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Heat Shock Factor 1 (HSF1) is the master regulator of the heat shock response, a universal survival mechanism throughout eukaryotic species used to buffer potentially lethal proteotoxic conditions. HSF1's function in vivo is regulated by several factors, including post translational modifications and elevated temperatures, whereupon it forms trimers to bind with heat shock elements in DNA. Unsurprisingly, HSF1 is also extremely sensitive to elevated temperatures in vitro, which poses specific technical challenges when producing HSF1 using a recombinant expression system. Although there are several useful publications which outline steps taken for HSF1 expression and purification, studies that describe specific strategies and detailed protocols to overcome HSF1 trimerisation and degradation are currently lacking. Herein, we have reported our detailed experimental protocol for the expression and purification of monomeric human HSF1 (HsHSF1) as a major species. We also propose a refined method of inducing HsHSF1 activation in vitro, that we consider more accurately mimics HsHSF1 activation in vivo and is therefore more physiologically relevant.

Coral bleaching is the single largest global threat to coral reefs worldwide. Integrating the diverse body of work on coral bleaching is critical to understanding and combating this global problem. Yet investigating the drivers, patterns, and processes of coral bleaching poses a major challenge. A recent review of published experiments revealed a wide range of experimental variables used across studies. Such a wide range of approaches enhances discovery, but without full transparency in the experimental and analytical methods used, can also make comparisons among studies challenging. To increase comparability but not stifle innovation, we propose a common framework for coral bleaching experiments that includes consideration of coral provenance, experimental conditions, and husbandry. For example, reporting the number of genets used, collection site conditions, the experimental temperature offset(s) from the maximum monthly mean (MMM) of the collection site, experimental light conditions, flow, and the feeding regime will greatly facilitate comparability across studies. Similarly, quantifying common response variables of endosymbiont (Symbiodiniaceae) and holobiont phenotypes (i.e., color, chlorophyll, endosymbiont cell density, mortality, and skeletal growth) could further facilitate cross-study comparisons. While no single bleaching experiment can provide the data necessary to determine global coral responses of all corals to current and future ocean warming, linking studies through a common framework as outlined here, would help increase comparability among experiments, facilitate synthetic insights into the causes and underlying mechanisms of coral bleaching, and reveal unique bleaching responses among genets, species, and regions. Such a collaborative framework that fosters transparency in methods used would strengthen comparisons among studies that can help inform coral reef management and facilitate conservation strategies to mitigate coral bleaching worldwide

Coral bleaching is the single largest global threat to coral reefs worldwide. Integrating the diverse body of work on coral bleaching is critical to understanding and combating this global problem. Yet investigating the drivers, patterns, and processes of coral bleaching poses a major challenge. A recent review of published experiments revealed a wide range of experimental variables used across studies. Such a wide range of approaches enhances discovery, but without full transparency in the experimental and analytical methods used, can also make comparisons among studies challenging. To increase comparability but not stifle innovation, we propose a common framework for coral bleaching experiments that includes consideration of coral provenance, experimental conditions, and husbandry. For example, reporting the number of genets used, collection site conditions, the experimental temperature offset(s) from the maximum monthly mean (MMM) of the collection site, experimental light conditions, flow, and the feeding regime will greatly facilitate comparability across studies. Similarly, quantifying common response variables of endosymbiont (Symbiodiniaceae) and holobiont phenotypes (i.e., color, chlorophyll, endosymbiont cell density, mortality, and skeletal growth) could further facilitate cross-study comparisons. While no single bleaching experiment can provide the data necessary to determine global coral responses of all corals to current and future ocean warming, linking studies through a common framework as outlined here, would help increase comparability among experiments, facilitate synthetic insights into the causes and underlying mechanisms of coral bleaching, and reveal unique bleaching responses among genets, species, and regions. Such a collaborative framework that fosters transparency in methods used would strengthen comparisons among studies that can help inform coral reef management and facilitate conservation strategies to mitigate coral bleaching worldwide

Heat shock protein 90 (HSP90) regulates multiple signalling pathways critical for tumour growth. As such, HSP90 inhibitors have been shown to act as effective anticancer agents in preclinical studies but, for a number of reasons, the same effect has not been observed in the clinical trials to date. One potential reason for this may be the presence of de novo or acquired resistance within the tumours. To investigate mechanisms of resistance, we generated resistant cell lines through gradual dose escalation of the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The resultant resistant cell lines maintained their respective levels of resistance (7-240×) in the absence of 17-AAG and were also cross-resistant with other benzoquinone ansamycin HSP90 inhibitors. Expression of members of the histone deacetylase family (HDAC 1, 5, 6) was altered in the resistant cells. To determine whether HDAC activity contributed to resistance, pan-HDAC inhibitors (TSA and LBH589) and the class II HDAC-specific inhibitor SNDX275 were found to resensitize resistant cells towards 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin. Most significantly, resistant cells were also identified as cross-resistant towards structurally distinct HSP90 inhibitors such as radicicol and the second-generation HSP90 inhibitors CCT018159, VER50589 and AUY922. HDAC inhibition also resensitized resistant cells towards these classes of HSP90 inhibitors. In conclusion, we report that prolonged 17-AAG treatment results in acquired resistance of cancer cells towards not just 17-AAG but also to a spectrum of structurally distinct HSP90 inhibitors. This acquired resistance can be inhibited using clinically relevant HDAC inhibitors. This work supports the potential benefit of using HSP90 and HDAC inhibitors in combination within the clinical setting.

BACKGROUND: High mammographic density (HMD) not only confers a significantly increased risk of breast cancer (BC) but also is associated with BCs of more advanced stages. However, it is unclear whether BC progression and metastasis are stimulated by HMD. We investigated whether patient-derived HMD breast tissue could stimulate the progression of MCF10DCIS.com cells compared with patient-matched low mammographic density (LMD) tissue. METHODS: Sterile breast specimens were obtained immediately after prophylactic mastectomy from high-risk women (n = 10). HMD and LMD regions of each specimen were resected under radiological guidance. Human MCF10DCIS.com cells, a model of ductal carcinoma in situ (DCIS), were implanted into silicone biochambers in the groins of severe combined immunodeficiency mice, either alone or with matched LMD or HMD tissue (1:1), and maintained for 6 weeks. We assessed biochamber weight as a measure of primary tumour growth, histological grade of the biochamber material, circulating tumour cells and metastatic burden by luciferase and histology. All statistical tests were two-sided. RESULTS: HMD breast tissue led to increased primary tumour take, increased biochamber weight and increased proportions of high-grade DCIS and grade 3 invasive BCs compared with LMD. This correlated with an increased metastatic burden in the mice co-implanted with HMD tissue. CONCLUSIONS: Our study is the first to explore the direct effect of HMD and LMD human breast tissue on the progression and dissemination of BC cells in vivo. The results suggest that HMD status should be a consideration in decision-making for management of patients with DCIS lesions.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

The glycoprotein (GP) Ib /V/IX receptor complex is an important adhesion molecule, originally thought to be unique to the megakaryocytic lineage. Recent evidence now indicates that GPIb /V/IX may be more widely expressed. In this study we report the presence of all subunits of the complex on four breast cancer cell lines, and 51 / 80 primary breast tumours. The surface expression of GPIb /V/IX was confirmed by flow cytometry, and by immunoprecipitation of biotin surface-labelled tumour cells. Western blotting of cell lysates under reducing conditions revealed that tumour cell-GPIb alpha had a relative molecular weight of 95 kDa as compared to 135 kDa on platelets. Despite the discrepant protein size, molecular analyses on the tumour cell-GPIb alpha subunit using RT-PCR and DNA sequencing revealed 100% sequence homology to platelet GPIb alpha. Tumour cell-GPIb /V/IX was capable of binding human von Willebrand factor (vWf), and this binding caused aggregation of tumour cells in suspension. Tumour cells bound to immobilised vWf in the presence of EDTA and demonstrated prominent filapodial extensions indicative of cytoskeletal reorganisation. Furthermore, in a modified Boyden chamber assay, prior exposure to vWf or a GPIb alpha monoclonal antibody, AK2, enhanced cell migration. The presence of a functional GPIb /V/IX-like complex in tumour cells suggests that this complex may participate in the process of haematogenous breast cancer metastasis.