Noninvasive detection of any-stage cancer using free glycosaminoglycans.

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 (Nurine = 220 cancer vs. 360 healthy) and plasma (Nplasma = 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.

Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults.

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.

Mistranslation can promote the exploration of alternative evolutionary trajectories in enzyme evolution.

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.

Mistranslation drives the evolution of robustness in TEM-1 ß-lactamase.

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 effects of stabilizing and directional selection on phenotypic and genotypic variation in a population of RNA enzymes.

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.

Plasma and Urine Free Glycosaminoglycans as Monitoring and Predictive Biomarkers in Metastatic Renal Cell Carcinoma: A Prospective Cohort Study.

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.

Plasma and Urine Free Glycosaminoglycans as Monitoring Biomarkers in Nonmetastatic Renal Cell Carcinoma-A Prospective Cohort Study.

Background: No liquid biomarkers are approved in renal cell carcinoma (RCC), making early detection of recurrence in surgically treated nonmetastatic (M0) patients dependent on radiological imaging. Urine- and plasma free glycosaminoglycan profiles-or free GAGomes-are promising biomarkers reflective of RCC metabolism. Objective: To explore whether free GAGomes could detect M0 RCC recurrence noninvasively. Design setting and participants: Between June 2016 and February 2021, we enrolled a prospective consecutive series of patients elected for (1) partial or radical nephrectomy for clinical M0 RCC (cohort 1) or (2) first-line therapy following RCC metachronous metastatic recurrence (cohort 2) at Sahlgrenska University Hospital, Gothenburg, Sweden. The study population included M0 RCC patients with recurrent disease (RD) versus no evidence of disease (NED) in at least one follow-up visit. Plasma and urine free GAGomes-consisting of 40 chondroitin sulfate (CS), heparan sulfate, and hyaluronic acid (HA) features-were measured in a blinded central laboratory preoperatively and at each postoperative follow-up visit until recurrence or end of follow-up in cohort 1, or before treatment start in cohort 2. Outcome measurements and statistical analysis: We used Bayesian logistic regression to correlate GAGome features with RD versus NED and with various histopathological variables. We developed three recurrence scores (plasma, urine, and combined) proportional to the predicted probability of RD. We internally validated the area under the curve (AUC) using bootstrap resampling. We performed a decision curve analysis to select a cutoff and report the corresponding net benefit, sensitivity, and specificity of each score. We used univariable analyses to correlate each preoperative score with recurrence-free survival (RFS). Results and limitations: Of 127 enrolled patients in total, 62 M0 RCC patients were in the study population (median age: 63 year, 35% female, and 82% clear cell). The median follow-up time was 3 months, totaling 72 postoperative visits -17 RD and 55 NED cases. RD was compatible with alterations in 14 (52%) of the detectable GAGome features, mostly free CS. Eleven (79%) of these correlated with at least one histopathological variable. We developed a plasma, a urine, and a combined free CS RCC recurrence score to diagnose RD versus NED with AUCs 0.91, 0.93, and 0.94, respectively. At a cutoff equivalent to ≥30% predicted probability of RD, the sensitivity and specificity were, respectively, 69% and 84% in plasma, 81% and 80% in urine, and 80% and 82% when combined, and the net benefit was equivalent to finding an extra ten, 13, and 12 cases of RD per hundred patients without any unnecessary imaging for plasma, urine, and combined, respectively. The combined score was prognostic of RFS in univariable analysis (hazard ratio = 1.90, p = 0.02). Limitations include a lack of external validation. Conclusions: Free CS scores detected postsurgical recurrence noninvasively in M0 RCC with substantial net benefit. External validity is required before wider clinical implementation. Patient summary: In this study, we examined a new noninvasive blood and urine test to detect whether renal cell carcinoma recurred after surgery.

Orthogonal translation enables heterologous ribosome engineering in E. coli.

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.

Directed evolution of rRNA improves translation kinetics and recombinant protein yield.

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.

Analytical performance of a standardized kit for mass spectrometry-based measurements of human glycosaminoglycans.

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 free GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 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 free GAG disaccharides allowing for comparability of measurements and enabling translational research.

Synthesis, cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 pyrimidine nucleoside analogues.

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, IC(50)=4.3microM). However, this compound was also cytotoxic to human normal fibroblasts (WI 38). Its Z-isomer 7 showed highly specific antiproliferative activity against Hep G2 (IC(50)=18microM) and no cytotoxicity to WI 38. Moreover, compounds 3, 4 and 14 expressed some marginal inhibitory activity against HIV-1 and HIV-2.

Modern methods for laboratory diversification of biomolecules.

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.

Mistranslation can enhance fitness through purging of deleterious mutations.

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.

Screening of potential prodrugs on cells derived from Dupuytren's disease patients.

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-sulfonylpyrimidine 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-sulfonylpyrimidine 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.