A Near-Infrared Cell Tracker Reagent for Multiscopic In Vivo Imaging and Quantification of Leukocyte Immune Responses

The complexity of the tumor microenvironment necessitates that cell behavior is studied in a broad, multi-scale context. Although tomographic and microscopy-based far and near infrared fluorescence (NIRF, >650 nm) imaging methods offer high resolution, sensitivity, and depth penetration, there has been a lack of optimized NIRF agents to label and track cells in their native environments at different scales. In this study we labeled mammalian leukocytes with VivoTag 680 (VT680), an amine reactive N-hydroxysuccinimide (NHS) ester of a (benz) indolium-derived far red fluorescent probe. We show that VT680 diffuses into leukocytes within minutes, covalently binds to cellular components, remains internalized for days in vitro and in vivo, and does not transfer fluorescence to adjacent cells. It is biocompatible, keeps cells fully functional, and fluoresces at high intensities. In a tumor model of cytotoxic T lymphocyte (CTL) immunotherapy, we track and quantify VT680-labeled cells longitudinally at the whole-body level with fluorescence-mediated molecular tomography (FMT), within tissues at single cell resolutions by multiphoton and confocal intravital microscopy, and ex vivo by flow cytometry. Thus, this approach is suitable to monitor cells at multiple resolutions in real time in their native environments by NIR-based fluorescence imaging

Genetics of Mechanosensation in the Heart

Mechanosensation (the ultimate conversion of a mechanical stimulus into a biochemical signal) as well as mechanotransduction (transmission of mechanically induced signals) belong to the most fundamental processes in biology. These effects, because of their dynamic nature, are particularly important for the cardiovascular system. Therefore, it is not surprising that defects in cardiac mechanosensation, are associated with various types of cardiomyopathy and heart failure. However, our current knowledge regarding the genetic basis of impaired mechanosensation in the cardiovascular system is beginning to shed light on this subject and is at the centre of this brief review

Behavioural and neuroanatomical correlates of auditory speech analysis in primary progressive aphasias

Background Non-verbal auditory impairment is increasingly recognised in the primary progressive aphasias (PPAs) but its relationship to speech processing and brain substrates has not been defined. Here we addressed these issues in patients representing the non-fluent variant (nfvPPA) and semantic variant (svPPA) syndromes of PPA. Methods We studied 19 patients with PPA in relation to 19 healthy older individuals. We manipulated three key auditory parameters—temporal regularity, phonemic spectral structure and prosodic predictability (an index of fundamental information content, or entropy)—in sequences of spoken syllables. The ability of participants to process these parameters was assessed using two-alternative, forced-choice tasks and neuroanatomical associations of task performance were assessed using voxel-based morphometry of patients’ brain magnetic resonance images. Results Relative to healthy controls, both the nfvPPA and svPPA groups had impaired processing of phonemic spectral structure and signal predictability while the nfvPPA group additionally had impaired processing of temporal regularity in speech signals. Task performance correlated with standard disease severity and neurolinguistic measures. Across the patient cohort, performance on the temporal regularity task was associated with grey matter in the left supplementary motor area and right caudate, performance on the phoneme processing task was associated with grey matter in the left supramarginal gyrus, and performance on the prosodic predictability task was associated with grey matter in the right putamen. Conclusions Our findings suggest that PPA syndromes may be underpinned by more generic deficits of auditory signal analysis, with a distributed cortico-subcortical neuraoanatomical substrate extending beyond the canonical language network. This has implications for syndrome classification and biomarker development

Inter-rater agreement in the assessment of abnormal chest X-ray findings for tuberculosis between two Asian countries

<p>Abstract</p> <p>Background</p> <p>Inter-rater agreement in the interpretation of chest X-ray (CXR) films is crucial for clinical and epidemiological studies of tuberculosis. We compared the readings of CXR films used for a survey of tuberculosis between raters from two Asian countries.</p> <p>Methods</p> <p>Of the 11,624 people enrolled in a prevalence survey in Hanoi, Viet Nam, in 2003, we studied 258 individuals whose CXR films did not exclude the possibility of active tuberculosis. Follow-up films obtained from accessible individuals in 2006 were also analyzed. Two Japanese and two Vietnamese raters read the CXR films based on a coding system proposed by Den Boon et al. and another system newly developed in this study. Inter-rater agreement was evaluated by kappa statistics. Marginal homogeneity was evaluated by the generalized estimating equation (GEE).</p> <p>Results</p> <p>CXR findings suspected of tuberculosis differed between the four raters. The frequencies of infiltrates and fibrosis/scarring detected on the films significantly differed between the raters from the two countries (<it>P </it>< 0.0001 and <it>P </it>= 0.0082, respectively, by GEE). The definition of findings such as primary cavity, used in the coding systems also affected the degree of agreement.</p> <p>Conclusions</p> <p>CXR findings were inconsistent between the raters with different backgrounds. High inter-rater agreement is a component necessary for an optimal CXR coding system, particularly in international studies. An analysis of reading results and a thorough discussion to achieve a consensus would be necessary to achieve further consistency and high quality of reading.</p

Differential Effects of HIF-1 Inhibition by YC-1 on the Overall Outcome and Blood-Brain Barrier Damage in a Rat Model of Ischemic Stroke

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular adaptation to hypoxia and has been suggested as a potent therapeutic target in cerebral ischemia. Here we show in an ischemic stroke model of rats that inhibiting HIF-1 and its downstream genes by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) significantly increases mortality and enlarges infarct volume evaluated by MRI and histological staining. Interestingly, the HIF-1 inhibition remarkably ameliorates ischemia-induced blood-brain barrier (BBB) disruption determined by Evans blue leakage although it does not affect brain edema. The result demonstrates that HIF-1 inhibition has differential effects on ischemic outcomes and BBB permeability. It indicates that HIF-1 may have different functions in different brain cells. Further analyses show that ischemia upregulates HIF-1 and its downstream genes erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glucose transporter (Glut) in neurons and brain endothelial cells and that YC-1 inhibits their expression. We postulate that HIF-1-induced VEGF increases BBB permeability while certain other proteins coded by HIF-1's downstream genes such as epo and glut provide neuroprotection in an ischemic brain. The results indicate that YC-1 lacks the potential as a cerebral ischemic treatment although it confers certain protection to the cerebral vascular system

Ontogeny-Driven rDNA Rearrangement, Methylation, and Transcription, and Paternal Influence

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures

A Novel System of Cytoskeletal Elements in the Human Pathogen Helicobacter pylori

Pathogenicity of the human pathogen Helicobacter pylori relies upon its capacity to adapt to a hostile environment and to escape from the host response. Therefore, cell shape, motility, and pH homeostasis of these bacteria are specifically adapted to the gastric mucus. We have found that the helical shape of H. pylori depends on coiled coil rich proteins (Ccrp), which form extended filamentous structures in vitro and in vivo, and are differentially required for the maintenance of cell morphology. We have developed an in vivo localization system for this pathogen. Consistent with a cytoskeleton-like structure, Ccrp proteins localized in a regular punctuate and static pattern within H. pylori cells. Ccrp genes show a high degree of sequence variation, which could be the reason for the morphological diversity between H. pylori strains. In contrast to other bacteria, the actin-like MreB protein is dispensable for viability in H. pylori, and does not affect cell shape, but cell length and chromosome segregation. In addition, mreB mutant cells displayed significantly reduced urease activity, and thus compromise a major pathogenicity factor of H. pylori. Our findings reveal that Ccrp proteins, but not MreB, affect cell morphology, while both cytoskeletal components affect the development of pathogenicity factors and/or cell cycle progression

Maternal Wnt/β-Catenin Signaling Coactivates Transcription through NF-κB Binding Sites during Xenopus Axis Formation

Maternal Wnt/β-Catenin signaling establishes a program of dorsal-specific gene expression required for axial patterning in Xenopus. We previously reported that a subset of dorsally expressed genes depends not only on Wnt/β-Catenin stimulation, but also on a MyD88-dependent Toll-like receptor/IL1-receptor (TLR/IL1-R) signaling pathway. Here we show that these two signal transduction cascades converge in the nucleus to coactivate gene transcription in blastulae through a direct interaction between β-Catenin and NF-κB proteins. A transdominant inhibitor of NF-κB, ΔNIκBα, phenocopies loss of MyD88 protein function, implicating Rel/NF-κB proteins as selective activators of dorsal-specific gene expression. Sensitive axis formation assays in the embryo demonstrate that dorsalization by Wnt/β-Catenin requires NF-κB protein activity, and vice versa. Xenopus nodal-related 3 (Xnr3) is one of the genes with dual β-Catenin/NF-κB input, and a proximal NF-κB consensus site contributes to the regional activity of its promoter. We demonstrate in vitro binding of Xenopus β-Catenin to several XRel proteins. This interaction is observed in vivo upon Wnt-stimulation. Finally, we show that a synthetic luciferase reporter gene responds to both endogenous and exogenous β-Catenin levels in an NF-κB motif dependent manner. These results suggest that β-Catenin acts as a transcriptional co-activator of NF-κB-dependent transcription in frog primary embryonic cells