Stability of Adult Emergence and Activity/Rest Rhythms in Fruit Flies Drosophila melanogaster under Semi-Natural Condition

Here we report the results of a study aimed at examining stability of adult emergence and activity/rest rhythms under semi-natural conditions (henceforth SN), in four large outbred fruit fly Drosophila melanogaster populations, selected for emergence in a narrow window of time under laboratory (henceforth LAB) light/dark (LD) cycles. When assessed under LAB, selected flies display enhanced stability in terms of higher amplitude, synchrony and accuracy in emergence and activity rhythms compared to controls. The present study was conducted to assess whether such differences in stability between selected and control populations, persist under SN where several gradually changing time-cues are present in their strongest form. The study revealed that under SN, emergence waveform of selected flies was modified, with even more enhanced peak and narrower gate-width compared to those observed in the LAB and compared to control populations in SN. Furthermore, flies from selected populations continued to exhibit enhanced synchrony and accuracy in their emergence and activity rhythms under SN compared to controls. Further analysis of zeitgeber effects revealed that enhanced stability in the rhythmicity of selected flies under SN was primarily due to increased sensitivity to light because emergence and activity rhythms of selected flies were as stable as controls under temperature cycles. These results thus suggest that stability of circadian rhythms in fruit flies D. melanogaster, which evolved as a consequence of selection for emergence in a narrow window of time under weak zeitgeber condition of LAB, persists robustly in the face of day-to-day variations in cycling environmental factors of nature.     

Arrival Time Correction for Dynamic Susceptibility Contrast MR Permeability Imaging in Stroke Patients

Purpose

To determine if applying an arrival time correction (ATC) to dynamic susceptibility contrast (DSC) based permeability imaging will improve its ability to identify contrast leakage in stroke patients for whom the shape of the measured curve may be very different due to hypoperfusion.

Materials and Methods

A technique described in brain tumor patients was adapted to incorporate a correction for delayed contrast delivery due to perfusion deficits. This technique was applied to the MRIs of 9 stroke patients known to have blood-brain barrier (BBB) disruption on T1 post contrast imaging. Regions of BBB damage were compared with normal tissue from the contralateral hemisphere. Receiver operating characteristic (ROC) analysis was performed to compare the detection of BBB damage before and after ATC.

Results

ATC improved the area under the curve (AUC) of the ROC from 0.53 to 0.70. The sensitivity improved from 0.51 to 0.67 and the specificity improved from 0.57 to 0.66. Visual inspection of the ROC curve revealed that the performance of the uncorrected analysis was worse than random guess at some thresholds.

Conclusions

The ability of DSC permeability imaging to identify contrast enhancing tissue in stroke patients improved considerably when an ATC was applied. Using DSC permeability imaging in stroke patients without an ATC may lead to false identification of BBB disruption.     

Regulation of global gene expression in human Loa loa infection is a function of chronicity

Background

Human filarial infection is characterized by downregulated parasite-antigen specific T cell responses but distinct differences exist between patients with longstanding infection (endemics) and those who acquired infection through temporary residency or visits to filarial-endemic regions (expatriates).

Methods and Findings

To characterize mechanisms underlying differences in T cells, analysis of global gene expression using human spotted microarrays was conducted on CD4⁺ and CD8⁺ T cells from microfilaremic Loa loa-infected endemic and expatriate patients. Assessment of unstimulated cells showed overexpression of genes linked to inflammation and caspase-associated cell death, particularly in endemics, and enrichment of the Th1/Th2 canonical pathway in endemic CD4⁺ cells. However, pathways within CD8⁺ unstimulated cells were most significantly enriched in both patient groups. Antigen (Ag)-driven gene expression was assessed to microfilarial Ag (MfAg) and to the nonparasite Ag streptolysin O (SLO). For MfAg-driven cells, the number of genes differing significantly from unstimulated cells was greater in endemics compared to expatriates (p<0.0001). Functional analysis showed a differential increase in genes associated with NFkB (both groups) and caspase activation (endemics). While the expatriate response to MfAg was primarily a CD4⁺ pro-inflammatory one, the endemic response included CD4⁺ and CD8⁺ cells and was linked to insulin signaling, histone complexes, and ubiquitination. Unlike the enrichment of canonical pathways in CD8⁺ unstimulated cells, both groups showed pathway enrichment in CD4⁺ cells to MfAg. Contrasting with the divergent responses to MfAg seen between endemics and expatriates, the CD4⁺ response to SLO was similar; however, CD8⁺ cells differed strongly in the nature and numbers (156 [endemics] vs 36 [expatriates]) of genes with differential expression.

Conclusions

These data suggest several important pathways are responsible for the different outcomes seen among filarial-infected patients with varying levels of chronicity and imply an important role for CD8⁺ cells in some of the global changes seen with lifelong exposure.     

Endothelial progenitor cells (EPCs) as gene carrier system for rat model of human glioma

Background

Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1) intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS) to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2) whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities.

Methods and Results

Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m) in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS). Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors.

Conclusion

EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for glioma therapy as well as imaging probes.     

Large-Scale Development of Cost-Effective Single-Nucleotide Polymorphism Marker Assays for Genetic Mapping in Pigeonpea and Comparative Mapping in Legumes

Single-nucleotide polymorphisms (SNPs, >2000) were discovered by using RNA-seq and allele-specific sequencing approaches in pigeonpea (Cajanus cajan). For making the SNP genotyping cost-effective, successful competitive allele-specific polymerase chain reaction (KASPar) assays were developed for 1616 SNPs and referred to as PKAMs (pigeonpea KASPar assay markers). Screening of PKAMs on 24 genotypes [23 from cultivated species and 1 wild species (Cajanus scarabaeoides)] defined a set of 1154 polymorphic markers (77.4%) with a polymorphism information content (PIC) value from 0.04 to 0.38. One thousand and ninety-four PKAMs showed polymorphisms between parental lines of the reference mapping population (C. cajan ICP 28 × C. scarabaeoides ICPW 94). By using high-quality marker genotyping data on 167 F₂ lines from the population, a comprehensive genetic map comprising 875 PKAMs with an average inter-marker distance of 1.11 cM was developed. Previously mapped 35 simple sequence repeat markers were integrated into the PKAM map and an integrated genetic map of 996.21 cM was constructed. Mapped PKAMs showed a higher degree of synteny with the genome of Glycine max followed by Medicago truncatula and Lotus japonicus and least with Vigna unguiculata. These PKAMs will be useful for genetics research and breeding applications in pigeonpea and for utilizing genome information from other legume species.     

Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging

We report on a new straightforward magnetic cell-labeling approach that combines three US Food and Drug Administration (FDA)-approved drugs--ferumoxytol, heparin and protamine--in serum-free medium to form self-assembling nanocomplexes that effectively label cells for in vivo magnetic resonance imaging (MRI). We observed that the ferumoxytol-heparin-protamine (HPF) nanocomplexes were stable in serum-free cell culture medium. HPF nanocomplexes show a threefold increase in T2 relaxivity compared to ferumoxytol. Electron microscopy showed internalized HPF in endosomes, which we confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells or T cells when compared to controls. In vivo MRI detected 1,000 HPF-labeled cells implanted in rat brains. This HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.