A new mouse model for renal lesions produced by intravenous injection of diphtheria toxin A-chain expression plasmid

BACKGROUND: Various animal models of renal failure have been produced and used to investigate mechanisms underlying renal disease and develop therapeutic drugs. Most methods available to produce such models appear to involve subtotal nephrectomy or intravenous administration of antibodies raised against basement membrane of glomeruli. In this study, we developed a novel method to produce mouse models of renal failure by intravenous injection of a plasmid carrying a toxic gene such as diphtheria toxin A-chain (DT-A) gene. DT-A is known to kill cells by inhibiting protein synthesis. METHODS: An expression plasmid carrying the cytomegalovirus enhancer/chicken β-actin promoter linked to a DT-A gene was mixed with lipid (FuGENE™6) and the resulting complexes were intravenously injected into adult male B6C3F1 mice every day for up to 6 days. After final injection, the kidneys of these mice were sampled on day 4 and weeks 3 and 5. RESULTS: H-E staining of the kidney specimens sampled on day 4 revealed remarkable alterations in glomerular compartments, as exemplified by mesangial cell proliferation and formation of extensive deposits in glomerular basement membrane. At weeks 3 and 5, gradual recovery of these tissues was observed. These mice exhibited proteinuria and disease resembling sub-acute glomerulonephritis. CONCLUSIONS: Repeated intravenous injections of DT-A expression plasmid DNA/lipid complex caused temporary abnormalities mainly in glomeruli of mouse kidney. The disease in these mice resembles sub-acute glomerulonephritis. These DT-A gene-incorporated mice will be useful as animal models in the fields of nephrology and regenerative medicine

Differentiation enhances aminolevulinic acid-dependent photodynamic treatment of LNCaP prostate cancer cells

Photodynamic therapy using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) may be applied to the treatment of neoplasms in a variety of organs. In order to enhance existing regimens of photodynamic therapy, we investigated the effects of adding differentiation therapy to photodynamic therapy in human prostate cancer cells in vitro. The objective of differentiation therapy per se is to reverse the lack of differentiation in cancer cells using pharmacological agents. The motivation for this study was to exploit the differentiation-dependent expression of some heme enzymes to enhance tumour cell toxicity of ALA-photodynamic therapy. A short course of differentiation therapy was applied to increase PpIX formation during subsequent ALA exposure. Using the synthetic androgen R1881, isomers of retinoic acid, and analogues of vitamin D for 3 to 4 days, exogenous ALA-dependent PpIX formation in LNCaP cells was increased, along with markers for growth arrest and for differentiation. As a consequence of higher PpIX levels, cytotoxic effects of visible light exposure were also enhanced. Short-term differentiation therapy increased not only the overall PpIX production but also reduced that fraction of cells that contained low PpIX levels as demonstrated by flow cytometry and fluorescence microscopy. This study suggests that it will be feasible to develop protocols combining short-term differentiation therapy with photodynamic therapy for enhanced photosensitisation

Decomposition of Gene Expression State Space Trajectories

Representing and analyzing complex networks remains a roadblock to creating dynamic network models of biological processes and pathways. The study of cell fate transitions can reveal much about the transcriptional regulatory programs that underlie these phenotypic changes and give rise to the coordinated patterns in expression changes that we observe. The application of gene expression state space trajectories to capture cell fate transitions at the genome-wide level is one approach currently used in the literature. In this paper, we analyze the gene expression dataset of Huang et al. (2005) which follows the differentiation of promyelocytes into neutrophil-like cells in the presence of inducers dimethyl sulfoxide and all-trans retinoic acid. Huang et al. (2005) build on the work of Kauffman (2004) who raised the attractor hypothesis, stating that cells exist in an expression landscape and their expression trajectories converge towards attractive sites in this landscape. We propose an alternative interpretation that explains this convergent behavior by recognizing that there are two types of processes participating in these cell fate transitions—core processes that include the specific differentiation pathways of promyelocytes to neutrophils, and transient processes that capture those pathways and responses specific to the inducer. Using functional enrichment analyses, specific biological examples and an analysis of the trajectories and their core and transient components we provide a validation of our hypothesis using the Huang et al. (2005) dataset

A CHIME/FRB Study of Burst Rate and Morphological Evolution of the Periodically Repeating FRB 20180916B

FRB 20180916B is a repeating fast radio burst (FRB) with a 16.3 day periodicity in its activity. In this study, we present morphological properties of 60 FRB 20180916B bursts detected by CHIME/FRB between 2018 August and 2021 December. We recorded raw voltage data for 45 of these bursts, enabling microseconds time resolution in some cases. We studied variation of spectro-temporal properties with time and activity phase. We find that the variation in dispersion measure (DM) is ≲1 pc cm−3 and that there is burst-to-burst variation in scattering time estimates ranging from ∼0.16 to over 2 ms, with no discernible trend with activity phase for either property. Furthermore, we find no DM and scattering variability corresponding to the recent change in rotation measure from the source, which has implications for the immediate environment of the source. We find that FRB 20180916B has thus far shown no epochs of heightened activity as have been seen in other active repeaters by CHIME/FRB, with its burst count consistent with originating from a Poissonian process. We also observe no change in the value of the activity period over the duration of our observations and set a 1σ upper limit of 1.5 × 10−4 day day−1 on the absolute period derivative. Finally, we discuss constraints on progenitor models yielded by our results, noting that our upper limits on changes in scattering and DM as a function of phase do not support models invoking a massive binary companion star as the origin of the 16.3 day periodicity.</p

Protocol Dependence of Sequencing-Based Gene Expression Measurements

RNA Seq provides unparalleled levels of information about the transcriptome including precise expression levels over a wide dynamic range. It is essential to understand how technical variation impacts the quality and interpretability of results, how potential errors could be introduced by the protocol, how the source of RNA affects transcript detection, and how all of these variations can impact the conclusions drawn. Multiple human RNA samples were used to assess RNA fragmentation, RNA fractionation, cDNA synthesis, and single versus multiple tag counting. Though protocols employing polyA RNA selection generate the highest number of non-ribosomal reads and the most precise measurements for coding transcripts, such protocols were found to detect only a fraction of the non-ribosomal RNA in human cells. PolyA RNA excludes thousands of annotated and even more unannotated transcripts, resulting in an incomplete view of the transcriptome. Ribosomal-depleted RNA provides a more cost-effective method for generating complete transcriptome coverage. Expression measurements using single tag counting provided advantages for assessing gene expression and for detecting short RNAs relative to multi-read protocols. Detection of short RNAs was also hampered by RNA fragmentation. Thus, this work will help researchers choose from among a range of options when analyzing gene expression, each with its own advantages and disadvantages

Dosimetric evaluation of Acuros XB Advanced Dose Calculation algorithm in heterogeneous media

<p>Abstract</p> <p>Background</p> <p>A study was realised to evaluate and determine relative figures of merit of a new algorithm for photon dose calculation when applied to inhomogeneous media.</p> <p>Methods</p> <p>The new Acuros XB algorithm implemented in the Varian Eclipse treatment planning system was compared against a Monte Carlo method (VMC++), and the Analytical Anisotropic Algorithm (AAA). The study was carried out in virtual phantoms characterized by simple geometrical structures. An insert of different material and density was included in a phantom built of skeletal-muscle and HU = 0 (setting "A"): Normal Lung (lung, 0.198 g/cm³); Light Lung (lung, 0.035 g/cm³); Bone (bone, 1.798 g/cm³); another phantom (setting "B") was built of adipose material and including thin layers of bone (1.85 g/cm³), adipose (0.92 g/cm³), cartilage (1.4745 g/cm³), air (0.0012 g/cm³). Investigations were performed for 6 and 15 MV photon beams, and for a large (13 × 13 cm²) and a small (2.8 × 13 cm²) field.</p> <p>Results</p> <p>Results are provided in terms of depth dose curves, transverse profiles and Gamma analysis (3 mm/3% and 2 mm/2% distance to agreement/dose difference criteria) in planes parallel to the beam central axis; Monte Carlo simulations were assumed as reference. Acuros XB gave an average gamma agreement, with a 3 mm/3% criteria, of 100%, 86% and 100% for Normal Lung, Light Lung and Bone settings, respectively, and dose to medium calculations. The same figures were 86%, 11% and 100% for AAA, where only dose rescaled to water calculations are possible.</p> <p>Conclusions</p> <p>In conclusion, Acuros XB algorithm provides a valid and accurate alternative to Monte Carlo calculations for heterogeneity management.</p

Phagocytosis of Streptococcus pyogenes by all-trans retinoic acid-differentiated HL-60 cells: roles of azurophilic granules and NADPH oxidase.

BACKGROUND: New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule-phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules. CONCLUSIONS/SIGNIFICANCE: The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion

Role of RecA and the SOS Response in Thymineless Death in Escherichia coli

Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans