The EBI2 receptor is coexpressed with CCR5 in CD4+ T cells and boosts HIV-1 R5 replication.

OBJECTIVE: CCR5, a G protein-coupled receptor (GPCR), is used by most HIV strains as a coreceptor. In this study, we looked for other GPCRs able to modify HIV-1 infection. DESIGN: We analyzed the effects of one GPCR coexpressed with CCR5, EBI2, on HIV-1 replicative cycle. METHODS: We identified GPCRs expressed in primary CD4+CCR5+ T cells by multi-RT-qPCR. We studied GPCR dimerization by FRET technology. Cell lines expressing EBI2 were established by transduction with HIV vectors. HIV-1 entry was quantified with virions harboring ß-lactamase fused to the viral protein vpr, early and late HIV-1 transcriptions by qPCR, NFkB nuclear activation by immunofluorescence and transfection, and viral production by measuring p24 concentration in culture supernatant by ELISA. RESULTS: We showed that EBI2 is naturally expressed in primary CD4+CCR5+ T cells, and that CCR5 and EBI2 heterodimerize. We observed that this coexpression reduced viral entry by 50%. The amount of HIV reverse transcripts was similar in cells expressing or not EBI2. Finally, the presence of EBI2 induced the translocation of NFkB and activated HIV-1 genome expression. Globally, the result was a drastic HIV-1 R5, but not X4, overproduction in EBI2-transduced cells. CONCLUSIONS: EBI2 expression in CD4+CCR5+ cells boosts HIV-1 R5 productive infection. As the natural ligand for EBI2 is present in blood and lymphoid tissues, the constant EBI2 activation might increase HIV replication in CD4+ T cells. It might be of interest to test the effect of EBI2 antagonists on the residual viral production persisting in patients aviremic under treatment.

AutoRG: An automatized reach-to-grasp platform technology for assessing forelimb motor function, neural circuit activation, and cognition in rodents.

BACKGROUND: Rodent reach-to-grasp function assessment is a translationally powerful model for evaluating neurological function impairments and recovery responses. Existing assessment platforms are experimenter-dependent, costly, or low-throughput with limited output measures. Further, a direct histologic comparison of neural activation has never been conducted between any novel, automated platform and the well-established single pellet skilled reach task (SRT). NEW METHOD: To address these technological and knowledge gaps, we designed an open-source, low-cost Automatized Reach-to-Grasp (AutoRG) pull platform that reduces experimenter interventions and variability. We assessed reach-to-grasp function in rats across seven progressively difficult stages using AutoRG. We mapped AutoRG and SRT-activated motor circuitries in the rat brain using volumetric imaging of the immediate early gene-encoded Arc (activity-regulated cytoskeleton-associated) protein. RESULTS: Rats demonstrated robust forelimb reaching and pulling behavior after training in AutoRG. Reliable force versus time responses were recorded for individual reach events in real time, which were used to derive several secondary functional measures of performance. Moreover, we provide the first demonstration that for a training period of 30 min, AutoRG and SRT both engage similar neural responses in the caudal forelimb area (CFA), rostral forelimb area (RFA), and sensorimotor area (S1). CONCLUSION: AutoRG is the first low-cost, open-source pull system designed for the scale-up of volitional forelimb motor function testing and characterization of rodent reaching behavior. The similarities in neuronal activation patterns observed in the rat motor cortex after SRT and AutoRG assessments validate the AutoRG as a rigorously characterized, scalable alternative to the conventional SRT and expensive commercial systems.

The dark side of the black caiman: Shedding light on species dietary ecology and movement in Agami Pond, French Guiana.

The black caiman is one of the largest neotropical top predators, which means that it could play a structuring role within swamp ecosystems. However, because of the difficulties inherent to studying black caimans, data are sorely lacking on many aspects of their general biology, natural history, and ecology, especially in French Guiana. We conducted a detailed study of the Agami Pond black caiman population using a multidisciplinary approach. The aim was to better understand the species' dietary ecology and movements in the pond, and thus its functional role in pond system. We gathered natural history data, tracked caiman movements using satellite transmitters, and characterized feeding ecology via stable isotope analysis. Our study was carried out over three sampling periods and spanned both wet and dry seasons, which differ in their hydrological and ecological conditions. Our results show that black caiman abundance and age demographics differed between seasons in Agami Pond. In the dry season, Agami Pond is one of the only areas within the marsh to hold water. It thus contains large quantities of different fish species, which form the basis of the black caiman's diet. Caiman body size, a proxy for age class, was around 1.5 meters. During the wet season, which corresponds to the breeding period for migratory birds (e.g., Agami herons), adult black caimans are present in Agami Pond. Adults were most abundant in the inundated forest. There, most individuals measured up to 2 meters. They also exhibited a particular "predatory" behavior near bird nests, preying on fallen chicks and adults. Juveniles and subadults were present during both seasons in the pond's open waters. These behavioral observations were backed up by stable isotope analysis, which revealed ontogenetic variation in the caiman's isotopic values. This isotopic variation reflected variation in diet that likely reduced intraspecific competition between adults and young. The telemetry and microchip data show that different age classes had different movement patterns and that seasonal variation in the pond may influence caiman prey availability and reproductive behavior. The new information gathered should help predict this species' responses to potential ecosystem disturbance (e.g., water pollution, habitat destruction) and inform the development of an effective conservation plan that involves locals and wildlife officials.

Reversing HIV latency via sphingosine-1-phosphate receptor 1 signaling.

OBJECTIVE: In this study, we looked for a new family of latency reversing agents. DESIGN: We searched for G-protein-coupled receptors (GPCR) coexpressed with the C-C chemokine receptor type 5 (CCR5) in primary CD4 T cells that activate infected cells and boost HIV production. METHODS: GPCR coexpression was unveiled by reverse transcriptase-PCR. We used fluorescence resonance energy transfer to analyze the dimerization with CCR5 of the expressed GPCR. Viral entry was measured by flow cytometry, reverse transcription by quantitative PCR, nuclear factor-kappa B translocation by immunofluorescence, long terminal repeat activation using a gene reporter assay and viral production by p24 quantification. RESULTS: Gαi-coupled sphingosine-1-phophate receptor 1 (S1P1) is highly coexpressed with CCR5 on primary CD4 T cells and dimerizes with it. The presence of S1P1 had major effects neither on viral entry nor on reverse transcription. Yet, S1P1 signaling induced NFκB activation, boosting the expression of the HIV LTR. Consequently, in culture medium containing sphingosine-1-phophate, the presence of S1P1 enhanced the replication of a CCR5-, but also of a CXCR4-using HIV-1 strain. The S1P1 ligand FTY720, a drug used in multiple sclerosis treatment, inhibited HIV-1 productive infection of monocyte-derived dendritic cells and of severe combined immunodeficiency mice engrafted with human peripheral blood mononuclear cells. Conversely, S1P1 agonists were able to force latently infected peripheral blood mononuclear cells and lymph node cells to produce virions in vitro. CONCLUSION: Altogether these data indicate that the presence of S1P1 facilitates HIV-1 replicative cycle by boosting viral genome transcription, S1P1 antagonists have anti-HIV effects and S1P1 agonists are HIV latency reversing agents.

The two human CXCR4 isoforms display different HIV receptor activities: consequences for the emergence of X4 strains.

CXCR4 is a chemokine receptor that plays key roles with its specific ligand, CXCL12, in stem cell homing and immune trafficking. It is also used as a coreceptor by some HIV-1 strains (X4 strains), whereas other strains (R5 strains) use an alternative coreceptor, CCR5. X4 strains mainly emerge at late stages of the infection and are linked to disease progression. Two isoforms of this coreceptor have been described in humans: CXCR4-A and CXCR4-B, corresponding to an unspliced and a spliced mRNA, respectively. In this study, we show that CXCR4-B, but not CXCR4-A, mediates an efficient HIV-1 X4 entry and productive infection. Yet, the chemotactic activity of CXCL12 on both isoforms was similar. Furthermore, HIV-R5 infection favored CXCR4-B expression over that of CXCR4-A. In vitro infection with an R5 strain increased CXCR4-B/CXCR4-A mRNA ratio in PBMCs, and this ratio correlated with HIV RNA plasma level in R5-infected individuals. In addition, the presence of the CXCR4-B isoform favored R5 to X4 switch more efficiently than did CXCR4-A in vitro. Hence, the predominance of CXCR4-B over CXCR4-A expression in PBMCs was linked to the ability of circulating HIV-1 strains to use CXCR4, as determined by genotyping. These data suggest that R5 to X4 switch could be favored by R5 infection-induced overexpression of CXCR4-B. Finally, we achieved a specific small interfering RNA-mediated knockdown of CXCR4-B. This represents a proof of concept for a possible gene-therapeutic approach aimed at blocking the HIV coreceptor activity of CXCR4 without knocking down its chemotactic activity.

Effect of adjuvants on the humoral immune response to congopain in mice and cattle.

BACKGROUND: We investigated several adjuvants for their effects on the humoral immune response in both mice and cattle using the central domain of congopain (C2), the major cysteine protease of Trypanosoma congolense, as a model for developing a vaccine against animal trypanosomosis. The magnitude and sustainability of the immune response against C2 and the occurrence of a booster effect of infection, an indirect measure of the presence of memory cells, were determined by ELISA, while spectrofluorometry was used to determine and measure the presence of enzyme-inhibiting antibodies. RESULTS: Mice immunized with recombinant C2 in TiterMax™, Adjuphos™, purified saponin Quil A™ or Gerbu™ showed the best response according to the evaluation criteria and the latter three were chosen for the cattle vaccination study. The cattle were challenged with T. congolense four and a half months after the last booster. Cattle immunized with recombinant C2 in purified saponin Quil A™ showed the best antibody response according to the measured parameters. CONCLUSIONS: We identified purified saponin Quil A™ as a good adjuvant for immunizations with C2. The results from this study will be useful in future attempts to develop an effective anti-disease vaccine against African trypanosomosis.

Hemispheric asymmetry in non-linear interdependence of EEG in post-traumatic stress disorder.

AIM: While volumetric and metabolic imaging on post-traumatic stress disorder (PTSD) patients has been intensively performed, few studies using electroencephalograms (EEG) have been done as yet. The aim of the present study was to investigate abnormalities in functional connectivity of cortical networks in PTSD. METHODS: Non-linear interdependence (NI), a measure of bidirectional, non-linear information transmission between two time series, was used. Resting EEG were recorded for 18 PTSD patients and 18 sex-matched healthy subjects on 16 channels with their eyes closed. RESULTS: The NI patterns in PTSD patients were hemisphere asymmetric: an increase in NI in the fronto-parieto-temporal regions of the left hemisphere (F7, F3, T3, C3, T5 and P3) and a decrease in the fronto-parieto-occipital regions of the right hemisphere (F4, C4, P4 and O2). The non-linearity of NI in EEG, estimated from the surrogate data method, exhibited an increase in the PTSD patients as compared with that of healthy subjects, particularly in the left hemispheric cortex. CONCLUSION: Abnormal functional connectivity in PTSD can be assessed using NI, a measure of multi-channel EEG.

Quantification of brain macrostates using dynamical nonstationarity of physiological time series.

The brain shows complex, nonstationarity temporal dynamics, with abrupt micro- and macrostate transitions during its information processing. Detecting and characterizing these transitions in dynamical states of the brain is a critical issue in the field of neuroscience and psychiatry. In the current study, a novel method is proposed to quantify brain macrostates (e.g., sleep stages or cognitive states) from shifts of dynamical microstates or dynamical nonstationarity. A ``dynamical microstate'' is a temporal unit of the information processing in the brain with fixed dynamical parameters and specific spatial distribution. In this proposed approach, a phase-space-based dynamical dissimilarity map (DDM) is used to detect transitions between dynamically stationary microstates in the time series, and Tsallis time-dependent entropy is applied to quantify dynamical patterns of transitions in the DDM. We demonstrate that the DDM successfully detects transitions between microstates of different temporal dynamics in the simulated physiological time series against high levels of noise. Based on the assumption of nonlinear, deterministic brain dynamics, we also demonstrate that dynamical nonstationarity analysis is useful to quantify brain macrostates (sleep stages I, II, III, IV, and rapid eye movement (REM) sleep) from sleep EEGs with an overall accuracy of 77%. We suggest that dynamical nonstationarity is a useful tool to quantify macroscopic mental states (statistical integration) of the brain using dynamical transitions at the microscopic scale in physiological data.

Substrate-specific modulation of a multisubstrate proteinase. C-terminal processing of fibrillar procollagens is the only BMP-1-dependent activity to be enhanced by PCPE-1.

Members of the bone morphogenetic protein-1/tolloid (BMP-1/Tld) family of metalloproteinases, also known as procollagen C-proteinases (PCPs), control multiple biological events (including matrix assembly, cross-linking, cell adhesion/migration and pattern formation) through enzymatic processing of several extracellular substrates. PCP activities on fibrillar procollagens can be stimulated by another family of extracellular proteins, PCP enhancers (PCPE-1, PCPE-2), which lack intrinsic enzymatic activity. While PCPs have multiple substrates, the extent to which PCPEs is involved in the processing of proteins other than fibrillar procollagens is unknown. In the experiments reported here, PCPE-1 was found to have no effect on the in vitro BMP-1 processing of procollagen VII, the procollagen V N-propeptide, the laminin 5 gamma2 chain, osteoglycin, prolysyl oxidase, or chordin. In contrast, PCPE-1 enhanced C-terminal processing of human fibrillar procollagen III but only when this substrate was in its native, disulfide-bonded conformation. Surprisingly, processing of procollagen III continued to be enhanced when essentially all the triple-helical region was removed. These and previous results (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869; Bernocco, S., Steiglitz, B. M., Svergun, D. I., Petoukhov, M. V., Ruggiero, F., Ricard-Blum, S., Ebel, C., Geourjon, C., Deleage, G., Font, B., Eichenberger, D., Greenspan, D. S., and Hulmes, D. J. S. (2003) J. Biol. Chem. 278, 7199-7205) indicate that the mechanism of PCPE-1 action involves recognition sites in both the C-propeptide domain and in the C-telopeptide region of the procollagen molecule. PCPEs therefore define a new class of extracellular adaptor proteins that stimulate proteinase activity in a substrate-specific manner, thereby providing a new target for the selective regulation of PCP activity on fibrillar procollagen substrates.

Deletion of epidermal growth factor-like domains converts mammalian tolloid into a chordinase and effective procollagen C-proteinase.

Bone morphogenetic protein (BMP)-1 and mammalian tolloid (mTld) are Ca(2+)-dependent metalloproteinases that result from alternative splicing of the bmp1 gene. They have different proteinase activities, e.g. BMP-1 effectively cleaves procollagen (an extracellular matrix protein) and chordin (a BMP antagonist), whereas mTld is a poor procollagen proteinase and will not cleave chordin in the absence of twisted gastrulation. This is perplexing because mTld (being the longer variant) might be expected to cleave all substrates cleaved by BMP-1. Studies have shown that the minimal structure for procollagen proteinase activity is proteinase-CUB1-CUB2 (BMP-1DeltaEC3) and therefore lacking the epidermal growth factor (EGF)-like domain thought to account for the Ca(2+) dependence of BMP-1. In this study we generated three deletion mutants of mTld that lacked either one or both EGF-like domains (referred to as "mTld-DeltaEGF"). The mutated proteins were poorly but sufficiently secreted from 293-EBNA cells for in vitro assays of procollagen and chordin cleavage. Most surprisingly, the mTld-DeltaEGF mutants required Ca(2+) for proteolytic activity, thereby showing that the EGF-like domains do not account for the Ca(2+) dependence of BMP-1/mTld. Moreover, the mTld-DeltaEGFs are effective procollagen proteinases and cleave chordin. Furthermore, BMP-1DeltaEC3 cleaves chordin and requires Ca(2+) for activity. Studies using nondenaturing gels showed that mTld molecules lacking EGF-like domains have a loose conformation such that in the presence of Ca(2+) binding sites for chordin and procollagen on the "BMP-1-part" of the molecule are exposed. We propose that the EGF-like domains could hold CUB4/5 domains in locations that exclude substrates cleavable by BMP-1.