Encoding a magic state with beyond break-even fidelity.

To run large-scale algorithms on a quantum computer, error-correcting codes must be able to perform a fundamental set of operations, called logic gates, while isolating the encoded information from noise1-8. We can complete a universal set of logic gates by producing special resources called magic states9-11. It is therefore important to produce high-fidelity magic states to conduct algorithms while introducing a minimal amount of noise to the computation. Here we propose and implement a scheme to prepare a magic state on a superconducting qubit array using error correction. We find that our scheme produces better magic states than those that can be prepared using the individual qubits of the device. This demonstrates a fundamental principle of fault-tolerant quantum computing12, namely, that we can use error correction to improve the quality of logic gates with noisy qubits. Moreover, we show that the yield of magic states can be increased using adaptive circuits, in which the circuit elements are changed depending on the outcome of mid-circuit measurements. This demonstrates an essential capability needed for many error-correction subroutines. We believe that our prototype will be invaluable in the future as it can reduce the number of physical qubits needed to produce high-fidelity magic states in large-scale quantum-computing architectures.

Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube.

Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in the specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here, we optimized a single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild-type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate that each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies that rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the retinoic acid response.

Computational design of Periplasmic binding protein biosensors guided by molecular dynamics.

Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein's output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.

Wnt and TGFß coordinate growth and patterning to regulate size-dependent behaviour.

Differential coordination of growth and patterning across metazoans gives rise to a diversity of sizes and shapes at tissue, organ and organismal levels. Although tissue size and tissue function can be interdependent1-5, mechanisms that coordinate size and function remain poorly understood. Planarians are regenerative flatworms that bidirectionally scale their adult body size6,7 and reproduce asexually, via transverse fission, in a size-dependent manner8-10. This model offers a robust context to address the gap in knowledge that underlies the link between size and function. Here, by generating an optimized planarian fission protocol in Schmidtea mediterranea, we show that progeny number and the frequency of fission initiation are correlated with parent size. Fission progeny size is fixed by previously unidentified mechanically vulnerable planes spaced at an absolute distance along the anterior-posterior axis. An RNA interference screen of genes for anterior-posterior patterning uncovered components of the TGFß and Wnt signalling pathways as regulators of the frequency of fission initiation rather than the position of fission planes. Finally, inhibition of Wnt and TGFß signalling during growth altered the patterning of mechanosensory neurons-a neural subpopulation that is distributed in accordance with worm size and modulates fission behaviour. Our study identifies a role for TGFß and Wnt in regulating size-dependent behaviour, and uncovers an interdependence between patterning, growth and neurological function.

Taking connected mobile-health diagnostics of infectious diseases to the field.

Mobile health, or 'mHealth', is the application of mobile devices, their components and related technologies to healthcare. It is already improving patients' access to treatment and advice. Now, in combination with internet-connected diagnostic devices, it offers novel ways to diagnose, track and control infectious diseases and to improve the efficiency of the health system. Here we examine the promise of these technologies and discuss the challenges in realizing their potential to increase patients' access to testing, aid in their treatment and improve the capability of public health authorities to monitor outbreaks, implement response strategies and assess the impact of interventions across the world.

Genome-wide quantification of contributions to sexual fitness identifies genes required for spore viability and health in fission yeast.

Numerous genes required for sexual reproduction remain to be identified even in simple model species like Schizosaccharomyces pombe. To address this, we developed an assay in S. pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wild type. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities and can be rescued by conditioned medium. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

PDBench: evaluating computational methods for protein-sequence design.

SUMMARY: Ever increasing amounts of protein structure data, combined with advances in machine learning, have led to the rapid proliferation of methods available for protein-sequence design. In order to utilize a design method effectively, it is important to understand the nuances of its performance and how it varies by design target. Here, we present PDBench, a set of proteins and a number of standard tests for assessing the performance of sequence-design methods. PDBench aims to maximize the structural diversity of the benchmark, compared with previous benchmarking sets, in order to provide useful biological insight into the behaviour of sequence-design methods, which is essential for evaluating their performance and practical utility. We believe that these tools are useful for guiding the development of novel sequence design algorithms and will enable users to choose a method that best suits their design target. AVAILABILITY AND IMPLEMENTATION: https://github.com/wells-wood-research/PDBench. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The impact of COVID-19 on medication reviews in English primary care. An OpenSAFELY-TPP analysis of 20 million adult electronic health records.

AIMS: The COVID-19 pandemic caused significant disruption to routine activity in primary care. Medication reviews are an important primary care activity ensuring safety and appropriateness of prescribing. A disruption could have significant negative implications for patient care. Using routinely collected data, our aim was first to describe codes used to record medication review activity and then to report the impact of COVID-19 on the rates of medication reviews. METHODS: With the approval of NHS England, we conducted a cohort study of 20 million adult patient records in general practice, in-situ using the OpenSAFELY platform. For each month, between April 2019 and March 2022, we report the percentage of patients with a medication review coded monthly and in the previous 12 months with breakdowns by regional, clinical and demographic subgroups and those prescribed high-risk medications. RESULTS: In April 2019, 32.3% of patients had a medication review coded in the previous 12 months. During the first COVID-19 lockdown, monthly activity decreased (-21.1% April 2020), but the 12-month rate was not substantially impacted (-10.5% March 2021). The rate of structured medication review in the last 12 months reached 2.9% by March 2022, with higher percentages in high-risk groups (care home residents 34.1%, age 90+ years 13.1%, high-risk medications 10.2%). The most used medication review code was Medication review done 314530002 (59.5%). CONCLUSIONS: There was a substantial reduction in the monthly rate of medication reviews during the pandemic but rates recovered by the end of the study period. Structured medication reviews were prioritized for high-risk patients.

Surgical experience and identification of errors in laparoscopic cholecystectomy.

BACKGROUND: Surgical errors are acts or omissions resulting in negative consequences and/or increased operating time. This study describes surgeon-reported errors in laparoscopic cholecystectomy. METHODS: Intraoperative videos were uploaded and annotated on Touch SurgeryTM Enterprise. Participants evaluated videos for severity using a 10-point intraoperative cholecystitis grading score, and errors using Observational Clinical Human Reliability Assessment, which includes skill, consequence, and mechanism classifications. RESULTS: Nine videos were assessed by 8 participants (3 junior (specialist trainee (ST) 3-5), 2 senior trainees (ST6-8), and 3 consultants). Participants identified 550 errors. Positive relationships were seen between total operating time and error count (r2 = 0.284, P < 0.001), intraoperative grade score and error count (r2 = 0.578, P = 0.001), and intraoperative grade score and total operating time (r2 = 0.157, P < 0.001). Error counts differed significantly across intraoperative phases (H(6) = 47.06, P < 0.001), most frequently at dissection of the hepatocystic triangle (total 282; median 33.5 (i.q.r. 23.5-47.8, range 15-63)), ligation/division of cystic structures (total 124; median 13.5 (i.q.r. 12-19.3, range 10-26)), and gallbladder dissection (total 117; median 14.5 (i.q.r. 10.3-18.8, range 6-26)). There were no significant differences in error counts between juniors, seniors, and consultants (H(2) = 0.03, P = 0.987). Errors were classified differently. For dissection of the hepatocystic triangle, thermal injuries (50 in total) were frequently classified as executional, consequential errors; trainees classified thermal injuries as step done with excessive force, speed, depth, distance, time or rotation (29 out of 50), whereas consultants classified them as incorrect orientation (6 out of 50). For ligation/division of cystic structures, inappropriate clipping (60 errors in total), procedural errors were reported by junior trainees (6 out of 60), but not consultants. For gallbladder dissection, inappropriate dissection (20 errors in total) was reported in incorrect planes by consultants and seniors (6 out of 20), but not by juniors. Poor economy of movement (11 errors in total) was reported more by consultants (8 out of 11) than trainees (3 out of 11). CONCLUSION: This study suggests that surgical experience influences error interpretation, but the benefits for surgical training are currently unclear.

A generative adversarial approach to facilitate archival-quality histopathologic diagnoses from frozen tissue sections.

In clinical diagnostics and research involving histopathology, formalin-fixed paraffin-embedded (FFPE) tissue is almost universally favored for its superb image quality. However, tissue processing time (>24 h) can slow decision-making. In contrast, fresh frozen (FF) processing (<1 h) can yield rapid information but diagnostic accuracy is suboptimal due to lack of clearing, morphologic deformation and more frequent artifacts. Here, we bridge this gap using artificial intelligence. We synthesize FFPE-like images ("virtual FFPE") from FF images using a generative adversarial network (GAN) from 98 paired kidney samples derived from 40 patients. Five board-certified pathologists evaluated the results in a blinded test. Image quality of the virtual FFPE data was assessed to be high and showed a close resemblance to real FFPE images. Clinical assessments of disease on the virtual FFPE images showed a higher inter-observer agreement compared to FF images. The nearly instantaneously generated virtual FFPE images can not only reduce time to information but can facilitate more precise diagnosis from routine FF images without extraneous costs and effort.

Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering.

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

Adjuvant therapy in patients with sarcomatoid renal cell carcinoma: post hoc analysis from Eastern Cooperative Oncology Group-American College of Radiology Imaging Network (ECOG-ACRIN) E2805.

OBJECTIVES: To study the effects of adjuvant therapy in patients with sarcomatoid renal cell carcinoma (sRCC) enrolled in the randomised phase III clinical trial E2805. PATIENTS AND METHODS: The original trial (E2805) was a randomised, double-blinded phase III clinical trial comparing outcomes in 1943 patients with RCC accrued between 2006 and 2010 and treated with up to 1 year of adjuvant placebo, sunitinib, or sorafenib. The present study analyses the cohort of patients with sRCC that participated in E2805. RESULTS: A total of 171 patients (8.8%) had sarcomatoid features. Of these, 52 patients received sunitinib, 58 received sorafenib, and 61 received placebo. Most patients were pT3-4 (71.1%, 63.7%, and 70.5%, respectively); 17.3%, 19.0%, and 27.9% had pathologically positive lymph nodes; and 59.6%, 62.1%, and 62.3% of the patients were University of California Los Angeles (UCLA) Integrated Staging System (UISS) very-high risk. In 49% of patients with subsequent development of metastatic disease, recurrence occurred in the lung, followed by 30% in the lymph nodes, and 13% in the liver. There was a high local recurrence rate in the renal bed (16%, 29%, and 18%, respectively). The 5-year disease-free survival (DFS) rates were 33.6%, 36.0%, and 27.8%, for sunitinib, sorafenib and placebo, respectively (hazard ratio [HR] 0.74, 95% confidence interval [CI] 0.45-1.20 for sunitinib vs placebo, and HR 0.82, 95% CI 0.53-1.28 for sorafenib vs placebo). CONCLUSIONS: Adjuvant therapy with sunitinib or sorafenib did not show an improvement in DFS or OS in patients with sRCC.

Definitive radiotherapy for extracranial oligoprogressive metastatic renal cell carcinoma as a strategy to defer systemic therapy escalation.

OBJECTIVE: To study whether delivering definitive radiotherapy (RT) to sites of oligoprogression in metastatic renal cell carcinoma (mRCC) enabled deferral of systemic therapy (ST) changes without compromising disease control or survival. PATIENTS AND METHODS: We identified patients with mRCC who received RT to three or fewer sites of extracranial progressive disease between 2014 and 2019 at a large tertiary cancer centre. Inclusion criteria were: (1) controlled disease for ≥3 months before oligoprogression, (2) all oligoprogression sites treated with a biologically effective dose of ≥100 Gy, and (3) availability of follow-up imaging. Time-to-event end-points were calculated from the start of RT. RESULTS: A total of 72 patients were identified (median follow-up 22 months, 95% confidence interval [CI] 19-32 months), with oligoprogressive lesions in lung/mediastinum (n = 35), spine (n = 30), and non-spine bone (n = 5). The most common systemic therapies before oligoprogression were none (n = 33), tyrosine kinase inhibitor (n = 23), and immunotherapy (n = 13). At 1 year, the local control rate was 96% (95% CI 87-99%); progression-free survival (PFS), 52% (95% CI 40-63%); and overall survival, 91% (95% CI 82-96%). At oligoprogression, ST was escalated (n = 16), maintained (n = 49), or discontinued (n = 7), with corresponding median (95% CI) PFS intervals of 19.7 (8.2-27.2) months, 10.1 (6.9-13.2) months, and 9.8 (2.4-28.9) months, respectively. Of the 49 patients maintained on the same ST at oligoprogression, 21 did not subsequently have ST escalation. CONCLUSION: Patients with oligoprogressive mRCC treated with RT had comparable PFS regardless of ST strategy, suggesting that RT may be a viable approach for delaying ST escalation. Randomised controlled trials comparing treatment of oligoprogression with RT vs ST alone are needed.

Fluorescence fluctuation-based super-resolution microscopy: Basic concepts for an easy start.

Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of approximately half of the wavelength of visible light, that is, within the range of 200 to 350 nm. Fluorescence fluctuation-based super-resolution microscopy (FF-SRM) is a term used to encompass a collection of image analysis techniques that rely on the statistical processing of temporal variations of the fluorescence signal. FF-SRM aims to reduce the uncertainty of the location of fluorophores within an image, often improving spatial resolution by several tens of nanometers. FF-SRM is suitable for live-cell imaging due to its compatibility with most fluorescent probes and relatively simple instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a range of FF-SRM methods published to date are described. Their operational parameters are explained and guidance for their selection is provided.

Due to light's wave nature, an optical microscope's resolution range is 200 to 350 nanometers. Several techniques enhance resolution; this work encompasses several fluorescence fluctuation super-resolution (FF-SMR) methods capable of achieving nanoscopic scales. FF-SRM is known to be suitable for fixed or live-cell imaging and compatible with most conventional microscope setups found in a laboratory. However, each FF-SRM approach has its strengths and weaknesses, which depend directly on the underlying principles through which enhanced spatial resolution is achieved. Therefore, the basic concepts and principles behind diverse FF-SRM methods are revisited in this review. In addition, their operational parameters are explained, and guidance for their selection is provided for microscopists interested in FF-SRM.

De Novo Designed Peptide and Protein Hairpins Self-Assemble into Sheets and Nanoparticles.

The design and assembly of peptide-based materials has advanced considerably, leading to a variety of fibrous, sheet, and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here a de novo peptide system is presented that forms nanoparticles or sheets depending on the strategic placement of a "disulfide pin" between two elements of secondary structure that drive self-assembly. Specifically, homodimerizing and homotrimerizing de novo coiled-coil α-helices are joined with a flexible linker to generate a series of linear peptides. The helices are pinned back-to-back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling indicates, and advanced microscopy shows, that the proximally pinned hairpins self-assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.

BAlaS: fast, interactive and accessible computational alanine-scanning using BudeAlaScan.

MOTIVATION: In experimental protein engineering, alanine-scanning mutagenesis involves the replacement of selected residues with alanine to determine the energetic contribution of each side chain to forming an interaction. For example, it is often used to study protein-protein interactions. However, such experiments can be time-consuming and costly, which has led to the development of programmes for performing computational alanine-scanning mutagenesis (CASM) to guide experiments. While programmes are available for this, there is a need for a real-time web application that is accessible to non-expert users. RESULTS: Here, we present BAlaS, an interactive web application for performing CASM via BudeAlaScan and visualizing its results. BAlaS is interactive and intuitive to use. Results are displayed directly in the browser for the structure being interrogated enabling their rapid inspection. BAlaS has broad applications in areas, such as drug discovery and protein-interface design. AVAILABILITY AND IMPLEMENTATION: BAlaS works on all modern browsers and is available through the following website: https://balas.app. The project is open source, distributed using an MIT license and is available on GitHub (https://github.com/wells-wood-research/balas).

Outcomes of Percutaneous Thermal Ablation for Biopsy-Proven T1a Renal Cell Carcinoma in Patients With Other Primary Malignancies.

OBJECTIVE. The purpose of this article is to investigate the oncologic effectiveness and survival outcomes of percutaneous image-guided thermal ablation for clinical T1a renal cell carcinoma (RCC) in patients with other primary nonrenal malignancies. MATERIALS AND METHODS. We reviewed records of patients with histologically proven T1a RCC (< 4.0 cm) treated with thermal ablation over a period of 10 years between January 2005 and December 2014. We recorded past or current history of primary malignancy other than RCC, status of the primary malignancy, tumor histology (in remission or under therapy), and whether patient was currently alive or not, and if not, the date and reason of death. Three cohorts were studied: patients with RCC only (group A), patients with RCC and other primary malignancy in remission (group B), and patients with RCC and other primary malignancy under treatment (group C). The Kaplan-Meier product-limit estimator was used to estimate the survival rates. RESULTS. One hundred nine patients met the inclusion criteria (109 lesions, 110 ablation procedures). There were 46, 45, and 18 patients in the A, B, and C groups, respectively. The 5-year survival was 87%, 63%, and 40% for groups A, B, and C, respectively. The local recurrence-free survival for the whole sample was 95% at 3, 5, and 10 years. The disease-free survival was 96%, 93%, and 91% at 3, 5, and 10 years. Although a significant difference is noted between the three cohorts in overall survival (p = .02); for RCC, there were no significance differences in the local recurrence-free, disease-free, metastasis-free, and cancer-specific survivals. In addition, there was no difference in outcomes for patients in group B (in remission) when compared with those in group C (under treatment). CONCLUSION. Thermal ablation is an effective and safe modality of treatment of T1a RCC in patients with other primary malignancies that are in remission or under treatment.

Initial testing of pegfilgrastim (Neulasta Onpro) on-body injector in multiple radiological imaging environments.

PURPOSE: An increasing number of implantable or external devices can impact whether patients can receive radiological imaging examinations. This study examines and tests the Neulasta (pegfilgrastim) Onpro on-body injector in multiple imaging environments. METHODS: The injector was analyzed for four imaging modalities with testing protocols and strategies developed for each modality. In x-ray and computed tomography (CT), scans with much higher exposure than clinical protocols were performed with the device attached to an anthropomorphic phantom. The device was monitored until the completion of drug delivery. For magnetic resonance imaging (MRI), the device was assessed using a hand-held magnet and underwent the magnetically induced displacement testing in a 1.5T clinical MRI scanner room. For ultrasound, magnetic field changes were measured around an ultrasound scanner system with three transducers. RESULTS: For x-ray and CT no sign of device error was identified during or after the high radiation exposure scans. Drug delivery was completed at expected timing with expected volume. For MRI the device showed significant attractive force towards the hand-held magnet and a 50-degree deflection angle at 50 cm from the opening of the scanner bore. No further assessment from the gradient or radiofrequency field was deemed necessary. For ultrasound the maximum magnetic field change from baseline was measured to be +11.7 µT in comparison to +74.2 µT at 4 inches from a working microwave. CONCLUSIONS: No device performance issue was identified under the extreme test conditions in x-ray or CT. The device was found to be MR Unsafe. Magnetic field changes around an ultrasound system met the limitation set by manufacture. Patient ultrasound scanning is considered safe as long as the transducers do not inadvertently loosen the device.

Prolyl Hydroxylase 3 Knockdown Accelerates VHL-Mutant Kidney Cancer Growth In Vivo.

Von Hippel Lindau (VHL) inactivation, which is common in clear cell renal cell carcinoma (ccRCC), leads directly to the disruption of oxygen homoeostasis. VHL works through hypoxia-inducible factors (HIFs). Within this VHL-HIF system, prolyl hydroxylases (PHDs) are the intermediary proteins that initiate the degradation of HIFs. PHD isoform 3's (PHD3) role in ccRCC growth in vivo is poorly understood. Using viral transduction, we knocked down the expression of PHD3 in the human ccRCC cell line UMRC3. Compared with control cells transduced with scrambled vector (UMRC3-SC cells), PHD3-knockdown cells (UMRC3-PHD3KD cells) showed increased cell invasion, tumor growth, and response to sunitinib. PHD3 knockdown reduced HIF2α expression and increased phosphorylated epidermal growth factor (EGFR) expression in untreated tumor models. However, following sunitinib treatment, expression of HIF2α and phosphorylated EGFR were equivalent in both PHD3 knockdown and control tumors. PHD3 knockdown changed the overall redox state of the cell as seen by the increased concentration of glutathione in PHD3 knockdown tumors relative to control tumors. UMRC3-PHD3KD cells had increased proliferation in cell culture when grown in the presence of hydrogen peroxide compared to UMRC3-SC control cells. Our findings illustrate (1) the variable effect of PHD3 on HIF2α expression, (2) an inverse relationship between PHD3 expression and tumor growth in ccRCC animal models, and (3) the role of PHD3 in maintaining the redox state of UMRC3 cells and their proliferative rate under oxidative stress.