Smoothing a rugged protein folding landscape by sequence-based redesign

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as $\alpha_1$-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create $\textit{conserpin}$, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.BTP is a Medical Research Council Career Development Fellow. AAN and JJH are supported by the Wellcome Trust (grant number WT 095195). SM acknowledges fellowship support from the Australian Research Council (FT100100960). NAB is an Australian Research Council Future Fellow (110100223). GIW is an Australian Research Council Discovery Outstanding Researcher Award Fellow (DP140100087). AMB is a National Health and Medical Research Senior Research Fellow (1022688). JCW is an NHMRC Senior Principal Research fellow and also acknowledges the support of an ARC Federation Fellowship. We thank the Australian Synchrotron for beam-time and technical assistance. This work was supported by the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE) (www.massive.org.au). We acknowledge the Monash Protein Production Unit and Monash Macromolecular Crystallization Facilit

Smoothing a rugged protein folding landscape by sequence-based redesign

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics

Adipose-Derived Stem Cells Spontaneously Express Neural Markers When Grown in a PEG-Based 3D Matrix.

Neurological diseases are among the leading causes of disability and death worldwide and remain difficult to treat. Tissue engineering offers avenues to test potential treatments; however, the development of biologically accurate models of brain tissues remains challenging. Given their neurogenic potential and availability, adipose-derived stem cells (ADSCs) are of interest for creating neural models. While progress has been made in differentiating ADSCs into neural cells, their differentiation in 3D environments, which are more representative of the in vivo physiological conditions of the nervous system, is crucial. This can be achieved by modulating the 3D matrix composition and stiffness. Human ADSCs were cultured for 14 days in a 1.1 kPa polyethylene glycol-based 3D hydrogel matrix to assess effects on cell morphology, cell viability, proteome changes and spontaneous neural differentiation. Results showed that cells continued to proliferate over the 14-day period and presented a different morphology to 2D cultures, with the cells elongating and aligning with one another. The proteome analysis revealed 439 proteins changed in abundance by >1.5 fold. Cyclic nucleotide 3'-phosphodiesterase (CNPase) markers were identified using immunocytochemistry and confirmed with proteomics. Findings indicate that ADSCs spontaneously increase neural marker expression when grown in an environment with similar mechanical properties to the central nervous system

Neural Marker Expression in Adipose-Derived Stem Cells Grown in PEG-Based 3D Matrix Is Enhanced in the Presence of B27 and CultureOne Supplements.

Adipose-derived stem cells (ADSCs) have incredible potential as an avenue to better understand and treat neurological disorders. While they have been successfully differentiated into neural stem cells and neurons, most such protocols involve 2D environments, which are not representative of in vivo physiology. In this study, human ADSCs were cultured in 1.1 kPa polyethylene-glycol 3D hydrogels for 10 days with B27, CultureOne (C1), and N2 neural supplements to examine the neural differentiation potential of ADSCs using both chemical and mechanical cues. Following treatment, cell viability, proliferation, morphology, and proteome changes were assessed. Results showed that cell viability was maintained during treatments, and while cells continued to proliferate over time, proliferation slowed down. Morphological changes between 3D untreated cells and treated cells were not observed. However, they were observed among 2D treatments, which exhibited cellular elongation and co-alignment. Proteome analysis showed changes consistent with early neural differentiation for B27 and C1 but not N2. No significant changes were detected using immunocytochemistry, potentially indicating a greater differentiation period was required. In conclusion, treatment of 3D-cultured ADSCs in PEG-based hydrogels with B27 and C1 further enhances neural marker expression, however, this was not observed using supplementation with N2

Novel facultative Methylocella strains are active methane consumers at terrestrial natural gas seeps

Natural gas seeps contribute to global climate change by releasing substantial amounts of the potent greenhouse gas methane and other climate-active gases including ethane and propane to the atmosphere. However, methanotrophs, bacteria capable of utilising methane as the sole source of carbon and energy, play a significant role in reducing the emissions of methane from many environments. Methylocella-like facultative methanotrophs are a unique group of bacteria that grow on other components of natural gas (i.e. ethane and propane) in addition to methane but a little is known about the distribution and activity of Methylocella in the environment. The purposes of this study were to identify bacteria involved in cycling methane emitted from natural gas seeps and, most importantly, to investigate if Methylocella-like facultative methanotrophs were active utilisers of natural gas at seep sites

Efficacy and toxicity outcomes for patients treated with focal salvage high dose rate brachytherapy for locally recurrent prostate cancer

Introduction Isolated local recurrence of prostate cancer following primary radiotherapy or brachytherapy may be treated with focal salvage high dose rate brachytherapy, although there remains an absence of high quality evidence to support this approach. Methods Men with prostate cancer treated consecutively between 2015 and 2018 using 19 Gy in a single fraction high dose rate brachytherapy (HDR) for locally recurrent prostate cancer were identified from an institutional database. Univariable analysis was performed to evaluate the relationship between patient, disease and treatment factors with biochemical progression free survival (bPFS). Results 43 patients were eligible for evaluation. Median follow up duration was 26 months (range 1–60). Median bPFS was 35 months (95% confidence interval 25.6–44.4). Kaplan-Meier estimates for bPFS at 1, 2 and 3 years post salvage were 95.2%, 70.6% and 41.8% respectively. On univariable Cox regression analysis, only nadir PSA was significantly associated with bPFS although the majority of patients were also treated with androgen deprivation therapy. Only one late grade 3 genitourinary toxicity was observed. Conclusion Focal salvage HDR brachytherapy may provide good biochemical control with a low risk of severe toxicity. Further evaluation within clinical trials are needed to establish its role in the management of locally recurrent prostate cancer

Ten-year longitudinal health-related quality of life following iodine-125 brachytherapy monotherapy for localized prostate cancer

Purpose This prospective longitudinal study quantifies health-related quality of life (HRQoL) up to 10 years following permanent iodine-125 (125I) prostate brachytherapy alone for localized prostate cancer. Material and methods In total, 120 patients completed a validated expanded prostate cancer index composite (EPIC) questionnaire pre-treatment and at 8 time points after treatment (6 weeks, 6, 10, 18 months, and 2, 3, 5, 10 years). At each time point, clinically relevant small, moderate, and severe declines in HRQoL were defined as 0.2-0.5 SD, 0.5-0.8 SD, and > 0.8 SD of baseline function for each of urinary, bowel, and sexual domains, respectively. Results Response rates in the first two years were > 90%, but thereafter dropped to 75% and 48% at 5 and 10 years, respectively. 50 patients (41.6%) responded at all stages. Maximal deterioration in mean urinary and sexual summary scores was noted 6 weeks after implant, with severe urinary symptoms and moderate bowel/sexual symptoms. At 6 months, urinary and bowel quality of life (QoL) had improved to mild impairment, which then fully resolved at 10 months. Sexual QoL remained mildly impaired throughout the 10 years of follow-up. At 10 years, new mild impairment of urinary and bowel QoL was found. Conclusions Clinically mild changes in urinary, bowel, and sexual QoL are found 10 years after 125I monotherapy. The impairment in sexual function persists from treatment, but urinary and bowel symptoms are new at 10 years

A comparison of outcomes for patients with intermediate and high risk prostate cancer treated with low dose rate and high dose rate brachytherapy in combination with external beam radiotherapy

Introduction There is evidence to support use of external beam radiotherapy (EBRT) in combination with both low dose rate brachytherapy (LDR–EBRT) and high dose rate brachytherapy (HDR–EBRT) to treat intermediate and high risk prostate cancer. Methods Men with intermediate and high risk prostate cancer treated using LDR–EBRT (treated between 1996 and 2007) and HDR–EBRT (treated between 2007 and 2012) were identified from an institutional database. Multivariable analysis was performed to evaluate the relationship between patient, disease and treatment factors with biochemical progression free survival (bPFS). Results 116 men were treated with LDR-EBRT and 171 were treated with HDR–EBRT. At 5 years, bPFS was estimated to be 90.5% for the LDR–EBRT cohort and 77.6% for the HDR–EBRT cohort. On multivariable analysis, patients treated with HDR–EBRT were more than twice as likely to experience biochemical progression compared with LDR–EBRT (HR 2.33, 95% CI 1.12–4.07). Patients with Gleason ≥8 disease were more than five times more likely to experience biochemical progression compared with Gleason 6 disease (HR 5.47, 95% CI 1.26–23.64). Cumulative incidence of ≥grade 3 genitourinary and gastrointestinal toxicities for the LDR–EBRT and HDR–EBRT cohorts were 8% versus 4% and 5% versus 1% respectively, although these differences did not reach statistical significance. Conclusion LDR–EBRT may provide more effective PSA control at 5 years compared with HDR–EBRT. Direct comparison of these treatments through randomised trials are recommended to investigate this hypothesis further