Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples.

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

VP22 light controlled delivery of oligonucleotides to ocular cells in vitro and in vivo.

PURPOSE: To study VP22 light controlled delivery of antisense oligonucleotide (ODN) to ocular cells in vitro and in vivo. METHODS: The C-terminal half of VP22 was expressed in Escherichia coli, purified and mixed with 20 mer phosphorothioate oligonucleotides (ODNs) to form light sensitive complex particles (vectosomes). Uptake of vectosomes and light induced redistribution of ODNs in human choroid melanoma cells (OCM-1) and in human retinal pigment epithelial cells (ARPE-19) were studied by confocal and electron microscopy. The effect of vectosomes formed with an antisense ODN corresponding to the 3'-untranslated region of the human c-raf kinase gene on the viability and the proliferation of OCM-1 cells was assessed before and after illumination. Cells incubated with vectosomes formed with a mismatched ODN, a free antisense ODN or a free mismatched ODN served as controls. White light transscleral illumination was carried out 24 h after the intravitreal injection of vectosomes in rat eyes. The distribution of fluorescent vectosomes and free fluorescent ODN was evaluated on cryosections by fluorescence microscopy before, and 1 h after illumination. RESULTS: Overnight incubation of human OCM-1 and ARPE-19 cells with vectosomes lead to intracellular internalization of the vectosomes. When not illuminated, internalized vectosomes remained stable within the cell cytoplasm. Disruption of vectosomes and release of the complexed ODN was induced by illumination of the cultures with a cold white light or a laser beam. In vitro, up to 60% inhibition of OCM-1 cell proliferation was observed in illuminated cultures incubated with vectosomes formed with antisense c-raf ODN. No inhibitory effect on the OCM-1 cell proliferation was observed in the absence of illumination or when the cells are incubated with a free antisense c-raf ODN and illuminated. In vivo, 24 h after intravitreal injection, vectosomes were observed within the various retinal layers accumulating in the cytoplasm of RPE cells. Transscleral illumination of the injected eyes with a cold white light induced disruption of the vectosomes and a preferential localization of the "released" ODNs within the cell nuclei of the ganglion cell layer, the inner nuclear layer and the RPE cells. CONCLUSIONS: In vitro, VP22 light controlled delivery of ODNs to ocular cells nuclei was feasible using white light or laser illumination. In vivo, a single intravitreal injection of vectosomes, followed by transscleral illumination allowed for the delivery of free ODNs to retinal and RPE cells.

Genome-wide association study of recurrent major depressive disorder in two European case-control cohorts.

Major depressive disorder (MDD) is a highly prevalent disorder with substantial heritability. Heritability has been shown to be substantial and higher in the variant of MDD characterized by recurrent episodes of depression. Genetic studies have thus far failed to identify clear and consistent evidence of genetic risk factors for MDD. We conducted a genome-wide association study (GWAS) in two independent datasets. The first GWAS was performed on 1022 recurrent MDD patients and 1000 controls genotyped on the Illumina 550 platform. The second was conducted on 492 recurrent MDD patients and 1052 controls selected from a population-based collection, genotyped on the Affymetrix 5.0 platform. Neither GWAS identified any SNP that achieved GWAS significance. We obtained imputed genotypes at the Illumina loci for the individuals genotyped on the Affymetrix platform, and performed a meta-analysis of the two GWASs for this common set of approximately half a million SNPs. The meta-analysis did not yield genome-wide significant results either. The results from our study suggest that SNPs with substantial odds ratio are unlikely to exist for MDD, at least in our datasets and among the relatively common SNPs genotyped or tagged by the half-million-loci arrays. Meta-analysis of larger datasets is warranted to identify SNPs with smaller effects or with rarer allele frequencies that contribute to the risk of MDD.

Growth promoting signaling by tenascin-C [corrected].

Tenascin-C is an adhesion-modulating extracellular matrix molecule that is highly expressed in tumor stroma and stimulates tumor cell proliferation. Adhesion of T98G glioblastoma cells to a fibronectin substratum is inhibited by tenascin-C. To address the mechanism of action, we performed a RNA expression analysis of T89G cells grown in the presence or absence of tenascin-C and found that tenascin-C down-regulates tropomyosin-1. Upon overexpression of tropomyosin-1, cell spreading on a fibronectin/tenascin-C substratum was restored, indicating that tenascin-C destabilizes actin stress fibers through down-regulation of tropomyosin-1. Tenascin-C also increased the expression of the endothelin receptor type A and stimulated the corresponding mitogen-activated protein kinase signaling pathway, which triggers extracellular signal-regulated kinase 1/2 phosphorylation and c-Fos expression. Tenascin-C additionally caused down-regulation of the Wnt inhibitor Dickkopf 1. In consequence, Wnt signaling was enhanced through stabilization of beta-catenin and stimulated the expression of the beta-catenin target Id2. Finally, our in vivo data derived from astrocytoma tissue arrays link increased tenascin-C and Id2 expression with high malignancy. Because increased endothelin and Wnt signaling, as well as reduced tropomyosin-1 expression, are closely linked to transformation and tumorigenesis, we suggest that tenascin-C specifically modulates these signaling pathways to enhance proliferation of glioma cells.

Mating system parameters in a high density population of andirobas in the Amazon forest

The objective of this work was to estimate the mating system parameters of a andiroba (Carapa guianensis) population using microsatellite markers and the mixed and correlated mating models. Twelve open‑pollinated progeny arrays of 15 individuals were sampled in an area with C. guianensis estimated density of 25.7 trees per hectare. Overall, the species has a mixed reproductive system, with a predominance of outcrossing. The multilocus outcrossing rate (t m = 0.862) was significantly lower than the unity, indicating that self‑pollination occurred. The rate of biparental inbreeding was substantial (t m ‑ t s = 0.134) and significantly different from zero. The correlation of selfing within progenies was high (r s = 0.635), indicating variation in the individual outcrossing rate. Consistent with this result, the estimate of the individual outcrossing rate ranged from 0.598 to 0.978. The multilocus correlation of paternity was low (r p(m) = 0.081), but significantly different from zero, suggesting that the progenies contain full‑sibs. The coancestry within progenies (Θ = 0.185) was higher and the variance effective size (Ne(v) = 2.7) was lower than expected for true half‑sib progenies (Θ = 0.125; Ne(v) = 4). These results suggest that, in order to maintain a minimum effective size of 150 individuals for breeding, genetic conservation, and environmental reforestation programs, seeds from at least 56 trees must be collected.

Real-time quantitative PCR assay for measurement of bird telomeres

We present the application of a real-time quantitative PCR assay, previously developed to measure relative telomere length in humans and mice, to two bird species, the zebra finch Taeniopygia guttata and the Alpine swift Apus melba. This technique is based on the PCR amplification of telomeric (TTAGGG)(n) sequences using specific oligonucleotide primers. Relative telomere length is expressed as the ratio (T/S) of telomere repeat copy number (T) to control single gene copy number (S). This method is particularly useful for comparisons of individuals within species, or where the same individuals are followed longitudinally. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a single control gene. In both species, we validated our PCR measurements of relative telomere length against absolute measurements of telomere length determined by the conventional method of quantifying telomere terminal restriction fragment (TRF) lengths using both the traditional Southern blot analysis (Alpine swifts) and in gel hybridization (zebra finches). As found in humans and mice, telomere lengths in the same sample measured by TRF and PCR were well correlated in both the Alpine swift and the zebra finch.. Hence, this PCR assay for measurement of bird telomeres, which is fast and requires only small amounts of genomic DNA, should open new avenues in the study of environmental factors influencing variation in telomere length, and how this variation translates into variation in cellular and whole organism senescence.

A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer.

To better understand the relationship between tumor-host interactions and the efficacy of chemotherapy, we have developed an analytical approach to quantify several biological processes observed in gene expression data sets. We tested the approach on tumor biopsies from individuals with estrogen receptor-negative breast cancer treated with chemotherapy. We report that increased stromal gene expression predicts resistance to preoperative chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC) in subjects in the EORTC 10994/BIG 00-01 trial. The predictive value of the stromal signature was successfully validated in two independent cohorts of subjects who received chemotherapy but not in an untreated control group, indicating that the signature is predictive rather than prognostic. The genes in the signature are expressed in reactive stroma, according to reanalysis of data from microdissected breast tumor samples. These findings identify a previously undescribed resistance mechanism to FEC treatment and suggest that antistromal agents may offer new ways to overcome resistance to chemotherapy.

High-SNR analytical performance of spatial multiplexing MIMO systems with CSI

In this paper, we investigate the average andoutage performance of spatial multiplexing multiple-input multiple-output (MIMO) systems with channel state information at both sides of the link. Such systems result, for example, from exploiting the channel eigenmodes in multiantenna systems. Dueto the complexity of obtaining the exact expression for the average bit error rate (BER) and the outage probability, we deriveapproximations in the high signal-to-noise ratio (SNR) regime assuming an uncorrelated Rayleigh flat-fading channel. Moreexactly, capitalizing on previous work by Wang and Giannakis, the average BER and outage probability versus SNR curves ofspatial multiplexing MIMO systems are characterized in terms of two key parameters: the array gain and the diversity gain. Finally, these results are applied to analyze the performance of a variety of linear MIMO transceiver designs available in the literature.

The IST METRA Project

This article summarizes the main achievementsof the Multi-Element Transmit andReceive Antennas (METRA) Project, an ISTresearch and technological development project carried out between January 2000 and June 2001 by Universitat Politècnica de Catalunya, the Center for Personkommunikation of Aalborg University, Nokia Networks, Nokia Mobile Phones, and Vodafone Group Research and Development.The main objective of METRA was the performanceevaluation of multi-antenna terminals incombination with adaptive antennas at the basestation in UMTS communication systems. 1 AMIMO channel sounder was developed that providedrealistic multi-antenna channel measurements.Using these measured data, stochasticchannel models were developed and properly validated.These models were also evaluated inorder to estimate their corresponding channelcapacity. Different MIMO configurations andprocessing schemes were developed for both theFDD and TDD modes of UTRA, and their linkperformance was assessed. Performance evaluationwas completed by system simulations thatillustrated the benefits of MIMO configurationsto the network operator. Implementation cost vs.performance improvement was also covered bythe project, including the base station and terminalmanufacturer and network operator viewpoints.Finally, significant standards contributionswere generated by the project and presented to the pertinent 3GPP working groups.

Smart antennas in software radio base stations

The application of adaptive antenna techniques to fixed-architecture base stations has been shown to offer wide-ranging benefits, including interference rejection capabilities or increased coverage and spectral efficiency.Unfortunately, the actual implementation ofthese techniques to mobile communication scenarios has traditionally been set back by two fundamental reasons. On one hand, the lack of flexibility of current transceiver architectures does not allow for the introduction of advanced add-on functionalities. On the other hand, theoften oversimplified models for the spatiotemporal characteristics of the radio communications channel generally give rise toperformance predictions that are, in practice, too optimistic. The advent of software radio architectures represents a big step toward theintroduction of advanced receive/transmitcapabilities. Thanks to their inherent flexibilityand robustness, software radio architecturesare the appropriate enabling technology for theimplementation of array processing techniques.Moreover, given the exponential progression ofcommunication standards in coexistence andtheir constant evolution, software reconfigurabilitywill probably soon become the only costefficientalternative for the transceiverupgrade. This article analyzes the requirementsfor the introduction of software radio techniquesand array processing architectures inmultistandard scenarios. It basically summarizesthe conclusions and results obtained withinthe ACTS project SUNBEAM,1 proposingalgorithms and analyzing the feasibility ofimplementation of innovative and softwarereconfigurablearray processing architectures inmultistandard settings.

Joint array combining and MLSE for single-user receivers in multipath Gaussian multiuser channels

The well-known structure of an array combiner along with a maximum likelihood sequence estimator (MLSE) receiveris the basis for the derivation of a space-time processor presentinggood properties in terms of co-channel and intersymbol interferencerejection. The use of spatial diversity at the receiver front-endtogether with a scalar MLSE implies a joint design of the spatialcombiner and the impulse response for the sequence detector. Thisis faced using the MMSE criterion under the constraint that thedesired user signal power is not cancelled, yielding an impulse responsefor the sequence detector that is matched to the channel andcombiner response. The procedure maximizes the signal-to-noiseratio at the input of the detector and exhibits excellent performancein realistic multipath channels.

ML estimator and hybrid beamformer for multipath and interference mitigation in GNSS receivers

This paper addresses the estimation of the code-phase(pseudorange) and the carrier-phase of the direct signal received from a direct-sequence spread-spectrum satellite transmitter. Thesignal is received by an antenna array in a scenario with interferenceand multipath propagation. These two effects are generallythe limiting error sources in most high-precision positioning applications.A new estimator of the code- and carrier-phases is derivedby using a simplified signal model and the maximum likelihood(ML) principle. The simplified model consists essentially ofgathering all signals, except for the direct one, in a component withunknown spatial correlation. The estimator exploits the knowledgeof the direction-of-arrival of the direct signal and is much simplerthan other estimators derived under more detailed signal models.Moreover, we present an iterative algorithm, that is adequate for apractical implementation and explores an interesting link betweenthe ML estimator and a hybrid beamformer. The mean squarederror and bias of the new estimator are computed for a numberof scenarios and compared with those of other methods. The presentedestimator and the hybrid beamforming outperform the existingtechniques of comparable complexity and attains, in manysituations, the Cramér–Rao lower bound of the problem at hand.

Space-time front-ends for RAKE receivers in the FDD mode of UTRA

A unified and general vision of different space-time processors is presented. Many popular receivers can beaccomodated, like V-RAKE receivers, weighted V-RAKE, or spatial narrowband beamforming. By makingappropriate assumptions on the space/time characteristic of the interference it is possible to enhance theperformance of the receiver through spatial/temporal pre-processors. These receivers will be tested in the FDDmode of UTRA.

Hybrid turbo FEC/ARQ systems and distributed space-time coding for cooperative transmission

Cooperative transmission can be seen as a "virtual" MIMO system, where themultiple transmit antennas are in fact implemented distributed by the antennas both at the source and the relay terminal. Depending on the system design, diversity/multiplexing gainsare achievable. This design involves the definition of the type of retransmission (incrementalredundancy, repetition coding), the design of the distributed space-time codes, the errorcorrecting scheme, the operation of the relay (decode&forward or amplify&forward) and thenumber of antennas at each terminal. Proposed schemes are evaluated in different conditionsin combination with forward error correcting codes (FEC), both for linear and near-optimum(sphere decoder) receivers, for its possible implementation in downlink high speed packetservices of cellular networks. Results show the benefits of coded cooperation over directtransmission in terms of increased throughput. It is shown that multiplexing gains areobserved even if the mobile station features a single antenna, provided that cell wide reuse of the relay radio resource is possible.

The role of Receptor Activator of NFKB Ligand (RANKL) in mammary gland development

Abstract : The female reproductive hormones estrogen, progesterone and prolactin control postnatal breast development and are important to breast carcinogenesis. The mechanisms by which they elicit proliferation and morphogenesis remain poorly understood. Using the mouse as a model to study the molecular mechanisms through which hormones elicit morphogenetic changes in the mammary gland in vivo, we found the Receptor Activator of NFκB Ligand, a Tumor Necrosis Factor family member, to be strongly induced by progesterone. Recent publications suggested that hormone dependant RANKURANK signals are involved in the terminal differentiation of mammary gland alveolar buds into lobulo-alveolar structures competent for lactation. I show that in the absence of epithelial RANKL a distinct earlier stage of mammary gland development, side branch formation, is blocked. RANKL acts as a major mediator downstream of progesterone; it is required for progesterone-induced paracrine proliferation and completely rescues the mutant phenotype when ectopically expressed in progesterone receptor (PR) KO mammary epithelia. RANKL is not required for cell autonomous division of estrogen receptor alpha (ERa) /PR positive cells. Cyclin D1, previously implicated as a mediator of RANKL, is not affected by ablation of RANKL and is not required for RANKL-induced paracrine proliferation but for the cell autonomous proliferation. Gene expression arrays to find specific RANKL downstream targets have identified Id4, ElfS and one secreted metalloprotease (Adamtsl8) as potential candidates validated by Q-RT-PCR. Interestingly, Id4 and Adamtsl8 are expressed by the myoepithelial cells. Their expression additionally coincides with RANKL mRNA expression at mid pregnancy, possibly implying a functional contribution of both genes to RANKL mediated sidebranch formation. ElfS in contrast, is found to be strongly expressed by the end of pregnancy supporting recent findings of a prolactin mediated regulation. As for RANKL, this gene was in particular induced in luminal cells. Taken together, I report that progesterone is the major proliferative stimulus in the adult mammary gland eliciting proliferation of ERaJPR positive cells by a cell autonomous, cyclin D1-dependent and a paracrine RANKL-dependent mechanism. My work moreover suggests, that RANKL acts as a major orchestrator affecting different downstream mediators, through which progesterone exerts its effects concomitantly on different cellular compartments. Résumé : Les hormones sexuelles telles que l'oestrogène, la progestérone et la prolactine contrôlent le développement postnatal du sein et sont impliquées dans la cazcinogenèse. Les mécanismes par lesquels elles induisent la prolifération et la morphogénèse demeurent incompris. En utilisant la souris comme modèle, J'ai trouvé que le ligand activateur du récepteur de NFκB, une protéine de la famille du facteur de nécrose des tumeurs, peut être fortement induit par la progestérone. Les publications récentes ont suggéré que cette protéine est nécessaire à la fin de la grossesse, quand les cellules sécrétrices du lait apparaissent. Par des techniques de transplantation d'épithélium, je montre contrairement aux études précédentes, qu'en l'absence de RANKL dans l'épithélium une partie distincte du développement mammaire, la formation de branches latérales, est bloquée. La progestérone agit de manière pazacrine par l'intermédiaire de 12ANKL pour induire la prolifération tandis que la mort cellulaire n'est pas affectée. De plus, l'injection d'une protéine recombinante RANKL dans une souris mutante pour le récepteur à la progestérone induit la prolifération des cellules épithéliales en l'absence de grossesse ; la surexpression de RANKL dans ces mêmes mutants mène à une réversion complète du phénotype. Mes expériences démontrent que la progestérone induit deux types distincts de prolifération. Un premier type direct dans laquelle les cellules positives au récepteur à la progestérone prolifèrent. Cette division cellulaire est alors indépendante de RANKL mais dépendante de la cycline D1. Le second type de prolifération est induit par un mécanisme pazacrine et dépend de RANKL mais pas de la cycline D1. Ici, les cellules négatives au récepteur à la progestérone prolifèrent. Pour détecter des gènes cibles de la voie de signalisation du RANKL, un profil d'expression des gènes a été généré. Les facteurs de transcription Id4, EIf5 et une métalloprotéase sécrétée (Adamtsl8) ont été identifiés en tant que cibles potentielles. D'autres analyses de validation démontrent qu'Id4, Adamtsl8, RANKL mais pas E1f5 sont fortement exprimés au cours de la grossesse, coïncidant avec la formation de branchements latéraux induit par progestérone. EIf5 s'est avéré être exprimé vers la fin de la grossesse appuyant des résultats récents proposant une régulation par la prolactine. Le système canalaire mammaire se compose de couches cellulaires: une couche interne de cellules luminales et une externe de cellules myoépithéliale. Les expériences génétiques d'expression ont révélé que RANKL. et E1f5 sont exprimés dans la partie luminale tandis qu'Id4 et Adamtsl8 sont dans les cellules myoépithéliales. En conclusion, je prouve que la progestérone est le stimulus principal induisant la prolifération dans la glande mammaire d'adulte. Deux mécanismes de prolifération sont impliqués: l'un direct dépendant de la cycline Dl et l'autre paracrine dépendant de RANKI.. Mon travail suggère par ailleurs que RANKL agit en tant que médiateur important, par lequel la progestérone exerce ses effets sur différents compartiments cellulaires tels que la coordination de la prolifération des cellules épithéliales avec la réorganisation de la matrice extracellulaire et de la membrane basale exigées pour la morphogénèse du système canalaire latéral.