Functional Characterisation of the Circular RNA, , in Huntington's Disease.

Trinucleotide repeat disorders comprise ~20 severe, inherited, human neuromuscular and neurodegenerative disorders, which result from an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington's disease (HD), results from expansion of the CAG repeat region in exon 1 of the gene via an unknown mechanism. Since non-coding RNAs have been implicated in the initiation and progression of many diseases, herein we focused on a circular RNA (circRNA) molecule arising from non-canonical splicing (backsplicing) of pre-mRNA.

Characterising Distinct Migratory Profiles of Infiltrating T-Cell Subsets in Human Glioblastoma.

Glioblastoma is the most common and aggressive form of primary brain cancer, with no improvements in the 5-year survival rate of 4.6% over the past three decades. T-cell-based immunotherapies such as immune-checkpoint inhibitors and chimeric antigen receptor T-cell therapy have prolonged the survival of patients with other cancers and have undergone early-phase clinical evaluation in glioblastoma patients.

A deep convolutional neural network for segmentation of whole-slide pathology images identifies novel tumour cell-perivascular niche interactions that are associated with poor survival in glioblastoma.

Background: Glioblastoma is the most aggressive type of brain cancer with high-levels of intra- and inter-tumour heterogeneity that contribute to its rapid growth and invasion within the brain. However, a spatial characterisation of gene signatures and the cell types expressing these in different tumour locations is still lacking.

Methods: We have used a deep convolutional neural network (DCNN) as a semantic segmentation model to segment seven different tumour regions including leading edge (LE), infiltrating tumour (IT), cellular tumour (CT), cellular tumour microvascular proliferation (CTmvp), cellular tumour pseudopalisading region around necrosis (CTpan), cellular tumour perinecrotic zones (CTpnz) and cellular tumour necrosis (CTne) in digitised glioblastoma histopathological slides from The Cancer Genome Atlas (TCGA).

A Drug Screening Pipeline Using 2D and 3D Patient-Derived In Vitro Models for Pre-Clinical Analysis of Therapy Response in Glioblastoma.

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6-9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microenvironment are required.

3D-printed microplate inserts for long term high-resolution imaging of live brain organoids.

Background: Organoids are a reliable model used in the study of human brain development and under pathological conditions. However, current methods for brain organoid culture generate tissues that range from 0.5 to 2 mm of size, which need to be constantly agitated to allow proper oxygenation.

SRRM4 Expands the Repertoire of Circular RNAs by Regulating Microexon Inclusion.

High-throughput RNA sequencing (RNA-seq) and dedicated bioinformatics pipelines have synergized to identify an expansive repertoire of unique circular RNAs (circRNAs), exceeding 100,000 variants. While the vast majority of these circRNAs comprise canonical exonic and intronic sequences, microexons (MEs)-which occur in 30% of functional mRNA transcripts-have been entirely overlooked. CircRNAs which contain these known MEs (ME-circRNAs) could be identified with commonly utilized circRNA prediction pipelines, CIRCexplorer2 and CIRI2, but were not previously recognized as ME-circRNAs.

Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy.

Objectives: Targeted immunotherapies such as chimeric antigen receptor (CAR)-T cells are emerging as attractive treatment options for glioblastoma, but rely on identification of a suitable tumor antigen. We validated a new target antigen for glioblastoma, fibroblast activation protein (FAP), by undertaking a detailed expression study of human samples.

Methods: Glioblastoma and normal tissues were assessed using immunostaining, supported by analyses of published transcriptomic datasets.

DMAPT is an Effective Radioprotector from Long-Term Radiation-Induced Damage to Normal Mouse Tissues .

While radiotherapy is widely used in cancer treatment, the benefits can be limited by radiation-induced damage to neighboring healthy tissues. We previously demonstrated in mice that the anti-inflammatory compound dimethylaminoparthenolide (DMAPT) selectively induces radiosensitivity in prostate tumor tissue from transgenic adenocarcinoma of mouse prostate (TRAMP) mice, while simultaneously protecting healthy tissues from 6 Gy whole-body radiation-induced apoptosis. Here, we examined the radioprotective effect of DMAPT on fibrosis in normal tissues after a partial-body fractionated radiation protocol that more closely mimics the image-guided fractionated radiotherapy protocols used clinically.

The Combination of Metformin and Valproic Acid Has a Greater Anti-tumoral Effect on Prostate Cancer Growth than Either Drug Alone.

Background/aim: The hypoglycemic drug metformin (MET) and the anti-epileptic drug valproic acid (VPA) have individually shown anti-tumor effects in prostate cancer in vitro. The present study intended to investigate the efficacy of the combination of MET and VPA in prostate cancer treatment in a pre-clinical xenograft model.

Materials And Methods: Prostate cancer cell lines (LNCaP and PC-3) were inoculated under the skin of BALB/c nude mice.

Chronic low dose ethanol induces an aggressive metastatic phenotype in TRAMP mice, which is counteracted by parthenolide.

Despite advances in prostate cancer therapy, dissemination and growth of metastases results in shortened survival. Here we examined the potential anti-cancer effect of the NF-κB inhibitor parthenolide (PTL) and its water soluble analogue dimethylaminoparthenolide (DMAPT) on tumour progression and metastasis in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model of prostate cancer. Six-week-old male TRAMP mice received PTL (40 mg/kg in 10% ethanol/saline), DMAPT (100 mg/kg in sterile water), or vehicle controls by oral gavage thrice weekly until palpable tumour formation.

The Combination of Metformin and Valproic Acid Induces Synergistic Apoptosis in the Presence of p53 and Androgen Signaling in Prostate Cancer.

We investigated the potential of combining the hypoglycemic drug metformin (MET) and the antiepileptic drug valproic acid (VPA), which act via different biochemical pathways, to provide enhanced antitumor responses in prostate cancer. Prostate cancer cell lines (LNCaP and PC-3), normal prostate epithelial cells (PrEC), and patient-derived prostate tumor explants were treated with MET and/or VPA. Proliferation and apoptosis were assessed.

Parthenolide Selectively Sensitizes Prostate Tumor Tissue to Radiotherapy while Protecting Healthy Tissues In Vivo.

Radiotherapy is widely used in cancer treatment, however the benefits can be limited by radiation-induced damage to neighboring normal tissues. Parthenolide (PTL) exhibits anti-inflammatory and anti-tumor properties and selectively induces radiosensitivity in prostate cancer cell lines, while protecting primary prostate epithelial cell lines from radiation-induced damage. Low doses of radiation have also been shown to protect from subsequent high-dose-radiation-induced apoptosis as well as DNA damage.

Temporal Responses to X-Radiation Exposure in Spleen in the pKZ1 Mouse Recombination Assay.

The in vivo mouse transgenic pKZ1 chromosomal inversion assay is a sensitive assay that responds to very low doses of DNA-damaging agents. pKZ1 inversions are measured as the frequency of cells expressing E. coli β-galactosidase protein, which can only be produced from an inverted pKZ1 transgene.

A single whole-body low dose X-irradiation does not affect L1, B1 and IAP repeat element DNA methylation longitudinally.

The low dose radioadaptive response has been shown to be protective against high doses of radiation as well as aging-induced genomic instability. We hypothesised that a single whole-body exposure of low dose radiation would induce a radioadaptive response thereby reducing or abrogating aging-related changes in repeat element DNA methylation in mice. Following sham or 10 mGy X-irradiation, serial peripheral blood sampling was performed and differences in Long Interspersed Nucleic Element 1 (L1), B1 and Intracisternal-A-Particle (IAP) repeat element methylation between samples were assessed using high resolution melt analysis of PCR amplicons.

Protection from radiation-induced apoptosis by the radioprotector amifostine (WR-2721) is radiation dose dependent.

The radioprotective agent amifostine is a free radical scavenger that can protect cells from the damaging effects of ionising radiation when administered prior to radiation exposure. However, amifostine has also been shown to protect cells from chromosomal mutations when administered after radiation exposure. As apoptosis is a common mechanism by which cells with mutations are removed from the cell population, we investigated whether amifostine stimulates apoptosis when administered after radiation exposure.

The methylation of DNA repeat elements is sex-dependent and temporally different in response to X radiation in radiosensitive and radioresistant mouse strains.

The effects of ionizing radiation on DNA methylation are of importance due to the role that DNA methylation plays in maintaining genome stability, and the presence of aberrant DNA methylation in many cancers. There is limited evidence that radiation-sensitivity may influence the modulation of DNA methylation by ionizing radiation, resulting in a loss of methylation. The BALB/c, CBA and C57Bl/6 strains are the most commonly utilized mouse strains in radiation research and are classified as radiation sensitive (BALB/c and CBA) or radiation resistant (C57Bl/6).

Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis.

We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2'-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS).

If bystander effects for apoptosis occur in spleen after low-dose irradiation in vivo then the magnitude of the effect falls within the range of normal homeostatic apoptosis.

To test whether bystander effects occur in vivo after low doses of radiation relevant to occupational and population exposure, we exposed mice to whole-body X-radiation doses (0.01 and 1 mGy) where only a proportion of cells would receive an electron track. We used a precise method to analyze the apoptosis frequency in situ in spleen tissue sections at 7 h and 1, 3 and 7 days after irradiation to determine whether an increase in apoptosis above that predicted by direct effects was observed.

An adoptive transfer method to detect low-dose radiation-induced bystander effects in vivo.

The potential for irradiated cells to induce biological effects in their unirradiated neighbors (known as the bystander effect) has been observed repeatedly in vitro. However, whether bystander effects occur in vivo under the specific conditions relevant to low-dose radiation protection is still unclear. To test this, the fate of bystander cells in the mouse spleen was examined using an adoptive transfer method designed to replicate the rare, irradiated cells in an organ that might be expected after a low-dose-rate, low-LET radiation exposure.

Multimeric interactions between complement factor H and its C3d ligand provide new insight on complement regulation.

Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations.

Functional and structural implications of the complement factor H Y402H polymorphism associated with age-related macular degeneration.

Purpose: A Tyr-to-His (Y402H) sequence variant in the factor H (FH) and factor H-like protein (FHL-1) gene is strongly associated with an increased susceptibility for age-related macular degeneration (AMD). The purpose of this study was to understand how the Y402H variant in FH/FHL-1 contributes to the pathogenesis of AMD and, in particular, whether interactions mediated by FH/FHL-1, including binding to C-reactive protein (CRP), group A streptococcal M protein (GAS M6), heparin, and retinal pigment epithelial cells (RPE), are affected.

Methods: FH was purified from sera of patients homozygous for FH(Y402) or (H402), and recombinant FH fragments representing FHL-1 were generated.

The regulatory SCR-1/5 and cell surface-binding SCR-16/20 fragments of factor H reveal partially folded-back solution structures and different self-associative properties.

Factor H (FH) is a plasma glycoprotein that plays a central role in regulation of the alternative pathway of complement. It is composed of 20 short complement regulator (SCR) domains. The SCR-1/5 fragment is required for decay acceleration and cofactor activity, while the SCR-16/20 fragment possesses binding sites for complement C3d and heparin.

Localization of the third heparin-binding site in the human complement regulator factor H1.

Complement factor H (fH) plays a pivotal role in regulating the alternative pathway, allowing complement activation to proceed on foreign surfaces, whilst protecting surrounding host cell surfaces from complement-mediated damage. Host cell recognition is mediated by polyanions such as sialic acid and glycosaminoglycans (GAGs), which promote a high affinity interaction between fH and C3b deposited on host cell surfaces. Factor H is composed of 20 short consensus repeats (SCRs); two heparin-binding sites have been identified within SCR 7 and SCR 20 and a third site is thought to exist within or near SCR 13.

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein.

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins.