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PURPOSE No liquid biomarkers are approved in metastatic renal cell carcinoma (mRCC) despite the need to predict and monitor response noninvasively to tailor treatment choices. Urine and plasma free glycosaminoglycan profiles (GAGomes) are promising metabolic biomarkers in mRCC. The objective of this study was to explore if GAGomes could predict and monitor response in mRCC. PATIENTS AND METHODS We enrolled a single-center prospective cohort of patients with mRCC elected for first-line therapy (ClinicalTrials.gov identifier: NCT02732665 ) plus three retrospective cohorts (ClinicalTrials.gov identifiers: NCT00715442 and NCT00126594 ) for external validation. Response was dichotomized as progressive disease (PD) versus non-PD every 8‐12 weeks. GAGomes were measured at treatment start, after 6-8 weeks, and every third month in a blinded laboratory. We correlated GAGomes with response and developed scores to classify PD versus non-PD, which were used to predict response at treatment start or after 6-8 weeks. RESULTS Fifty patients with mRCC were prospectively included, and all received tyrosine kinase inhibitors (TKIs). PD correlated with alterations in 40% of GAGome features. We developed plasma, urine, and combined glycosaminoglycan progression scores that monitored PD at each response evaluation visit with the area under the receiving operating characteristic curve (AUC) of 0.93, 0.97, and 0.98, respectively. For internal validation, the scores predicted PD at treatment start with the AUC of 0.66, 0.68, and 0.74 and after 6-8 weeks with the AUC of 0.76, 0.66, and 0.75. For external validation, 70 patients with mRCC were retrospectively included and all received TKI-containing regimens. The plasma score predicted PD at treatment start with the AUC of 0.90 and at 6-8 weeks with the AUC of 0.89. The pooled sensitivity and specificity were 58% and 79% at treatment start. Limitations include the exploratory study design. CONCLUSION GAGomes changed in association with mRCC response to TKIs and may provide biologic insights into mRCC mechanisms of response.

In bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo., Ribosome kinetics are rate-limiting for protein synthesis. Here the authors evolve diverse 16S rRNAs for enhanced protein synthesis rates and genetic code expansion efficiencies in vivo.

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells., Synthetic biologists often co-opt heterologous parts to affect new functions in living cells, yet such an approach has rarely been extended to structural components of the ribosome. Here, the authors describe generalizable methods to express ribosomes from divergent microbes in E. coli and maximize their function.

Cancer mortality is exacerbated by late-stage diagnosis. Liquid biopsies based on genomic biomarkers can noninvasively diagnose cancers. However, validation studies have reported ~10% sensitivity to detect stage I cancer in a screening population and specific types, such as brain or genitourinary tumors, remain undetectable. We investigated urine and plasma free glycosaminoglycan profiles (GAGomes) as tumor metabolism biomarkers for multi-cancer early detection (MCED) of 14 cancer types using 2,064 samples from 1,260 cancer or healthy subjects. We observed widespread cancer-specific changes in biofluidic GAGomes recapitulated in an in vivo cancer progression model. We developed three machine learning models based on urine ( N urine = 220 cancer vs. 360 healthy) and plasma ( N plasma = 517 vs. 425) GAGomes that can detect any cancer with an area under the receiver operating characteristic curve of 0.83–0.93 with up to 62% sensitivity to stage I disease at 95% specificity. Undetected patients had a 39 to 50% lower risk of death. GAGomes predicted the putative cancer location with 89% accuracy. In a validation study on a screening-like population requiring ≥ 99% specificity, combined GAGomes predicted any cancer type with poor prognosis within 18 months with 43% sensitivity (21% in stage I; N = 121 and 49 cases). Overall, GAGomes appeared to be powerful MCED metabolic biomarkers, potentially doubling the number of stage I cancers detectable using genomic biomarkers.

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration - but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research.

Abstract Precision oncology aims to tailor clinical decisions specifically to patients with the objective of improving treatment outcomes. This can be achieved by leveraging omics information for accurate molecular characterization of tumors. Tumor tissue biopsies are currently the main source of information for molecular profiling. However, biopsies are invasive and limited in resolving spatiotemporal heterogeneity in tumor tissues. Alternative non-invasive liquid biopsies can exploit patient’s body fluids to access multiple layers of tumor-specific biological information (genomes, epigenomes, transcriptomes, proteomes, metabolomes, circulating tumor cells, and exosomes). Analysis and integration of these large and diverse datasets using statistical and machine learning approaches can yield important insights into tumor biology and lead to discovery of new diagnostic, predictive, and prognostic biomarkers. Translation of these new diagnostic tools into standard clinical practice could transform oncology, as demonstrated by a number of liquid biopsy assays already entering clinical use. In this review, we highlight successes and challenges facing the rapidly evolving field of cancer biomarker research. Lay Summary Precision oncology aims to tailor clinical decisions specifically to patients with the objective of improving treatment outcomes. The discovery of biomarkers for precision oncology has been accelerated by high-throughput experimental and computational methods, which can inform fine-grained characterization of tumors for clinical decision-making. Moreover, advances in the liquid biopsy field allow non-invasive sampling of patient’s body fluids with the aim of analyzing circulating biomarkers, obviating the need for invasive tumor tissue biopsies. In this review, we highlight successes and challenges facing the rapidly evolving field of liquid biopsy cancer biomarker research.

Plasma and urine glycosaminoglycans (GAGs) are long linear sulfated polysaccharides recognized as potential non-invasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition, the so-called GAGome, a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their urine and plasma free GAGomes using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS) method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine, and hemoglobin or erythrocyte counts in plasma. We found higher free GAGome concentration – but not composition - in males. Partitioned by gender, we established reference intervals for all detectable free GAGome features in urine and plasma. We carried out a transference analysis in healthy individuals from two distinct geographical sites, including the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal plasma and urine free GAGomes reference intervals and represents a critical resource for physiology and biomarker research.

Darwinian evolution preferentially follows mutational pathways whose individual steps increase fitness. Alternative pathways with mutational steps that do not increase fitness are less accessible. Here, we show that mistranslation, the erroneous incorporation of amino acids into nascent proteins, can increase the accessibility of such alternative pathways and, ultimately, of high fitness genotypes. We subject populations of the beta‐lactamase TEM‐1 to directed evolution in Escherichia coli under both low‐ and high‐mistranslation rates, selecting for high activity on the antibiotic cefotaxime. Under low mistranslation rates, different evolving TEM‐1 populations ascend the same high cefotaxime‐resistance peak, which requires three canonical DNA mutations. In contrast, under high mistranslation rates they ascend three different high cefotaxime‐resistance genotypes, which leads to higher genotypic diversity among populations. We experimentally reconstruct the adaptive DNA mutations and the potential evolutionary paths to these high cefotaxime‐resistance genotypes. This reconstruction shows that some of the DNA mutations do not change fitness under low mistranslation, but cause a significant increase in fitness under high‐mistranslation, which helps increase the accessibility of different high cefotaxime‐resistance genotypes. In addition, these mutations form a network of pairwise epistatic interactions that leads to mutually exclusive evolutionary trajectories towards different high cefotaxime‐resistance genotypes. Our observations demonstrate that protein mistranslation and the phenotypic mutations it causes can alter the evolutionary exploration of fitness landscapes and reduce the predictability of evolution., High mistranslation can increase the fitness benefits of adaptive DNA mutations, and thus help open evolutionary paths to alternative high‐fitness genotypes (left panel) that would be inaccessible under low mistranslation (right panel).

Glycosaminoglycans (GAGs) are long linear sulfated polysaccharides implicated in processes linked to disease development such as mucopolysaccharidosis, respiratory failure, cancer, and viral infections, thereby serving as potential biomarkers. A successful clinical translation of GAGs as biomarkers depends on the availability of standardized GAG measurements. However, owing to the analytical complexity associated with the quantification of GAG concentration and structural composition, a standardized method to simultaneously measure multiple GAGs is missing. In this study, we sought to characterize the analytical performance of a ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS)-based kit for the quantification of 17 GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 GAG disaccharides. In native human samples, here using urine as a reference matrix, the analytical performance of the kit was acceptable for the quantification of CS disaccharides. Intra- and inter-laboratory tests performed in an external laboratory demonstrated robust reproducibility of GAG measurements showing that the kit was acceptably standardized. In conclusion, these results indicated that the UHPLC-MS/MS kit was standardized for the simultaneous measurement of GAG disaccharides allowing for comparability of measurements and enabling translational research.SummaryAnalytical performance of a kit for standardized GAG measurements, based on an established UHPLC-MS/MS method

Genetic variation fuels Darwinian evolution, yet spontaneous mutation rates are maintained at low levels to ensure cellular viability. Low mutation rates preclude the exhaustive exploration of sequence space for protein evolution and genome engineering applications, prompting scientists to develop methods for efficient and targeted diversification of nucleic acid sequences. Directed evolution of biomolecules relies upon the generation of unbiased genetic diversity to discover variants with desirable properties, whereas genome-engineering applications require selective modifications on a genomic scale with minimal off-targets. Here, we review the current toolkit of mutagenesis strategies employed in directed evolution and genome engineering. These state-of-the-art methods enable facile modifications and improvements of single genes, multicomponent pathways, and whole genomes for basic and applied research, while simultaneously paving the way for genome editing therapeutic interventions.

3034 Background: Non-invasive liquid biopsies promise to enable early cancer detection and improve patient outcomes. However, virtually all liquid biopsies rely on genomic biomarkers, with limited sensitivity to early-stage tumors and poor detection of cancers shedding little cell-free DNA, like genitourinary or brain tumors. Here, we explored the use of plasma and urine glycosaminoglycan (GAGs) profiles, or GAGomes, as biomarkers reflective of tumor metabolism to serve as an alternative pan-cancer liquid biopsy. Methods: In this case-control study, we enrolled retrospective and prospective cohorts from Sweden and Italy. Included cases were treatment-naïve early-stage/low-grade cancers or metastatic/high-grade cancers across 14 histological types. Included controls were healthy 22-78 y/o adults with no history of cancer. We measured GAGomes – encompassing 17 chondroitin sulfate (CS), heparan sulfate (HS), and hyaluronate (HA) disaccharides - using a standardized UHPLC-MS/MS-based kit in a central blind laboratory. We tested the top GAGome features different in cancer using Bayesian estimation. These were used to design one plasma and one urine GAG score for the binary classification of cancer vs. control in a discovery set. We computed the area-under-the-curve (AUC), and sensitivity at 98% specificity of each GAG score in the validation set. A subset analysis was performed in early-stage/low-grade cancers only. In the subset of cases with survival records, we used multivariable Cox regression to estimate the hazard ratio (HR) for overall survival (OS) on each GAG score adjusted for cancer type, age, and gender. Results: GAGomes were measured in 753 plasma samples (460 cancers across 14 types, median age = 66 y/o, 51% female vs. 293 healthy adults, median age = 58 y/o, 57% female) and 559 urine samples (219 cancers across 5 types, median age = 69 y/o, 23% female vs. 340 healthy adults, median age = 56 y/o, 60% female). In the discovery set, the urine GAG score had an AUC = 0.80 (95% CI: 0.74-0.85, 124 cancers across 5 types vs. 184 controls) while the plasma GAG score had an AUC = 0.82 (95% CI: 0.78-0.86, 153 cancers across 14 types vs. 282 controls). In the validation set, the urine GAG score had an AUC = 0.78 (95% CI: 0.71-0.84, 95 cancers across 5 types vs. 156 controls) with 35% sensitivity at 98% specificity. The plasma GAG score had an AUC = 0.84 (95% CI: 0.79-0.88, 178 cancers across 14 types vs. 140 controls) with 41% sensitivity at 98% specificity. In the subset of early-stage/low-grade cancers, the AUC was 0.78 and 0.72 in plasma and urine, respectively. The plasma and urine GAG scores were independent predictors of OS regardless of cancer type (HR = 1.39, p = 0.005 in plasma [ N = 283, 11 types, 67 deaths, median follow-up 17 months] and HR = 1.53, p = 0.016 in urine [ N = 161, 4 types, 32 deaths, median follow-up 15 months]). Conclusions: GAGomes were sensitive non-invasive metabolic biomarkers for any-stage cancer, including genitourinary and brain tumors.

Phenotypic mutations are amino acid changes caused by mistranslation. How phenotypic mutations affect the adaptive evolution of new protein functions is unknown. Here we evolve the antibiotic resistance protein TEM-1 towards resistance on the antibiotic cefotaxime in an Escherichia coli strain with a high mistranslation rate. TEM-1 populations evolved in such strains endow host cells with a general growth advantage, not only on cefotaxime but also on several other antibiotics that ancestral TEM-1 had been unable to deactivate. High-throughput sequencing of TEM-1 populations shows that this advantage is associated with a lower incidence of weakly deleterious genotypic mutations. Our observations show that mistranslation is not just a source of noise that delays adaptive evolution. It could even facilitate adaptive evolution by exacerbating the effects of deleterious mutations and leading to their more efficient purging. The ubiquity of mistranslation and its effects render mistranslation an important factor in adaptive protein evolution., Mistranslation results in amino acid changes in proteins known as phenotypic mutations and these occur at a much higher rate than DNA mutations. Here, the authors show that mistranslation can increase the response to directional selection by exacerbating the fitness effects of deleterious DNA mutations.

Graphical abstract, A series of the novel C-5 alkynyl pyrimidine nucleoside analogues (1–14) in which the sugar moiety was replaced by the conformationally restricted Z- and E-2-butenyl spacer between the phthalimido and pyrimidine ring were synthesized by using Sonogashira cross-coupling reaction. Cytostatic activity evaluation of the novel compounds showed that E-isomers exhibited, in general, better cytostatic activities than the corresponding Z-isomers. E-isomer 14 exhibited the best cytostatic effect against all evaluated malignant cell lines, particularly against hepatocellular carcinoma (Hep G2, IC50 = 4.3 μM). However, this compound was also cytotoxic to human normal fibroblasts (WI 38). Its Z-isomer 7 showed highly specific antiproliferative activity against Hep G2 (IC50 = 18 μM) and no cytotoxicity to WI 38. Moreover, compounds 3, 4 and 14 expressed some marginal inhibitory activity against HIV-1 and HIV-2.

How biological systems such as proteins achieve robustness to ubiquitous perturbations is a fundamental biological question. Such perturbations include errors that introduce phenotypic mutations into nascent proteins during the translation of mRNA. These errors are remarkably frequent. They are also costly, because they reduce protein stability and help create toxic misfolded proteins. Adaptive evolution might reduce these costs of protein mistranslation by two principal mechanisms. The first increases the accuracy of translation via synonymous “high fidelity” codons at especially sensitive sites. The second increases the robustness of proteins to phenotypic errors via amino acids that increase protein stability. To study how these mechanisms are exploited by populations evolving in the laboratory, we evolved the antibiotic resistance gene TEM-1 in Escherichia coli hosts with either normal or high rates of mistranslation. We analyzed TEM-1 populations that evolved under relaxed and stringent selection for antibiotic resistance by single molecule real-time sequencing. Under relaxed selection, mistranslating populations reduce mistranslation costs by reducing TEM-1 expression. Under stringent selection, they efficiently purge destabilizing amino acid changes. More importantly, they accumulate stabilizing amino acid changes rather than synonymous changes that increase translational accuracy. In the large populations we study, and on short evolutionary timescales, the path of least resistance in TEM-1 evolution consists of reducing the consequences of translation errors rather than the errors themselves.

The distribution of variation in a quantitative trait and its underlying distribution of genotypic diversity can both be shaped by stabilizing and directional selection. Understanding either distribution is important because it determines a population's response to natural selection. Unfortunately existing theory makes conflicting predictions about how selection shapes these distributions and very little pertinent experimental evidence exists. Here we study a simple genetic system an evolving RNA enzyme (ribozyme) in which a combination of high throughput genotyping and measurement of a biochemical phenotype allow us to address this question. We show that directional selection compared to stabilizing selection increases the genotypic diversity of an evolving ribozyme population. In contrast it leaves the variance in the phenotypic trait unchanged. © 2013 Springer Science+Business Media New York.

A series of the novel pyrimidine (3-6) and purine (12-15, 18-21) acyclic nucleoside analogues in which the sugar moiety was replaced by a sterically constrained Z-4-amino-, 4-aminohydrochloride-2-butenyl, or aliphatic 4-aminohydrochloride-2-butyl moiety were synthesized and evaluated for their anti-viral and cytostatic activity potency. Cytostatic evaluation of the novel compounds on selected panel of human tumour-cell lines showed that the majority of compounds exerted a non-specific anti-proliferative effect at the highest tested concentration (i.e. 1 x 10(-4) M) against all cell lines. Nevertheless, a rather moderate but selective anti-proliferative effects on HeLa cell cultures in comparison to normal fibroblasts WI 38, were observed for compounds 15 and 21. No anti-viral activity was observed, except for compounds 3, 4, 5 and 19 that showed anti-HIV activity at 50% effective concentration ranging between 10 and 96 mu M. ispartof: Medicinal Chemistry Research vol:20 issue:3 pages:280-286 status: published

Dupuytren’s Disease (DD) is a fibroproliferative disorder, the cure for which is still limited to surgical excision of the affected fascia, often leading to high recurrence rates. Due to this fact, non-surgical treatments are being investigated, among them those targeting molecular processes of proliferation and differentiation in Dupuytren’ s cell cultures. Drugs with antiproliferative action may be valuable in DD treatment. Through characterization of changes on DD-specific cells, we therefore decided to test the therapeutic potential of new cytostatic drugs for DD treatment and/or for reduction of post-operative recurrence rates. The N-sulphonylpyrimidine derivative, amidino-substituted benzimidazo[1, 2-a]quinoline, and amidino dihydrothienothienyl[2, 3-c]quinolone hydrochloride, known to affect proliferation processes, were tested for their antiproliferative activity on primary fibroblasts/myofibroblasts cell cultures derived from the palmar fascia of patients with DD. Only amidino dihydrothienothienyl[2, 3-c]quinolone hydrochloride acted in a highly specific manner on cells derived from diseased fascia of DD patients and exhibited a low cytotoxic effect. This result might be a consequence of its specific activity on cytoskeleton changes occurring in differentiating cells. A similar short-term differential antiproliferative effect was observed by the N-sulphonylpyrimidine derivative that was however completely lost after 6- and 14-day treatments. The amidino-substituted benzimidazo[1, 2-a]quinoline exerted a strong non-specific, dose-related antiproliferative activity on cell types.