed to endotoxin (less than 4 pg/mL) during the isolation and the binding test, and Fg binding was not altered by preincubation of MNL with lipopolysaccharide. The binding of Fg was inhibited by anti-Mac-1 antibodies (OKM1). Antibodies to surface-bound Fg were able to induce luminol-dependent chemiluminescence, indicating that Fg binding sites have receptor function. Emission of a signal depended on MNL exposure to Fg, on specific, divalent antibodies, but not on the antibody Fc portion. These data show that human monocytes constitutively express specific Fg receptors and suggest that Mac-1, a member of the integrin superfamily, is involved in Fg recognition.

Plasma Levels of MASP-1 and MASP-2 are Elevated in Type 1 Diabetes and Correlate with Glycaemic Control

There is increasing evidence that the complement system plays an important role in diabetes and the development of diabetic vascular complications. In particular, mannan-binding lectin (MBL) levels are elevated in diabetes patients, and diabetes patients with diabetic nephropathy have higher MBL levels than diabetes patients with normal renal function. The MBL-associated serine proteases (MASPs) MASP-1, MASP-2, and MASP-3, and MBL-associated protein MAp44 have not yet been studied in diabetes patients. We therefore measured plasma levels of MASP-1, MASP-2, MASP-3, and MAp44 in 30 children with type 1 diabetes mellitus (T1DM) and 17 matched control subjects, and in 45 adults with T1DM and 31 matched control subjects. MASP-1 and MASP-2 levels were significantly higher in children and adults with T1DM than in their respective control groups, whereas MASP-3 and MAp44 levels did not differ between patients and controls. MASP-1 and MASP-2 levels correlated with HbA1c, and MASP levels decreased when glycaemic control improved. Since MASP-1 and MASP-2 have been shown to directly interact with blood coagulation, elevated levels of these proteins may play a role in the enhanced thrombotic environment and consequent vascular complications in diabetes.

Inhibition of HIV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A.

Human immunodeficiency virus 1 (HIV-1) multiplication depends on a cellular protein, cyclophilin A (CyPA), that gets integrated into viral particles. Because CyPA is not required for cell viability, we attempted to block its synthesis in order to inhibit HIV-1 replication. For this purpose, we used antisense U7 small nuclear RNAs (snRNAs) that disturb CyPA pre-mRNA splicing and short interfering RNAs (siRNAs) that target CyPA mRNA for degradation. With dual-specificity U7 snRNAs targeting the 3' and 5' splice sites of CyPA exons 3 or 4, we obtained an efficient skipping of these exons and a strong reduction of CyPA protein. Furthermore, short interfering RNAs targeting two segments of the CyPA coding region strongly reduced CyPA mRNA and protein levels. Upon lentiviral vector-mediated transduction, prolonged antisense effects were obtained for both types of antisense RNAs in the human T-cell line CEM-SS. These transduced CEM-SS cells showed a delayed, and for the siRNAs also reduced, HIV-1 multiplication. Since the two types of antisense RNAs function by different mechanisms, combining the two approaches may result in a synergistic effect.

3-β-d-Glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc) is an insect detoxification product of maize 1,4-benzoxazin-3-ones

In order to defend themselves against arthropod herbivores, maize plants produce 1,4-benzoxazin-3-ones (BXs), which are stored as weakly active glucosides in the vacuole. Upon tissue disruption, BXs come into contact with β-glucosidases, resulting in the release of active aglycones and their breakdown products. While some aglycones can be reglucosylated by specialist herbivores, little is known about how they detoxify BX breakdown products. Here we report on the structure of an N-glucoside, 3-β-d-glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc), purified from Spodoptera frugiperda faeces. In vitro assays showed that MBOA-N-Glc is formed enzymatically in the insect gut using the BX breakdown product 6-methoxy-2-benzoxazolinone (MBOA) as precursor. While Spodoptera littoralis and S. frugiperda caterpillars readily glucosylated MBOA, larvae of the European corn borer Ostrinia nubilalis were hardly able to process the molecule. Accordingly, Spodoptera caterpillar growth was unaffected by the presence of MBOA, while O. nubilalis growth was reduced. We conclude that glucosylation of MBOA is an important detoxification mechanism that helps insects tolerate maize BXs.

Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores

In monocotyledonous plants, 1,4-benzoxazin-3-ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4-benzoxazin-3-ones in plant–insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4-benzoxazin-3-ones at the plant–insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4-benzoxazin-3-ones and active extracts from mutant plants lacking 1,4-benzoxazin-3-ones. Our data show that maize plants possess a two-step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2-β-d-glucopyranosyloxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA-Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA) is released by plant-derived β-glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4-benzoxazin-3-ones such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant–insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.

Inhibition of photosynthesis by high temperature in oak (Quercus pubescens L.) leaves grown under natural conditions closely correlates with a reversible heat-dependent reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase

Inhibition of the net photosynthetic CO2 assimilation rate (Pn) by high temperature was examined in oak (Quercus pubescens L.) leaves grown under natural conditions. Combined measurements of gas exchange and chlorophyll (Chl) a fluorescence were employed to differentiate between inhibition originating from heat effects on components of the thylakoid membranes and that resulting from effects on photosynthetic carbon metabolism. Regardless of whether temperature was increased rapidly or gradually, Pn decreased with increasing leaf temperature and was more than 90% reduced at 45 °C as compared to 25 °C. Inhibition of Pn by heat stress did not result from reduced stomatal conductance (gs), as heat-induced reduction of gs was accompanied by an increase of the intercellular CO2 concentration (Ci). Chl a fluorescence measurements revealed that between 25 and 45 °C heat-dependent alterations of thylakoid-associated processes contributed only marginally, if at all, to the inhibition of Pn by heat stress, with photosystem II being remarkably well protected against thermal inactivation. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased from about 90% at 25 °C to less than 30% at 45 °C. Heat stress did not affect Rubisco per se, since full activity could be restored by incubation with CO2 and Mg2+. Western-blot analysis of leaf extracts disclosed the presence of two Rubisco activase polypeptides, but heat stress did not alter the profile of the activase bands. Inhibition of Pn at high leaf temperature could be markedly reduced by artificially increasing Ci. A high Ci also stimulated photosynthetic electron transport and resulted in reduced non-photochemical fluorescence quenching. Recovery experiments showed that heat-dependent inhibition of Pn was largely, if not fully, reversible. The present results demonstrate that in Q. pubescens leaves the thylakoid membranes in general and photosynthetic electron transport in particular were well protected against heat-induced perturbations and that inhibition of Pn by high temperature closely correlated with a reversible heat-dependent reduction of the Rubisco activation state.