Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E-coli

Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression

Centralized repeated resectability assessment of patients with colorectalliver metastases during first-line treatment : prospective study

Y Background: Metastasectomy is probably underused in metastatic colorectal cancer. The aim of this study was to investigate the effect of centralized repeated assessment on resectability rate of liver metastases. Methods: The prospective RAXO study was a nationwide study in Finland. Patients with treatable metastatic colorectal cancer at any site were eligible. This planned substudy included patients with baseline liver metastases between 2012 and 2018. Resectability was reassessed by the multidisciplinary team at Helsinki tertiary referral centre upfront and twice during first-line systemic therapy. Outcomes were resectability rates, management changes, and survival. Results: Of 812 patients included, 301 (37.1 per cent) had liver-only metastases. Of these, tumours were categorized as upfront resectable in 161 (53.5 per cent), and became amenable to surgery during systemic treatment in 63 (20.9 per cent). Some 207 patients (68.7 per cent) eventually underwent liver resection or ablation. At baseline, a discrepancy in resectability between central and local judgement was noted for 102 patients (33.9 per cent). Median disease-free survival (DFS) after first resection was 20 months and overall survival (OS) 79 months. Median OS after diagnosis of metastatic colorectal cancer was 80, 32, and 21 months in R0-1 resection, R2/ablation, and non-resected groups, and 5-year OS rates were 68, 37, and 9 per cent, respectively. Liver and extrahepatic metastases were present in 511 patients. Of these, tumours in 72 patients (14.1 per cent) were categorized as upfront resectable, and 53 patients (10.4 per cent) became eligible for surgery. Eventually 110 patients (21.5 per cent) underwent liver resection or ablation. At baseline, a discrepancy between local and central resectability was noted for 116 patients (22.7 per cent). Median DFS from first resection was 7 months and median OS 55 months. Median OS after diagnosis of metastatic colorectal cancer was 79, 42, and 17 months in R0-1 resection, R2/ablation, and non-resected groups, with 5-year OS rates of 65, 39, and 2 per cent, respectively. Conclusion: Repeated centralized resectability assessment in patients with colorectal liver metastases improved resection and survival rates.Peer reviewe

Mobile Sensing at the Service of Mental Well-being: a Large-scale Longitudinal Study

Measuring mental well-being with mobile sensing has been an increasingly active research topic. Pervasiveness of smartphones combined with the convenience of mobile app distribution platforms (e.g., Google Play) provide a tremendous opportunity to reach out to millions of users. However, the studies at the confluence of mental health and mobile sensing have been longitudinally limited, controlled, or confined to a small number of participants. In this paper we report on what we believe is the largest longitudinal in-the-wild study of mood through smartphones. We describe an Android app to collect participants’ self-reported moods and system triggered experience sampling data while passively measuring their physical activity, sociability, and mobility via their device’s sensors. We report the results of a large-scale analysis of the data collected for about three years from $\sim$ 18; 000 users. The paper makes three primary contributions. First, we show how we used physical and software sensors in smartphones to automatically and accurately identify routines. Then, we demonstrate the strong correlation between these routines and users’ personality, well-being perception, and other psychological variables. Finally, we explore predictability of users’ mood using their passive sensing data. Our findings show that, especially for weekends, mobile sensing can be used to predict users’ mood with an accuracy of about 70%. These results have the potential to impact the design of future mobile apps for mood/behavior tracking and interventions.This work was supported by the EPSRC through Grants UBHAVE (EP/I032673/1) and GALE (EP/K019392)

Centralized repeated resectability assessment of patients with colorectalliver metastases during first-line treatment: prospective study

Background: Metastasectomy is probably underused in metastatic colorectal cancer. The aim of this study was to investigate the effect of centralized repeated assessment on resectability rate of liver metastases.Methods: The prospective RAXO study was a nationwide study in Finland. Patients with treatable metastatic colorectal cancer at any site were eligible. This planned substudy included patients with baseline liver metastases between 2012 and 2018. Resectability was reassessed by the multidisciplinary team at Helsinki tertiary referral centre upfront and twice during first-line systemic therapy. Outcomes were resectability rates, management changes, and survival.Results: Of 812 patients included, 301 (37.1 per cent) had liver-only metastases. Of these, tumours were categorized as upfront resectable in 161 (53.5 per cent), and became amenable to surgery during systemic treatment in 63 (20.9 per cent). Some 207 patients (68.7 per cent) eventually underwent liver resection or ablation. At baseline, a discrepancy in resectability between central and local judgement was noted for 102 patients (33.9 per cent). Median disease-free survival (DFS) after first resection was 20 months and overall survival (OS) 79 months. Median OS after diagnosis of metastatic colorectal cancer was 80, 32, and 21 months in R0-1 resection, R2/ablation, and non-resected groups, and 5-year OS rates were 68, 37, and 9 per cent, respectively. Liver and extrahepatic metastases were present in 511 patients. Of these, tumours in 72 patients (14.1 per cent) were categorized as upfront resectable, and 53 patients (10.4 per cent) became eligible for surgery. Eventually 110 patients (21.5 per cent) underwent liver resection or ablation. At baseline, a discrepancy between local and central resectability was noted for 116 patients (22.7 per cent). Median DFS from first resection was 7 months and median OS 55 months. Median OS after diagnosis of metastatic colorectal cancer was 79, 42, and 17 months in R0-1 resection, R2/ablation, and non-resected groups, with 5-year OS rates of 65, 39, and 2 per cent, respectively.Conclusion: Repeated centralized resectability assessment in patients with colorectal liver metastases improved resection and survival rates.</p

Humanization, radiolabeling and biodistribution studies of an igg1-type antibody targeting uncomplexed psa for theranostic applications

Metastatic castration-resistant prostate cancer is today incurable. Conventional imaging methods have limited detection, affecting their ability to give an accurate outcome prognosis, and current therapies for metastatic prostate cancer are insufficient. This inevitably leads to patients relapsing with castration-resistant prostate cancer. Targeting prostate-specific antigens whose expression is closely linked to the activity in the androgen receptor pathway, and thus the pathogenesis of prostate cancer, is a possible way to increase specificity and reduce off-target effects. We have humanized and evaluated radioimmunoconjugates of a previously murine antibody, m5A10, targeting PSA intended for theranostics of hormone-refractory prostate cancer. The humanized antibody h5A10 was expressed in mammalian HEK293 cells transfected with the nucleotide sequences for the heavy and light chains of the antibody. Cell culture medium was filtered and purified by Protein G chromatography, and the buffer was changed to PBS pH 7.4 by dialysis. Murine and humanized 5A10 were conjugated with p-SCN-Bn-CHX-A”-DTPA. Surface plasmon resonance was used to characterize the binding to PSA of the immunoconjugates. Immunoconjugates were labeled with either indium-111 or lutetium-177. Biodistribution studies of murine and humanized 5A10 were performed in mice with LNCaP xenografts. 5A10 was successfully humanized, and in vivo targeting showed specific binding in xenografts. The results thus give an excellent platform for further theranostic development of humanized 5A10 for clinical applications

Contracting and Asset Management: Establishing an Asset Specificity Framework for Determining the Optimal Management of Tourism Infrastructure

Infrastructure and engineering asset management strategies are offered to develop the asset base of the tourism industry. This paper explores options for asset management approaches to develop and extend strategic decision-making for infrastructure and engineered assets in the tourism sector

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis

ecognition of extracellular peptides by plasma membrane‐localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE‐9. GRI is cleaved by METACASPASE‐9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane‐localized, atypical leucine‐rich repeat receptor‐like kinase POLLEN‐SPECIFIC RECEPTOR‐LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS‐dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor