Chloroquine, the Coronavirus Crisis, and Neurodegeneration: A Perspective

On the verge of the ongoing coronavirus pandemic, in vitro data suggested that chloroquine, and its analog hydroxychloroquine, may be useful in controlling SARS-CoV-2 infection. Efforts are ongoing in order to test this hypothesis in clinical trials. Some studies demonstrated no evidence of efficacy, whereas in some cases results were retracted after reporting. Despite the lack of scientific validation, support for the use of these compounds continues from various influencers. At the cellular level, the lysosomotropic drug chloroquine accumulates in acidic organelles where it acts as an alkalizing agent with possible downstream effects on several cellular pathways. In this perspective, we discuss a possible modulatory role of these drugs in two shared features of neurodegenerative diseases, the cellular accumulation of aberrantly folded proteins and the contribution of neuroinflammation in this pathogenic process. Certainly, the decision on the use of chloroquine must be determined by its efficacy in the specific clinical situation. However, at an unprecedented time of a potential widespread use of chloroquine, we seek to raise awareness of its potential impact in ongoing clinical trials evaluating disease-modifying therapies in neurodegeneration

Brain-derived neurotrophic factor expression predicts adverse pathological & clinical outcomes in human breast cancer

Introduction Brain-derived neurotrophic factor (BDNF) has established physiological roles in the development and function of the vertebrate nervous system. BDNF has also been implicated in several human malignancies, including breast cancer (BC). However, the precise biological role of BDNF and its utility as a novel biomarker have yet to be determined. The objective of this study was to determine the mRNA and protein expression of BDNF in a cohort of women with BC. Expression levels were compared with normal background tissues and evaluated against established pathological parameters and clinical outcome over a 10 year follow-up period. Methods BC tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription, BDNF transcript levels were determined using real-time quantitative PCR. BDNF protein expression in mammary tissues was assessed with standard immuno-histochemical methodology. Expression levels were analyzed against tumour size, grade, nodal involvement, TNM stage, Nottingham Prognostic Index (NPI) and clinical outcome over a 10 year follow-up period. Results Immuno-histochemical staining revealed substantially greater BDNF expression within neoplastic cells, compared to normal mammary epithelial cells. Significantly higher mRNA transcript levels were found in the BC specimens compared to background tissues (p = 0.007). The expression of BDNF mRNA was demonstrated to increase with increasing NPI; NPI-1 vs. NPI-2 (p = 0.009). Increased BDNF transcript levels were found to be significantly associated with nodal positivity (p = 0.047). Compared to patients who remained disease free, higher BDNF expression was significantly associated with local recurrence (LR) (p = 0.0014), death from BC (p = 0.018) and poor prognosis overall (p = 0.013). After a median follow up of 10 years, higher BDNF expression levels were significantly associated with reduced overall survival (OS) (106 vs. 136 months, p = 0.006). BDNF emerged as an independent prognostic variable in multivariate analysis for disease free survival (DFS) (p = 0.026) and approached significance for OS (p = 0.055). Conclusion BDNF expression was found to be significantly higher in BC specimens compared to normal tissue. Higher transcript levels were significantly associated with unfavourable pathological parameters including nodal positivity and increasing NPI; and adverse clinical outcomes including LR, death from BC, poor prognosis, reduced DFS and OS. BDNF offers utility as a prognostic marker and potential for targeted therapeutic strategies

Identification of target-specific bioisosteric fragments from ligand-protein crystallographic data

Bioisosteres are functional groups or atoms that are structurally different but that can form similar intermolecular interactions. Potential bioisosteres were identified here from analysing the X-ray crystallographic structures for sets of different ligands complexed with a fixed protein. The protein was used to align the ligands with each other, and then pairs of ligands compared to identify substructural features with high volume overlap that occurred in approximately the same region of geometric space. The resulting pairs of substructural features can suggest potential bioisosteric replacements for use in lead-optimisation studies. Experiments with 12 sets of ligand-protein complexes from the Protein Data Bank demonstrate the effectiveness of the procedure

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Summary: Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development. : Maffioletti et al. generate human 3D artificial skeletal muscles from healthy donors and patient-specific pluripotent stem cells. These human artificial muscles accurately model severe genetic muscle diseases. They can be engineered to include other cell types present in skeletal muscle, such as vascular cells and motor neurons. Keywords: skeletal muscle, pluripotent stem cells, iPS cells, myogenic differentiation, tissue engineering, disease modeling, muscular dystrophy, organoid

Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Background Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility. Methods A panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1wt or ESR1Y537S, modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies. Results Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001. Conclusions Our data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER

A neuroprotective astrocyte state is induced by neuronal signal EphB1 but fails in ALS models

Astrocyte responses to neuronal injury may be beneficial or detrimental to neuronal recovery, but the mechanisms that determine these different responses are poorly understood. Here we show that ephrin type-B receptor 1 (EphB1) is upregulated in injured motor neurons, which in turn can activate astrocytes through ephrin-B1-mediated stimulation of signal transducer and activator of transcription-3 (STAT3). Transcriptional analysis shows that EphB1 induces a protective and anti-inflammatory signature in astrocytes, partially linked to the STAT3 network. This is distinct from the response evoked by interleukin (IL)-6 that is known to induce both pro inflammatory and anti-inflammatory processes. Finally, we demonstrate that the EphB1–ephrin-B1 pathway is disrupted in human stem cell derived astrocyte and mouse models of amyotrophic lateral sclerosis (ALS). Our work identifies an early neuronal help-me signal that activates a neuroprotective astrocytic response, which fails in ALS, and therefore represents an attractive therapeutic target.This work has been funded by Medical Research Council (MR/P008658/1 for A.L.), the Walker Fund (A.L.), John van Geest Fund (A.L.) and the Wellcome Trust (101149/Z/13/A for R.P.). We also acknowledge the DPUK/MRC platform for provision of the Opera Phenix for high-throughput iPSC analysis and the support by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. We are grateful for the support from the Grant Agency of the Czech Republic (GACR 17-21146 S, S.F.). Dr Christopher Sibley is supported by the Edmond Lilly Safra Fellowship, Dr Rickie Patani holds a Wellcome Trust Clinician Scientist Fellowship and Dr András Lakatos holds an MRC Clinician Scientist Fellowship

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development. Maffioletti et al. generate human 3D artificial skeletal muscles from healthy donors and patient-specific pluripotent stem cells. These human artificial muscles accurately model severe genetic muscle diseases. They can be engineered to include other cell types present in skeletal muscle, such as vascular cells and motor neurons

Primary radiotherapy and deep inferior epigastric perforator flap reconstruction for patients with breast cancer (PRADA): a multicentre, prospective, non-randomised, feasibility study

BACKGROUND: Radiotherapy before mastectomy and autologous free-flap breast reconstruction can avoid adverse radiation effects on healthy donor tissues and delays to adjuvant radiotherapy. However, evidence for this treatment sequence is sparse. We aimed to explore the feasibility of preoperative radiotherapy followed by skin-sparing mastectomy and deep inferior epigastric perforator (DIEP) flap reconstruction in patients with breast cancer requiring mastectomy. METHODS: We conducted a prospective, non-randomised, feasibility study at two National Health Service trusts in the UK. Eligible patients were women aged older than 18 years with a laboratory diagnosis of primary breast cancer requiring mastectomy and post-mastectomy radiotherapy, who were suitable for DIEP flap reconstruction. Preoperative radiotherapy started 3-4 weeks after neoadjuvant chemotherapy and was delivered to the breast, plus regional nodes as required, at 40 Gy in 15 fractions (over 3 weeks) or 42·72 Gy in 16 fractions (over 3·2 weeks). Adverse skin radiation toxicity was assessed preoperatively using the Radiation Therapy Oncology Group toxicity grading system. Skin-sparing mastectomy and DIEP flap reconstruction were planned for 2-6 weeks after completion of preoperative radiotherapy. The primary endpoint was the proportion of open breast wounds greater than 1 cm width requiring a dressing at 4 weeks after surgery, assessed in all participants. This study is registered with ClinicalTrials.gov, NCT02771938, and is closed to recruitment. FINDINGS: Between Jan 25, 2016, and Dec 11, 2017, 33 patients were enrolled. At 4 weeks after surgery, four (12·1%, 95% CI 3·4-28·2) of 33 patients had an open breast wound greater than 1 cm. One (3%) patient had confluent moist desquamation (grade 3). There were no serious treatment-related adverse events and no treatment-related deaths. INTERPRETATION: Preoperative radiotherapy followed by skin-sparing mastectomy and immediate DIEP flap reconstruction is feasible and technically safe, with rates of breast open wounds similar to those reported with post-mastectomy radiotherapy. A randomised trial comparing preoperative radiotherapy with post-mastectomy radiotherapy is required to precisely determine and compare surgical, oncological, and breast reconstruction outcomes, including quality of life. FUNDING: Cancer Research UK, National Institute for Health Research

Hyperpolarised 13C MRI: a new horizon for non-invasive diagnosis of aggressive breast cancer

Hyperpolarised 13C MRI (HP-MRI) is a novel imaging technique that allows real-time analysis of metabolic pathways in vivo. 1 The technology to conduct HP-MRI in humans has recently become available and is starting to be clinically applied. As knowledge of molecular biology advances, it is increasingly apparent that cancer cell metabolism is related to disease outcomes, with lactate attracting specific attention. 2 Recent reviews of breast cancer screening programs have raised concerns and increased public awareness of over treatment. The scientific community needs to shift focus from improving cancer detection alone to pursuing novel methods of distinguishing aggressive breast cancers from those which will remain indolent. HP-MRI offers the opportunity to identify aggressive tumour phenotypes and help monitor/predict therapeutic response. Here we report one of the first cases of breast cancer imaged using HP-MRI alongside correlative conventional imaging, including breast MRI