OptiJ: Open-source optical projection tomography of large organ samples

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 µm resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples

Gastrointestinal Dysfunction in a Parkinson’s Disease Rat Model and the Changes of Dopaminergic, Nitric Oxidergic, and Cholinergic Neurotransmitters in Myenteric Plexus

This study aims to explore the gastrointestinal dysfunction and the changes of dopaminergic, nitric oxidergic, and cholinergic neurons in the myenteric plexus of a Parkinson’s disease (PD) rat model. A PD rat model was induced through unilateral substantia nigra administration of 6-hydroxydopamine. Four weeks later, the feces in 1 h and residual solid food in stomach at 2 h after feeding were measured. Changes in tyrosine hydroxylase (TH) in substantial nigra, TH, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) in gastric antrum and colon tissue were examined by immunohistochemistry. Reverse transcription (RT) polymerase chain reaction (PCR) and Western blot were used to evaluate and compare the levels of messenger RNA (mRNA) and protein expression of TH, ChAT, and nNOS in the GI tract between normal and 6-hydroxydopamine-lesioned rats. Compared with control samples, the number of TH+ cells in the damaged side of substantia nigra of 6-hydroxydopamine-lesioned rats decreased significantly (P < 0.01). The weight and water content of the fecal matter decreased (P < 0.01), and the percentage of residual solid food increased (P < 0.01). The average integrated optical densities of TH-positive areas in the gastric antrum and colon tissue increased significantly (P < 0.01), nNOS decreased significantly (P < 0.01), and there were no significant changes in ChAT (P > 0.05). TH and nNOS mRNA levels in the gastric antrum and proximal colon decreased (P < 0.01), there were no significant changes in ChAT mRNA levels (P > 0.05). The protein levels of TH in the GI tract were significantly increased (P < 0.01), nNOS significantly decreased (P < 0.01), and ChAT had no significant changes (P > 0.05). 6-Hydroxydopamine-lesioned rats had delayed gastric emptying and constipation that might be related to the gastrointestinal TH increase and nNOS decrease. These symptoms were not related to changes in cholinergic transmitters

DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential.

Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.The Balasubramanian laboratory is core-funded by Cancer Research UK (C14303/A17197) and further supported by a Cancer Research UK programme grant (C9681/A18618). S.B. is a Wellcome Trust Senior Investigator (099232/Z/12/Z)

PTTG1 Attenuates Drug-Induced Cellular Senescence

As PTTG1 (pituitary tumor transforming gene) abundance correlates with adverse outcomes in cancer treatment, we determined mechanisms underlying this observation by assessing the role of PTTG1 in regulating cell response to anti-neoplastic drugs. HCT116 cells devoid of PTTG1 (PTTG1−/−) exhibited enhanced drug sensitivity as assessed by measuring BrdU incorporation in vitro. Apoptosis, mitosis catastrophe or DNA damage were not detected, but features of senescence were observed using low doses of doxorubicin and TSA. The number of drug-induced PTTG1−/− senescent cells increased ∼4 fold as compared to WT PTTG1-replete cells (p<0.001). p21, an important regulator of cell senescence, was induced ∼3 fold in HCT116 PTTG1−/− cells upon doxorubicin or Trichostatin A treatment. Binding of Sp1, p53 and p300 to the p21 promoter was enhanced in PTTG1−/− cells after treatment, suggesting transcriptional regulation of p21. p21 knock down abrogated the observed senescent effects of these drugs, indicating that PTTG1 likely suppresses p21 to regulate drug-induced senescence. PTTG1 also regulated SW620 colon cancer cells response to doxorubicin and TSA mediated by p21. Subcutaneously xenografted PTTG1−/− HCT116 cells developed smaller tumors and exhibited enhanced responses to doxorubicin. PTTG1−/− tumor tissue derived from excised tumors exhibited increased doxorubicin-induced senescence. As senescence is a determinant of cell responses to anti-neoplastic treatments, these findings suggest PTTG1 as a tumor cell marker to predict anti-neoplastic treatment outcomes

Quarantine for pandemic influenza control at the borders of small island nations

Background: Although border quarantine is included in many influenza pandemic plans, detailed guidelines have yet to be formulated, including considerations for the optimal quarantine length. Motivated by the situation of small island nations, which will probably experience the introduction of pandemic influenza via just one airport, we examined the potential effectiveness of quarantine as a border control measure. Methods: Analysing the detailed epidemiologic characteristics of influenza, the effectiveness of quarantine at the borders of islands was modelled as the relative reduction of the risk of releasing infectious individuals into the community, explicitly accounting for the presence of asymptomatic infected individuals. The potential benefit of adding the use of rapid diagnostic testing to the quarantine process was also considered. Results: We predict that 95% and 99% effectiveness in preventing the release of infectious individuals into the community could be achieved with quarantine periods of longer than 4.7 and 8.6 days, respectively. If rapid diagnostic testing is combined with quarantine, the lengths of quarantine to achieve 95% and 99% effectiveness could be shortened to 2.6 and 5.7 days, respectively. Sensitivity analysis revealed that quarantine alone for 8.7 days or quarantine for 5.7 days combined with using rapid diagnostic testing could prevent secondary transmissions caused by the released infectious individuals for a plausible range of prevalence at the source country (up to 10%) and for a modest number of incoming travellers (up to 8000 individuals). Conclusion: Quarantine atthe borders of island nations could contribute substantially to preventing the arrival of pandemic influenza (or at least delaying the arrival date). For small island nations we recommend consideration of quarantine alone for 9 days or quarantine for 6 days combined with using rapid diagnostic testing (if available). © 2009 Nishiura et al; licensee BioMed Central Ltd.published_or_final_versio

Safety and Immunogenicity of a Malaria Vaccine, Plasmodium falciparum AMA-1/MSP-1 Chimeric Protein Formulated in Montanide ISA 720 in Healthy Adults

The P. falciparum chimeric protein 2.9 (PfCP-2.9) consisting of the sequences of MSP1-19 and AMA-1 (III) is a malaria vaccine candidate that was found to induce inhibitory antibodies in rabbits and monkeys. This was a phase I randomized, single-blind, placebo-controlled, dose-escalation study to evaluate the safety and immunogenicity of the PfCP-2.9 formulated with a novel adjuvant Montanide ISA720. Fifty-two subjects were randomly assigned to 4 dose groups of 10 participants, each receiving the test vaccine of 20, 50, 100, or 200 µg respectively, and 1 placebo group of 12 participants receiving the adjuvant only.The vaccine formulation was shown to be safe and well-tolerated, and none of the participants withdrew. The total incidence of local adverse events (AEs) was 75%, distributed among 58% of the placebo group and 80% of those vaccinated. Among the vaccinated, 65% had events that were mild and 15% experienced moderate AEs. Almost all systemic adverse reactions observed in this study were graded as mild and required no therapy. The participants receiving the test vaccine developed detectable antibody responses which were boosted by the repeated vaccinations. Sixty percent of the vaccinated participants had high ELISA titers (>1∶10,000) of antigen-specific antibodies which could also recognize native parasite proteins in an immunofluorescence assay (IFA).This study is the first clinical trial for this candidate and builds on previous investigations supporting PfCP-2.9/ISA720 as a promising blood-stage malaria vaccine. Results demonstrate safety, tolerability (particularly at the lower doses tested) and immunogenicity of the formulation. Further clinical development is ongoing to explore optimizing the dose and schedule of the formulation to decrease reactogenicity without compromising immunogenicity.

Inducing Ni Sensitivity in the Ni Hyperaccumulator Plant Alyssum inflatum Nyárády (Brassicaceae) by Transforming with CAX1, a Vacuolar Membrane Calcium Transporter

The importance of calcium in nickel tolerance was studied in the nickel hyperaccumulator plant Alyssum inflatum by gene transformation of CAX1, a vacuolar membrane transporter that reduces cytosolic calcium. CAX1 from Arabidopsis thaliana with a CaMV35S promoter accompanying a kanamycin resistance gene was transferred into A. inflatum using Agrobacterium tumefaciens. Transformed calli were subcultured three times on kanamycin-rich media and transformation was confirmed by PCR using a specific primer for CAX1. At least 10 callus lines were used as a pool of transformed material. Both transformed and untransformed calli were treated with varying concentrations of either calcium (1–15 mM) or nickel (0– 500 lM) to compare their responses to those ions. Increased external calcium generally led to increased callus biomass, however, the increase was greater for untransformed callus. Further, increased external calcium led to increased callus calcium concentrations. Transformed callus was less nickel tolerant than untransformed callus: under increasing nickel concentrations callus relative growth rate was significantly less for transformed callus. Transformed callus also contained significantly less nickel than untransformed callus when exposed to the highest external nickel concentration (200 lM). We suggest that transformation with CAX1 decreased cytosolic calcium and resulted in decreased nickel tolerance. This in turn suggests that, at low cytosolic calcium concentrations, other nickel tolerance mechanisms (e.g., complexation and vacuolar sequestration) are insufficient for nickel tolerance. We propose that high cytosolic calcium is an important mechanism that results in nickel tolerance by nickel hyperaccumulator plants

G-quadruplex structures mark human regulatory chromatin

G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5' UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as $\textit{MYC}$. Strikingly, $\textit{de novo}$ and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor-induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.European Molecular Biology Organization (EMBO Long-Term Fellowship), University of Cambridge, Cancer Research UK (Grant ID: C14303/A17197), Wellcome Trust (Grant ID: 099232/z/12/z