Every year, more than 200,000 Americans die from sepsis, a severe illness caused by bacterial infection of the bloodstream. In the United States, 2%–3% of all hospital admissions are for sepsis, which has a mortality rate of about 30%. In sepsis, pathogens that have entered the bloodstream through a wound or from an infected organ induce a systemic inflammatory response, which can cause circulatory system damage, multiple organ dysfunction, and death, particularly in people with other medical conditions.Treatment for sepsis includes antibiotics to clear the initiating bacteria and to provide medical support for damaged organs—mechanical ventilation for lung dysfunction, for example. However, the prognosis for patients would be much better if organ dysfunction could be prevented, so researchers, including Samir Parikh, Vikas Sukhatme, and their colleagues, are investigating how organs become damaged during sepsis. Parikh's team now report results suggesting that a protein called angiopoietin-2 may play a pivotal role in acute respiratory distress syndrome (ARDS), a condition that affects about 40% of patients with sepsis and worsens their prognosis.ARDS starts with protein-rich fluid leaking out of the blood capillaries of the lung into the alveoli, the terminal airspaces where gas exchange normally occurs. The presence of this fluid in the alveoli impairs gas exchange, and prevents the lungs from expanding and contracting normally during breathing. But what causes fluid to leak out of the vasculature? Capillaries are normally lined with endothelial cells linked together with adhesive junctions to form an impermeable barrier. The development of this barrier is controlled in part by two peptides called angiopoietin-1 (Ang-1) and angoipoietin-2 (Ang-2). Both peptides bind as ligands to a signaling receptor called Tie-2. Ang-1 functions as an agonist; its binding activates the Tie-2 receptor. In contrast, Ang-2 is an antagonist, whose binding blocks signal transduction. Animal experiments have shown that the two ligands have opposite functions in vascular development: Ang-1 promotes the stabilization of nascent blood vessels; Ang-2 disrupts this action. These findings, the fact that Tie-2 expression may be abnormal in bronchopulmonary dysplasia , and the high level of Tie-2 expression in lungs prompted Parikh's team to ask if the Tie-2 signaling pathway is affected in sepsis and, specifically, if excess Ang-2 levels might promote the vascular leakage that underlies ARDS.The researchers measured serum Ang-2 concentrations in 22 patients admitted to the hospital with sepsis, as well as in 29 control individuals hospitalized with other conditions. Admission levels of Ang-2 were higher in patients with septic shock (characterized by low blood pressure) or multiorgan failure than in control patients or in patients with sepsis alone. Over time, Ang-2 levels mirrored the clinical course of illness in individual patients, increasing as they became more sick and declining as they recovered. Finally, Ang-2 levels correlated with impaired lung function. These results are consistent with the idea that increased circulating Ang-2 might contribute to vascular leakage in sepsis.To test their hypothesis further, the researchers turned to thin sheets (monolayers) of cultured endothelial cells. The addition of patient serum containing high levels of Ang-2 (but not serum containing low levels) to these monolayers caused changes in the actomyosin cytoskeleton (a network of proteins that forms the cellular skeleton). These structural changes, which resulted in the endothelial cells contracting and gaps forming between them, could be replicated by the addition of Ang-2 alone, and were accompanied by increased permeability of the endothelial monolayer. Adding Ang-1 reversed the effects of Ang-2 on the endothelial cells. Further experiments indicated that Ang-2 activated a protein called Rho-kinase and the subsequent addition of phosphate groups to myosin light chain, a protein involved in cell contraction. In the last set of experiments, the researchers showed that systemic administration of Ang-2 to healthy mice increased vascular permeability and caused fluid extravasation in their lungs, changes reminiscent of early ARDS.These results support a role of Ang-2 in sepsis-associated ARDS, and suggest new targets for the treatment of sepsis. Up until now, the emphasis has been on damping down the systemic immune response, but drugs that do this often have unwanted side effects. Targeting Tie-2 signaling might be a cleaner way to deal with the leaky vasculature that causes ARDS and other aspects of sepsis. In addition, suggest Parikh and colleagues, serial Ang-2 measurements might identify which patients at risk of developing ARDS would benefit most from therapy aimed at preventing vascular leakage. But, they acknowledge, further experiments are needed to prove conclusively that Ang-2 plays a causal role in human ARDS, to clarify why the lungs are especially susceptible to systemic excess levels of Ang-2, and to test whether interfering with Ang-2 function can yield therapeutic benefits.


The Origin of Species, evolutionary biologists are still working out the conditions and processes that give rise to new species. Central to the identity of a species—whether species exist in nature or as theoretical constructs, as some argue—is the ability to interbreed and produce fertile offspring. Several mechanisms can act as reproductive barriers, either before or after fertilization, to prevent gene flow between populations and set the stage for speciation.Nearly 150 years after Darwin published One postfertilization mechanism in plants, called hybrid necrosis, arises from interactions between genes that cause misshapen, yellow, or damaged leaves and stunted growth, much like an infection. Hybrid necrosis occurs in crosses within and between species, suggesting that similar evolutionary processes may be at work at different times as the genes of the interbreeding species drift apart.Conditions like hybrid necrosis, according to the classic Dobzhansky-Muller model of hybrid incompatibility, arise from deleterious interactions between genes inherited from the parents. As parental lineages diverge, the theory goes, each line evolves independent mutations that are harmless in the parent but prove detrimental when co-expressed in the hybrid. The evolutionary forces driving this divergence are not well understood, but adaptive evolution has been implicated in generating hybrid incompatibility, based on evidence that known incompatibility genes evolve rapidly.Arabidopsis thaliana, the favorite model organism of plant biologists. The researchers show that, in four cases of necrosis, the disorder results from the activation of genes normally involved in local responses to a pathogen attack, such as killing infected cells. In the hybrid, the genes interact to trigger a systemic immune response, leading to an autoimmunity-like condition characterized by cell death throughout the plant. Further evidence for this scenario comes from the identification of a common plant disease-resistance gene as a causative agent in one case of necrosis.In a new study, Kirsten Bomblies et al. investigate the genetic basis of hybrid necrosis in A. thaliana strains, Uk-1 and Uk-3, developed severe necrosis and failed to flower. Although all the offspring became necrotic at the mild temperature that A. thaliana encounters in the wild, they flowered at higher temperatures, allowing the researchers to collect seeds for genetic analysis.Bomblies et al. began investigating genetic incompatibilities when they noticed that hybrid progeny of two DM1) was derived from the Uk-3 strain; the second, DM2, came from the Uk-1 plants. To isolate and analyze these potential incompatibility regions, the researchers first conducted a series of backcrosses, crossing the Uk-1/Uk-3 hybrids with a standard A. thaliana lab strain (Col-0) to transfer the unlinked loci to the same genomic background. They selected plants with clear signs of necrosis for subsequent crosses to ensure that they retained both loci. Over a few more backcrosses, and with the help of molecular markers, they generated Col-0 strains carrying either the Uk-1 region or the Uk-3 region and cloned each region for further analysis.The researchers scanned the genomes of the afflicted plants for genetic variations that might contribute to necrosis and identified two regions, one inherited from each parent strain, located on different chromosomes. One locus  appeared normal. But when they crossed a Col-0 strain carrying the Uk-3 region with a Col-0/Uk-1 line, the offspring had the same necrotic symptoms as the Uk-1/Uk-3 hybrids. These experiments demonstrated not only that interactions between the Uk-3 region and the Uk-1 region cause necrosis but that a gene located within the Uk-3 region is all that is required for necrosis when the Uk-1 region is present.DM1 region from the Uk-3 strain contains seven genes, including an NB-LRR gene; NB-LRR receptors are the most common type of disease-resistance genes in plants.  NB-LRR mutations have previously been shown to cause lesions and retarded growth, consistent with inappropriate activation of plant immune responses and similar to necrosis in Uk-1/Uk-3 hybrids, implicating the NB-LRR gene as a candidate gene for DM1. By inhibiting the expression of several genes in the DM1 region of one strain, the researchers confirmed that the NB-LRR gene was indeed the culprit. Only when NB-LRR was inactivated did hybrid plants appear normal, confirming the gene’s role in autoimmunity and necrosis. Supporting this conclusion, microarray analysis of leaves from sick plants showed a substantial increase in activity for genes involved in the immune response to infection, compared with normal plants. A similar bias was found in the gene expression profiles of other hybrids showing necrosis symptoms.TheNB-LRR variant arose and persists in wild Arabidopsis plants, the researchers investigated the prevalence of hybrid necrosis among wild strains. About 2% of wild crosses developed the same necrotic symptoms as the Uk-1/Uk-3 hybrids, owing to five different sets of incompatible genes. And again, necrosis occurred at the environmentally relevant mild temperatures but not at higher temperatures.Having determined that the DM2 region of Uk-1 interacts with the DM1 Uk-3 region, but they point out that DM2 also contains NB-LRR genes. Among the possible scenarios this suggests is that selection pressures on disease-resistance genes engaged in host battles with pathogens caused the genes to diverge in the parental lineages to the point where they became incompatible in the hybrid genome. This link between hybrid necrosis and disease-resistance pathways has also been suggested in tobacco and tomato plants, indicating that this mechanism may act to erect postfertilization gene-flow barriers in diverse plant species. By establishing A. thaliana as a promising model for elucidating the agents of hybrid necrosis and other genetic incompatibilities, this study provides a framework for more fully exploring the role of disease-resistance genes in necrosis and the evolutionary forces that erect gene-flow barriers on the path to speciation.Altogether, these results show how a genetic incompatibility syndrome arises when two genes that are normally harmless in a parental lineage turn out to have disastrous consequences for hybrid offspring. The researchers have not yet determined which gene in the


Comparative genomic analysis using cDNA microarray is a new approach and a useful tool to identify important genetic sequences or genes that are conserved throughout evolution. Identification of these conserved sequences will help elucidate important molecular mechanisms or pathways common to many species. For example, the stockpiled transcripts in the oocyte necessary for successful fertilization and early embryonic development still remain relatively unknown. The objective of this study was to identify genes expressed in oocytes and conserved in three evolutionarily distant species.SMFN , Spin (Spindlin), and PRMT1 (Protein arginine methyltransferase 1) are transcripts present in oocytes and conserved in three evolutionarily distant species.In this study we report the construction of a multi-species cDNA microarray containing 3,456 transcripts from three distinct oocyte-libraries from bovine, mouse and Xenopus laevis. Following the cross-species hybridizations, data analysis revealed that 1,541 positive hybridization signals were generated by oocytes of all three species, and 268 of these are preferentially expressed in the oocyte. Data reproducibility analyses comparing same-species to cross-species hybridization indicates that cross-species hybridizations are highly reproducible, thus increasing the confidence level in their specificity. A validation by RT-PCR using gene- and species-specific primers confirmed that cross-species hybridization allows the production of specific and reliable data. Finally, a second validation step through gene-specific microarray hybridizations further supported the validity of our cross-species microarray results. Results from these cross-species hybridizations on our multi-species cDNA microarray revealed that Cross-species hybridization using a multi-species cDNA microarray is a powerful tool for the discovery of genes involved in evolutionarily conserved molecular mechanisms. The present study identified conserved genes in the oocytes of three distant species that will help understand the unique role of maternal transcripts in early embryonic development. Mos (Moloney sarcoma oncogene) . By comparing between hybridizations the average log signal intensities of two clones from each species corresponding to the same Zp family member (n = 6), it is possible to observe that all the clones corresponding to this gene are consistent, not only between same-species, but also among the three different species  and had to be preferentially expressed in the oocyte. Next, the candidate had to have a known ortholog gene in the two other species, an essential criterion if we wanted to design species-specific primers. Finally, we selected candidates that were not yet reported to be expressed in the oocyte for the two other species since we wanted to know if this cross-species hybridization approach allowed the production of precise and reliable data across species that could lead to the discovery of novel transcripts present in the oocyte.The RT-PCR amplifications were performed on cDNA generated from oocytes total RNA using gene- and species-specific primers Table . For eacThe second step in this validation process was to perform gene-specific microarray hybridization on the multi-species cDNA microarray. Following the RT-PCR amplification reaction, the amplicons were labeled and hybridized to our multi-species microarray slide in order to assess the efficiency of gene-specific but cross-species hybridizations. This experiment was performed in three replicates and hybridizations were performed with either probes of both species simultaneously or only one species at a time. We reasoned that the validation could be considered successful only if the signals detected significantly above background corresponded to the selected candidate spotted on the array. This validation process revealed that all three candidates showed specific cross-species hybridization on our multi-species microarray slide supporting once again the idea that our cross-species hybridizations are specific even between not so closely related species Fig. .The microarray gene chip platform is a powerful tool allowing for the analysis of thousands of genes simultaneously. In this study, we explored the technical feasibility of utilizing cross-species hybridizations to identify genes expressed in the oocyte and that are conserved across three species. Our experimental strategy was twofold. The first goal was to test the possibility of cross-species hybridization of three distantly related vertebrates on cDNA arrays, and most importantly, our second goal was to identify genes expressed in oocytes of all three species. The rationale was to use a subtractive strategy to produce libraries enriched in transcripts preferentially expressed in oocytes, and to use the homology requirement for high specificity microarray hybridization to identify those transcripts that are conserved through evolution. Moreover, we believed that transcripts fulfilling both criteria, specificity to the oocyte and evolutionarily conserved, are potentially important maternal genes involved in key functions of oocyte maturation and early development. By working simultaneously with three evolutionarily distant species, it increased our efficiency at identifying novel oocyte-specific genes and elucidating the important evolutionarily conserved mechanisms in different species. Also, this approach facilitates the identification of new genes not previously identified due to their low expression level in a particular species.One of the unique features of preimplantation embryo development is that it occurs in the presence of maternally stored RNAs in oocytes as the embryonic genome has yet to be activated. These transcripts have specific functions either in oogenesis, oocyte maturation, fertilization and/or the early phase of preimplantation development. Only a few of these genes are well known and have been characterized, as Sharov et al., have demonstrated that, in the mouse, 119 out of the 196 oocyte-specific ESTs were unknown genes in 2003 . IdentifIn the present study we designed a cDNA multi-species microarray containing 3,456 transcripts from bovine, mouse, and Xenopus laevis oocytes. Transcripts found on the array were randomly selected from oocyte-subtracted libraries constructed in a previous study . The useIn this study, results obtained from the reproducibility analysis increased the confidence in the data generated from our hybridizations. The CCs calculated with all the clones on the array showed that signal intensities from all three probes used were highly reproducible (0.934 – 0.957). For the bovine and mouse probes, cross-species hybridizations showed a slightly lower correlation coefficient compared with the same-species experiments. On the other hand, Xenopus laevis clones always generated the lowest CCs even with the Xenopus laevis probe, which might be in part related to a lower amount of cDNA spotted onto the array. Nevertheless, the correlation coefficients for the cross-species hybridization were sufficiently high to assure that reproducibility between replicated experiments is acceptable, increasing our confidence in the validity of cross-species hybridizations results. Also, the sequence mismatches present between the three species should be taken into account since they probably contribute to the lower correlations observed in cross-species hybridizations.The degree of homology between probes and targets when performing cross-species hybridizations is extremely variable. In the presence of sequence mismatches, relative hybridization intensities will reflect both differences in transcript abundance levels, as well as differences in hybridization kinetics. In addition, it can even be variable between two different cross-species hybridizations, especially when the studied species are not equally divergent. Due to these limitations, the goal of this study was not to assess gene expression levels but instead, to survey the products of three subtracted libraries in order to identify transcripts present in oocytes of all three species. Nevertheless, sequence homology had to be sufficiently high in the target region to result in a proper hybridization, since mismatches will inevitably occur in evolutionarily distant animals. To assess this issue, all transcripts identified as being preferentially expressed in the oocyte and present in all three species were compared to identify possible hits with the other two species using the GenBank database. On average, our bovine sequences showed an 86% and 80% identity rate with the mouse and Xenopus laevis sequences, respectively and between the mouse and Xenopus, an 80% identity rate was observed. In the last few years, a number of studies have successfully used cross-species hybridizations ,34-40. JFurthermore, the present study has demonstrated that cross-hybridization results can be confirmed by both RT-PCR reactions and gene-specific hybridizations. In order to ascertain that the observed signals were not originating from the annealing of random non-specific sequences, specificity validation was conducted through a simple detection test using species- and gene-specific primers and by gene-specific microarray hybridization. The labeled amplification products corresponding to three independent single genes showed positive signals only for their corresponding target even across species, lending support to the validity of our cross-species hybridizations.SMFN, also known as CGI-114, which is homologue of Orn, a 3-prime-to-5-prime exoribonuclease of E. coli. The ORN protein is known to attack the free 3-prime hydroxyl group on single-stranded RNA, releasing 5-prime mononucleotides in a sequential manner [GeneID: 25996] . The similarity between the mouse Smfn gene sequence and our bovine clone sequence is relatively high; 91% on 304 bp. A Blast search of our bovine clone sequence against Xenopus laevis sequence revealed that a cDNA clone IMAGE 7205916 [GenBank: BC087528] has an acceptable identity rate with our bovine clone; 79% on 206 bp. This clone is reported to be testis specific [Unigene: Xl.9259]. Like in the mouse, no expression was reported in the Xenopus laevis oocyte for this transcript. With our cross-species microarray hybridization we were able to detect the presence of SMFN transcripts in bovine, mouse and Xenopus laevis oocytes, and this was also confirmed by RT-PCR and gene-specific cross-species microarray hybridizations.Three candidate genes, one from each species, were selected for these validation processes. Our first candidate gene used for validation was bovine : 25996] . In humaecycling . In the Spin (Spindlin), an abundant maternal transcript present in the unfertilized egg and 2-cell, but not 8-cell mouse embryo . This predicted sequence has 94% similarity (234 bp) with our mouse Spin clone. Since it is a predicted sequence, no report of its expression pattern is available. A Blast search of our mouse clone sequence against Xenopus laevis sequences revealed that a cDNA clone IMAGE 6324148 [GenBank: BC097748] has an acceptable identity rate with our clone; 84% on 146 bp. No report of the expression pattern for the bovine and Xenopus laevis are currently available. Once again, with our cross-species microarray hybridization we were able to detect the presence of SPIN transcripts in bovine, mouse and Xenopus laevis oocytes, and this was also confirmed by RT-PCR and gene-specific cross-species microarray hybridizations.Our second candidate gene used for validation was mouse e embryo . SPIN pre embryo . Also, fe embryo . The SPIPRMT1), an xCirp2-binding protein. The methylation of xCIRP2 (cold-inducible RNA binding protein 2) by PRMT1 results in the accumulation of xCIRP2 in the cytoplasm . Mouse Heterogeneous nuclear ribonucleoproteins methyltransferase-like 2 (Hrmt1l2) also possess a high identity rate with Xenopus PRMT1; 78% on 413 bp. Mouse Hrmt1l2 gene is reported to be expressed in a variety of tissues including testis, ovary and embryo, but there are no reports indicating its specific expression in the oocyte [Unigene: Mm.21911]. Once more, we were able to detect the presence of PRMT1 transcripts in bovine, mouse and Xenopus laevis oocytes, and this was also confirmed by RT-PCR and gene-specific cross-species microarray hybridizations.The third and last candidate gene used for validation is the Xenopus laevis Protein arginine methyltransferase 1  was used as an exogenous positive control (n = 260).The multi-species cDNA microarray used in this study contained transcripts from three oocyte-specific libraries constructed previously using Suppressive Subtractive Hybridization (SSH) . BrieflyDNA sequencing was performed as previously described . The resForward-subtracted PCR products from the subtracted libraries (oocyte minus somatic tissues) corresponding to oocyte-libraries were used as probes to hybridize the cDNA multi-species microarray as previously described . BrieflySlides were prehybridized with DIG buffer  supplemented with yeast tRNA  and Cot-1 DNA  for 1 hour at 37°C. Slides were then hybridized overnight at 37°C with labeled purified probes added to fresh prehybridization solution. Hybridizations were performed in the ArrayBooster using the Advacard AC3C . Slides were then washed once with 1 × SSC-0.2% SDS for 10 min at RT, 1 × SCC-0.2% SDS for 10 min at 55°C, and for 5 min at RT with 0.1 × SCC-0.2% SDS. Hybridizations were always performed with probes corresponding to two different species, in an all pair design . A dye swap experiments was included for each sample to take into consideration the variation in dye incorporation efficiency. Moreover, two biological replicates for each sample were used. Thus, for each species, the hybridizations were carried out twice with one dye and twice with the other dye, giving four technical replicate hybridizations per species. Since each clone was replicated four times on the microarray, a total of 16 data points were generated for all the candidates per species-specific hybridization.GFP cDNA fragments were added to the probes in equal amounts, before labeling, to use as positive controls.Considering that the same spots were also hybridized during cross-species hybridizations, 48 data points were generated for each spotted clone (4 spotted clones × 4 hybridizations × 3 species). Quality control was performed through the addition of 424 negative and 260 positive controls included on the array. Slides were scanned using the VersArray ChipReader System  and visualized with the ChipReader software . Microarray image processing was performed with the ArrayPro Analyzer software . Local background was subtracted and data were normalized (LOWESS). Microarray experiments presented in this study adhere to the standards proposed by the Microarray Gene Expression Data Society . Raw andFirst, data were log transformed before proceeding with a simple analysis where thoughtful criteria were applied to minimize, to the extent possible, the false positive rate. The analysis consisted of a pretreatment to eliminate uninformative data according to a calculated threshold; t = m + 2 × sd . Transcripts above the calculated threshold were considered as present in the bovine, mouse, or Xenopus laevis oocytes. A second independent analysis with the NIA Array Analysis tool was also conducted ,54. BrieSubsequently, clones for which all three species and all replicates were above the calculated threshold were selected to generate a list of transcripts common in oocytes of all three species .To further characterize this subpopulation of transcripts common in oocytes of all three species, another classification was performed in order to identify the ones that are preferentially expressed in oocytes. This was done by comparing results from a previous study  where trData reproducibility was assessed by calculating the correlation coefficients between signal intensities across replicated experiments in a pair-wise manner with the NIA Array Analysis tool . For eacGAPD  amplifications were carried out as a positive control amplification to assure the quality of the cDNAs used for this experiment. GAPD PCR products were also labeled to use as a positive control of hybridization to assure the quality of the hybridization.To validate the presence of oocyte-expressed genes detected in all three species, RT-PCR analysis, using gene- and species-specific primers, were performed on three different selected candidate genes Table . BrieflyMV designed, optimized and carried out the protocols, analyzed the results and wrote the manuscript. CR was involved in the conceptualization of this project and critically reviewed the manuscript for important intellectual content. SM was involved in the microarray data analysis and critically reviewed the manuscript. MFP provided support in the preparation of this manuscript and critically reviewed the manuscript for important intellectual content. MAS was involved in the conceptualization of this project, the preparation of the manuscript and provided mentorship. All authors read and approved the final manuscript.


Studies in rodents show that olfactory processing in the principal neurons of olfactory bulb (OB) and piriform cortex (PC) is controlled by local inhibitory interneurons, and glutamate NMDA receptor plays a role in this inhibitory control. It is not clear if findings from studies in rodents translate to olfactory processing in nonhuman primates (NHPs). In this study, the effect of the glutamate NMDA receptor antagonist MK801 on odorant-induced olfactory responses in the OB and PC of anesthetized NHPs (rhesus monkeys) was investigated by cerebral blood volume (CBV) fMRI. Isoamyl-acetate was used as the odor stimulant. For each NHP, sixty fMRI measurements were made during a 4-h period, with each 4-min measurement consisting of a 1-min baseline period, a 1-min odor stimulation period, and a 2-min recovery period. MK801 (0.3 mg/kg) was intravenously delivered 1 hour after starting fMRI. Before MK801 injection, olfactory fMRI activations were observed only in the OB, not in the PC. After MK801 injection, olfactory fMRI activations in the OB increased, and robust olfactory fMRI activations were observed in the PC. The data indicate that MK801 enhances the olfactory responses in both the OB and PC. The enhancement effects of MK801 are most likely from its blockage of NMDA receptors on local inhibitory interneurons and the attenuation of the inhibition onto principal neurons. This study suggests that the mechanism of local inhibitory control of principal neurons in the OB and PC derived from studies in rodents translates to NHPs. The olfactory processing pathway in mammals begins at olfactory receptor neurons (ORNs) in nasal epithelium. Axons of ORNs project into olfactory bulb (OB) where they synapse with mitral cells. Mitral cells are the principal neurons in the OB and they send excitatory projections to the principal neurons  in the piriform cortex (PC) . The priBecause of the inhibitions on the principal neurons from local inhibitory neurons, odorant-induced olfactory responses in the principal neurons of the OB and PC depend on not only their excitatory inputs, but also the inhibitory inputs from local inhibitory interneurons. Studies from rodents show that glutamate NMDA receptor antagonists MK801 (Dizocilpine) or APV  can enhance the olfactory responses in the principal neurons by reducing the interneuron-induced inhibition on principal neurons. This reduction of the inhibition is from the blockage of the NMDA receptor-mediated transmission from the principal neurons to interneurons , 4, 9–11Even though NHPs have played a vital role in understanding the neural processing in humans, the olfactory processing in NHPs, especially the effects of pharmacological agents on the olfactory processing in NHPs have not been well studied. Techniques which can measure the olfactory response in the olfactory system of NHPs are limited. An fMRI technique which can measure the olfactory response in NHPs has been successfully developed recently, and the odorant-induced olfactory fMRI activations can be robustly observed in the OB of NHPs . This fMin vivo in rats . In . In anesTo examine the consistency of olfactory fMRI responses, the 4-h experiment period was divided into four 1-h periods. The time courses of fMRI signals in the OB and PC  from thet-tests, all p<0.04).To analyze the statistical significance of the MK801 effect on olfaction, the mean amplitudes of the fMRI activations during the odor stimulation were calculated from the time courses shown in The major findings of this study are: 1) Comparable to MK801 effect on the olfactory processing in rats , MK801 ep = 0.004), and the difference (9 breaths/min) was higher than the MK801-induced respiration rate increase (6 breaths/min). In this study, MK801 increased the respiration rate . RespiraAs indicated in In the OB, mitral cells  and granule cells (interneurons) form reciprocal dendrodendritic synapses , 3. OdorIn the PC, the principal neurons  receive excitatory input from mitral cells and inhibitory input from the local inhibitory interneurons . The neuIn this study, PC shows no fMRI activations without MK801 delivery Figs , which iAfter MK801 delivery, it interrupts the glutamate NMDA transmission to the interneurons, the NMDA receptor-mediated activations in the interneurons is blocked , 10, 32,NMDA receptor not only plays a role in the neural transmission between principal neurons and interneurons, in-vitro electrophysiology and local pharmacology studies suggest that it also plays a role in the neural transmission between the principal neurons of the different olfactory structures , 33, 34.We demonstrate for the first time that the odorant-induced olfactory responses in the OB and PC in NHPs are increased by MK801. The observation described in this report suggests that fMRI is a valuable tool to study the mechanism of the olfactory processing in NHPs.


Effective poststroke motor rehabilitation depends on repeated limb practice with voluntary efforts. An electromyography (EMG)-driven neuromuscular electrical stimulation (NMES)-robot arm was designed for the multi-joint physical training on the elbow, the wrist, and the fingers.To investigate the training effects of the device-assisted approach on subacute stroke patients and to compare the effects with those achieved by the traditional physical treatments.n = 14) or the time-matched traditional therapy . For the evaluation of the training effects, clinical assessments including Fugl-Meyer Assessment (FMA), Modified Ashworth Score (MAS), Action Research Arm Test (ARAT), and Function Independence Measurement (FIM) were conducted before, after the rehabilitation training, and 3 months later. Session-by-session EMG parameters in the NMES-robot group, including normalized co-contraction Indexes (CI) and EMG activation level of target muscles, were used to monitor the progress in muscular coordination patterns.This study was a pilot randomized controlled trial with a 3-month follow-up. Subacute stroke participants were randomly assigned into two groups, and then received 20-session upper limb training with the EMG-driven NMES-robotic arm  > 0.279] for both groups. Significant improvement in FMA wrist/hand was only observed in the NMES-robot group  after the treatments. Significant reduction in MAS wrist was observed in the NMES-robot group after the training  and the effects were maintained for 3 months. MAS scores in the control group were elevated following training , and remained at an elevated level when assessed 3 months later. The EMG parameters indicated a release of muscle co-contraction in the muscle pairs of biceps brachii and flexor carpi radialis and biceps brachii and triceps brachii, as well as a reduction of muscle activation level in the wrist flexor in the NMES-robot group.Significant improvements were obtained in FMA (full score and shoulder/elbow), ARAT, and FIM [The NMES-robot-assisted training was effective for early stroke upper limb rehabilitation and promoted independence in the daily living comparable to the traditional physical therapy. It could achieve higher motor outcomes at the distal joints and more effective release in muscle tones than the traditional therapy.ClinicalTrials.gov, identifier NCT02117089; date of registration: April 10, 2014. Stroke is one of the leading causes of permanent disability in adults . ApproxiEffective motor restoration after stroke depends on repeated and intensive practice of the paralyzed limbs with voluntary efforts , 12, 13.Rehabilitation robots can assist human therapists to conduct the intensive and repeated physical training with different numbers and sizes of electrical motors. Various robots have been designed for poststroke upper limb rehabilitation –21. AmonIn our previous works, we designed a series of voluntary intention-driven rehabilitation robotics for physical training at the elbow, the wrist, and fingers , 28–31. The integrated EMG-driven NMES-robot training system Figure  can assiFour-channel NMES was applied on the muscles of biceps brachii (BIC) during elbow flexion, triceps brachii (TRI) during elbow extension, flexor carpi radialis (FCR) for wrist flexion, and the last channel on both the extensor carpi ulnaris (ECU) and extensor digitorum (ED) for wrist extension and the associated hand open . The muscles of the ECU and ED are close to each other anatomically with narrow muscle bellies on the dorsal side of the forearm, and can be recruited together by just one-channel surface NMES . They weAssistance from both the robot and NMES modules was under the control of EMG signals from the target muscles and helped the participant to conduct the phasic and sequential limb tasks, i.e., (1) elbow extension, (2) wrist extension and hand open, (3) wrist flexion, and (4) elbow flexion. EMG-triggered control was adopted in this work, i.e., in each motion phase, once the EMG activation level of a driving muscle exceeded a preset threshold , the reThe study was approved by the Human Subjects Ethics Subcommittee of Hong Kong Polytechnic University and Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. We screened the stroke inpatients in the teaching hospital, and recruited participants with upper limb motor deficits satisfying the following inclusion criteria: (1) had a singular and unilateral brain lesion due to a stroke acquired within 4 months; (2) had standard medical care and sustained in a stable condition; (3) had enough cognition to understand the content or purpose of the study and follow simple instructions as assessed by the Mini-Mental State Examination (MMSE > 21) ; (4) motThe study was a pilot randomized controlled trial with a 3-month follow-up (3MFU). Inpatients after stroke were screened by a collaborative clinician according to the inclusion criteria 7–10 days before the start of the training, in a project period of 24 months. The potential participants were told about the training program of the study, and the recruited participants gave the written consent on the participation in the training program which could be either the device-assisted training or the traditional treatment before the randomization. Then, the recruited participants were randomly assigned into two groups, according to a computer-based random number generator, i.e., the computer program generated either “1”  or “2” (the control group) with an equal probability of 0.5 . The recruitment of the subjects was in a relatively sequential way due to the availability of the training device (only one set for the respective left and right sides) and the hospital stay of the participants in the management. Once both sides of the robotic arms were occupied for the training, the recruitment was suspended. In the subject screening period, the clinician also needed to take into account of the availability of the device for left or right hemiplegia in the coming possible NMES-robot-assisted training. Figure In the experimental group, i.e., the NMES-robot group, each participant received 20-session upper limb training assisted by the NMES-robot system with an intensity of 5 sessions/week, 1 session/day (from Monday to Friday), finished in one month. In each session, a participant was instructed to conduct the device-assisted and repeated limb motions, i.e., (1) elbow extension, (2) wrist extension and hand open, (3) wrist flexion, and (4) elbow flexion for totally 40 min. There was a break of 10 min after a practice of 20 min to avoid the muscle fatigue. In this work, the training loads between the NMES-robot group and the control group were matched by the time and the frequency of the sessions for the upper limb training. The upper limb rehabilitation time of a session for the NMES-robot group was deducted from the routine upper limb training  by human therapists. The routine upper limb treatment included muscle stretching, passive/assistive ROM and occupational treatments such as feeding/eating, grooming practices. In the practical operation in this work, the participants in the NMES-robot group were transferred to another treatment room to receive the 40-min device-assisted training, and then returned to receive the rest of the routine physical treatments on the upper limb. For most of the participants in the NMES-robot group, they only practiced the muscle stretching and passive ROM after returned from the NMES-robot training for around 10–15 min, due to the fatigue experienced in the upper limb. The participants in the control group only received the routine rehabilitation therapies on the upper limb .In this study, functional evaluation for each participant on their paretic upper limbs were scored by the following assessments: the FMA , applied for performance-based measurement of motosensory functions on the poststroke hemiplegia ; the ActIn each training session for the NMES-robot group, a participant was first instructed to perform a bare-arm evaluation task before the device-assisted rehabilitation training, in order to trace the evolution of the muscle coordination during the recovery as we did in the robot-assisted upper limb training on chronic stroke previously . The evai, EMGi(t) was the EMG linear envelope with respect to the maximal value recorded during the bare-arm evaluation tasks and maximum voluntary contractions in each session, and T was the length of the signal as we did previously  normalized EMG activation level of each muscle; and (2) normalized co-contraction index (CI) between the muscle pairs. The processing methods of the normalized EMG activation level was calculated as follows, i.e.,eviously , 28. In eviously , 28.ijA(t) represented the overlapping activity , EMGj(t)]) of the EMG linear envelopes for muscle i and j, and T was the length of the signal, EMGi,j(t) are the EMG envelopes as in Eq. The CI between a pair of muscles could be expressed as:t-test with an insignificant statistical difference (P > 0.05) on all clinical assessments  across the 20 sessions were also analyzed by one-way ANOVA for the investigation of recovery process across the whole training sessions in the NMES-robot group. The primary outcomes of the study were the FMA and MAS clinical scores; and the other clinical scores and EMG parameters were the secondary outcomes. It was because FMA could reflect task-specified voluntary motor functions in the whole upper limb and MAS could reflect the variation of muscle spasticity at different joints in the upper limb compared to other clinical scores. The levels of statistical significance were indicated at 0.05, 0.01, and 0.001 in this study.The baselines of the two groups were first compared by independent n = 14) and the control group (n = 10). The demographic data of the participants after the randomization are presented in Table We screened 78 stroke inpatients in the wards of the hospital, and 54 of them did not meet the inclusion criteria with single or multiple reasons of 1) unstable clinical symptoms for continuous and long-term physical training, (2) secondary stroke, (3) cognitive impairment, (4) severe motor impairment (full FMA < 15), and (5) no detectable EMG in a driving muscle (<3 SD of the baseline). A total of 24 participants fulfilled the inclusion criteria and were recruited in this study. They were randomly assigned into two groups: the NMES-robot group , and the improvement could be maintained for 3 months .Figure P < 0.001, EF = 0.542 two-way ANCOVA, Table P ≤ 0.001, one-way ANOVA with Bonferroni post hoc test).Function Independence Measurement scores in both the NMES-robot and control groups are shown in Figure P < 0.001, EF > 0.176, Table P < 0.01, EF > 0.152, Table P < 0.05, one-way ANOVA with Bonferroni post hoc tests, Table P = 0.048, EF = 0.145, Table P > 0.05, one-way ANOVA, Table P < 0.01, EF > 0.359, one-way ANCOVA, Table P < 0.01, EF > 0.334, one-way ANCOVA, Table Figure P < 0.05, one-way ANOVA with Bonferroni post hoc tests). The EMG activation levels increased in the first few training sessions and reached the peak around the third session. Subsequently, the values decreased in the following 17 sessions and finally reached a plateau in the last five training sessions. No significant variation was observed in other target muscles . Figure P < 0.05, one-way ANOVA with Bonferroni post hoc test). The CI values of both muscle pairs reached the peak within the first eight training sessions and then continually decreased in the following process, then reached a steady level in the last three sessions. No significant change was observed in CI values of other muscle pairs.Figure The results of this study indicated that in the early stage after stroke, motor function in the paretic upper limbs of stroke participants could be significantly improved through both traditional rehabilitation treatment and upper limb training by the EMG-driven NMES-robotic system. The ARAT and FIM scores suggested that the effects of early intervention using EMG-driven NMES-robot training were comparable to the effects of traditional treatments; these findings reflected the improved upper limb function, particularly, in the hand (by ARAT), and improved independence of ADL (by FIM) observed in this study. Although no specific tasks were assigned to the finger joints in this study, the increase in the ARAT scores after NMES-robot training also indicated the voluntary motor improvement in the fingers after the treatments. We noticed that there was no significant improvement in the FIM scores observed after NMES-robot training for chronic stroke patients in our previous study . HoweverAs indicated by the FMA scores, the NMES-robotic arm developed in this study could assist stroke patients to improve the motor function of their entire paretic upper limb, with voluntary effort. Both the NMES-robot training and traditional physical therapies improved the participants’ motor function at the shoulder and elbow, and this improvement was maintained even after 3 months. However, the NMES-robot group achieved greater improvement in the FMA shoulder/elbow and wrist/hand scores than did the control group. Two possible reasons for the improvements in the entire upper limb in the NMES-robot group, especially the shoulder (no actuated assistance in the training), motor function are as follows: (1) The participants’ shoulder-related muscles for arm lifting were practiced during the training process supported by the hanging system used in this study and (2) when the muscle around a joint was trained, the adjacent proximal joint would be simultaneously improved, as indicated in our previous study ; accordiAfter the traditional rehabilitation treatments, the participants’ muscle tone (spasticity) increased significantly at all three parts . This could be because of the following: (1) the muscle tone was gradually generated through spontaneous recovery process, following the pathological sequence after stroke ; 2) com com2) reThe improvement in upper limb motor function in the NMES-robot group was reflected by the clinical scores, and the session-by-session recovery progress was revealed through the EMG parameters. The reduced EMG activation levels of FCR implied a release of muscle spasticity at the wrist, which was consistent with the variation in the MAS wrist scores. Most of the patients reached a steady state after the 15th training session. The reduction of FCR was also related to the decrease in the CI values of FCR&BIC, indicating a release in the co-contraction patterns between the elbow and wrist joints. These joints could be moved more independently during arm withdrawing/flexing motions. In addition, a significant reduction in CI values between the BIC&TRI muscle pair was observed, suggesting improved coordination between the flexors and extensors at the elbow joint and an improved independence in muscle contraction over the 20 training sessions. The EMG activation levels of the FCR increased in the first 3–4 training sessions, and the CI values of BIC&TRI muscle pairs reached the peak within the first eighth training session. This was reasonable because in subacute stroke, most of the patients experience muscle weakness in the very beginning, and the muscle strength then recovers through both spontaneous processes and physical training. In addition, the participants needed to adapt to the training process in the first several training sessions. The results of the EMG parameters suggested that NMES-robot training could help release the muscle spasticity and promote muscle coordination within and across different joints effectively, particularly at the wrist in this work.The main limitation of this study is the small sample size. Despite the relatively small populations recruited, we observed consistent results on the motor improvements achieved in the NMES-robot group by clinical assessments and EMG parameters. The EMG parameters were only recorded for the NMES-robot group to provide the understanding on the evolutionary process of the muscular activities under the training program. Unbalanced arms were obtained after the randomization in this work, mainly due to the limited project period and the small sample size recruited in a relatively sequential way. Randomized clinical trials with larger scales  will be conducted to consolidate the rehabilitation effectiveness of the EMG-driven NMES-robot-assisted upper limb training in the future.In this work, the EMG-driven NMES-robot arm was applied for multi-joint coordinated upper limb rehabilitation on subacute stroke participants in comparison with the traditional physical therapy. Both of the treatments could significantly promote the independence in daily activities with comparable intensities. The NMES-robot-assisted training could be more effective in releasing muscle tones and in improving the muscle coordination in the whole upper limb, because of the intensive practices on both the proximal and the distal joints delivered in a short period of rehabilitation. All the training effects achieved by the NMES-robot-assisted rehabilitation could be maintained for 3 months after the training. The NMES-robot-assisted upper limb training could be complementary to the traditional manual training to cope with the shortage of the human rehabilitation professionals in the industry.The study was carried out in accordance with the human ethic guidelines of the Human Subjects Ethics Subcommittee of Hong Kong Polytechnic University and Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. All participants recruited in the study gave written informed consent in accordance with the Declaration of Helsinki before the start of the training. The medical status of the participants was monitored by the routine management in the hospital.QQ and QL contributed in the NMES-robot arm assisted training experiment, data collection and analysis, and manuscript drafting. SN and WP contributed in the clinical trial design and subject management. YZ contributed in the data analysis. XH conceived of the study and coordinated the whole project, including the clinical trial design, human subject experiments, and manuscript drafting.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.


Antiretroviral therapy cannot cure HIV-1 infection due to the persistence of a small number of latently infected cells harboring replication-competent proviruses. Measuring persistent HIV-1 is challenging, as it consists of a mosaic population of defective and intact proviruses that can shift from a state of latency to active HIV-1 transcription. Due to this complexity, most of the current assays detect multiple categories of persistent HIV-1, leading to an overestimate of the true size of the latent reservoir. Here, we review the development of the viral outgrowth assay, the gold-standard quantification of replication-competent proviruses, and discuss the insights provided by full-length HIV-1 genome sequencing methods, which allowed us to unravel the composition of the proviral landscape. In this review, we provide a dissection of what defines HIV-1 persistence and we examine the unmet needs to measure the efficacy of interventions aimed at eliminating the HIV-1 reservoir. Early s T cells –8. Howev T cells –17. ThisPersistent HIV-1 is an all-encompassing term that includes all forms of integrated proviral DNA that persist in infected individuals on the time-scale of years despite fully suppressive ART . The degree to which post-transcriptional blocks to HIV-1 gene expression enforce a state of latency in vivo is not yet completely understood, but is under active investigation.Given the complexity of HIV-1 persistence, it is critical that we employ accurate and precise definitions in our discussion. HIV-1 reservoir(s). While several cell populations have been proposed as HIV-1 reservoirs, the only population yet convincingly shown to be a long-term reservoir for HIV-1 are resting memory CD4+ T cells harboring latent but replication-competent HIV-1. We term these cells the latent reservoir. In Fig. While many forms of persistent proviral DNA may impact the health of infected individuals, only replication-competent proviruses capable of viral rebound after ART cessation are a barrier to curing HIV-1 infection , 6. Coll+ T lymphoblasts were the preferred target of infection. Activated CD4+ T lymphoblasts are short-lived effector cells of the immune system, and landmark viral dynamics studies indicated that most productively infected cells displayed a short half-life [Early studies defining the tropism of HIV-1 indicated that activated CD4alf-life –32. Thes+ T cells from viremic donors, but that these quiescent proviruses could be induced by activation to produce infectious virus [Nonetheless, experimental observations early in the HIV/AIDS epidemic were consistent with some form of HIV-1 persistence. A number of studies indicated that the fraction of mononuclear cells which contained proviral DNA far exceeded those that were positive for HIV-1 RNA , suggestus virus –5. Follous virus , 8, 34. + T cells harboring latent HIV-1: the quantitative viral outgrowth assay (QVOA) [+ T cells are purified from the blood of HIV+ donors on suppressive ART and then plated in a limiting dilution format. Because these resting cells do not produce virus without T cell activation [Critical to these early discoveries was the method developed to quantify the frequency of resting CD4y (QVOA) . In this+ T cells to reverse HIV-1 latency, facilitation of outgrowth of reactivated replication-competent proviruses, and detection of that outgrowth. Various modifications of each of these steps have been successfully implemented.In essence, successful performance of the QVOA requires complete and reproducible activation of input CD4+ T cells to demonstrate the existence of quiescent but replication-competent proviruses. These resting memory CD4+ T cells were defined by the absence of accepted cell-surface markers of T cell activation . However, given that the latent reservoir in resting CD4+ T cells has now been established as a mechanism of HIV-1 persistence, the purification of resting memory CD4+ T cells is not required. In fact, in some instances the purification of resting memory CD4+ T cells for QVOA analysis may not be advisable . The QVOA is now routinely performed on bulk CD4+ T cells from HIV-1+ donors. In this situation, some infected CD4+ T lymphoblasts may also be present. Most of these cells have a short half-life, but in patients on ART they are likely derived from in vivo activation of infected resting CD4+ T cells.In the original studies, the QVOA was performed on resting memory CD4+ T cells via stimulation with the mitogen PHA and allogeneic, irradiated feeder PBMCs. These conditions were selected on the basis of previous reports that they induced activation of roughly 100% of resting T cells [The standard QVOA protocol , 35, 36  T cells . However T cells , CD3 and T cells , and pho T cells . A large T cells . This al T cells .After successful reactivation of latent proviruses via T cell stimulation, released virus is propagated by the addition of cells susceptible to HIV-1 infection. This step is critical, as the exponential outgrowth of reactivated proviruses definitively establishes these proviruses as replication-competent. While PHA-stimulated, CD8-depleted PBMCs obtained from uninfected donors have traditionally been used to promote viral outgrowth, alternatives have been described. These include the use of bulk PBMCs from uninfected donors  and the Replication-competent proviruses must then be detected to determine the frequency of latent HIV-1 infection. Traditionally, the QVOA is performed in a limiting dilution format and viral outgrowth is detected via p24 antigen ELISA, with the frequency of latent infection calculated based on which well dilutions are positive for outgrowth using limiting dilution . Using tTo reduce the time required to complete the QVOA, alternative methods for detecting viral outgrowth have been described. RT-qPCR detection of HIV-1 RNA in culture supernatant at assay day 7 was shown to be comparable to p24 antigen ELISA at day 14 at identifying outgrowth-positive wells , signifi+ T cells, but the assay has also been modified to enable the interrogation of latent HIV-1 in other cell types. A viral outgrowth assay for latent SIV in resting macaque CD4+ T cells has been previously reported [The QVOA enabled the identification of the latent reservoir in resting memory CD4reported , 47, andreported . While t+ T cells from HIV-1 infected donors on ART are subjected to multiple rounds of T cell activation and virus expansion. In most cases, restimulation of cells from wells negative for outgrowth in the initial round of T cell activation increased the total number of latent proviruses detected by the QVOA. These findings demonstrate that not all replication-competent proviruses are reactivated by a given round of T cell activation, and suggest that cells carrying replication-competent HIV-1 can be activated and proliferate without producing virus [+ T cells harboring replication-competent proviruses may not respond uniformly to a given round of T cell activation. These findings are consistent with other recent reports in vivo [While the QVOA provides definitive evidence of the persistence of latent HIV-1, recent studies demonstrate that the assay does not capture all replication-competent proviruses in the latent reservoir. Two recent studies by our group demonstrate that additional viral outgrowth can be induced with multiple rounds of T cell activation , 17. In ng virus , 17, 49. in vivo  and ex v in vivo . The fin in vivo . This ha+ T cells are present at a higher frequency than detected by the standard QVOA. The standard QVOA can thus be considered a definitive minimal estimate for the size of the latent reservoir, capturing only a fraction of the latent reservoir with a single round of stimulation and outgrowth. Multiple-stimulation QVOA captures an ever-increasing fraction of the latent reservoir with each successive round of T cell activation and outgrowth. It is conceivable that after a sufficient number of rounds of T cell activation and outgrowth, the multiple-stimulation QVOA would capture all latently infected cells, but this is not experimentally feasible. Critically, these findings indicate that the latent reservoir is not well defined by the proviruses that can be reactivated and captured in the standard or multiple-stimulation QVOA. Rather, the latent reservoir may be better defined as those proviruses that are genetically intact and thus capable of replication in vivo. Under this definition, the latent reservoir can be best interrogated using molecular approaches that distinguish genetically intact proviruses.Multiple-stimulation QVOA studies have demonstrated that latently infected CD46 resting CD4+ T cells contain HIV-1 proviruses, but only a fraction of these proviruses are replication-competent HIV-1 [+ T cells. This large difference fueled interest in better understanding the proviral landscape. Several methods have been developed to capture the sequence of individual proviruses to distinguish defective proviruses from those intact ones that are potentially capable of replication.In infected individuals on ART, 300/10nt HIV-1 . The freThe importance of such sequencing based approaches is underscored by the surprising results of two recent studies that used a novel and unbiased single genome amplification method to define the sequence landscape of persistent proviruses and examine the nature of defects present in replication incompetent proviruses , 13. In Using this initial single proviral-genome sequencing method, we examined the sequences of the proviruses present in QVOA wells that were negative for viral outgrowth . The vas+ T cells taken directly from infected individuals on ART. In this study, the composition of the proviral landscape was compared between viremic individuals and individuals who initiated ART during either acute or chronic infection [A follow-up study employing the same method interrogated the proviral genomes from HIV-1 infected individuals present in unstimulated resting CD4nfection . Even inThe relevance of defective HIV-1 proviruses to the development and assessment of cure strategies is currently being investigated. A recent study demonstrated that hypermutated proviruses and proviruses that have defects in the major splice donor site and/or the packaging signal can be transcribed and that the resulting RNAs can spliced and translated . AdditioFull-length proviral sequencing provides many advantages compared to other methods that probe sub-genomic regions. As discussed above, the proviral landscape is extremely heterogeneous due to HIV-1 intra-host diversity, internal deletions, and deamination-induced hypermutation. As a consequence, all sub-genomic amplification strategies introduce bias, resulting in sequence data that may not be representative of the actual population of HIV-1 proviruses present in a given sample. In particular, sub-genomic PCR studies can vastly overestimate the frequency of latently infected cells by detecting proviruses with lethal defects outside of the region amplified. They also underestimate the total number of infected cells because they fail to amplify proviruses with deletions overlapping the region amplified. Some sub-genomic sequencing assays can overestimate the fraction of identical sequences resulting from clonal expansion, especially in samples with reduced HIV-1 diversity, such as culture supernatants from QVOA or cells from patients treated during the early stages of acute infection .de-novo assembly mapping reads against a reference genome. The output proviral sequences are then sorted based on the presence of defects that would preclude replication. This new approach is cost-effective and it simplifies the sequencing of difficult regions compared to Sanger approaches while yielding enough sequencing data to study the composition of the proviral landscape in CD4+ T cell subsets of interest. The study from Lee and colleagues applied this new sequencing approach to polarized phenotypic subsets of cells sorted based on intracellular cytokine staining [+ T cells, which predominate in ART-treated subjects. Recently, Hiener et al. used the FLIP assay  to investigate the composition of proviral DNA in CD4+ T cells across the differentiation subsets  [+ T cells showing the highest levels of intact genomes, contributing to a median of 75% of all intact proviruses. Surprisingly, central memory CD4+ T cells had the lowest levels of intact proviruses. Follow-up studies may be needed to investigate whether sub-optimal sampling and margination of central memory CD4+ T cells in secondary lymphoid tissues could have affected these observations.On the other hand, full-length sequencing is time consuming and labor intensive, often requiring custom design of additional primers and multiple sequencing reactions. Two recent studies applied Illumina-based next generation sequencing (NGS) to full-length HIV-1 proviral sequencing , 16. Bri Memory) . This asThe studies from Lee et al. and Hiener et al. pioneered the use of NGS to characterize HIV-1 persistence and contributed to our understanding of the complexity of the proviral landscape. However, the composition of the HIV-1 reservoir is extremely variable, and it can be difficult to generalize the findings observed in a limited number of subjects in a single time point. In addition, the abundance and species richness of HIV-1 infected clones is still unpredictable and a small number of highly expanded clones can skew the global picture within each patient at a given time point.The development of assays that measure HIV-1 persistence has been invaluable in furthering our understanding of the impact of ART on viral replication and the nature of the latent reservoir. However, current methods have several limitations and the quantification of the latent reservoir remains challenging.Limit of detection and dynamic range are two of the biggest constraints of current assays, especially in the context of interventions based on purging of the reservoir or on very early treatment. Hematopoietic stem cell transplantation (HSCT) can lead to dramatic reductions in the size of the latent reservoir (up to 4 logs) –57, makiThese difficulties can be extrapolated to larger clinical trials involving new molecules aimed at reducing the latent reservoir, such as latency reversing agents (LRA), therapeutic vaccines, CAR T cells, and/or ADCC-competent broadly neutralizing anti-HIV-1 antibodies. In such trials, both the magnitude of changes in reservoir size and the scale of response to a given treatment might vary across study participants, requiring large sample sizes to demonstrate efficacy. In this context, the assay(s) used to quantify HIV-1 persistence would need adequate throughput to process hundreds of samples and be capable of reliably detecting even modest changes in reservoir size. As discussed above, approaches that allow the quantification of replication-competent proviruses are labor and time consuming, which makes them impractical for large cohort studies. PCR-based assays that measure HIV-1 DNA have a higher throughput, but cannot distinguish replication competent from defective proviruses. Infected cells carrying intact versus defective proviruses may respond very differently to a given intervention, and total HIV-1 DNA data could be difficult to interpret or even misleading, given the “background noise” of defective proviruses.Continuous efforts are needed to develop new approaches that can measure and characterize that true “minimal residual disease” that must be targeted by future curative strategies. We propose that the next generation of assays designed to measure the HIV-1 reservoir should have a low and “modular” limit of detection, a wide dynamic range, the capacity to distinguish intact proviruses, and should be scalable for studies with large samples sizes. As the need for such assays in clinical trials grows, rigorous studies to qualify or validate such assays will be important, and the clinical utility of any given assay will need to be formally established. It is our view that efforts to design and implement new assays for the HIV-1 latent reservoir will ultimately help to enable the discovery and development of new therapeutics aimed at curing the infection.


Glyco-gold nanoparticles combine in a single entity the peculiar properties of gold nanoparticles with the biological activity of carbohydrates. The result is an exciting nanosystem, able to mimic the natural multivalent presentation of saccharide moieties and to exploit the peculiar optical properties of the metallic core. In this review, we present recent advances on glyco-gold nanoparticle applications in different biological fields, highlighting the key parameters which inspire the glyco nanoparticle design. Carbohydrates are well recognized as crucial biomolecules for their capability to produce an enormous number of biocodes which are translated into their ability to promote or suppress a plethora of biological events . NeverthThe increasing ability to manipulate material at the nanosize allowed the development of many glyco nanoparticles helpful in a wide range of applications, from the drug delivery to the imaging –7. AmongThe present review focuses its attention only toward the synthesis of gold-based nanoparticles coated with carbohydrates. A first introduction related to the main protocols to produce glyco-gold nanoparticles is followed by a section where the main parameters which should be taken into account for designing the best performing nanosystem are discussed. Then, three sections summarize the achievements and ongoing researches published in the last four years, from 2013 to 2016 with a special focus on glyco-gold nanoparticle applications .Despite the great inertness of this noble metal, Au can form stable bonds with sulfur-containing compounds  –10 and i3+ reduction by means of NaBH4 in the presence of thiol-ending neoglycoconjugates. Following this approach, a rapid synthesis of AuNPs characterized by elevated glycan density on the NP surface and a 1–10 nm diameter range can be obtained . Th. Th88]. E.coli-specific killing GAuNP nanocomposite, carrying either a targeting agent towards E. coli pili and an antibacterial polymer, resulted able to bind E. coli specifically and to kill the attached bacteria, by enhancing the local concentration of biocidal agent on the bacterial surface [In search of novel antimicrobial antigens, chitosan-streptomycin GAuNPs (CA NPs), i.e., carbohydrate-antibiotic conjugates, were prepared and resulted able to overcome antibiotic resistance of microbial biofilms, since CA NPs render streptomycin more accessible to biofilms, thereby more available to interact with biofilm bacteria . Similar surface .GAuNPs find interesting application also in the treatment of HIV infection. β-D-Glucose coated AuNPs have been decorated with the antiviral drugs abacavir (ABC) and lamivudine (3TC) and used as delivery systems. These co-drug structures were able to release the drugs in acidic conditions to inhibit viral replication in cellular assays .N-isopropylacrylamide) (PNIPAM)-coated AuNPs, compatible with SAIB matrix. The nanogel had good opacity for 2D X-ray visualization and was tested in vivo in immunocompetent healthy NMRI-mice and in humans [3+ have been developed and used as magnetic resonance imaging (MRI) contrast agents. Recently, gold based nanosystems containing Gd3+ were developed. Arus et al. described the synthesis of water dispersible GAuNPs decorated with Gd complexes [3+ acted as MRI contrast agent. The authors settled an ex vivo experiment which let them to use small amounts of contrast agent , avoiding toxicology and in vivo test. Moreover, GAuNPs showed their extreme versatility in a very interesting paper reported by Penadés and collaborators [GAuNPs represent a promising candidate for developing theranostic agents, able to carry drugs to a selected tissue and to act as a sensitive probe. In fact, the metallic core of GAuNPs has been exploited to design and develop promising early detecting agents of cancer or inflammation. In this contest, Andresen and co-workers produced a nanogel injectable in humans to detect cancer cells. The nanogel was composed by sucrose acetate isobutyltyrate (SAIB) and poly functionalized AuNPs were found able to elicit a pro-inflammatory response in dendritic cells via interaction with the lectin DC-SIGN, as indicated by the up-regulation of several maturation markers and increased secretion of pro-inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) [f, a five-membered form of galactose found in nonmammalian pathogenic species, i.e., the fungus Aspergillus, is able to modulate the innate immune response via dendritic cells, suggesting Galf-GNPs as versatile tools in functional studies to address the role of Galf in host-pathogen interaction.Glycan-conjugated globular shaped nanoparticles have a higher surface area to volume ratio in comparison to other glycoclusters, i.e., glycolipid micelles and glycoproteins, which allows an ordered, controlled and stable tridimensional presentation of the ligands, providing higher ligand binding capacity ,96. For  (TNF-α) . These dx trisaccharide [DC-SIGN targeting through GAuNPs has also been exploited differently in a study on GAuNPs functionalized with α-fucosylamide, an efficacious synthetic DC-SIGN ligand, analogue of the natural fucose-containing Lewisccharide . This paAuNPs engineering is offering significant contribution to immunology also in vaccine development. The repetitive antigen display is the key point related to this nanotechnology-based approach, which aim to trigger the production of specific and functional antibodies that prevent initial infection limiting pathogen/viral dissemination. Recent publications suggest that many different aspects are becoming clear and have to be underlined.9GlcNAc2 was identified as a conserved motif on the HIV surface: this result stemmed from the observation that partial structures of this glycan could be recognized by broadly neutralizing antibodies, found in infected patients. These data suggested the use of these self-glycans for the design of effective vaccine and inspired the preparation of new generation GAuNPs, coated with synthetic partial structures of Man9 multimerized on the same GAuNP, which provided better binding to the anti-HIV antibody 2G12 compared to GAuNPs carrying only one individual oligomannoside [The design of anti HIV-1 vaccines depends on the identification of sugar epitopes of the surface envelope glycoprotein of HIV-1, capable of eliciting a protective response. GAuNPs coated with high-mannose type oligosaccharide of HIV-1 gp120, were prepared as glycoconjugate systems able to mimic the natural presentation of gp-120 high-mannose glycans. It was demonstrated that they were able to inhibit DC-SIGN-mediated HIV-1 infections and to interfere with the antibody/gp120 binding process. Most of these systems proved able to elicit the production of carbohydrate-specific antibodies in animals, though the generated IgGs turned out to be unable to neutralize the virus. More recently, the Manannoside . This reA different approach towards the development of a synthetic HIV vaccine candidate based on GAuNPs, exploit the glyco-nanosystem as a tool which modulate and control the biologically active conformation of a significant epitope . In thisAnother example on the importance of the way in which glycoconjugates are presented through NPs, regards the design and construction of GAuNPs decorated with mucin-related glycans for cancer immunotherapy . A monomIn a different example, Barchi and co-workers focused on the Thomsen Friedenreich tumor-associated antigen (TFag) and synthesized TFag-amino acid-coated AuNPs by means of a thiol-polyethylene glycol spacer. Among the synthesized GAuNPs, TFag-Thr-AuNPs showed, in vitro, an increased cytotoxic activity toward lymphoma cells, resulting active at a very low micromolar range . With thThe potential of AuNPs as antigen carriers for the development of synthetic vaccines is still being investigated in the context of bacterial infections. NPs are indeed promising vector systems to explore vaccination against infectious diseases.91-98Listeria peptide antigen as a system to target DCs, since Listeria protection is largely dependent on T-cells activation [Listeria. The system was effective in mice immunization either using a DC vaccine or a standard immunization approach. The data were followed by a study on pregnant mice vaccination which demonstrated the ability of this AuNP based vaccine system to protect neonates born to vaccinated mothers from the bacteria [Recently, a study on the identification of an optimal vaccine formulation to protect against listeriosis, proposed the use of GAuNPs loaded with glucose and the LLOtivation . The prebacteria .In another study, the impact of multivalency on the ability of GAuNPs to induce immune cell responses in vitro was studied using highly pure NPs coated with non-immunoactive mono- and disaccharides related to the capsular polysaccharide of serogroup A of Neisseria meningitides . The resS. pneumoniae (Pn) CPS type 19F and 14 [In another recent study on the development of synthetic nanosystems as potential carbohydrate-based vaccine, GAuNPs were loaded with synthetic oligosaccharide fragments corresponding to the repeating units of F and 14 . This neThe GAuNP technology has also been applied in the regulation of important genes of the apoptotic pathway. New GAuNP systems displaying cMyc targeting siRNA resulted capable of inducing apoptosis via hyperactivation of cell death receptors and caspase pathways .Glyco-gold nanoparticles represent one of the most versatile, exciting and promising hybrid nanosystems. Combining the vast properties of the noble metal at its nano-size with the fundamental biological role covered by carbohydrates, GAuNPs hold great potentiality in bio-nanomedicine. In this review, we have attempted to summarize the last results obtained in this field, anticipating the numerous applications with a detailed description and implication of each part of the hybrid system. A careful GAuNP design must take into consideration all the parameters which characterize the final glyco-nanoparticle. Well planned GAuNPs offer a unique chance to researchers to mimic the nature’s glycocalix and to explore the world of weak carbohydrate interactions, opening the way to innovative diagnostic tools or new therapeutic or theranostic agents.


Here, we explore the possibility to further increase the fine-mapping resolution of these QTL via a pedigree-based imputation strategy that aims to better capture the genetic diversity in the divergently selected, but outbred, founder lines. The founders of the intercross were high-density genotyped, and then pedigree-based imputation was used to assign genotypes throughout the pedigree. Imputation increased the marker density 20-fold in the selected QTL, providing 6911 markers for the subsequent analysis. Both single-marker association and multi-marker backward-elimination analyses were used to explore regions associated with 56-d body weight. The approach revealed several statistically and population structure independent associations and increased the mapping resolution. Further, most QTL were also found to contain multiple independent associations to markers that were not fixed in the founder populations, implying a complex underlying architecture due to the combined effects of multiple, linked loci perhaps located on independent haplotypes that still segregate in the selected lines.The Virginia chicken lines have been divergently selected for juvenile body weight for more than 50 generations. Today, the high- and low-weight lines show a >12-fold difference for the selected trait, 56-d body weight. These lines provide unique opportunities to study the genetic architecture of long-term, single-trait selection. Previously, several quantitative trait loci (QTL) contributing to weight differences between the lines were mapped in an F Due to their multi-factorial nature and despite the ability to obtain data on both genome-wide genetic markers and phenotypes from large numbers of individuals, it is challenging to disentangle their genetic architecture by analyzing data from the commercial populations. An alternate strategy is to make use of experimental populations resulting from long-term selection experiments, where the focus has been to develop divergent lines from a common base population using more coherent selection criteria. Such populations will display larger phenotypic differences than populations subjected to composite, commercial breeding programs and hence facilitate in-depth studies of the genetic basis underlying the selection response and general genetic architecture of these traits , divergent, single-trait selection for 56-d high (HWS) or low (LWS) body weight in chickens  mapping in an Ftercross . Explora2 intercross, we developed, genotyped and phenotyped for body weight at 56 d of age (BW56), a nine-generation AIL. This large AIL originated from the same founders as the F2 intercross, but was selectively genotyped at a higher resolution (∼1 marker/cM) in nine QTL .To replicate and fine-map the body weight QTL inferred in the Fe.g., QTL introgression . In total, the AIL population consisted of 1536 F0–F8 individuals with complete records on pedigree and genotypes (see Genotyping), and 1348 F2–F8 individuals with juvenile body weight (BW56) records.An AIL was founded by reciprocal crosses of 29 HWS and 30 LWS founder birds from generation 40 (The complete AIL pedigree (1536 birds) had earlier been genotyped in nine selected QTL for 304 SNP-markers that passed quality control as described in galGal3). In the ordered marker set, the SNP-chip markers were evenly distributed in the intervals between the sparser set of markers genotyped across the entire AIL.All genotyped markers in the QTL  were firUsing the software ChromoPhase  in the scan, the marker with the strongest association was added as a covariate in the model. This procedure was repeated until no additional significant markers were detected. The markers from this analysis with an allele-frequency > 0.10 in the population were subjected to the full backward-elimination analysis described in the next section.Next, a forward-selection analysis was performed by scanning across all markers within each QTL using Model 1. If any of the markers were nominally significant  level. The analysis was performed using bootstrapping with 1000 resamples. Markers with an RMIP (Resample Model Inclusion Probability) > 0.46, as suggested for an AIL generation F18  as imple-package . The addFile S1 contains detailed descriptions of all supplemental data files. File S2 contains the genotypes, File S3 the pedigree, and File S4 the phenotypes.Genotype, phenotype, and pedigree data are included in the supplemental files. Supplemental Material, We compared the results of the imputation-based association analyses with the previously reported results from the linkage-based analysis of the same nine QTL in Growth9 on GGA7   increases weight by 18.9 (SE 5.6) g, and it still segregates at an intermediary frequency (P = 0.50) in the LWS line. Previously, IGFBP2) gene in the middle of the major association peak at 24.8 Mb. In addition to the strong association around 24 Mb, the association analysis also highlights two additional regions (centered around 18 and 29 Mb). A single imputed marker  it did not contribute to the founder line difference. Two linked imputed markers are kept in the third region , but segregates at an intermediary frequency in the HWS line (P = 0.61). At the second marker, the major allele in the HWS (P = 0.74) segregates at an intermediary frequency in the LWS (P = 0.41). Due to the close linkage between the associated markers, it is difficult to interpret their individual effects and disentangle whether the detected associations are due to the LD-pattern of multiple closely linked loci or a single locus with multiple segregating alleles. The peak in the F2 QTL overlaps the major 23.7–26.4 Mb association peak detected in this analysis . The strongest of these association-peaks was located near the peak detected using the earlier linkage-based analysis. Several of the significant associated markers were located in this region (173.6–175.3 Mb). A candidate gene for growth, Asparagine-linked glycosylation 11 homolog gene (ALG11), is located at 174.6 Mb and has a strong mutation in its regulatory region , with the last genotyped marker (rs14196021) located at 65.5 Mb. The distance between the markers, and the region of low association between them in the single-marker analysis  is not mapped to the chicken genome and no significant signal was found using a denser marker-map by Growth2 QTL in the F2 analyses  in the multi-locus analysis and this peak was inside the original F2 QTL [101.6–131.9 Mb;   amounting to about a fifth of that estimated in the F2 population  amounting to about a fifth of the 92 g estimated in the original F2 analysis , the association-peaks are shifted when comparing results from both studies. Further work is needed to explore whether this reflects separate loci with distinct genetic architectures that could only be detected with the respective methods, or if they reflect a signal of the same underlying causal locus.Overall, the location of the association signals in this study overlapped well with the top signals in the earlier linkage analyses. However, in two of the QTL , the current fine-mapping analysis suggests that one, or both, founder lines segregate for multiple functional alleles in the QTL. To compare the estimates from the line-cross analysis in the F2 and the association analyses in the AIL F2–F8 generations, they need to be compared using a common reference. Here, we did this by estimating how much the associated markers in each QTL were expected to contribute to the founder line difference under the assumption that they act completely additively. Under this assumption, their contribution would equal twice the sum of the allele-substitution effects of the markers in a QTL weighted by their respective allele-frequency differences between the founder lines. That is, if the markers are fixed for alternative alleles in the founder lines they would contribute two allele-substitution effects to the founder line difference, whereas they would contribute nothing if the allele was present at equal frequencies in both founder lines. This estimate is conservative as, for example, dominance leads to an underestimation of the contribution of the locus. This as, in the presence of a dominant allele, one line will not need to be fixed to contribute most of its effect because this is also displayed in the heterozygotes for that allele. When comparing estimates this way, the combined effects of the associated markers in each of the QTL contribute from 10 to 75% of that estimated in the F2 by 2. As discussed above, further analyses in other populations with more informative genetic markers using other statistical methods are, however, required to explore this further.Here, we estimated the additive genetic effects of the fine-mapped regions using data from the FOur analyses suggest that there is extensive within-line segregation in the QTL regions. One possible explanation for the slow fixation in these loci could be that the beneficial alleles at the linked fine-mapped loci were located on different haplotypes at the onset of selection. Due to the low selection pressure on each QTL region resulting from the highly polygenic architecture of the selected trait, the close linkage between the loci contributing to the QTL, and the small effective breeding population, the probability that beneficial recombinant haplotypes are selected and increase in frequency in the population should be low. Another alternative explanation could be that the effects of the linked loci are dependent on the genetic background (epistasis) or dominance, which might have affected the selection pressure on individual contributing loci. As we did not explore the contributions by dominance or epistasis in this study, further work would be necessary to evaluate their contributions to the low fixation in the QTL.A key for successful imputation of the high-density marker set throughout the AIL pedigree is that the haplotypes across these markers are correctly estimated in the founders. There are several properties of the Virginia lines that improve haplotype estimation from high-density genotypes. First, as the number of generations since the lines diverged is relatively few (40 generations), most new haplotypes will result from recombination of original haplotypes, rather than by new mutations. Second, the strong artificial selection imposed on the populations since they were founded is likely to have further reduced haplotype diversity across the genome. This is likely the reason that many selective-sweeps across long haplotypes have been found to be fixed, or nearly fixed, across the genome within and between the lineages . Given tGenotype data are available for all individuals in the AIL pedigree. The dense marker backbone (∼1 marker/cM) from the first genotyping of the AIL (The highly polygenic genetic architecture in this population is consistent with what has been revealed in other fine-mapping analyses in deep intercrosses (Here, the association analysis was performed using a linear model including fixed effects of genotype, sex, and AIL generation. Sex and generation were included as both these environmental factors had significant effects on BW56 (In conclusion, this study shows that the proposed imputation-based association-mapping strategy, and further model selection by backward-elimination in a bootstrap-based framework, is useful for identifying independent association signals within and across the nine evaluated QTL. The association-peaks were narrower than those obtained in the earlier performed linkage analysis, often highlighting regions down to 2–3 Mb in length allowing the identification of multiple association signals in several QTL. This suggests that the association-based strategy has higher resolution, as well as provides an improved power to disentangle the effects of multiple linked loci inside QTL, compared to linkage-based fine-mapping. Combining traditional linkage-based approaches to analyze outbred advanced intercross populations with imputation-based association-mapping approaches might thus be an important and cost-effective approach to improve the efficiency in postassociation bioinformatics analyses and functional explorations aiming to identify candidate mutations. A previous candidate gene study based on the nine QTL fine-mapped here has already reported some interesting mutations in growth-related genes (www.g3journal.org/lookup/suppl/doi:10.1534/g3.116.036012/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.