Evolution from adherent to suspension: systems biology of HEK293 cell line development

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin

The reliability, validity and sensitivity of a novel soccer-specific reactive repeated-sprint test (RRST).

PURPOSE: The aim of this study was to determine the reliability, validity and sensitivity of a reactive repeated-sprint test (RRST). METHODS: Elite (n = 72) and sub-elite male (n = 87) and elite female soccer players (n = 12) completed the RRST at set times during a season. Total distance timed was 30 m and the RRST performance measure was the total time (s) across eight repetitions. Competitive match running performance was measured using GPS and high-intensity running quantified (≥ 19.8 km h(-1)). RESULTS: Test-retest coefficient of variation in elite U16 and sub-elite U19 players was 0.71 and 0.84 %, respectively. Elite U18 players' RRST performances were better (P < 0.01) than elite U16, sub-elite U16, U18, U19 and elite senior female players (58.25 ± 1.34 vs 59.97 ± 1.64, 61.42 ± 2.25, 61.66 ± 1.70, 61.02 ± 2.31 and 63.88 ± 1.46 s; ES 0.6-1.9). For elite U18 players, RRST performances for central defenders (59.84 ± 1.35 s) were lower (P < 0.05) than full backs (57.85 ± 0.77 s), but not attackers (58.17 ± 1.73 s) or central and wide midfielders (58.55 ± 1.08 and 58.58 ± 1.89 s; ES 0.7-1.4). Elite U16 players demonstrated lower (P < 0.01) RRST performances during the preparation period versus the start, middle and end of season periods (61.13 ± 1.53 vs 59.51 ± 1.39, 59.25 ± 1.42 and 59.20 ± 1.57 s; ES 1.0-1.1). Very large magnitude correlations (P < 0.01) were observed between RRST performance and high-intensity running in the most intense 5-min period of a match for both elite and sub-elite U18 players (r = -0.71 and -0.74), with the best time of the RRST also correlating with the arrowhead agility test for elite U16 and U18 players (r = 0.84 and 0.75). CONCLUSION: The data demonstrate that the RRST is a reliable and valid test that distinguishes between performance across standard, position and seasonal period

Intra-tumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing

A single tumor may contain cells with different somatic mutations. By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis. In contrast, the extent of epigenetic intra-tumor heterogeneity and how it influences tumor biology is under-explored. We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing. We used deep single molecule bisulfite sequencing and sample-specific DNA barcodes to determine the spectrum of MLH1 promoter methylation across an average of 1000 molecules in each of 33 individual samples in parallel, including endometrial cancer, matched blood and normal endometrium. This first glimpse, deep into each tumor, revealed unexpectedly heterogeneous patterns of methylation at the MLH1 promoter within a subset of endometrial tumors. This high-resolution analysis allowed us to measure the clonality of methylation in individual tumors and gain insight into the accumulation of aberrant promoter methylation on both alleles during tumorigenesis

TBCRC 019: A phase II trial of nanoparticle albumin-bound paclitaxel with or without the anti-death receptor 5 monoclonal antibody tigatuzumab in patients with triple negative breast cancer

Purpose: Tigatuzumab (TIG), an agonistic anti-DR5 antibody, triggers apoptosis in DR5+ human tumor cells without crosslinking. TIG has strong in vitro/in vivo activity against basal-like breast cancer cells enhanced by chemotherapy agents. This study evaluates activity of TIG and chemotherapy in patients with metastatic triple-negative breast cancer (TNBC). Experimental Design: Randomized 2:1 phase II trial of albumin-bound paclitaxel (nab-PAC) ± TIG in patients with TNBC stratified by prior chemotherapy. Patients received nab-PAC weekly × 3 ± TIG every other week, every 28 days. Primary objective was within-arm objective response rate (ORR). Secondary objectives were safety, progression-free survival (PFS), clinical benefit, and TIG immunogenicity. Metastatic research biopsies were required. Results: Among 64 patients (60 treated; TIG/nab-PAC n = 39 and nab-PAC n = 21), there were 3 complete remissions (CR), 8 partial remissions (PR; 1 almost CR), 11 stable diseases (SD), and 17 progressive diseases (PD) in the TIG/nab-PAC arm (ORR, 28%), and no CRs, 8 PRs, 4 SDs, and 9 PDs in the nab-PAC arm (ORR, 38%). There was a numerical increase in CRs and several patients had prolonged PFS (1,025+, 781, 672, 460, 334) in the TIG/nab-PAC arm. Grade 3 toxicities were 28% and 29%, respectively, with no grade 4–5. Exploratory analysis suggests an association of ROCK1 gene pathway activation with efficacy in the TIG/nab-PAC arm. Conclusions: ORR and PFS were similar in both. Preclinical activity of TIG in basal-like breast cancer and prolonged PFS in few patients in the combination arm support further investigation of anti-DR5 agents. ROCK pathway activation merits further evaluation

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio

Match running performance and physical capacity profiles of U8 and U10 soccer players

Aim This study aimed to characterize match running performance of very young soccer players and evaluate the relationship between these data and physical capacities and technical skills. Methods Distances covered at different speed thresholds were measured during 31 official matches using GPS technology in U10 (n = 12; age 10.1 ± 0.1 years) and U8 (n = 15; age 7.9 ± 0.1 years) national soccer players. Counter movement jump performance (CMJ), 20 m shuttle running (20 m-SR), linear sprint performance (10, 20, 30 m), shuttle (SHDT) and slalom dribble tests (SLDT) were performed to determine the players physical capacities and technical skills. Results Physical capacities and technical skills were higher in U10 versus U8 players [P 0.05, ES: 0.74). The U10 players covered more total (TD) and high-intensity running distance (HIRD) than their younger counterparts did (P 0.05, ES: 0.99). TD and HIRD covered across the three 15 min periods of match play did not decline (P > 0.05, ES: 0.02–0.55). Very large magnitude correlations were observed between the U8 and U10 players performances during the 20 m-SR versus TD (r = 0.79; P < 0.01) and HIRD (r = 0.82; P < 0.01) covered during match play. Conclusions Data demonstrate differences in match running performance and physical capacity between U8 and U10 players, and large magnitude relationships between match play measures and physical test performances. These findings could be useful to sports science staff working within the academies

A novel hybrid promoter responsive to pathophysiological and pharmacological regulation

The aim of this study was to construct a promoter containing DNA motifs for an endogenous transcription factor associated with inflammation along with motifs for pharmacological regulation factors. We demonstrate in transfected cells that expression of a gene of interest is induced by hypoxic conditions or through pharmacological induction, and also show pharmacological repression. In vivo studies utilised electroporation of plasmid to mouse paws, a delivery method shown to be effective by bioluminescence imaging. For gene therapy, the promoter was used to drive expression of IL-1Ra in a paw inflammation model with therapeutic effect observed which was further enhanced when the promoter was additionally induced with a pharmacological activator. One of the most important observations from this study was that promoter induction by hypoxia or inflammation could be prevented by the pharmacological repressor in the absence of doxycycline. These studies demonstrate that hybrid promoters enable pharmacological adjustment to the pathophysiological level of gene expression and, importantly, that they allow termination of gene expression even in the presence of pathophysiological stimuli

Skeletal muscle and performance adaptations to high-intensity training in elite male soccer players: speed endurance runs versus small-sided game training.

PURPOSE: To examine the skeletal muscle and performance responses across two different exercise training modalities which are highly applied in soccer training. METHODS: Using an RCT design, 39 well-trained male soccer players were randomized into either a speed endurance training (SET; n = 21) or a small-sided game group (SSG; n = 18). Over 4 weeks, thrice weekly, SET performed 6-10 × 30-s all-out runs with 3-min recovery, while SSG completed 2 × 7-9-min small-sided games with 2-min recovery. Muscle biopsies were obtained from m. vastus lateralis pre and post intervention and were subsequently analysed for metabolic enzyme activity and muscle protein expression. Moreover, the Yo-Yo Intermittent Recovery level 2 test (Yo-Yo IR2) was performed. RESULTS: Muscle CS maximal activity increased (P < 0.05) by 18% in SET only, demonstrating larger (P < 0.05) improvement than SSG, while HAD activity increased (P < 0.05) by 24% in both groups. Na(+)-K(+) ATPase α1 subunit protein expression increased (P < 0.05) in SET and SSG (19 and 37%, respectively), while MCT4 protein expression rose (P < 0.05) by 30 and 61% in SET and SSG, respectively. SOD2 protein expression increased (P < 0.05) by 28 and 37% in SET and SSG, respectively, while GLUT-4 protein expression increased (P < 0.05) by 40% in SSG only. Finally, SET displayed 39% greater improvement (P < 0.05) in Yo-Yo IR2 performance than SSG. CONCLUSION: Speed endurance training improved muscle oxidative capacity and exercise performance more pronouncedly than small-sided game training, but comparable responses were in muscle ion transporters and antioxidative capacity in well-trained male soccer players

FGFR1-Induced Epithelial to Mesenchymal Transition through MAPK/PLCγ/COX-2-Mediated Mechanisms

Tumour invasion and metastasis is the most common cause of death from cancer. For epithelial cells to invade surrounding tissues and metastasise, an epithelial-mesenchymal transition (EMT) is required. We have demonstrated that FGFR1 expression is increased in bladder cancer and that activation of FGFR1 induces an EMT in urothelial carcinoma (UC) cell lines. Here, we created an in vitro FGFR1-inducible model of EMT, and used this model to identify regulators of urothelial EMT. FGFR1 activation promoted EMT over a period of 72 hours. Initially a rapid increase in actin stress fibres occurred, followed by an increase in cell size, altered morphology and increased migration and invasion. By using site-directed mutagenesis and small molecule inhibitors we demonstrated that combined activation of the mitogen activated protein kinase (MAPK) and phospholipase C gamma (PLCγ) pathways regulated this EMT. Actin stress fibre formation was regulated by PLCγ activation, and was also important for the increase in cell size, migration and altered morphology. MAPK activation regulated migration and E-cadherin expression, indicating that combined activation of PLCγand MAPK is required for a full EMT. We used expression microarrays to assess changes in gene expression downstream of these signalling cascades. COX-2 was transcriptionally upregulated by FGFR1 and caused increased intracellular prostaglandin E2 levels, which promoted migration. In conclusion, we have demonstrated that FGFR1 activation in UC cells lines promotes EMT via coordinated activation of multiple signalling pathways and by promoting activation of prostaglandin synthesis