An Immune Atlas of Clear Cell Renal Cell Carcinoma.

Immune cells in the tumor microenvironment modulate cancer progression and are attractive therapeutic targets. Macrophages and T cells are key components of the microenvironment, yet their phenotypes and relationships in this ecosystem and to clinical outcomes are ill defined. We used mass cytometry with extensive antibody panels to perform in-depth immune profiling of samples from 73 clear cell renal cell carcinoma (ccRCC) patients and five healthy controls. In 3.5 million measured cells, we identified 17 tumor-associated macrophage phenotypes, 22 T cell phenotypes, and a distinct immune composition correlated with progression-free survival, thereby presenting an in-depth human atlas of the immune tumor microenvironment in this disease. This study revealed potential biomarkers and targets for immunotherapy development and validated tools that can be used for immune profiling of other tumor types.

Cell-free gene expression: an expanded repertoire of applications.

Cell-free biology is the activation of biological processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent technical renaissance has facilitated high-yielding (grams of protein per litre), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biological design. These advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits.

Three-dimensional structure-guided evolution of a ribosome with tethered subunits.

RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.

Primary retroperitoneal lymph node dissection for metastatic non-seminomatous germ cell tumours: outcomes and adjuvant chemotherapy.

OBJECTIVES: To compare the outcomes and treatment burden of primary retroperitoneal lymph node dissection (pRPLND) alone versus pRPLND + adjuvant chemotherapy (AC) in patients with pathological stage II (PSII) non-seminomatous germ cell tumours (NSGCT). PATIENTS AND METHODS: Retrospective review of the Princess Margaret Cancer Center eTestes cancer database identified patients with PSII NSGCT after pRPLND between 1995 and 2020. The primary outcome was relapse-free survival (RFS). Secondary outcomes included disease-specific survival (DSS), burden of relapse treatment, and factors associated with relapse. RESULTS: A total of 109 PSII patients were included in the study. There were 96 patients treated with pRPLND alone and 13 treated with pRPLND + AC. The median follow-up was 61 months. The 5-year RFS was 72% for the pRPLND-only group vs 92% for the pRPLND + AC group (hazard ratio [HR] 4.372, 95% confidence interval [CI] 0.59-32.36; P = 0.11). Within the pRPLND-only group the 5-year RFS differed by pN stage (pN1 = 94% vs pN2/N3 = 67%, P = 0.03). Despite a higher relapse rate within the pRPLND-only group, the DSS was similar at 5 years (98% pRPLND only vs 100% pRPLND + AC, P = 0.48). Only 24 (25%) of the patients in the pRPLND-only group required any subsequent chemotherapy. Despite achieving similar survival, the cumulative post-RPLND treatment burden was less for the pRPLND-only group than the pRPLND+AC group overall (average 1.23 vs 2.46 cycles of chemotherapy per patient in group). CONCLUSION: The majority of patients with PSII NSGCT treated with pRPLND alone do not experience a recurrence or require chemotherapy. Despite a lower relapse risk when AC is given, no difference in survival was seen but higher chemotherapy burden was entertained. AC may constitute overtreatment for most patients with PSII NSGCT treated with pRPLND.

Long-term outcomes and cost savings of office fulguration of papillary Ta low-grade bladder cancer.

OBJECTIVES: To assess whether office-based fulguration (OF) under local anaesthesia for small, recurrent, pathological Ta low-grade (LG) non-muscle-invasive bladder cancer (NMIBC) is an effective alternative to transurethral resection of bladder tumour (TURBT), avoiding the costs and risks of procedure, and anesthesia. PATIENTS AND METHODS: Of 521 patients with primary TaLG NMIBC, this retrospective study included 270 patients who underwent OF during follow-up for recurrent, small, papillary LG-appearing tumours at a university centre (University Health Network, University of Toronto, Canada). We assessed the cumulative incidence of cancer-specific mortality (CSM) and disease progression (to MIBC or metastases), as well as possible direct cost savings. RESULTS: In the 270 patients with recurrent TaLG NMIBC treated with OF, the mean (sd) age was 64.9 (13.3) years, 70.8% were men, and 60.3% had single tumours. The mean (sd, range) number of OF procedures per patient was 3.1 (3.2, 1-22). The median (interquartile range) follow-up was 10.1 (5.8-16.2) years. Patients also underwent a mean (sd) of 3.6 (3.0) TURBTs during follow-up in case of numerous or bulkier recurrence. In all, 44.4% of patients never received intravesical therapy. The 10-year incidence of CSM and progression were 0% and 3.1% (95% confidence interval 0.8-5.4%), respectively. Direct cost savings in Ontario were estimated at $6994.14 (Canadian dollars) per patient over the study follow-up. CONCLUSIONS: This study supports that properly selected patients with recurrent, apparent TaLG NMIBC can be safely managed with OF under local anaesthesia with occasional TURBT for larger or numerous recurrent tumours, without compromising long-term oncological outcomes. This approach could generate substantial cost-saving to healthcare systems, is patient-friendly, and could be adopted more widely.

Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences.

RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a 'Quick' method to generate a single PAM prediction or in a 'Comprehensive' method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.

Computationally-guided design and selection of high performing ribosomal active site mutants.

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rRNA engineering.

Radical cystectomy versus trimodality therapy for muscle-invasive bladder cancer: a multi-institutional propensity score matched and weighted analysis.

BACKGROUND: Previous randomised controlled trials comparing bladder preservation with radical cystectomy for muscle-invasive bladder cancer closed due to insufficient accrual. Given that no further trials are foreseen, we aimed to use propensity scores to compare trimodality therapy (maximal transurethral resection of bladder tumour followed by concurrent chemoradiation) with radical cystectomy. METHODS: This retrospective analysis included 722 patients with clinical stage T2-T4N0M0 muscle-invasive urothelial carcinoma of the bladder (440 underwent radical cystectomy, 282 received trimodality therapy) who would have been eligible for both approaches, treated at three university centres in the USA and Canada between Jan 1, 2005, and Dec 31, 2017. All patients had solitary tumours less than 7 cm, no or unilateral hydronephrosis, and no extensive or multifocal carcinoma in situ. The 440 cases of radical cystectomy represent 29% of all radical cystectomies performed during the study period at the contributing institutions. The primary endpoint was metastasis-free survival. Secondary endpoints included overall survival, cancer-specific survival, and disease-free survival. Differences in survival outcomes by treatment were analysed using propensity scores incorporated in propensity score matching (PSM) using logistic regression and 3:1 matching with replacement and inverse probability treatment weighting (IPTW). FINDINGS: In the PSM analysis, the 3:1 matched cohort comprised 1119 patients (837 radical cystectomy, 282 trimodality therapy). After matching, age (71·4 years [IQR 66·0-77·1] for radical cystectomy vs 71·6 years [64·0-78·9] for trimodality therapy), sex (213 [25%] vs 68 [24%] female; 624 [75%] vs 214 [76%] male), cT2 stage (755 [90%] vs 255 [90%]), presence of hydronephrosis (97 [12%] vs 27 [10%]), and receipt of neoadjuvant or adjuvant chemotherapy (492 [59%] vs 159 [56%]) were similar between groups. Median follow-up was 4·38 years (IQR 1·6-6·7) versus 4·88 years (2·8-7·7), respectively. 5-year metastasis-free survival was 74% (95% CI 70-78) for radical cystectomy and 75% (70-80) for trimodality therapy with IPTW and 74% (70-77) and 74% (68-79) with PSM. There was no difference in metastasis-free survival either with IPTW (subdistribution hazard ratio [SHR] 0·89 [95% CI 0·67-1·20]; p=0·40) or PSM (SHR 0·93 [0·71-1·24]; p=0·64). 5-year cancer-specific survival for radical cystectomy versus trimodality therapy was 81% (95% CI 77-85) versus 84% (79-89) with IPTW and 83% (80-86) versus 85% (80-89) with PSM. 5-year disease-free survival was 73% (95% CI 69-77) versus 74% (69-79) with IPTW and 76% (72-80) versus 76% (71-81) with PSM. There were no differences in cancer-specific survival (IPTW: SHR 0·72 [95% CI 0·50-1·04]; p=0·071; PSM: SHR 0·73 [0·52-1·02]; p=0·057) and disease-free survival (IPTW: SHR 0·87 [0·65-1·16]; p=0·35; PSM: SHR 0·88 [0·67-1·16]; p=0·37) between radical cystectomy and trimodality therapy. Overall survival favoured trimodality therapy (IPTW: 66% [95% CI 61-71] vs 73% [68-78]; hazard ratio [HR] 0·70 [95% CI 0·53-0·92]; p=0·010; PSM: 72% [69-75] vs 77% [72-81]; HR 0·75 [0·58-0·97]; p=0·0078). Outcomes for radical cystectomy and trimodality therapy were not statistically different among centres for cancer-specific survival and metastasis-free survival (p=0·22-0·90). Salvage cystectomy was done in 38 (13%) trimodality therapy patients. Pathological stage in the 440 radical cystectomy patients was pT2 in 124 (28%), pT3-4 in 194 (44%), and 114 (26%) node positive. The median number of nodes removed was 39, the soft tissue positive margin rate was 1% (n=5), and the perioperative mortality rate was 2·5% (n=11). INTERPRETATION: This multi-institutional study provides the best evidence to date showing similar oncological outcomes between radical cystectomy and trimodality therapy for select patients with muscle-invasive bladder cancer. These results support that trimodality therapy, in the setting of multidisciplinary shared decision making, should be offered to all suitable candidates with muscle-invasive bladder cancer and not only to patients with significant comorbidities for whom surgery is not an option. FUNDING: Sinai Health Foundation, Princess Margaret Cancer Foundation, Massachusetts General Hospital.

Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer interferometry.

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free expression as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

A dynamic kinetic model captures cell-free metabolism for improved butanol production.

Cell-free systems are useful tools for prototyping metabolic pathways and optimizing the production of various bioproducts. Mechanistically-based kinetic models are uniquely suited to analyze dynamic experimental data collected from cell-free systems and provide vital qualitative insight. However, to date, dynamic kinetic models have not been applied with rigorous biological constraints or trained on adequate experimental data to the degree that they would give high confidence in predictions and broadly demonstrate the potential for widespread use of such kinetic models. In this work, we construct a large-scale dynamic model of cell-free metabolism with the goal of understanding and optimizing butanol production in a cell-free system. Using a combination of parameterization methods, the resultant model captures experimental metabolite measurements across two experimental conditions for nine metabolites at timepoints between 0 and 24 h. We present analysis of the model predictions, provide recommendations for butanol optimization, and identify the aldehyde/alcohol dehydrogenase as the primary bottleneck in butanol production. Sensitivity analysis further reveals the extent to which various parameters are constrained, and our approach for probing valid parameter ranges can be applied to other modeling efforts.

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping.

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

Prognostic impact of variant histologies in urothelial bladder cancer treated with radical cystectomy.

OBJECTIVES: To evaluate variant histologies (VHs) for disease-specific survival (DSS) in patients with invasive urothelial bladder cancer (BCa) undergoing radical cystectomy (RC). MATERIALS AND METHODS: We analysed a multi-institutional cohort of 1082 patients treated with upfront RC for cT1-4aN0M0 urothelial BCa at eight centres. Univariable and multivariable Cox' regression analyses were used to assess the effect of different VHs on DSS in overall cohort and three stage-based analyses. The stages were defined as 'organ-confined' (≤pT2N0), 'locally advanced' (pT3-4N0) and 'node-positive' (pTanyN1-3). RESULTS: Overall, 784 patients (72.5%) had pure urothelial carcinoma (UC), while the remaining 298 (27.5%) harboured a VH. Squamous differentiation was the most common VH, observed in 166 patients (15.3%), followed by micropapillary (40 patients [3.7%]), sarcomatoid (29 patients [2.7%]), glandular (18 patients [1.7%]), lymphoepithelioma-like (14 patients [1.3%]), small-cell (13 patients [1.2%]), clear-cell (eight patients [0.7%]), nested (seven patients [0.6%]) and plasmacytoid VH (three patients [0.3%]). The median follow-up was 2.3 years. Overall, 534 (49.4%) disease-related deaths occurred. In uni- and multivariable analyses, plasmacytoid and small-cell VHs were associated with worse DSS in the overall cohort (both P = 0.04). In univariable analyses, sarcomatoid VH was significantly associated with worse DSS, while lymphoepithelioma-like VH had favourable DSS compared to pure UC. Clear-cell (P = 0.015) and small-cell (P = 0.011) VH were associated with worse DSS in the organ-confined and node-positive cohorts, respectively. CONCLUSIONS: More than 25% of patients harboured a VH at time of RC. Compared to pure UC, clear-cell, plasmacytoid, small-cell and sarcomatoid VHs were associated with worse DSS, while lymphoepithelioma-like VH was characterized by a DSS benefit. Accurate pathological diagnosis of VHs may ensure tailored counselling to identify patients who require more intensive management.

Cell-free Biosynthesis of Peptidomimetics.

A wide variety of peptidomimetics (peptide analogs) possessing innovative biological functions have been brought forth as therapeutic candidates through cell-free protein synthesis (CFPS) systems. A key feature of these peptidomimetic drugs is the use of non-canonical amino acid building blocks with diverse biochemical properties that expand functional diversity. Here, we summarize recent technologies leveraging CFPS platforms to expand the reach of peptidomimetics drugs. We also offer perspectives on engineering the translational machinery that may open new opportunities for expanding genetically encoded chemistry to transform drug discovery practice beyond traditional boundaries.

Linking the Salmonella enterica 1,2-Propanediol Utilization Bacterial Microcompartment Shell to the Enzymatic Core via the Shell Protein PduB.

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here, we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is critical for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy, we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and system-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis. IMPORTANCE MCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here, we provide evidence of a potential mechanism in S. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

Rapid and Sensitive Detection of Antigen from SARS-CoV-2 Variants of Concern by a Multivalent Minibinder-Functionalized Nanomechanical Sensor.

New platforms for the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are urgently needed. Here we report the development of a nanomechanical sensor based on the deflection of a microcantilever capable of detecting the SARS-CoV-2 spike (S) glycoprotein antigen using computationally designed multivalent minibinders immobilized on a microcantilever surface. The sensor exhibits rapid (<5 min) detection of the target antigens down to concentrations of 0.05 ng/mL (362 fM) and is more than an order of magnitude more sensitive than an antibody-based cantilever sensor. Validation of the sensor with clinical samples from 33 patients, including 9 patients infected with the Omicron (BA.1) variant observed detection of antigen from nasopharyngeal swabs with cycle threshold (Ct) values as high as 39, suggesting a limit of detection similar to that of the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate the use of minibinders and nanomechanical sensors for the rapid and sensitive detection of SARS-CoV-2 and potentially other disease markers.

In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

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.

Cytoreductive nephrectomy and exposure to sunitinib - a post hoc analysis of the Immediate Surgery or Surgery After Sunitinib Malate in Treating Patients With Metastatic Kidney Cancer (SURTIME) trial.

OBJECTIVE: To analyse if exposure to sunitinib in the Immediate Surgery or Surgery After Sunitinib Malate in Treating Patients With Metastatic Kidney Cancer (SURTIME) trial, which investigated opposite sequences of cytoreductive nephrectomy (CN) and systemic therapy, is associated with the overall survival (OS) benefit observed in the deferred CN arm. PATIENTS AND METHODS: A post hoc analysis of SURTIME trial data. Variables analysed included number of patients receiving sunitinib, time from randomisation to start sunitinib, overall response rate by Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1, and duration of drug exposure and dose in the intention-to-treat population of the immediate and deferred arm. Descriptive methods and 95% confidence-intervals (CI) were used. RESULTS: In the deferred arm, 97.7% (95% CI 89.3-99.6%; n = 48) received sunitinib vs 80% (95% CI 66.9-88.7%, n = 40) in the immediate arm. Following immediate CN, 19.6% progressed 4 weeks after CN and the median time to start sunitinib was 39.5 vs 4.5 days in the deferred arm. At week 16, 46.0% had progressed at metastatic sites in the immediate CN arm vs 32.7% in the deferred arm. Sunitinib dose reductions, escalations and interruptions were not statistically significantly different between arms. Among patients who received sunitinib in the immediate or deferred arm the median total sunitinib treatment duration was 172.5 vs 248 days. Reduction of target lesions was more profound in the deferred arm. CONCLUSIONS: In comparison to the deferred CN approach, immediate CN impairs administration, onset, and duration of sunitinib. Starting with systemic therapy leads to early and more profound disease control and identification of progression prior to planned CN, which may have contributed to the observed OS benefit.

Strategies for in vitro engineering of the translation machinery.

Engineering the process of molecular translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chemical biology to generate novel biological insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies-purified and extract-based systems-and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including transfer RNAs, translation factors, and the ribosome. Finally, future directions and enabling technological advances for the field are discussed.

Mutational characterization and mapping of the 70S ribosome active site.

The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications.