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1.  Different Arms of the Adaptive Immune System Induced by a Combination Vaccine Work in Concert to Provide Enhanced Clearance of Influenza 
PLoS ONE  2014;9(12):e115356.
Current split influenza virus vaccines that induce strain-specific neutralising antibodies provide some degree of protection against influenza infection but there is a clear need to improve their effectiveness. The constant antigenic drift of influenza viruses means that vaccines are often not an exact match to the circulating strain and so levels of relevant antibodies may not be sufficiently high to afford protection. In the situation where the emergent influenza virus is completely novel, as is the case with pandemic strains, existing vaccines may provide no benefit. In this study we tested the concept of a combination vaccine consisting of sub-optimal doses of split influenza virus vaccine mixed with a cross-protective T-cell inducing lipopeptide containing the TLR2 ligand Pam2Cys. Mice immunised with combination vaccines showed superior levels of lung viral clearance after challenge compared to either split virus or lipopeptide alone, mediated through activation of enhanced humoral and/or additional cellular responses. The mechanism of action of these vaccines was dependent on the route of administration, with intranasal administration being superior to subcutaneous and intramuscular routes, potentially through the induction of memory CD8+ T cells in the lungs. This immunisation strategy not only provides a mechanism for minimising the dose of split virus antigen but also, through the induction of cross-protective CD8+ T cells, proves a breadth of immunity to provide potential benefit upon encounter with serologically diverse influenza isolates.
PMCID: PMC4270762  PMID: 25522323
2.  Patterns of gene expression associated with recovery and injury in heat-stressed rats 
BMC Genomics  2014;15(1):1058.
The in vivo gene response associated with hyperthermia is poorly understood. Here, we perform a global, multiorgan characterization of the gene response to heat stress using an in vivo conscious rat model.
We heated rats until implanted thermal probes indicated a maximal core temperature of 41.8°C (Tc,Max). We then compared transcriptomic profiles of liver, lung, kidney, and heart tissues harvested from groups of experimental animals at Tc,Max, 24 hours, and 48 hours after heat stress to time-matched controls kept at an ambient temperature. Cardiac histopathology at 48 hours supported persistent cardiac injury in three out of six animals. Microarray analysis identified 78 differentially expressed genes common to all four organs at Tc,Max. Self-organizing maps identified gene-specific signatures corresponding to protein-folding disorders in heat-stressed rats with histopathological evidence of cardiac injury at 48 hours. Quantitative proteomics analysis by iTRAQ (isobaric tag for relative and absolute quantitation) demonstrated that differential protein expression most closely matched the transcriptomic profile in heat-injured animals at 48 hours. Calculation of protein supersaturation scores supported an increased propensity of proteins to aggregate for proteins that were found to be changing in abundance at 24 hours and in animals with cardiac injury at 48 hours, suggesting a mechanistic association between protein misfolding and the heat-stress response.
Pathway analyses at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism and activation of the unfolded protein response in heat-stressed rats with histopathological evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1058) contains supplementary material, which is available to authorized users.
PMCID: PMC4302131  PMID: 25471284
Heat stress; Transcriptomics; Proteomics; Systems biology; Protein aggregation
3.  Project Kealahou: Improving Hawai‘i's System of Care for At-Risk Girls and Young Women through Gender-Responsive, Trauma-Informed Care 
Project Kealahou (PK) is a six-year, federally-funded program aimed at improving services and outcomes for Hawai‘i's female youth who are at risk for running away, truancy, abuse, suicide, arrest and incarceration. PK builds upon two decades of sustained cross-agency efforts among the state's mental health, juvenile justice, education, and child welfare systems to promote system-of-care (SOC) principles of community-based, individualized, culturally and linguistically competent, family driven, youth-guided, and evidence-based services. In addition, PK emphasizes trauma-informed and gender-responsive care in serving its target population of females ages 11–18 years who have experienced psychological trauma.
Results from the first four years of the implementation of PK in the Department of Health's (DOH) Child and Adolescent Mental Health Division (CAMHD) highlight the serious familial, socioeconomic, functional, and interpersonal challenges faced by the young women who receive services in Hawai‘i's SOC. Despite the challenges faced by PK youth and their families, preliminary results of the evaluation of PK show significant improvements across multiple clinical and functional domains of service recipients. A financial analysis indicates that these outcomes were obtained with a minimal overall increase in costs when compared to standard care alone. Overall, these results suggest that PK may offer a cost effective way to improve access, care, and outcomes for at-risk youth and their families in Hawai‘i.
PMCID: PMC4300548  PMID: 25628971
Trauma; Youth; Girls; Mental Health; System of Care (SOC); Community Mental Health Initiative (CMHI); Trauma-Informed Care; Gender-Responsive Care
4.  The membrane protein Pannexin1 forms two open channel conformations depending on the mode of activation 
Science signaling  2014;7(335):ra69.
Pannexin1 (Panx1) participates in several signaling events that involve ATP release, including the innate immune response, ciliary beat in airway epithelia and oxygen supply in the vasculature. The view that Panx1 forms a large ATP-release channel has been challenged by the association of a low conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K+), Panx1 formed a high conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K+. The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single particle electron microscopic analysis revealed that K+ stimulated the formation of channels with a larger pore diameter than those formed in the absence of K+. These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties.
PMCID: PMC4243966  PMID: 25056878
5.  Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells* 
The Journal of Biological Chemistry  2014;289(52):35695-35710.
Background: The interaction of platelet CLEC-2 with Podoplanin is critical for development of the lymphatics.
Results: CLEC-2 forms a central cluster upon engagement with Podoplanin, which clusters Podoplanin. Clustering is dependent on Syk and is critical for adhesion.
Conclusion: Clustering regulates the interaction of platelets with lymphatic endothelial cells.
Significance: These findings account for the similar lymphatic phenotype of CLEC-2- and Syk-deficient mice.
The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.
PMCID: PMC4276840  PMID: 25368330
Endothelial Cell; Lipid Bilayer; Platelet; Receptor; Tyrosine-Protein Kinase (Tyrosine Kinase); CLEC-2; ITAM; Podoplanin; Src Family Kinase; Syk
6.  Identification of evolutionarily conserved amino acid residues in homeodomain of KNOX proteins for intercellular trafficking 
Plant Signaling & Behavior  2014;9:e28355.
Maize KNOTTED (KN1) homeodomain (HD) protein is a well-known mobile transcription factor crucial for stem cell maintenance. Recent studies have revealed that the trihelical HD of KNOTTED1-like homeobox (KNOX) proteins is necessary and sufficient for selective cell-to-cell trafficking. Also, the efficient trafficking ability for HD is likely to be acquired during the evolution of early nonvascular land plants. Here, using the point-mutated HD of KN1 and SHOOT MERISTEMLESS (STM) in the trichome rescue system, together with molecular structure modeling, we have found the evolutionarily conserved amino acid residues, such as arginine in helix α1 and leucine in helix α3, which are essential for intercellular trafficking. Our studies provided important clues for the 3-dimensional protein structure required for cell-to-cell movement of non-cell-autonomous transcription factors.
PMCID: PMC4091555  PMID: 24603432
homeodomain; intercellular protein trafficking; non-cell-autonomous; plasmodesmata; transcription factor
7.  Progression of carcinogen‐induced fibrosarcomas is associated with the accumulation of naïve CD4+ T cells via blood vessels and lymphatics 
The tumor microenvironment comprises newly formed blood and lymphatic vessels which shape the influx, retention and departure of lymphocytes within the tumor mass. Thus, by influencing the intratumoral composition of lymphocytes, these vessels affect the manner in which the adaptive immune system responds to the tumor, either promoting or impairing effective antitumor immunity. In our study, we utilized a mouse model of carcinogen‐induced fibrosarcoma to examine the composition of tumor‐infiltrating lymphocytes during tumor progression. In particular, we sought to determine whether CD4+Foxp3+ regulatory T cells (Tregs) became enriched during tumor progression thereby contributing to tumor‐driven immunosuppression. This was not the case as the proportion of Tregs and effector CD4+ T cells actually declined within the tumor owing to the unexpected accumulation of naïve T cells. However, we found no evidence for antigen‐driven migration of these T cells or for their participation in an antitumor immune response. Our data support the notion that lymphocytes can enter tumors via aberrantly formed blood and lymphatic vessels. Such findings suggest that targeting both the tumor vasculature and lymphatics will alter the balance of lymphocyte subpopulations that enter the tumor mass. A consideration of this aspect of tumor immunology may be critical to the success of solid cancer immunotherapies.
What's new?
It is well known that a tumors' microenvironment can impair the anti‐tumor immune response. The culprits are usually assumed to be various suppressor cells and cytokines. In this study, however, the authors found that seemingly innocuous, naïve T cells may also play a significant role—simply by accumulating and possibly out‐competing activated effector cells within the tumor. A better understanding of the signals produced by the tumor microenvironment may allow researchers to alter this T‐cell pool, thus enhancing the immune response.
PMCID: PMC4114538  PMID: 24142504
T cells; carcinogen; lymphatics
8.  Activation of the Interferon Induction Cascade by Influenza A Viruses Requires Viral RNA Synthesis and Nuclear Export 
Journal of Virology  2014;88(8):3942-3952.
We have examined the requirements for virus transcription and replication and thus the roles of input and progeny genomes in the generation of interferon (IFN)-inducing pathogen-associated molecular patterns (PAMPs) by influenza A viruses using inhibitors of these processes. Using IFN regulatory factor 3 (IRF3) phosphorylation as a marker of activation of the IFN induction cascade that occurs upstream of the IFN-β promoter, we demonstrate strong activation of the IFN induction cascade in A549 cells infected with a variety of influenza A viruses in the presence of cycloheximide or nucleoprotein (NP) small interfering RNA (siRNA), which inhibits viral protein synthesis and thus complementary ribonucleoprotein (cRNP) and progeny viral RNP (vRNP) synthesis. In contrast, activation of the IFN induction cascade by influenza viruses was very effectively abrogated by treatment with actinomycin D and other transcription inhibitors, which correlated with the inhibition of the synthesis of all viral RNA species. Furthermore, 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole, an inhibitor that prevents viral RNA export from the nucleus, was also a potent inhibitor of IRF3 activation; thus, both viral RNA synthesis and nuclear export are required for IFN induction by influenza A viruses. While the exact nature of the viral PAMPs remains to be determined, our data suggest that in this experimental system the major influenza A virus PAMPs are distinct from those of incoming genomes or progeny vRNPs.
IMPORTANCE The host interferon system exerts an extremely potent antiviral response that efficiently restricts virus replication and spread; the interferon response can thus dictate the outcome of a virus infection, and it is therefore important to understand how viruses induce interferon. Both input and progeny genomes have been linked to interferon induction by influenza viruses. However, our experiments in tissue culture cells show that viral RNA synthesis and nuclear export are required to activate this response. Furthermore, the interferon induction cascade is activated under conditions in which the synthesis of progeny genomes is inhibited. Therefore, in tissue culture cells, input and progeny genomes are not the predominant inducers of interferon generated by influenza A viruses; the major viral interferon inducer(s) still remains to be identified.
PMCID: PMC3993719  PMID: 24478437
9.  ATP and potassium ions: a deadly combination for astrocytes 
Scientific Reports  2014;4:4576.
The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.
PMCID: PMC3974143  PMID: 24694658
10.  Whole adult organism transcriptional profiling of acute metal exposures in male Zebrafish 
A convergence of technological breakthroughs in the past decade has facilitated the development of rapid screening tools for biomarkers of toxicant exposure and effect. Platforms using the whole adult organism to evaluate the genome-wide response to toxicants are especially attractive. Recent work demonstrates the feasibility of this approach in vertebrates using the experimentally robust zebrafish model. In the present study, we evaluated gene expression changes in whole adult male zebrafish following an acute 24 hr high dose exposure to three metals with known human health risks. Male adult zebrafish were exposed to nickel chloride, cobalt chloride or sodium dichromate concentrations corresponding to their respective 96 hr LC20, LC40 and LC60. Histopathology was performed on a subset of metal-exposed zebrafish to phenotypically anchor transcriptional changes associated with each metal.
Comparative analysis identified subsets of differentially expressed transcripts both overlapping and unique to each metal. Application of gene ontology (GO) and transcription factor (TF) enrichment algorithms revealed a number of key biological processes perturbed by metal poisonings and the master transcriptional regulators mediating gene expression changes. Metal poisoning differentially activated biological processes associated with ribosome biogenesis, proteosomal degradation, and p53 signaling cascades, while repressing oxygen-generating pathways associated with amino acid and lipid metabolism. Despite appreciable effects on gene regulation, nickel poisoning did not induce any morphological alterations in male zebrafish organs and tissues. Histopathological effects of cobalt remained confined to the olfactory system, while chromium targeted the gills, pharynx, and intestinal mucosa. A number of enriched transcription factors mediated the observed gene response to metal poisoning, including known targets such as p53, HIF1α, and the myc oncogene, and novel regulatory factors such as XBP1, GATA6 and HNF3β.
This work uses an experimentally innovative approach to capture global responses to metal poisoning and provides mechanistic insights into metal toxicity.
PMCID: PMC4007779  PMID: 24612858
Metals; Toxicity mechanisms; Zebrafish; Whole organism; Nickel; Chromium; Cobalt; Toxicogenomics
11.  SASI-Seq: sample assurance Spike-Ins, and highly differentiating 384 barcoding for Illumina sequencing 
BMC Genomics  2014;15:110.
A minor but significant fraction of samples subjected to next-generation sequencing methods are either mixed-up or cross-contaminated. These events can lead to false or inconclusive results. We have therefore developed SASI-Seq; a process whereby a set of uniquely barcoded DNA fragments are added to samples destined for sequencing. From the final sequencing data, one can verify that all the reads derive from the original sample(s) and not from contaminants or other samples.
By adding a mixture of three uniquely barcoded amplicons, of different sizes spanning the range of insert sizes one would normally use for Illumina sequencing, at a spike-in level of approximately 0.1%, we demonstrate that these fragments remain intimately associated with the sample. They can be detected following even the tightest size selection regimes or exome enrichment and can report the occurrence of sample mix-ups and cross-contamination.
As a consequence of this work, we have designed a set of 384 eleven-base Illumina barcode sequences that are at least 5 changes apart from each other, allowing for single-error correction and very low levels of barcode misallocation due to sequencing error.
SASI-Seq is a simple, inexpensive and flexible tool that enables sample assurance, allows deconvolution of sample mix-ups and reports levels of cross-contamination between samples throughout NGS workflows.
PMCID: PMC4008303  PMID: 24507442
Next-generation sequencing; Indexing; Barcode; Illumina; Sample assurance; Spike-in; Contamination; Sample identity
12.  TLR Agonists as Modulators of the Innate Immune Response and Their Potential as Agents Against Infectious Disease 
Immunotherapies that can either activate or suppress innate immune responses are being investigated as treatments against infectious diseases and the pathology they can cause. The objective of these therapies is to elicit protective immune responses thereby limiting the harm inflicted by the pathogen. The Toll-like receptor (TLR) signaling pathway plays critical roles in numerous host immune defenses and has been identified as an immunotherapeutic target against the consequences of infectious challenge. This review focuses on some of the recent advances being made in the development of TLR-ligands as potential prophylactic and/or therapeutic agents.
PMCID: PMC3939722  PMID: 24624130
TLR agonists; immunomodulation; innate immunity; toll-like receptors; cytokines and inflammation
13.  Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines 
PLoS ONE  2013;8(12):e83751.
Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.
PMCID: PMC3875483  PMID: 24386269
14.  The Human Interferon-Induced MxA Protein Inhibits Early Stages of Influenza A Virus Infection by Retaining the Incoming Viral Genome in the Cytoplasm 
Journal of Virology  2013;87(23):13053-13058.
The induction of an interferon-induced antiviral state is a powerful cellular response against viral infection that limits viral spread. Here, we show that a preexisting antiviral state inhibits the replication of influenza A viruses in human A549 cells by preventing transport of the viral genome to the nucleus and that the interferon-induced MxA protein is necessary but not sufficient for this process. This represents a previously unreported antiviral function of MxA against influenza A virus infection.
PMCID: PMC3838145  PMID: 24049170
15.  The chemokine CX3CL1 promotes trafficking of dendritic cells through inflamed lymphatics 
Journal of Cell Science  2013;126(22):5259-5270.
Tissue inflammation is characterised by increased trafficking of antigen-loaded dendritic cells (DCs) from the periphery via afferent lymphatics to draining lymph nodes, with a resulting stimulation of ongoing immune responses. Transmigration across lymphatic endothelium constitutes the first step in this process and is known to involve the chemokine CCL21 and its receptor CCR7. However, the precise details of DC transit remain obscure and it is likely that additional chemokine-receptor pairs have roles in lymphatic vessel entry. Here, we report that the transmembrane chemokine CX3CL1 (fractalkine) is induced in inflamed lymphatic endothelium, both in vitro in TNF-α-treated human dermal lymphatic endothelial cells (HDLECs) and in vivo in a mouse model of skin hypersensitivity. However, unlike blood endothelial cells, which express predominantly transmembrane CX3CL1 as a leukocyte adhesion molecule, HDLECs shed virtually all CX3CL1 at their basolateral surface through matrix metalloproteinases. We show for the first time that both recombinant soluble CX3CL1 and endogenous secreted CX3CL1 promote basolateral-to-luminal migration of DCs across HDLEC monolayers in vitro. Furthermore, we show in vivo that neutralising antibodies against CX3CL1 dramatically reduce allergen-induced trafficking of cutaneous DCs to draining lymph nodes as assessed by FITC skin painting in mice. Finally, we show that deletion of the CX3CL1 receptor in Cx3cr1−/− DCs results in markedly delayed lymphatic trafficking in vivo and impaired translymphatic migration in vitro, thus establishing a previously unrecognised role for this atypical chemokine in regulating DC trafficking through the lymphatics.
PMCID: PMC3828594  PMID: 24006262
Lymphatic; Chemokine; DC trafficking; Inflammation; Endothelial cell
16.  The food dye FD&C Blue No. 1 is a selective inhibitor of the ATP release channel Panx1 
The Journal of General Physiology  2013;141(5):649-656.
The food dye FD&C Blue No. 1 (Brilliant Blue FCF [BB FCF]) is structurally similar to the purinergic receptor antagonist Brilliant Blue G (BBG), which is a well-known inhibitor of the ionotropic P2X7 receptor (P2X7R). The P2X7R functionally interacts with the membrane channel protein pannexin 1 (Panx1) in inflammasome signaling. Intriguingly, ligands to the P2X7R, regardless of whether they are acting as agonists or antagonists at the receptor, inhibit Panx1 channels. Thus, because both P2X7R and Panx1 are inhibited by BBG, the diagnostic value of the drug is limited. Here, we show that the food dye BB FCF is a selective inhibitor of Panx1 channels, with an IC50 of 0.27 µM. No significant effect was observed with concentrations as high as 100 µM of BB FCF on P2X7R. Differing by just one hydroxyl group from BB FCF, the food dye FD&C Green No. 3 exhibited similar selective inhibition of Panx1 channels. A reverse selectivity was observed for the P2X7R antagonist, oxidized ATP, which in contrast to other P2X7R antagonists had no significant inhibitory effect on Panx1 channels.
Based on its selective action, BB FCF can be added to the repertoire of drugs to study the physiology of Panx1 channels. Furthermore, because Panx1 channels appear to be involved directly or indirectly through P2X7Rs in several disorders, BB FCF and derivatives of this “safe” food dye should be given serious consideration for pharmacological intervention of conditions such as acute Crohn’s disease, stroke, and injuries to the central nervous system.
PMCID: PMC3639576  PMID: 23589583
17.  Lipidated promiscuous peptide augments the expression of MHC-II molecules on dendritic cells and activates T cells 
Background & objectives:
In spite of the fact that BCG is the most widely used vaccine, tuberculosis (TB) continues to be a major killer disease in TB-endemic regions. Recently, many emerging evidences from the published literature indicate the role of environmental mycobacteria in blocking the processing and presentation of BCG antigens and thereby impairing with suboptimal generation of protective T cells. To surmount this problem associated with BCG, we constructed a novel lipopeptide (L91) by conjugating a promiscuous peptide consisting of CD4+ T-helper epitope of sequence of 91-110 of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys, an agonist of Toll-like receptor-2.
Mice were immunized subcutaneously with 20 nmol of L91, followed by a booster with 10 nmol, after an interval of 21 days of primary immunization. Animals were sacrificed after seven days of post-booster immunization. L91 induced immune response was characterized by the expression of MHC-II and CD74 on the surface of dendritic cells (DCs) by flowcytometry. Cytokines (IL-4, IL-10, IFN-γ) secretion and anti-peptide antibodies were measured by ELISA.
Self-adjuvanting lipopeptide vaccine (L91) was directly bound to MHC-II molecules and without requiring extensive processing for its presentation to T cells. It stimulated and activated dendritic cells and augmented the expression of MHC-II molecules. Further, it activated effector CD4 T cells to mainly secrete interferon (IFN)-γ but not interleukin (IL)-4 and IL-10. L91 did not elicit anti-peptide antibodies.
Interpretation & conclusions:
The findings suggest that L91 evokes maturation and upregulation of MHC class II molecules and promotes better antigen presentation and, therefore, optimum activation of T cells. L91 mainly induces effector Th1 cells, as evidenced by predominant release of IFN-γ, consequently can mount favourable immune response against M. tuberculosis. As L91 does not provoke the generation of anti-peptide antibodies, there is no fear of the efficacy of the vaccine being neutralized by pre-existing anti-mycobacterial antibodies in TB-endemic population. In conclusion, L91 may be considered as a future potential candidate vaccine against TB.
PMCID: PMC3928704  PMID: 24434326
BCG; L91; Pam2Cys; promiscuous peptides; Th1 – tuberculosis
18.  Src activation by β-adrenoreceptors is a key switch for tumor metastasis 
Nature communications  2013;4:1403.
Norepinephrine (NE) can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here, we identify Src as a key regulator of phosphoproteomic signaling networks activated in response to beta-adrenergic signaling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumor cell migration, invasion and growth. In human ovarian cancer samples, high tumoral NE levels were correlated with high pSrcY419 levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signaling in the tumor microenvironment.
PMCID: PMC3561638  PMID: 23360994
19.  A fluorometric assay for trehalose in the picomole range 
Plant Methods  2013;9:21.
Trehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.
A kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%. A. thaliana Col-0 rosettes contain about 20–30 nmol g-1FW of trehalose, increasing to about 50–60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.
The fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.
PMCID: PMC3698175  PMID: 23786766
Arabidopsis thaliana; Ramosa3; Trehalase; Trehalose; Zea mays
20.  Cryotomography of Budding Influenza A Virus Reveals Filaments with Diverse Morphologies that Mostly Do Not Bear a Genome at Their Distal End 
PLoS Pathogens  2013;9(6):e1003413.
Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.
Author Summary
Influenza viruses that have been cultivated in the laboratory usually produce particles that are spherical. However, viruses isolated from patients frequently produce long filamentous particles, as well as smaller elliptical particles that we term “bacilliform virions”. Long filaments may be important for cell-to-cell transmission or facilitate release of the smaller particles by disrupting the mucous layer of the respiratory tract. We have used three-dimensional electron microscopy to investigate the structure of influenza virus filaments ‘budding’ from cells. We found that many of the long filaments had a large bulbous head at the end furthest from the cell. Many of these bulbs were empty while some contained tubules that we believe are made of a scaffold-protein M1 that usually lines the inner surface of the viral membrane. Bacilliform virions contain genomes comprised of eight segments of RNA; these are each wrapped up in protein and packaged in an ordered manner. None of the bulb-headed filaments and very few narrower ones had this feature. We hypothesise that the diverse viral structures we have seen suggest distinct assembly pathways and moreover functions. Long filamentous structures that do not appear to contain genomes may combat the immune response or help the smaller virus particles spread.
PMCID: PMC3675018  PMID: 23754946
22.  Alterations in gene expression in Caenorhabditis elegans associated with organophosphate pesticide intoxication and recovery 
BMC Genomics  2013;14:291.
The principal toxicity of acute organophosphate (OP) pesticide poisoning is the disruption of neurotransmission through inhibition of acetylcholinesterase (AChE). However, other mechanisms leading to persistent effects and neurodegeneration remain controversial and difficult to detect. Because Caenorhabditis elegans is relatively resistant to OP lethality—particularly through the inhibition of AChE—studies in this nematode provide an opportunity to observe alterations in global gene expression following OP exposure that cannot be readily observed in less resistant organisms.
We exposed cultures of worms in axenic, defined medium to dichlorvos under three exposure protocols. In the first, worms were exposed continuously throughout the experiment. In the second and third, the worms were exposed for either 2 or 8 h, the dichlorvos was washed out of the culture, and the worms were allowed to recover. We then analyzed gene expression using whole genome microarrays from RNA obtained from worms sampled at multiple time points throughout the exposure. The worms showed a time-dependent increase in the expression of genes involved in stress responses. Early in the exposure, the predominant effect was on metabolic processes, while at later times, an immune-like response and cellular repair mechanisms dominated the expression pattern. Following removal of dichlorvos, the gene expression in the worms appeared to relatively rapidly return to steady-state levels.
The changes in gene expression observed in the worms following exposure to dichlorvos point towards two potential mechanisms of toxicity: inhibition of AChE and mitochondrial disruption.
PMCID: PMC3760450  PMID: 23631360
Caenorhabditis elegans; Organophosphate pesticide intoxication; Gene expression; Dichlorvos; Acetylcholinesterase inhibition; Mitochondrial disruption; Dichlorvos-induced developmental delay
23.  The effect of volume conductor modeling on the estimation of cardiac vectors in fetal magnetocardiography 
Physiological Measurement  2012;33(4):651-665.
Previous studies based on fetal magnetocardiographic (fMCG) recordings used simplified volume conductor models to estimate the fetal cardiac vector as an unequivocal measure of the cardiac source strength. However, the effect of simplified volume conductor modeling on the accuracy of the fMCG inverse solution remains largely unknown. Aiming to determine the sensitivity of the source estimators to the details of the volume conductor model, we performed simulations using fetal-maternal anatomical information from ultrasound images obtained in 20 pregnant women in various stages of pregnancy. The magnetic field produced by a cardiac source model was computed using the boundary element method for a piecewise homogeneous volume conductor with three nested compartments (fetal body, amniotic fluid and maternal abdomen) of different electrical conductivities. For late gestation, we also considered the case of a fourth highly insulating layer of vernix caseosa covering the fetus. The errors introduced for simplified volume conductors were assessed by comparing the reconstruction results obtained with realistic versus spherically symmetric models. Our study demonstrates a significant effect of simplified volume conductor modeling, resulting mainly in an underestimation of the cardiac vector magnitude and low goodness-of-fit. These findings are confirmed by the analysis of real fMCG data recorded in mid-gestation.
PMCID: PMC3351031  PMID: 22442179
fetal magnetocardiography; cardiac vector; boundary element method; volume conductor
24.  A conditional knockout resource for the genome–wide study of mouse gene function 
Nature  2011;474(7351):337-342.
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
PMCID: PMC3572410  PMID: 21677750
25.  A novel radiographic targeting guide for percutaneous placement of transfacet screws in the cervical spine with limited fluoroscopy: A cadaveric feasibility study 
We describe a technique for percutaneous transfacet screw placement in the cervical spine without the need for lateral-view fluoroscopy.
Previously established articular pillar morphometry was used to define the ideal trajectory for transfacet screw placement in the subaxial cervical spine. A unique targeting guide was developed to allow placement of Kirschner wires across the facet joint at 90° without the guidance of lateral-view fluoroscopy. Kirschner wires and cannulated screws were placed percutaneously in 7 cadaveric specimens. Placement of instrumentation was performed entirely under modified anteroposterior-view fluoroscopy. All specimens were assessed for acceptable screw placement by 2 fellowship-trained orthopaedic spine surgeons using computed tomography. Open dissection was used to confirm radiographic interpretation. Acceptable placement was defined as a screw crossing the facet joint, achieving purchase in the inferior and superior articular processes, and not violating critical structures. Malposition was defined as a violation of the transverse foramen, spinal canal, or nerve root or inadequate fixation.
A total of 48 screws were placed. Placement of 45 screws was acceptable. The 3 instances of screw malposition included a facet fracture, a facet distraction, and a C6-7 screw contacting the C7 nerve root in a specimen with a small C7 superior articular process.
Our data show that with the appropriate radiographic technique and a targeting guide, percutaneous transfacet screws can be safely placed at C3-7 without the need for lateral-view fluoroscopy during the targeting phase. Because of the variable morphometry of the C7 lateral mass, however, care must be taken when placing a transfacet screw at C6-7.
Clinical Relevance
This study describes a technique that has the potential to provide a less invasive strategy for posterior instrumentation of the cervical spine. Further investigation is needed before this technique can be applied clinically.
PMCID: PMC4300881
Transfacet screws; Cervical spine; Radiographic targeting guide; Minimally invasive surgery of the spine

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