The cerebellum is organized into parasagittal zones that control sensory-motor behavior. Although the architecture of adult zones is well understood, very little is known about how zones emerge during development. Understanding the process of zone formation is an essential step toward unraveling how circuits are constructed to support specific behaviors. Therefore, we focused this study on postnatal development to determine the spatial and temporal changes that establish zonal patterns during circuit formation. We used a combination of wholemount and tissue section immunohistochemistry in mice to show that the cytoskeletal protein neurofilament heavy chain (NFH) is a robust marker for postnatal cerebellar zonal patterning. The patterned expression of NFH is initiated shortly after birth, and compared to the domains of several known zonal markers such as zebrin II, HSP25, neurogranin, and phospholipase Cβ4 (PLCβ4), NFH does not exhibit transient expression patterns that are typically remodeled between stages, and the adult zones do not emerge after a period of uniform expression in all lobules. Instead, we found that throughout postnatal development NFH gradually reveals distinct zones in each cerebellar lobule. The boundaries of individual NFH zones sharpen over time, as zones are refined during the second and third weeks after birth. Double labeling with neurogranin and PLCβ4 further revealed that although the postnatal expression of NFH is spatially and temporally unique, its pattern of zones respects a fundamental and well-known molecular topography in the cerebellum. The dynamics of NFH expression support the hypothesis that adult circuits are derived from an embryonic map that is refined into zones during the first 3-weeks of life. The adult cerebellum is comprised of relatively few cell types that are found, with only few exceptions, throughout all ten of its lobules Larsell, . Howeverengrailed1/2, L7/Pcp2, White and Sillitoe, Recent work suggests that adult zones are likely derived from a simpler pattern of embryonic “clusters”  mice were maintained in our colony and used for all experiments. Pups were collected at P0, P1, P2, P3, P5, P7, P10, P12, P15, P17, and P20. For these studies, mice were considered adult after P28 because by this age cerebellar patterns are mature. Noon on the day a vaginal plug was detected was considered embryonic day (E) 0.5. At least three mice were used for each age.Mice were anesthetized with avertin and perfused with 4% paraformaldehyde (PFA) diluted in 0.1 M phosphate-buffered saline . The tissue was then post-fixed for 24–48 h in 4% PFA and then cryoprotected in a series of sucrose solutions . Serial 40 μm thick coronal and sagittal sections Figure  were cutWholemount immunohistochemistry for NFH was carried out as previously described  was purchased from Covance . Anti-SMI-32 recognizes the non-phosphorylated form of NFH (see manufacturer product datasheet for details), which on tissue sections labels the soma, dendrites, and axons of adult Purkinje cells  as a chromogen. These experiments were achieved using horseradish peroxidase (HRP) conjugated goat anti-rabbit or HRP-conjugated goat anti-mouse  secondary antibodies. Staining for fluorescence immunohistochemistry was carried out using Alexa 488- and 555-conjugated immunoglobulins , both diluted 1:1,500.Photomicrographs of tissue sections were captured using a Leica DFC360 FX (fluorescence) and DFC 490 (DAB reacted tissue sections) camera mounted on a Leica DM6000 microscope. Images of tissue sections were acquired and analyzed using Leica Application Suite and Leica Application Suite FX software. Photomicrographs of wholemount stained cerebella were captured with a Leica MZ16 FA stereomicroscope mounted with a Leica DFC3000 FX camera running Leica LAS software supplemented with the Leica Montage module. All raw data was imported into Adobe Photoshop CS4 and corrected for brightness and contrast levels only. Schematics were drawn in Adobe Illustrator CS4.In this study, we performed wholemount and tissue section immunohistochemistry to determine the spatial and temporal expression of NFH patterning during postnatal development of the mouse cerebellum. Two forms of NFH are expressed in the cerebellum: phosphorylated and non-phosphorylated  to gain a better understanding of how zones resolve during circuit formation. Notably, we discuss in detail our findings from P0 to P2 mice because several zonal markers including zebrin II, HSP25, and PLCβ4, initiate expression around birth. We examine P7 in detail not only because it marks the end of the first week, but also because it correlates with when the major mossy fiber tracts are resolving into zones  the intensity of NFH expression within zones increases and (2) zonal boundaries sharpen dramatically in both the vermis and the hemispheres. For example, at P20 the vermis of lobule IX is organized into clear zones, and the hemisphere zones extend over several lobules curving with the prominent contours of Crus II and the PML Figures  and 5. POur finding that NFH is expressed in specific zones from ~P2 onwards suggests that its pattern might respect the fundamental molecular topography that was previously observed during early postnatal development (Larouche et al., The postnatal morphogenesis of cerebellar zones is poorly understood. In this study, we found that NFH, a member of the neurofilament cytoskeletal protein family, is expressed within Purkinje cell zones during early postnatal development. We revealed that during the first three weeks of life the maturation of NFH expression into adult zones mainly involves boundary refinement rather than gross reorganization of the overall pattern. Moreover, we show that during development, NFH zones respect the same medial-lateral boundaries as two known markers of postnatal zones, neurogranin and PLCβ4. Despite the protracted period of boundary refinement, the pattern of NFH expression falls into a well-understood framework of cerebellar sagittal zones.We previously showed that, in the adult, NFH is expressed in a unique pattern of Purkinje cell zones (Demilly et al., L7/Pcp2-CreER allele, and lineage tracing using a viral strategy that labels Purkinje cells on the day they are born all suggest that positional information contained within Purkinje cell clusters is transferred to the pattern of adult Purkinje cell zones (Larouche et al., Ip3r1 reporter transgene allowed embryonic clusters to be followed into perinatal parasagittal zones (Fujita et al., Neurogranin and PLCβ4 expression, fate mapping using an The zonal organization of Purkinje cells is thought to guide the formation of patterned topographic circuits (Apps and Hawkes, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While the survivors are currently predicted to live past 60 years, they suffer higher incidence of age-related conditions including Alzheimer's disease (AD). This paper is centered on the mechanisms by which mitochondrial factors and oxidative stress may orchestrate an adaptive response directed to maintain basic cellular functions and survival in DS. In this context, the timing of therapeutic interventions should be carefully considered for the successful treatment of chronic disorders in the DS population.Genome-wide gene deregulation and oxidative stress appear to be critical factors determining the high variability of phenotypes in Down's syndrome (DS). Even though individuals with trisomy 21 exhibit a higher survival rate compared to other aneuploidies, most of them die  Down's syndrome (DS) or trisomy 21 is a prevalent genetic cause of intellectual disability due to full or partial triplication of chromosome 21 (HSA21). The presentation varies greatly between individuals. The molecular bases of this variation is “the gene dosage effect” caused by the extra chromosome 21, which leads to a global imbalance on gene expression , in, in29], ogenesis  [29].β subunit and complex III nuclear encoded core protein I [Increased oxidative stress in DS and AD correlates with a decrease in several mitochondrial components including complex IV nuclear encoded subunit IV, mtDNA encoded subunit I , complexrotein I , and mitrotein I . A recenrotein I . The genrotein I , 37, whirotein I , and therotein I , all of Based on the considerations above, it is conceivable that oxidative stress and redox changes play a dual role in DS. At low levels, they promote cellular proliferation, while at higher levels, they produce oxidative damage and initiate apoptosis . AdaptivSince mitochondria are the main source of ROS production, their role is essential in age-related oxidative damage. While abundant research supports the idea that reduced oxidative stress is associated with increased life span –46 severGPx and catalase are also Nrf2 target genes carrying ARE sequences in their promoters [A prominent sensor related to hormesis is the Keap1-Nrf2-ARE signaling complex. Under normal redox conditions, the transcription factor NFE2-related factor 2 (Nrf2) binds to Kelch-like ECH-associated protein 1 (Keap1) in the cytosol leading to its proteasomal degradation . Keap1 iromoters . Interesromoters .γ, PGC1α, and PI3K/Akt, all of which participate in mitochondrial biogenesis [β cells, endothelial cells, and fibroblasts, which is consistent with a cellular adaptation to reduce ROS production and prevent cellular injury [Nrf2 also interacts with PPARogenesis . Thus, togenesis  and mitoogenesis  in DS cer injury . HoweverOne consequence of the considerations above is that not only the compounds but also the timing of treatment options should be carefully considered in DS patients. For example, long-term treatments designed to reduce oxidative stress may not add any incremental benefit on top of the changes driven by hormesis. Interventions would be more effective if introduced at the very onset of stress or disease. In fact, recent findings indicate that exercise-induced oxidative stress ameliorates insulin resistance and generates an adaptive response enhancing the endogenous antioxidant defense capacity . HoweverIn conclusion, DS is the result of a whole genome imbalance caused by triplication of HSA21 genes. The severity and spectrum of the syndrome vary greatly. Besides oxidative damage, mtDNA mutations and mitochondrial dysfunction emerge as important modulators of DS phenotypes. This variability is further influenced by an adaptive cellular response to stress. A comprehensive and detailed analysis of signature pathways unique to hormesis will be required to fully assess the role of the adaptive response in DS . Key ele


Murabutide (MB) is a synthetic immunomodulator recognized by the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptor on mammalian cells. MB has previously been approved for testing in multiple human clinical trials to determine its value as an antiviral therapeutic, and as an adjuvant for injected vaccines. We have found a new use for this immunomodulator; it functions as a mucosal adjuvant that enhances immunogenicity of virus-like particles (VLP) administered intranasally. MB enhanced Norwalk virus (NV) VLP-specific IgG systemically and IgA production at distal mucosal sites following intranasal (IN) vaccination. A dose escalation study identified 100 µg as the optimal MB dosage in mice, based on the magnitude of VLP-specific IgG, IgG1, IgG2a and IgA production in serum and VLP-specific IgA production at distal mucosal sites. IN vaccination using VLP with MB was compared to IN delivery VLP with cholera toxin (CT) or gardiquimod (GARD) and to parenteral VLP delivery with alum; the MB groups were equivalent to CT and GARD and superior to alum in inducing mucosal immune responses and stimulated equivalent systemic VLP-specific antibodies. These data support the further testing of MB as a potent mucosal adjuvant for inducing robust and durable antibody responses to non-replicating subunit vaccines. The majority of US FDA approved vaccines are administered parenterally (subcutaneous or intramuscular routes) and induce systemic immune responses (measured by serum antibody production). This systemic IgG may participate in local immune responses at distal mucosal sites, however with reduced efficacy relative to secretory IgA (sIgA). Since many pathogens gain entry through mucosal sites, efforts have been made to induce robust sIgA throughout the common mucosal immune system (CMIS) by mucosal vaccination. The development of these mucosal vaccines has been limited by the lack of mucosal adjuvants that are both safe and potent inducers of mucosal and systemic immune responses.Most vaccines in use today are formulated with aluminum salts to enhance immunogenicity. Despite the long history of utilizing these aluminum salt formulations as adjuvants, the mechanism of action was not elucidated until several recent studies have implicated sensing by the nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) to mediate systemic immune responses characterized by the production of IgG1 and IgE The use of cholera toxin (CT) as a mucosal adjuvant is known to induce potent systemic and mucosal antigen-specific immune responses. However, its use in human clinical trials has resulted in high toxicity and is therefore unsuitable for use in humans In 1974, MDP had been identified as an immunostimulant that induced non-specific immune responses to antigens MB, like MDP, contains the minimal necessary conserved structural motif of peptidoglycan to be recognized by the NOD2 receptor on host cells Norwalk virus virus-like particles (NV VLP) can be used as a model mucosal subunit vaccine to test the adjuvanticity of novel immunopotentiators, formulations, or routes of vaccination Nicotinia benthamiana by Kentucky Bioprocessing  and clarified leaf extracts were filtered and fractionated as previously described Recombinant NV VLP were expressed in MB stock solution of 10 mg/ml was prepared by resuspending 5 mg lyophilized murabutide powder  in 500 µl sterile endotoxin free water and vortexed until fully resuspended. Stocks were stored long-term at −80°C and short-term at −20°C until use. Cholera toxin  and gardiquimod (InvivoGen) were prepared as previously described n = 7 per group) for study 1 and 3 groups for study 2 and allowed to acclimate for 1 week. In the first study, mice were vaccinated intranasally on day 0 and 21, as previously described All animals were housed in accordance with American Association for Laboratory Animal Care (AALAC) standards, provided unlimited access to food and water, and all procedures and handling for this study were approved by the Animal Welfare Act and Arizona State University (ASU) Institutional Animal Care and Use committee (IACUC) (Assurance # A3217-01). Prior to vaccination, 5-week-old inbred female BALB/c mice  were randomly distributed into 8 groups , and on days 12, 21, 42, and 56 for study 1 and on day 0 prior to vaccination (preimmune), and on days 12, 21, 42, 56, 84, and 112, as previously described. All mice were humanely euthanized on day 56 (study 1) and day 112 (study 2) in accordance with the Animal Welfare Act and ASU IACUC. Uterine lavages were collected post-mortem by opening the abdominal cavity, fully excising each uterine horn, and flushing each horn with 200 µl PBS, as previously described Insect cell-derived NV VLP were manufactured and purified as previously described 7 IgG secreting splenocytes.Spleens were harvested following euthanization on day 112 as previously described P value of <0.05.Prism software  was used to graph and evaluate statistical comparisons of all data. NV VLP-specific IgA and IgG antibody titers are expressed as geometric mean titers (GMT) for each vaccination group at each time point. Samples from the PBS groups of mock-vaccinated mice were negative by ELISA (not shown). All responders and nonresponders were included in the computation of the GMT and negative samples were assigned the value of 1.0 for calculation purposes. The Kruskal-Wallis one-way analysis of variance (ANOVA) followed by a Dunn’s post-test was used to compare ELISA values at each time point between individual treatment groups versus PBS alone. One-way analysis of variance (ANOVA) followed by Bonferroni’s post-test was used to compare ELISPOT values between individual treatment groups versus PBS alone. Statistical significance was considered to be a To evaluate the immunogenicity of MB as a mucosal adjuvant in a dose-escalation study (study 1), liquid formulations of VLP (25 µg) were administered intranasally on study day 0 and day 21 and compared to three doses of VLP and MB , VLP and CT, or delivered parenterally with alum . All vacThe antigen to adjuvant dose-response was evaluated with intranasal delivery of VLP (25 µg) codelivered with 25 µg, 100 µg, or 250 µg of MB. Following intranasal vaccination with VLP and MB (100 µg or 250 µg), mice (n = 7 each group) produced VLP-specific serum IgG and IgG1 by day 12 that were significantly higher than mock-vaccinated mice, and remained elevated for the duration of the study . The lowWe evaluated the ability of VLP codelivered with MB (25–250 µg), VLP with alum, VLP and CT, and VLP alone to induce VLP-specific IgA antibodies in the saliva and fecal extracts. Intranasal vaccination with VLP codelivered with MB at all dosages (25–250 µg) produced equivalent levels of VLP-specific salivary IgA relative to VLP and CT, and higher and more sustainable levels of VLP-specific salivary IgA compared to levels produced by VLP alone and by antigen delivered with alum . VLP delMice vaccinated with VLP codelivered with MB at all dosages tested and VLP and CT produced more sustainable levels of VLP-specific fecal IgA antibodies compared to mice vaccinated with either antigen alone or antigen and alum . Mice vaAs the respiratory tract is the primary site of vaccination, we evaluated the ability of VLP codelivered with MB  compared to VLP and CT, VLP alone, and VLP delivered parenterally with alum to induce production of VLP-specific IgA antibodies in the nasal cavity . VLP-speAs an additional measurement of mucosal immunity in the CMIS, we evaluated antibody production in the reproductive tract. Mice vaccinated with NV VLP codelivered with MB  and VLP and CT produced significant levels of NV VLP-specific vaginal IgA titers by day 12 and 21 and remained elevated throughout the study (P<0.05–0.01) . The aluVaccination with VLP and MB  and VLP and CT resulted in high levels of uterine IgA compared to PBS control- vaccinated groups and were 3- and 4- fold higher than levels produced by the alum-containing group or VLP alone, respectively . UterineTo determine the efficacy of MB as an adjuvant in eliciting an extended systemic and mucosal immune response, a parallel treatment study was conducted for 16 weeks (twice the length of the first study) and a time point used to evaluate long-term antibody responses Production of VLP-specific vaginal IgA in the VLP with MB (200 µg) group was significantly elevated throughout the duration of the study (day 112) relative to the PBS control-vaccinated group (P<0.01) . VLP witSubunit vaccines almost always require adjuvants to achieve robust and sustainable immune responses. However, many potent mucosal adjuvants, such as CT, may induce severe and toxic side effects, rendering them unsuitable for use in humans, while other adjuvants elicit only systemic immune responses Alum is one of the few adjuvants approved by the FDA for clinical use and is a potent adjuvant for induction of systemic IgG responses in a variety of vaccines Our data provide evidence that murabutide is a mucosal adjuvant since it significantly enhances the induction and sustained levels of serum IgA production in mice following vaccination with NV VLPs  and 5. FOur first study tested the ability of MB, delivered with NV VLP by IN vaccination, to elicit a more robust and durable systemic and mucosal immune response than that produced by vaccination with VLP alone, by parenteral vaccination with VLP plus alum or by codelivery with the mucosal adjuvant CT –4 This dIn our second study, we demonstrate that IN vaccination of VLP codelivered with MB not only elicits significant VLP-specific systemic and mucosal responses compared to VLP alone or PBS control-immunized mice, but the responses were sustained throughout this longer-term study (to day 112) –6. FurthIN vaccination with MB induces robust VLP-specific systemic and mucosal responses. The enhanced systemic and mucosal immune responses elicited by the co-delivery of MB with VLP were sustained for the duration of both studies and no adverse effects were observed in mice following intranasal MB administration. We have demonstrated superior induction of mucosal antibodies (IgA) after IN vaccination with NV VLP with MB in comparison to parenteral delivery of the VLP with alum or IN delivery of VLP alone and equivalent mucosal IgA induction to IN codelivery of VLP with CT or GARD. Furthermore, sustainability of the immune response generated by vaccination using MB as a mucosal adjuvant, and the prior history of using MB in human clinical trials, make this immunomodulator an attractive candidate adjuvant for other subunit antigens and/or vaccine formulations for mucosal immunization.


A proper balance between synapse assembly and disassembly is crucial for the formation of functional neuronal circuits and synaptic plasticity in the adult brain. During development, synaptogenesis generates a vast excess of synapses, which are subsequently eliminated. Importantly, aberrant synaptic disassembly during development underpins many neurological disorders. Wnt secreted proteins are robust synaptogenic factors that regulate synapse assembly and function in the developing and mature brain. Recent studies show that Wnt blockade with the antagonist Dickkopf-1 (Dkk1) induces the rapid disassembly of synapses in mature neurons. Importantly, Dkk1 mediates synaptic loss induced by Amyloid-ß, a key pathogenic molecule in Alzheimer’s disease (AD). These findings provide new insights into the potential contribution of dysfunctional Wnt signaling to synaptic loss observed in neurodegenerative diseases. In this review, we discuss the role of Wnt signaling in vertebrate synaptic assembly, function and maintenance, and consider how dysfunction of Wnt signaling could contribute to synaptic disassembly in neurodegenerative diseases such as AD. During early development, Wnts regulate critical cellular processes such as cell proliferation and cell fate, neuronal polarity and migration. In addition, Wnts modulate dendritogenesis, axon guidance and synaptogenesis  reveal that Wnt signaling activates a divergent-canonical pathway through Disheveled (Dvl) resulting in the inhibition of Glycogen synthase kinase-3 ß (Gsk3ß) to regulate axonal remodeling   receptors promotes presynaptic organization. Wnt3a and Wnt7a bind to the Fz1 and Fz5 receptors, respectively, to stimulate presynaptic assembly  CA1 and CA3 regions of the hippocampus  receptor clustering at inhibitory synapses through CaMKII signaling , Wnt release from motoneurons regulates postsynaptic differentiation on muscle cells  signals to both sides of the synapse  stimulates Wnt7a/b expression in the adult mouse hippocampus during the remodeling of mossy fiber terminals that contact CA3 dendrites. Importantly, blockade of endogenous Wnts by local microinjection of the Wnt antagonist Sfrp1 suppresses activity-dependent presynaptic remodeling and synaptogenesis in the CA3 region  and Parkinson’s disease (De Ferrari and Moon, in vitro (Shruster et al., A potential role for Wnt signaling in the pathogenesis of AD has been suggested by a number of recent studies (De Ferrari and Moon, Further links between dysfunction of Wnt signaling and AD comes from studies on the Wnt antagonist Dkk1. Dkk1 expression is elevated in human postmortem AD brains and in mouse models of AD (Caricasole et al., in vivo.How does Aβ induce Dkk1 expression? A recent report suggests that increased soluble Aβ induces the intracellular accumulation of the cell survival factor clusterin, possibly by preventing its neural secretion (Killick et al., Recent studies have shown that endogenous Dkk1 expression becomes progressively elevated in the aged brain and may be associated with general cognitive decline as well as susceptibility to age related pathological neurodegenerative disorder (Scott et al., The delicate balance between synapse assembly, disassembly and maintenance are highly regulated processes controlled by a complex molecular dialogue between pre- and postsynaptic neurons. Importantly, neuronal activity, which has profound effects on these processes, contributes to the expression and release of Wnt factors, which in turn modulate this trans-synaptic dialogue. Indeed, several studies have demonstrated that Wnts contribute to activity-mediated synapse formation during early neuronal circuit development and during experience-dependent plasticity.in vivo studies will provide key evidence for the contribution of dysfunction in Wnt signaling to synaptic degeneration in AD.Wnt-mediated synaptic maintenance is now emerging in relation to neurodegeneration. Indeed, the Wnt antagonist Dkk1, which has strong synaptic disassembly activity, is now linked to AD. Importantly, Dkk1 mediates the effect of Aβ on synaptic disassembly. Future It is an exciting time to study Wnt signaling in the nervous system as we begin to unravel the mechanisms by which Wnts modulate experience-mediated plasticity. Moreover, the potential role of Wnts in synaptic maintenance opens new avenues to develop therapeutic strategies for the treatment of neurodegenerative diseases at early stages. Finally, the finding that Dkk1 is elevated in the AD brain has sparked enthusiasm that this molecule could be used as a biomarker at early stages of AD or other neurodegenerative diseases.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently, aberrant methylation of CpG islands of cancer-related genes has emerged as an important epigenetic mechanism of their transcriptional dysregulation during tumour development . Therefo16INK4A is an important G1/S cell cycle regulator gene [The ptor gene . RASSF 1tor gene . The MGMTo date, little is known about the exact role of hypermethylation of these genes in pancreatic adenocarcinoma, as the molecular mechanisms underlying these neoplasms remain poorly understood. MSP has been designed to distinguish methylated from unmethylated DNA. ‘Hot start’ polymerase chain reaction (PCR) permits the Taq polymerase to begin the reaction only after the template and primers are in single-stranded form.2O. Ten microlitres of each second MSP reaction were directly loaded onto 2% agarose, stained with ethidium bromide and visualized under UV illumination. The presence of a visible MSP product in those lanes marked M indicated the presence of methylated alleles. The results on promoter methylation of all genes were correlated with known clinicopathological parameters.All double MSPs were performed with controls for unmethylated and methylated alleles and a control with H16INK4A gene was methylated in all 13 patients , quick and cost-effective test to analyse methylation status of CpG dinucleotides in a CpG island, making the technique suitable for high-throughput analysis of clinical samples. Our findings suggest that p


XMRV (xenotropic murine leukemia virus-related virus) was initially discovered in association with prostate cancer and later with chronic fatigue syndrome (CFS). Its association with CFS is now largely discredited, and current results support a laboratory origin for XMRV with no reproducible evidence for infection of humans. However, some results indicating the presence of XMRV in prostate cancer are difficult to attribute to sample contamination. Here we have sought biological evidence that might confirm the presence of XMRV in prostate cancer samples previously having tested positive.We have tested for infectious XMRV and neutralizing antibodies against XMRV in blood plasma from 29 subjects with prostate cancer, and for infectious XMRV in prostate secretions from another five prostate cancer subjects. Nine of these subjects had previously tested positive for XMRV by PCR or by virus assay. We did not detect XMRV or related retroviruses in any sample, and the neutralizing activities of the plasma samples were all very low, a result inconsistent with XMRV infection of the plasma donors.We find no evidence for XMRV infection of any human subject tested, either by assay for infectious virus or for neutralizing antibodies. Our results are consistent with the majority of published studies on XMRV, which find that XMRV is not present in humans. The observed low to undetectable XMRV neutralization by human plasma indicates a lack of innate restriction of XMRV replication by soluble factors in human blood. The retrovirus XMRV (xenotropic murine leukemia virus-related virus) was initially discovered in human prostate cancer samples Currently, a role for XMRV in CFS is largely disproven, and the original paper that found this association has been retracted Here we have analyzed blood plasma and expressed prostatic secretions (EPS) from prostate cancer patients, some of whom previously tested positive for XMRV +L− and marker rescue assays that have been shown to effectively detect XMRV +L− assay we used measures the ability of a retrovirus to infect and cause spread of the Moloney murine sarcoma virus present in PG-4 cat cells Mus dunni tail fibroblasts (dunni cells) transduced with a retroviral vector (LAPSN) that produces human placental alkaline phosphatase (AP). The dunni/LAPSN cells were exposed to test samples, were passaged for a month to allow virus spread, and were assayed for production of the LAPSN vector on naive dunni cells. Dunni cells were chosen for this assay because of their susceptibility to a wide range of murine leukemia viruses +L− assay to quantitate replication-competent XMRV after incubation with the samples, in comparison to XMRV incubated with culture medium as a control. In some experiments, we measured the ability of plasma to neutralize the LAPSN vector packaged in XMRV virions (XMRV-pseudotype LAPSN vector) as a surrogate for direct measurement of XMRV neutralization.To detect infectious XMRV and related retroviruses in patient plasma and EPS samples, we used S+L− assay. The cells were then assayed weekly for LAPSN production during passage of the cells for a month. LAPSN production was detected at one week following infection with 50 FFU of XMRV, at 2 weeks following infection with 10 and 5 FFU, and at 4 weeks for 1 of 2 plates infected with 1 FFU of XMRV. These results show that the marker rescue assay is approximately as sensitive as the S+L− assay for detection of XMRV. However, this marker rescue assay may be more sensitive than the S+L− assay for some retroviruses because of the known sensitivity of dunni cells to a wide range of murine retroviruses, while fewer types of murine retroviruses can infect the cat cells used in the S+L− assay.To determine the kinetics of virus spread and the sensitivity of the marker rescue assay, the assay was conducted by exposing dunni/LAPSN cells to 50, 25, 10, 5, 1, or 0 focus-forming units (FFU) of XMRV, as determined by S+L− assay. To detect neutralizing antibodies, plasma samples were incubated at 1∶10 and 1∶100 dilutions with XMRV-pseudotype LAPSN vector for 30 min at room temperature, and the remaining LAPSN virus was measured by AP+ focus assay. Only one of the ten plasma samples showed weak neutralizing activity . This neutralizing activity was eliminated by heat inactivation of the plasma, which inactivates complement, showing that no antibodies were present that could directly block virus infection.We first tested whether blood plasma from a set of ten prostate cancer patients, three of whom previously tested positive for XMRV by RT-PCR, contained replication-competent XMRV and/or neutralizing antibodies against XMRV . We did +L− or marker rescue assays, in plasma from any of the 21 patients tested, including four who previously tested positive for XMRV by PCR , showing that XMRV can survive and be detected in EPS fluid. However, no replication-competent virus was detected in any of 5 EPS samples from prostate cancer patients by S+L− or marker rescue assays (see We next tested EPS fluids obtained from excised prostate glands says see  [15].M. dunni endogenous retrovirus (MDEV) M. dunni cells was observed following treatment of the cells with 5-iodo-2′-deoxyuridine or hydrocortisone We did experience some false positive results with the marker rescue assay. In a few cases we detected LAPSN production following exposure of dunni/LAPSN cells to plasma, but viral interference analysis showed that LAPSN transduction was completely blocked in dunni cells expressing the +L− and dunni cell-based marker rescue assays that we used to detect virus are both capable of detecting xenotropic and polytropic retroviruses +L− assay can detect RD114 feline retrovirus, feline leukemia virus types A, B and C, gibbon ape leukemia virus, and Mus caroli endogenous retrovirus (McERV) Of the 29 plasma and 5 EPS samples from prostate cancer patients that we tested, none had a detectable level of replication-competent XMRV or related retroviruses. Included were two plasma samples (VP29 and VP35) from patients who tested XMRV positive by viral detection DNA microarray (Virochip) analysis in the original study To determine whether XMRV could persist in blood and to detect possible immune responses against XMRV, we assayed the ability of blood plasma to neutralize XMRV infectivity. We detected only minimal neutralization even under the most stringent condition of incubating a small amount of XMRV with undiluted, non-heat-inactivated plasma. At most, 75% of the XMRV was neutralized after incubation with plasma, suggesting that XMRV shed into the blood would have a long enough half-life to allow detection. Indeed, 7 of 21 plasma samples assayed in this way showed ≤10% neutralization , indicatSeveral factors may explain the differences in neutralizing antibody activities: i) heparin present in serum or plasma made from blood collected in heparinized tubes can nonspecifically inhibit virus infectivity, ii) repeated freezing and thawing of samples can inactivate complement resulting in reduced neutralization, iii) specific viral components of the virus used for neutralization studies can affect the results  samples used in the current study were obtained at the Cleveland Clinic following approval by the Cleveland Clinic Foundation Institutional Review Board. All samples were obtained from subjects with prostate cancer after written informed consent was obtained. Plasma samples were prepared from blood collected in standard EDTA tubes, EPS fluid was obtained by massage of excised prostate glands after prostatectomy, and all samples were stored at −70°C. Many of the plasma samples were frozen and thawed once for analysis, while others were frozen and thawed a limited number of times .M. dunni tail fibroblasts (dunni cells) 5 per 6-cm dish on day 1. On day 2, plasma and EPS samples were thawed and portions of each sample (or culture medium as a no plasma control) were incubated with a small volume of infectious XMRV (harvested from 22Rv1 cells) for 15 to 30 minutes at room temperature, while the other portions of the plasma and EPS samples were kept on ice. Virus-spiked and untreated samples were added to the PG-4 cells in the presence of 4 µg/ml Polybrene. Cells were fed on day 3, and foci were counted on day 4 or 5. In some experiments the plasma was heat-inactivated at 56°C for 30 min before assay for virus neutralization.PG-4 cells were seeded at 2.5×105 per 6-cm dish on day 1 and were exposed to test samples (blood plasma or EPS) in the presence of 4 µg/ml Polybrene on day 2. The cells were then passaged for a month at high density (to facilitate virus spread) by trypsinizing and reseeding the cells at a 1∶10 dilution every time the cells became confluent. LAPSN production was then measured by feeding confluent cells, harvesting the medium the next day, removing cells by filtration (0.2 µm-pore-size surfactant-free cellulose acetate filters) or by centrifugation , by adding medium samples with 4 µg/ml Polybrene to dunni or 293 cells seeded the day before at 5×104 per well of 12-well plates, and by staining the cells for AP two days later.Dunni and DU145 cells containing the LAPSN retroviral vector (dunni/LAPSN and DU145/LAPSN cells) were generated by exposing cells to helper-free LAPSN vector generated from PA317 retrovirus packaging cells M. dunni endogenous retrovirus (MDEV) from dunni cells. On day 1 the infected and uninfected dunni cells were seeded in 12-well dishes at 5×104 per well. On day 2, the medium was replaced with 1 ml of medium containing 4 µg of Polybrene and 0.1 ml of medium harvested from the marker rescue assay cells. On day 4, the cells were stained for AP. The MDEV-infected dunni cells are resistant to MDEV but permissive to XMRV while the XMRV-infected dunni cells are resistant to XMRV but permissive to MDEV.Replicating virus detected in the marker rescue assay was subjected to interference analysis using dunni cells chronically infected with either XMRV from 22Rv1 cells or with the


Our results are consistent with an ecosystem-wide pattern in communities' structure, with relatively high species turnover but functionally analogous leaf-litter ant communities' organization. Our results suggest directional shifts in the morphological space along the environmental gradient from overdispersed to aggregated (from North to South), suggesting that primary productivity and environmental heterogeneity  determine the distribution of traits and regulate the assembly rules, shaping local leaf-litter ant communities. Contrary to the expected and most common pattern along latitudinal gradients, the Atlantic Forest leaf litter ant communities show an inverse pattern in richness, that is, richer communities in higher than in lower latitudes. The morphological specialization of communities showed more morphologically distinct communities at low latitudes and species redundancy at high latitudes. We claim that an inverse latitudinal gradient in primary productivity and environmental heterogeneity across the Atlantic forest may affect morphological diversity and species richness, enhancing species coexistence mechanisms, and producing thus the observed patterns. We suggest that a functional framework based on flexible enough traits should be pursued to allow comparisons at local, regional and global levels.General principles that shape community structure can be described based on a functional trait approach grounded on predictive models; increased attention has been paid to factors accounting for the functional diversity of species assemblages and its association with species richness along environmental gradients. We analyze here the interaction between leaf-litter ant species richness, the local communities' morphological structure and fundamental niche within the context of a northeast-southeast latitudinal gradient in one of the world's most species-rich ecosystems, the Atlantic Forest, representing 2,700 km of tropical rainforest along almost 20 Functional diversity is an important concept in community ecology because it encompasses information on functional traits, absent in measures of species diversity Invertebrate traits have been rarely used to study community response to habitat gradients All necessary permits required for the described study were obtained, complying with all relevant regulations. All research was conducted with the approval by the Brazilian Institute of Environment and Renewable Natural Resources .We surveyed 26 Atlantic rain forest localities  as regul2 leaf litter samples were collected and mini-Winkler apparatuses were used to extract the ant fauna. At each site, a transect of 1,200 m was selected for the ant survey, starting at least 500 m from the forest edge. At each transect we marked 25 points set at 50 m intervals; at each point, two 1-m2 leaf litter samples were collected, one 25 m to the left and one 25 m to the right of the point, covering a projected sampled area of 12.5 hectares. In total, fifty leaf litter samples were collected at each site, vigorously sifted through a sieve of 1 cm grid size and transferred to mini-Winkler extractors, where the sifted leaf litter was held in a mesh sack and suspended in a larger cotton enclosure for 48 hours. The ants dropped out of the mesh sack to be collected live in plastic cups containing a humid sponge. The ants were then transferred to white trays and then to vials with 70% ethanol. Sampling was carried out between 9:00 a.m. and 4:00 p.m. The ants of all samples were sorted out and up to three workers of each morphospecies in each sample were mounted. Whenever possible, specimens were identified to species by comparing them to taxonomic descriptions and material deposited in the Museum of Zoology of the São Paulo University Formicidae collection, where voucher species were deposited. Specimens not identified to species were given a code for each distinct morphospecies.We carried out quantitative surveys in the years 2000 and 2002 summers (December-March), when the ant activity is believed to be relatively high. Along each site 50 1-mWe measured morphological variables of 1,944 individuals from 530 species, extracted from leaf litter samples collected in the Atlantic Forest localities. Sample sizes for morphological studies ranged from one to ten individuals per species (average  = 5/species), depending on availability and within species size ranges. There were 128 species represented by a single individual, 63 species by two, 44 species by three, 33 species by four, 215 species by five and 47 species by six or more measured individuals.We selected nine ant morphological characters relevant to indicate, isolated or in groups, habitat preference and resource use http://www.worldclim.org) at 30 s resolution. At each locality, five variables were chosen to represent climatic data: (1) annual mean temperature, (2) minimum temperature (the average minimum temperature of the coldest month), (3) maximum temperature (the average maximum temperature of the warmest month), (4) temperature annual range (defined as the difference between the maximum temperature of the warmest and coolest months); and (5) annual precipitation. We used (6) altitude (elevation above sea level) and (7) habitat area (hectares) as site characteristics . We used the FD_Ind function http://www.ecolag.univ-montp2.fr/software>) for computing the components of functional diversity. FDPG was computed using the treedive function in the vegan package Species richness was defined as the number of species recorded in each locality. We used the morphological traits to estimate morphological diversity. Trait variation or multivariate trait differences within a community is generically referred to as ‘functional diversity’ First, we assessed the relationship between morphological or taxonomic diversity and latitude with generalized linear models (GLMs). Model checking showed data heterogeneity (more variation at greater latitudes) and non-linearity related to altitude; so we used GAM models with Gaussian distribution, latitude and altitude as predictors and a fixed variance structure in the model, allowing for larger residual spread if altitude increases.Then, we used a multiple-predictors model to test the effects of climatic data , productivity (AET), habitat area and altitude on taxonomic and morphological diversity. The relationship between the morphological diversity indices and the predictors was assessed with regression models assuming a Gaussian error distribution. We used GLM Poisson with log-link function to model the relationship between species richness, rarefied species richness, abundance and the predictors. We used stepwise selection by AIC in the MASS package in R Because comparatively high altitude areas occur only in the southeastern of the Atlantic Forest, we refit the models excluding high altitude sites (eight sites higher than 400 m) to evaluate if the interpretation of latitudinal effects change when analyzing only lowland sites. When the deviance analysis suggested overdispersion in the single-predictor model, we adjusted standard errors using quasi-Poisson GLM.sor) weighted presence-absence and is defined as: Fsor  =  BL k1 k2/(BLk1 + BLk2)∧2, where BLk1 k2 is the total dendrogram branch length common to all species in communities k1 and k2, and BLk1 and BLk2 are the total dendrogram branch lengths common to the species within communities k1 and k2, respectively. The second and third used measures were the mean pairwise distance (FMPD) between all species and the mean nearest-neighbor distance (FMNTD) separating each species from its closest relative. We used the comdist and comdistnt functions in the picante package We used three metrics of functional beta diversity, each highlighting different aspects of functional turnover. The Functional Sorensen's Index  to calculate species turnover, which reflects the compositional dissimilarity between pairs of sites as the likelihood that a species occurs in these two sites: Ssor  =  (b+c)/2a+b+c For consistence with the FWe used distance-decay relationships to assess rates of decline in taxonomic and functional similarity as a function of geographic and environmental distances We combined the climatic and environmental data  in a principal component analysis to define the environmental gradient. The first principal component, used to examine the combined environmental turnover along the gradient, accounted for 60% of the variance in the environmental space and loaded the variables approximately equally , except precipitation (loading  = −0.074) and AET (loading  = 0.050). The environmental distance between sites was calculated as the absolute difference between the values of the PC-1 at these sites. The range of temperature for the latitudinal gradient was 9.6 to 18.7 degrees Celsius, while it was 9.6 to 18.4 degrees Celsius to the lowland areas.capscale function was used to carry out db-RDA and model selection was based on ordiR2step function in vegan package In addition to Mantel tests, we used distance-based redundancy analysis (dbRDA) coupled with permutation-based model selection to find the variables that most strongly relate to patterns of turnover along the latitudinal gradient We used two morphological structure metrics: the mean pairwise distance (MPD) and the mean nearest-neighbor distance (MNTD) among all species in an ant community to describe community assembly. The input data in the form of a distance matrix was built based on the Euclidean distance between species using the nine untransformed morphological ant traits.A null distribution was generated by randomizing the names of the species across the tips of a trait dendrogram to assess the significance of observed MPD and MNTD. We used standardized effect sizes (SES) to express morphological structure: positive or negative values of these indices indicate morphological clustering or overdispersion. SES values greater than 1.96 or below than 1.96 indicate higher than expected s or lower than expected morphological dissimilarity between species, respectively. Values in between indicate no morphological structure.Deviations from the null expectation can be used as evidence for action of a number of ecological processes determining the assembly of the local ant communities: (i) a regular or even dispersion of morphology is the predicted outcome of the classic concepts of competitive exclusion or niche partitioning We did not translate the predictions of existing ant diversity hypothesis into a set of estimates of the trait-based tests applied in this study. We were interested in the analysis of spatial variation in the morphological structure of leaf-litter ant communities, and not to infer the processes underlying filters, but rather to quantify their relative strength and direction. To reach this, we calculated MPD and MNTD indices of “random communities”, each defining a different species pool to the null communities: (1) unconstrained Atlantic Forest species pool ; (2) constrained within-region species pools (randomizing site composition across Atlantic Forest regions); (3) unconstrained within-guilds ; (4) constrained within-guilds (randomizing within-guilds composition across Atlantic Forest regions). In the unconstrained analysis, trait dissimilarity within communities (or guilds) is compared to the corresponding trait dissimilarity expected for the Atlantic Forest list; in the constrained analysis, the expected trait dissimilarity is calculated for Atlantic Forest species pools (different regions of the biome or subsets in the data set as determined by an UPGMA clustering based on Bray-Curtis distance index) that include the set of potential species for a site delimited likely by environmental and dispersal limitation filters. Visual examination of the cluster indicates that sites can be grouped into four groups: (1) north localities , (2) intermediate latitude areas  (3) low southeastern-south localities , and (4) high southeastern-south Atlantic Forest areas .The species pools' sizes were defined as the number of species in each group determined by the cluster analysis. In all randomizations we kept the number of species in a community (or sample) the same. Although our definition of species pool is simple   . Models PG), 5% (FRic), 9% (FEve), and 16% (FDiv) of the variation in functional diversity. Up to 53% of the variation in the functional specialization of the communities was explained by a combination of temperature, altitude, productivity and precipitation. Habitat area was consistently excluded in the best models suggesting no effect of habitat area in the scale of our study on functional or taxonomic diversities , with adjacent sites being most similar between each other than to any other site in terms of environmental conditions . We therOverall the morphological tests support random morphological structuring along the latitudinal gradient at the community level in both delimited and unconstrained species pools. Significantly aggregated communities (SES <−1.96) occur only at higher latitudes (southern and south of the Atlantic Forest) for the unconstrained analysis . The numWe recognize nine morphological guilds in the Atlantic Forest leaf-litter ant fauna Within-guilds morphological structure was in general random. When morphological structure was detected , the spaCommunity ecologists are increasingly using quantitative diversity components of assemblages in an integrative approach encompassing taxonomic, phylogenetic and functional diversity dimensions Our results show that facets of biodiversity respond differently to a tropical forest latitudinal gradient. Leaf-litter ant species richness  showed an inverse relationship with latitude, even after adjusted for the number of individuals. The functional specialization of the ant leaf-litter communities showed a classical linear response to the latitudinal gradient, increasing from high to low latitudes, defined as the average distance of its species to the center of gravity in the morphological space. In this case, species size, eye position and body shape change on average faster at low than in high latitudes in the Atlantic Forest, leading to communities made up of the same groups of species, although more functionally distinct at low latitudes. Functional richness (defined by two metrics) and functional evenness either were not or were weakly related to the latitudinal gradient, suggesting that the amount of functional space occupied by the assemblages and the regularity of the species abundances distribution in the morphological space The observed inverse latitudinal gradient of ant species richness is intriguing; although the observed pattern is supported even accounting for species occurrences or when high altitude areas were excluded from models. We hypothesized that the increase in leaf-litter ant species richness with latitude in the Atlantic Forest may reflect the synergy of two processes affecting spatial turnover. First, environmental heterogeneity may significantly influence community structure The cumulative effects of human activities along 500 years of Atlantic Forest occupation have led to a vast lowland habitat loss and hyper fragmentation in the northeastern of the Atlantic Forest Community turnover along a spatial gradient is an increasingly recognized biogeographic pattern known as distance decay in communities' similarity studies Our results show random patterns in morphology at community-level, suggesting that niche processes do not influence community assembly by acting on the measured traits. When communities are morphologically structured, we found that the most prevalent pattern is aggregation. We found weak support for niche-structured communities because at regional or local scales morphology was not different from random. Further, upon closer examination of trait dissimilarity constrained by Atlantic Forest regions, we did not find consistent patterns of trait diversity related to biotic processes. The unconstrained species pools analysis on the relationship between latitude and SES values (indicating the distribution of species within the morphospace), as shown in The use of guild classification based on morphology We have used morphological traits with known ecological function in generalist and specialist ant guilds Our dataset is unique in focusing an hyperdiverse invertebrate taxon, in covering a large extension in one of the world's most species-rich tropical rainforests and by combining complementary aspects of community structure, as compositional, functional and morphological data in a hotspot of biodiversity Figure S1Relationship between species richness and geographic range for each leaf-litter ant guild along the Brazilian Atlantic Forest. LEP =  Large-size epigaeic predators; MEP =  medium-size epigaeic predators; MHP =  medium-size hypogaeic predators; SHP =  small-size hypogaeic predators; SP =  specialized predators; DP =  Dacetine predators; G =  generalists species; SHG =  small-size hypogaeic generalists; FG =  fungus-growers.(TIFF)Click here for additional data file.Figure S2Relationship between morphological structure (MPD) for leaf-litter ant guilds and latitude along the Brazilian Atlantic Forest (unconstrained species pool). Guild codes like in (TIFF)Click here for additional data file.Figure S3Relationship between morphological structure (MNTD) for leaf-litter ant guilds and latitude along the Brazilian Atlantic Forest (unconstrained species pool). Guild codes like in (TIFF)Click here for additional data file.Figure S4Relationship between morphological structure (MPD) for leaf-litter ant guilds and latitude along the Brazilian Atlantic Forest (constrained species pool). Guild codes like in (TIFF)Click here for additional data file.Figure S5Relationship between morphological structure (MNTD) for leaf-litter ant guilds and latitude along the Brazilian Atlantic Forest (constrained species pool). Guild codes like that in (TIFF)Click here for additional data file.Table S1Atlantic Forest leaf-litter ant sampling sites, coordinates, site characteristics  and environmental parameters employed in the analyses. Lat =  Latitude; Long  =  Longitude; Area =  area (hectare); Alt  =  altitude (elevation above sea level); Prec  =  annual precipitation; MinT  =  minimum temperature (the average minimum temperature of the coldest month); MaxT  =  maximum temperature (the average maximum temperature of the warmest month); Trange  =  temperature annual range; Tmean  =  annual mean temperature; AET  =  annual actual evapotranspiration; PET  =  annual potential evapotranspiration.(PDF)Click here for additional data file.Table S2Leaf-litter ant species registered in the Brazilian Atlantic Forest, guild classification and morphological traits used in this study.Guilds: LEP =  Large-size epigaeic predators; MEP =  medium-size epigaeic predators; MHP =  medium-size hypogaeic predators; SHP =  small-size hypogaeic predators; SP =  specialized predators; DP =  Dacetine predators; G =  generalists species; SHG =  small-size hypogaeic generalists; FG =  fungus-growers; N =  army-ants; V =  arboricolous ants. Morphological traits: AL =  mesosoma length; PeL =  petiole length; PeH =  petiole height; FL =  hind femur length; MW =  mandible width; ID =  interocular distance; CL =  clypeus length; EL =  compound eye length; DEM =  distance of compound eye to mandible insertion.(PDF)Click here for additional data file.Table S3A) Generalized Additive Model (GAM) results for the leaf-litter ant taxonomic and functional diversity in 26 Atlantic Forest sites. The predictors are smoothers for Latitude and Altitude. e.d.f., Estimated degrees of freedom. (B) Generalized Linear Model (GLM) results for the leaf-litter ant diversity (taxonomic) in low land Atlantic Forest sites (<400 m). The predictors are Latitude and Habitat Area. d.f., Degrees of freedom.((PDF)Click here for additional data file.Table S4A) Multiple Predictor Models of taxonomic and functional diversities  for the leaf-litter ant fauna in low (<400 m) and high (>700 m) land Atlantic forest areas. Predictors are Temperature Annual Range, Altitude (m), AET, Precipitation and Habitat Area. Values in bold indicates significant variable contributing to the model . (B) Multiple Predictor Models of taxonomic and functional diversities  for the leaf-litter ant fauna in low land Atlantic forest areas (<400 m). Predictors are Temperature Annual Range, Altitude (m), AET, Precipitation and Habitat Area. Values in bold indicates significant variable contributing to the model .((PDF)Click here for additional data file.Table S5Partial Mantel correlations between community or functional similarity and environmental distance controlling for geographic distance (and vice versa).(PDF)Click here for additional data file.Table S6Summary of model selection on constrained ordination models (dbRDA) that describe the influence of environmental variables on the leaf-litter ant fauna along Atlantic Forest sites.(PDF)Click here for additional data file.Table S7Summary of GAMs to examine the relationship between the morphological structure of the leaf-litter ant assemblages and latitude in the Atlantic Forest.(PDF)Click here for additional data file.Table S8Summary of GAMs to examine the relationship between the morphological structure of the leaf-litter ant guilds and latitude in the Atlantic Forest.(PDF)Click here for additional data file.


Before (Pre-) and after (Post-) training, muscular levels of selected mRNAs were analyzed from resting muscle biopsies and RSA tested until exhaustion  with muscle perfusion assessed by near-infrared spectroscopy. From Pre- to Post-, the average power output of all sprints in RSA was increased (p<0.01) to the same extent  in RSH and in RSN but the number of sprints to exhaustion was increased in RSH  but not in RSN (9.3±4.2 vs. 8.9±3.5). mRNA concentrations of HIF-1α (+55%), carbonic anhydrase III (+35%) and monocarboxylate transporter-4 (+20%) were augmented (p<0.05) whereas mitochondrial transcription factor A (−40%), peroxisome proliferator-activated receptor gamma coactivator 1α (−23%) and monocarboxylate transporter-1 (−36%) were decreased (p<0.01) in RSH only. Besides, the changes in total hemoglobin variations (Δ[tHb]) during sprints throughout RSA test increased to a greater extent (p<0.01) in RSH. Our findings show larger improvement in repeated sprint performance in RSH than in RSN with significant molecular adaptations and larger blood perfusion variations in active muscles.While intermittent hypoxic training (IHT) has been reported to evoke cellular responses via hypoxia inducible factors (HIFs) but without substantial performance benefits in endurance athletes, we hypothesized that repeated sprint training in hypoxia could enhance repeated sprint ability (RSA) performed in normoxia via improved glycolysis and O This later mechanism is also influenced by the exercise intensity 2 demand tightly Hypoxic conditions can be characterized in working muscle by a decreased oxygen tension  Indeed, repeated sprints (RS) consist in maximal intensity exercise bouts with incomplete recoveries and could potentially challenge these adaptive mechanisms if performed in hypoxia. RS require huge energy amounts over short periods of time provided primarily by anaerobic glycolysis 2 extraction than their slow twitch counterparts 2 utilization by FT.Besides, fast-twitch fibers (FT), preferentially recruited while sprinting Nonetheless, to our knowledge, these mechanisms and the potential associated benefits for the ability to repeat sprints in normoxia have never been investigated in a randomized blind controlled study. The goal of our study was therefore to test putative additional benefit on systemic repeated sprint ability performance of RS training in hypoxia vs. normoxia and to assess blood perfusion and molecular responses at the muscular level.50 moderately trained male cyclists  volunteered in the study and provided their written informed consent after the state Medical Ethics Committee approved the protocol . Subjects were all non-smokers lowlanders. None of the subjects were acclimatized or recently exposed to altitude and were asked to avoid any exposure to an altitude of more than 1500 m during the protocol.Experimental protocol consisted in two testing sessions before (Pre-) and after (Post-) a four-week supervised specific training period (8 training sessions). Subjects were randomly assigned to one of the two treatment groups with repeated sprint training either in normoxia  or hypoxia . A control group  completed only Pre- and, after four weeks without specific training, Post-. Subjects were not specifically accustomed with 10 s cycling sprints but they were familiar with laboratory testing and longer high intensity cycling intervals. Protocol was run in single-blind fashion, as subjects both in RSH and RSN were told that all training sessions were held at altitude . When asked, 95% of the subjects in RSH and 85% in RSN declared that their training was performed in hypoxia. Analyzes of blood samples and muscle biopsies were performed double-blinded.iO2) was set to 14.6% to simulate an altitude of 3000 m and controlled regularly with an electronic device . In order to blind subjects to altitude, the system was also run for RSN with a normoxic airflow into the chamber. Air input flow (up to 1000 l/min) was sufficient for safe, comfortable and stable training conditions. Temperature inside the chamber was maintained at 25° in average and three fans and crushed ice buckets were used to cool subjects.Volunteers completed eight specific cycling training sessions with repeated sprints during four weeks starting the week after Pre- and finishing the week before Post-. Two sessions per week were completed in a hypoxic chamber  built in our laboratory at an altitude of 485 m . The hypoxic chamber is a well-ventilated 30 cu. m room (2.4 m×5.0 m×2.5 m) with transparent glass panels. The system consists in a compressor storing air in pressurized tanks with serial connection to air filters allowing oxygen reduction  in the air input flow to the chamber. For RSH, inspired oxygen fraction  was therefore advised. In all cases, subjects were asked to fill a nutritional diary for three days before each test and reproduce the last meals avoiding alcohol and caffeine intakes during the 24 h before each test. Tap water was provided Upon arrival in the morning, subjects were weighed and a 2,5 ml blood sample was then taken from the antecubital vein for immediate hematocrit and hemoglobin concentration ([Hb]) evaluation. Gold standard hematocrit measurement was done by centrifugation of heparinized capillaries during 5 min at 13000 rpm . [Hb] was assessed with a photometry device . Blood lactate concentration ([La]) was measured from a capillary fingertip sample  at rest and immediately after each test.vastus lateralis muscle using percutaneous needle biopsy technique  Resting skeletal muscle microbiopsy samples were taken under local anaesthesia  of the left leg from the belly of the RNA from skeletal muscle (approximately 25 mg of muscle) was extracted using a commercially available preparation, peqGOLD Tri-Fast . Five micrograms of RNA were reverse transcribed to cDNA using Random Hexamer primers  and AffinityScript Multiple Temperature Reverse Transcriptase , while quantitative PCR was performed using an MX3000p thermal cycler system and Brilliant SYBER Green QPCR Master Mix (Agilent Technologies). PCR primers sequences are listed in 20 mg of tissue were used to measure enzyme activities of interest in RSH and RSN but not in CON. Lactate dehydrogenase (LDH) activity was determined using a colorimetric method developed by BioAssay system (QuantiChrom) according to manufacturer’s instructions. The non-radioactive colorimetric LDH assay is based on the reduction of the tetrazolium salt MTT in a NADH-coupled enzymatic reaction to a reduced form of MTT which exhibits an absorption maximum at 565 nm .kg-1 with the ergometer in manufacturer’s “Wingate mode” unless otherwise specified. RPE and leg pain were assessed as described above.An electronically braked cycling ergometer  equipped with clip-on pedals was used for all performance tests. Seating and handlebar positions were adjusted individually for all subjects and reproduced for all tests. For each performance test, maximal instantaneous power output and pedaling frequency (PF) were recorded and average power output calculated. All tests were performed against an individual fixed torque of 0.8 Nmm. vastus lateralis on the right leg, at mid thigh with an interoptode spacing of 40 mm. The probe was attached to the skin with double-sided tape and firmly fastened with an opaque cotton elastic band wrapped around subjects’ thigh. Position of the probe was marked during Pre- with a permanent pen for accurate repositioning in the Post-trial. A standard differential pathlength factor (DPF) of 4.0 was used in lack of any clear standard value for human quadriceps muscle during cycling sprints 2Hb]), desoxyhemoglobin ([HHb]) and total hemoglobin/myoglobin ([tHb]) were determined. 2Hb] to blood flow variations 2Hb signals might be confounded by rapid blood volume changes during sprints av and Δ[tHb]av) were calculated. The same analysis was performed during the successive recovery phases. In addition, the Δ[tHb]/Power was calculated as the ratio between Δ[tHb] and mean power for each sprint as an index of blood perfusion relative to the intensity of each sprint.Muscle oxygenation was assessed using a near-infrared spectroscopy (NIRS) technique well described elsewhere Surface EMG of the vastus lateralis (VL) and biceps femoris (BF) muscles on the right leg were recorded during the tests using MP36 hardware  with specific electrodes  with a diameter of 35 mm pasted longitudinally on the distal part of the muscle belly with an center-to-center distance of 25 mm. A ground electrode was located on the patella. Prior to electrode placement, skin on the surface of these muscles was carefully shaved, abraded and cleaned (impedance <5 kΩ). EMG cables running inside subjects’ cycling tights prevented cables movement. The myoelectric signal was collected at 2000 Hz, amplified and filtered . EMG parameters were then computed in a dedicated software . During RS, each muscle activity was determined by calculating the mean value of the root mean square (RMS) of the signal for each sprint. Average values were then calculated for the entire duration (10 s) of each sprint.Approximately 20 min after the resting biopsy, subjects performed a 5 min warm-up at 120 W at their preferred PF followed by an isolated 10 s sprint, 4 min 50 s recovery at 120 W and a second isolated 10 s sprint. 15 s after this second sprint, subjects were asked to stop pedaling and rest keeping the right leg motionless and straight during 5 min for NIRS baseline measurement. Subjects then started pedaling for 45 s against a 20 W resistance at a PF of approximately 85 rpm followed immediately by the repeated sprint ability (RSA) test with a 1∶2 work-to-rest ratio . Immediately after each sprint, the ergometer switched automatically to a resistance of 20 W during the 20 s active recovery phase. Subjects were given very strong verbal encouragement and performed as many sprints as possible until exhaustion (i.e. task failure). To avoid any protective pacing strategy, average power during the first two sprints was controlled to reach at least 95% of the best average of the two isolated sprints, which was the case in all subjects. Subjects were never given any indication on the number of sprints performed. A minimal PF of 70 rpm after 5 s or less of sprinting was set as a criterion to stop subjects’ test. However, volitional exhaustion was observed in all subjects before this criterion was reached. Unfinished sprints  where not taken into consideration.2peak) was calculated as the highest 30 s average value during the test with a gas exchange analyzer . Although residual fatigue from the RSA test was probable, this test was performed for all subjects at the same moment of the testing sequence allowing for a good comparison of subjects’ aerobic capacity (average power during 3 min). For this test, the ergometer was set in linear mode (power output increases as PF increases).45 min after the RSA test, subjects performed a 3-min all-out test following a protocol well described elsewhere Prior to the first and last training sessions, subjects performed a standard 30 s Wingate test Data are presented as mean (SD). Performance and blood perfusion changes during RSA test were first evaluated with a 2-way (training group×sprint number) general linear model repeated-measures ANOVA with all pairwise multiple comparison procedures (Holm-Sidak method). Performance improvement, muscle oxygenation during RSA, mRNA concentrations, blood lactate and other single variables test were then evaluated with 2-way (training group×time (Pre- vs. Post-)) general linear model repeated-measures ANOVAs with all pairwise multiple comparison procedures (Holm-Sidak method). All analyses were made using Sigmaplot 11.0 software . Null hypothesis was rejected at p<0.05.Total work and training intensity during supervised RS training were similar during RSN and RSH, with only the mean heart rate being higher in RSH . In addiFrom Pre- to Post-, the average power of all sprints during the RSA test increased (p<0.01) to the same extent  in RSH and in RSN, respectively, but not in CON . The number of sprints prior to exhaustion was increased in RSH  but not in RSN  nor in CON . In Post- compared to Pre-, 10-s average power in the successive sprints was significantly improved till the ninth sprint in RSH and till the seventh in RSN. In RSH, this 10-s power output was significantly better (p<0.01) in the 10th and 11th sprints in Post- than the ninth sprint in Pre- . SignifiThe average power during the 3-min all-out test as well as hematocrit and hemoglobin concentration at rest were unchanged between Pre- and Post- in all groups  and 4. Rav increased to a greater extent  in RSH than in RSN and was not different in Pre- and in Post- in CON  and in RSN  but was not different in Pre- and in Post- in CON .Meanwhile, Δ[HHb]The average Δ[tHb]/Power ratio increased to a greater extent  in RSH  than in RSN  and was not different Pre- compared to Post- in CON .av increased to a similar extent in RSH and in RSN but was not different Pre- compared to Post- in CON  but not in RSN  nor in CON .Meanwhile, Δ[HHb]From Pre- to Post-, average RMS activity of the VL muscle was not different in RSH , in RSN  or in CON (0.53±0.21 vs. 0.51±0.19 mV). Similarly, from Pre- to Post-, average RMS activity of the BF muscle was not different in RSH , in RSN  or in CON . RMS activity of the VL across the RSA test decreased relatively to the first sprint (set to 100%) but to a similar extent in RSH , RSN  or in CON  in Pre- vs. Post-, respectively.The principal novel finding of this investigation is that repeated sprint training in hypoxia allows further enhancement of repeated‐sprint performance to exhaustion than the same training in normoxia. Second, in RSH, the amplitude of blood flow variations during sprint phases was increased. Third, the increased mRNA expression of factors involved in pH regulation and glycolysis as well as the decrease in factors involved in mitochondrial biogenesis after RSH suggest a potential enhancement of the glycolytic (but not oxidative) activity in muscle.RSH and RSN elicited similar significant power improvements (∼6–7%) but hypoxic training only additionally delayed the decrease of power output and therefore extended the number of sprints performed. The ability to reach a higher PF and maintain it high during the last 5 s of the successive sprints explains the power improvements both in RSH and RSN. Indeed, maximal power production (e.g. during repeated sprints) is related to the ability to recruit more FT at very high PF In addition, the aerobic performance  was not modified after training . Similarav was expected and could contribute to higher power production both in RSH and RSN. Then, during the repeated sprint test, fatigue appeared rapidly, highlighted by the power decrement  accumulates in muscle. Since increased Pi levels may decline force, especially in FT recruited during such fatiguing exercise av during sprints in RSH compared to RSN and the increase of the Δ[tHb] to power ratio, so it can be argued that blood perfusion was improved in RSH and could explain additional sprints performed in RSH.Δ[tHb] represents changes in blood volume in the muscle (i.e. blood perfusion) ∼33–35%,  througho2 delivery to working FT (compared to slow twitch (ST)) and their greater fractional O2 extraction if highly perfused 2 delivery to FT, minimizing substrate level phosphorylation and intracellular perturbation (e.g. Pi accumulation and decreased pH), thereby “making FT to behave more like their oxidatively efficient ST counterparts” Moreover, during sprinting, due to the high PF, FT are predominantly recruited Interestingly, a recent study underlined a higher decrease in power output but with similar [tHb] kinetics during a RSA test performed in hypoxia compared to normoxia 2 availability to demand ratio is lowered and muscle tissue oxygen delivery is maintained by an increased extraction of O2 in blood 2 delivery to the muscle av in RSH  increased variations of blood perfusion possibly delaying fatigue during a RSA test and ii) specific molecular adaptations large enough for inducing further improvement in systemic RSA performance. Our results suggest an improved vascular conductance in repeated sprints to exhaustion where fast-twitch fibers are likely better utilized. Fatigue could potentially be delayed through FT working with less anaerobic energy dependence. In parallel to the increased blood perfusion and potentially better waste metabolites removal, modifications at the molecular level support a shift towards improved anaerobic glycolytic activity following RS training in hypoxia only.In conclusion, this study reports the effectiveness of RSH for improving intermittent exercises performed in normoxia. The mechanisms are likely largely different than those previously associated to the inefficient IHT.


OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines  and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction) and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells. The endoplasmic reticulum (ER) is a vital organelle involved in secretory and membrane protein biosynthesis. When the homeostasis in the ER lumen is perturbed such that an accumulation of unfolded, misfolded or aggregated proteins occurs this creates a state of ER stress. Eukaryotic cells relieve this stress by inducing the unfolded protein response (UPR), which attempts to restore and maintain normal ER homeostasis and function In mammalian cells three principal, ubiquitously expressed ER stress sensors; PKR-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) mediate the UPR In addition to the three main ER stress sensors, additional proteins related to ATF6 such as Old Astrocyte Specifically Induced Substance (OASIS)  are expressed in certain cell types in vivoER stress has been shown to occur in cancer cells potentially due to the hypoxic conditions experienced by cancer cells Human glioma cell lines U373, A172 and U87 were obtained from Dr. James Rutka . Details for these established cell lines can be found in the following references Total RNA was isolated from human glioma and rat C6 cell lines using TRIzol reagent  followed by purification using the RNeasy RNA isolation kit . cDNA was synthesized using the One step RT-PCR kit (Qiagen) in a PTC-200  thermal cycler. Real-time PCR was performed as described previously Full length rat OASIS cDNA was synthesized from rat pancreatic islet total RNA and subcloned into pCR II Topo vector (Invitrogen) as described earlier 5) were transfected with 100 nM siRNA using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.Small interfering RNAs (siRNAs) consisting of synthetic annealed RNA duplexes to human OASIS were obtained from Invitrogen, Inc. An siRNA directed to green fluorescent protein (GFP) was used as a control. Cells (1×106) were transfected with 100 nM control or OASIS siRNA for 3 days and incubated at 37°C until cells reached 90% confluence to form a monolayer in a 6 well plate. A p200 pipette tip was used to create a uniform scratch of the cell monolayer followed by a wash with PBS. Fresh DMEM medium  was added and the cells were incubated for 24–48 h. Representative DIC images of wound healing were monitored with Olympus fluorescence inverted microscope (IX71). Wound closure was determined by quantifying the scratch area using ImageJ v1.42l analysis software.To monitor migration rate, U373 cells  for 15–20 min on ice. The lysate was centrifuged (10 min) and protein concentration measured using the BCA protein assay kit . Equivalent protein amounts were resolved using 10% SDS-PAGE and electro-transferred to Hybond nitrocellulose membranes . Immunodetection was performed with the following primary antibodies: rabbit anti-OASIS , mouse anti-KDEL, mouse anti-PDI , rabbit anti-cleaved caspase 3 , anti-γ-tubulin . The secondary antibodies, anti-mouse HRP  and anti-rabbit HRP  were used as required and detected by ECL kit . Immunoblots were scanned and protein intensities were quantified using Scion Image software .Cells were treated as described in the figure legends then fixed and processed for immunofluorescence as described in reference Where applicable results are presented as mean ±SEM. Statistical significance was assessed using the Student’s t-test  or ANOVA followed by Tukey post-hoc test as indicated in the figure legends (p<0.05 was considered significant).We investigated OASIS expression in three human glioma cell lines, U373, A172 and U87. The presence of OASIS mRNA in these cell lines was detected by RT-PCR. An ∼1.5 kbp OASIS cDNA was amplified in all three cell lines and in the rat C6 glioma cell line used as a positive control . By realTo examine OASIS protein expression, the human glioma cell lines were treated or not with tunicamycin (TM) or thapigargin (TG) and cell lysates were prepared. Rat C6 glioma cells transfected or not with rat OASIS were used for comparison. Immunoblot analysis of the cell lysates with anti-OASIS antibody showed barely detectable levels of the ∼85 kDa OASIS protein in all three cell lines under control conditions . With TMMouse OASIS has previously been shown to be glycosylated To address whether endogenous OASIS expressed in human glioma cell lines plays a role in the ER stress response and in extracellular matrix production, we knocked-down OASIS expression using siRNA. As shown in OASIS has also been implicated in modulating extracellular matrix components including chondroitin sulfate proteoglycans Another extracellular matrix component shown to be induced by OASIS in bone osteoblast cells is the collagen gene Col1a1 Glioma tumor cells are characterized by their highly invasive and infiltrative capacity. Given that OASIS knock-down resulted in reduced chondrotin sulfate proteoglycan protein expression we examined the migration rate of glioma cells using a wound scratch assay. U373 cells were transfected with control or OASIS siRNAs then a scratch wound was made to the cells and the area was monitored by DIC microscopy. Cells in which OASIS was knocked-down had reduced migration rate compared to control siRNA transfected cells . WhereasDecreased cell migration could result from reduced cellular growth (proliferation) or increased cell death resulting from apoptosis. We thus monitored cellular apoptosis in control and OASIS siRNA treated cells in the presence and absence of TG-induced ER stress. U373 and U87 human glioma lines were relatively resistant to apoptosis induced by TG requiring 48–72 h of treatment to detect cleaved capsase 3 . HoweverOASIS was first identified in mouse astrocytes and glioma cell lines and discovered to be an ER stress response protein To examine if OASIS modulates UPR genes such as chaperones we knocked down OASIS expression in the U373 and U87 cell lines. The cells were subsequently treated with TG to induce ER stress. Interestingly, knock-down of OASIS reduced the induction of both GRP78 and GRP94 proteins in response to ER stress. This is consistent with results in the literature that have found that GRP78 is a target gene of OASIS in rat C6 glioma cells Rat C6 glioma cells exposed to TG had increased levels of chondroitin sulfate in vitro migration assay identified that OASIS silenced cells have poor migration efficiency. The exact mechanism of this effect is unknown, although presumably OASIS is required for maintaining ECM components that are required for efficient cell migration. Gliomas are characterized by aggressive growth and invasiveness, which is closely related to cell-ECM interactions in vivoThe http://www.gbmbase.org) which focuses on glioblastoma multiforme research, indicates that OASIS and various ER stress response genes are changed in gliomas relative to control tissue  glioma expression database in vivo and this may contribute to more resistant and invasive cancers.In summary, we identified that the ER stress sensor OASIS is a glycoprotein that is differentially expressed in human glioma cell lines. OASIS protein is induced by ER stress and appears to contribute to both maximal induction of the UPR (chaperone capacity), as well as maintaining extracellular matrix (CSPG) protein expression in glioma lines that express this protein. Because of these effects, we hypothesize that gliomas that express OASIS may be better off under hypoxic conditions Figure S1Expression of selected ER stress and ECM genes in human glioblastoma multiforme (GBM) tumors(DOCX)Click here for additional data file.Table S1Expression of selected ER stress and ECM genes in human glioblastoma multiforme (GBM) tumors(DOCX)Click here for additional data file.


For this purpose, we first characterized the impact of ART on HIV RNA levels in multiple organs of bone marrow-liver-thymus (BLT) humanized mice and found that antiretroviral drug penetration and activity was sufficient to reduce, but not eliminate, HIV production in each tissue tested. For targeted cytotoxic killing of these persistent vRNA+ cells, we treated BLT mice undergoing ART with an HIV-specific immunotoxin. We found that compared to ART alone, this agent profoundly depleted productively infected cells systemically. These results offer proof-of-concept that targeted cytotoxic therapies can be effective components of HIV eradication strategies.Antiretroviral therapy (ART) can reduce HIV levels in plasma to undetectable levels, but rather little is known about the effects of ART outside of the peripheral blood regarding persistent virus production in tissue reservoirs. Understanding the dynamics of ART-induced reductions in viral RNA (vRNA) levels throughout the body is important for the development of strategies to eradicate infectious HIV from patients. Essential to a successful eradication therapy is a component capable of killing persisting HIV infected cells during ART. Therefore, we determined the  Antiretroviral therapy (ART) improves the quality of life for HIV infected individuals. However, ART is currently a lifelong commitment because HIV persists during treatment despite being suppressed below detection. If therapy is stopped, the HIV reappears. A concerted effort is ongoing to develop new eradication therapies to prevent virus rebound, but there are challenges to be overcome. Our work is a major step forward in this process. We measured persistent HIV throughout the body during ART using bone marrow/liver/thymus (BLT) humanized mice, a model validated to study HIV persistence. HIV infected BLT mice were treated with tenofovir, emtricitabine and raltegravir. Despite documented tissue penetration by these drugs, we found that HIV expression persists in cells isolated from all the tissues analyzed . We therefore complemented ART with an immunotoxin that specifically kills HIV expressing cells while leaving other cells untouched. Our results demonstrate a dramatic reduction in persistent HIV throughout the body resulting from the killing of virus producing cells. Thus, our study provides new insights into the locations of HIV persistence during ART and a demonstration that persistent HIV can be successfully targeted inside the body. To do so, we sought to analyze HIV persistence during ART in a comprehensive panel of tissues and organs over time – a study that cannot be performed in human subjects. For this reason, we used BLT humanized mice + T cell depletion and immune activation) recapitulate what is observed in HIV-infected patients The fact that tissue specific effects of ART on HIV persistence are poorly understood in patients For this study, BLT mice were treated with a triple combination antiretroviral drug regimen that included the nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate (TDF), the nucleoside reverse transcriptase inhibitor emtricitabine (FTC) and the integrase inhibitor raltegravir (RAL). This ART regimen was chosen because of its robust pharmacodynamic properties + T cells levels  In human peripheral blood s levels . We alsos levels    . This prDespite the lifesaving benefits of ART in HIV patients, relatively little is known regarding the organ specific impact of this therapy on HIV production and persistence HIV-infected cells persisting during ART represent suitable targets for cytotoxic HIV-specific immunotherapies such as the 3B3-PE38 immunotoxin. Since such therapies target HIV proteins expressed by infected cells, their efficacy requires active virus transcription. Therefore there is no predicted impact of immunotoxin treatment on the size of the transcriptionally silent latent HIV reservoir. For this reason, the HIV latent reservoir was not quantitated in this study and our conclusions are based on the quantitation of cell-associated vRNA and vRNA-producing cell numbers. Specifically, we demonstrate that 3B3-PE38 kills vRNA producing cells throughout the body such that the reduction of vRNA levels during combined therapy is more rapid than with ART only . A recenComparing and contrasting data from BLT mice with data from patients and NHP is essential to understanding the predictive nature of our model. The most notable difference between our study and those in patients and NHP is the duration of ART. Our study examined the impact of ART in BLT mice over ∼2 months, while patient and macaque studies have examined cell-associated vRNA levels during several years of ART ex vivo expanded natural or genetically modified T cells in vivo efficacy of any of these other strategies alone or in combinations.The results presented here provide proof of concept for targeted cytotoxic therapy as a successful complement to ART for the depletion of persisting HIV infected cells. In addition to the 3B3-PE38, alternative immunotoxins with different effector domains and targeting moieties are available in vivo efficacy evaluation of interventions to destroy HIV producing cells during therapy. Within this framework we were able to demonstrate that persistent HIV producing cells systemically present during ART were significantly depleted by an Env-targeting immunotoxin. Such targeted cytotoxic interventions may prove to be critically important components of an effective HIV eradication strategy.In summary, we determined that the systemic penetration of antiretroviral drugs into different organs throughout the body was sufficient to reduce cell-associated vRNA levels ART alone was unable to completely eliminate vRNA expressing cells in tissues. This observation provided a quantitative framework for the systemic All animal experiments were conducted following NIH guidelines for housing and care of laboratory animals and in accordance with The University of North Carolina at Chapel Hill (UNC Chapel Hill) regulations after review and approval by the UNC Chapel Hill Institutional Animal Care and Use Committee (permit number 12-171).+ cells (Advanced Bioscience Resources) and monitored for human reconstitution in peripheral blood by flow cytometry +/−15 SD) human CD45+ cells of which 58% (+/−22 SD) were human T cells. Of the human T cells, 81% (+/−6 SD) were human CD4+ T cells.BLT mice were prepared essentially as previously described JRCSF was monitored in peripheral blood by determining plasma levels of vRNA as described  using one-step reverse transcriptase real-time PCR [ABI custom TaqMan Assays-by-Design] Infection of BLT mice with HIV-1in situ hybridization (ISH), tissues were fixed overnight in 4% paraformaldehyde at 4°C and then transferred to 70% ethanol. Tissues were then embedded in paraffin for sectioning. After deparaffinization with xylene and rehydration through graded ethanols, tissue sections were treated with HCl, triethanolamine, digitonin and 4 µg/mL Proteinase K as previously described 35S labeled antisense riboprobe and 0.5 mM aurintricarboxylic acid in the hybridization mix. After extensive washes and ribonuclease treatment, tissue sections were dehydrated, coated in Ilford K5 emulsion diluted with glycerol and ammonium acetate, exposed at 4°C for 7–14 days, and developed and fixed per manufacturer's instructions. They were stained with Hematoxylin, dehydrated, and mounted with Permount. Non-infected control tissues and sense riboprobe controls were analyzed when appropriate. Photographic images using epifluorescence were taken with a digital camera and the tiffs were analyzed for the area of the sections and the area occupied by silver grains using Photoshop with Fovea Pro. Section weights were estimated from their 5-micron thickness and their area. These methods have been extensively reviewed For HIV RNA ART in infected BLT mice consisted of three antiretrovirals administered daily via intraperitoneal injection: tenofovir disoproxil fumarate , emtricitabine  and raltegravir  13C TFV and 13C 15N FTC). Following vortexing and centrifugation, the supernatant was removed and evaporated to dryness. The extracts were reconstituted with 1 mM ammonium phosphate prior to LC-MS/MS analysis. All compounds were eluted from a Phenomenex Synergi Polar-RP  analytical column. An API-5000 triple quadrupole mass spectrometer  was used to detect the analytes. Data were collected using AB Sciex Analyst Chromatography Software . The dynamic range of this assay was 1–1000 ng/mL for each compound using a 1/concentration2 weighted linear regression. Calibrators and quality control samples were within 15% of the nominal value for all compounds. Plasma samples with concentrations above the calibration range were diluted into the calibration range with blank plasma prior to reanalysis.Plasma and snap frozen tissue samples from BLT mice receiving daily ART were analyzed for drug concentrations. Quantification of FTC, tenofovir (TFV) and RAL plasma concentrations was performed by protein precipitation and LC-MS/MS analysis. 10 µl of each stored plasma sample was mixed with 75 µL of methanol containing the isotopically-labeled internal standards . Following vortexing and centrifugation, the supernatant was removed and evaporated to dryness. The extracts were reconstituted with 1 mM ammonium phosphate prior to LC-MS/MS analysis. TFV and FTC were eluted from a Waters Atlantis T3  analytical column. RAL was eluted from a Phenomenex Synergi Polar-RP  analytical column. An API- 5000 triple quadrupole mass spectrometer was used to detect all analytes. Data were collected using AB Sciex Analyst Chromatography Software. The dynamic range of this assay was 0.3–300 ng/mL of homogenate for each compound using a 1/concentration2 weighted linear regression. Based upon the mass of the tissue extracted, the concentrations were converted from ng/mL homogenate to ng/g tissue. Calibrators and quality control samples were within 15% of the nominal value all compounds.The quantitation of FTC, TFV and RAL in tissues started with the homogenization of each tissue in a 70% acetonitrile + 30% 1 mM ammonium phosphate solution. A portion of the resulting homogenate was extracted by protein precipitation with acetonitrile containing isotopically-labeled internal standards (All statistical tests performed using an alpha level of 0.05. Exact log rank tests were utilized to generate p values for