Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy

Collagen hydrogels are a rapidly expanding platform in bioengineering and soft materials engineering for novel applications focused on medical therapeutics, medical devices and biosensors. Observations linking microstructure to material properties and function enables rational design strategies to control this space. Visualisation of the microscale organisation of these soft hydrated materials presents unique technical challenges due to the relationship between hydration and the molecular organisation of a collagen gel. Scanning electron microscopy is a robust tool widely employed to visualise and explore materials on the microscale. However, investigation of collagen gel microstructure is difficult without imparting structural changes during preparation and/or observation. Electrons are poorly propagated within liquid-phase materials, limiting the ability of electron microscopy to interrogate hydrated gels. Sample preparation techniques to remove water induce artefactual changes in material microstructure particularly in complex materials such as collagen, highlighting a critical need to develop robust material handling protocols for the imaging of collagen hydrogels. Here a collagen hydrogel is fabricated, and the gel state explored under high-vacuum (10−6Pa) and low-vacuum (80–120Pa) conditions, and in an environmental SEM chamber. Visualisation of collagen fibres is found to be dependent on the degree of sample hydration, with higher imaging chamber pressures and humidity resulting in decreased feature fidelity. Reduction of imaging chamber pressure is used to induce evaporation of gel water content, revealing collagen fibres of significantly larger diameter than observed in samples dehydrated prior to imaging. Rapid freezing and cryogenic handling of the gel material is found to retain a porous 3D structure following sublimation of the gel water content. Comparative analysis of collagen hydrogel materials demonstrates the care needed when preparing hydrogel samples for electron microscopy

Delivery of temozolomide and N3-propargyl analog to brain tumors using an apoferritin nanocage

Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values 500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies

Immobilized Enzymes on Gold Nanoparticles: From Enhanced Stability to Cleaning of Heritage Textiles

Enzyme-based treatments are used in heritage conservation for the effective removal of glues and other damaging organic layers from the surfaces of historic and artisitc works. Despite their potential, however, the application of enzymatic treatments is currently limited due to their poor efficiency, and low operational and environmental stability. We demonstrate the use of α-amylase immobilized on gold nanoparticles to improve the efficacy of enzymatic treatments enchancing both the reactivity and the stability of the formulations. Gold nanoparticles coated with α-amylase exhibit significant advantages compared to free enzymes. We report up to 5-times greater resistance to environmental changes, up to 2-times higher efficacy towards removal of starch-based glues from textile, and deeper penetration through the fibres, without causing damage or inducing salt precipitation. These results offer exciting prospects for the development of novel enzymatic formulations, both for heritage conservation and the wider application of enzymes, such as in medicine, the detergent industry and green chemistry

Exome sequencing in classic hairy cell leukaemia reveals widespread variation in acquired somatic mutations between individual tumours apart from the signature BRAF V(600)E lesion

In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease

An integrated approach to determine interactive genotoxic and global gene expression effects of multiwalled carbon nanotubes (MWCNTs) and benzo[a]pyrene (BaP) on marine mussels: evidence of reverse ‘Trojan Horse’ effects

International audienc

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Ten-year mortality, disease progression, and treatment-related side effects in men with localised prostate cancer from the ProtecT randomised controlled trial according to treatment received

Background The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy. Objective To report outcomes according to treatment received in men in randomised and treatment choice cohorts. Design, setting, and participants This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy. Intervention Two cohorts included 1643 men who agreed to be randomised and 997 who declined randomisation and chose treatment. Outcome measurements and statistical analysis Analysis was carried out to assess mortality, metastasis and progression and health-related quality of life impacts on urinary, bowel, and sexual function using patient-reported outcome measures. Analysis was based on comparisons between groups defined by treatment received for both randomised and treatment choice cohorts in turn, with pooled estimates of intervention effect obtained using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores. Results and limitations According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and changes in the protocol for AM during the lengthy follow-up required in trials of screen-detected PCa. Conclusions Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group. Patient summary More than 95 out of every 100 men with low or intermediate risk localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are better after active monitoring, but the risks of spreading of prostate cancer are more common

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients