A Novel Tissue-Free Method to Estimate Tumor-Derived Cell-Free DNA Quantity Using Tumor Methylation Patterns.

Estimating the abundance of cell-free DNA (cfDNA) fragments shed from a tumor (i.e., circulating tumor DNA (ctDNA)) can approximate tumor burden, which has numerous clinical applications. We derived a novel, broadly applicable statistical method to quantify cancer-indicative methylation patterns within cfDNA to estimate ctDNA abundance, even at low levels. Our algorithm identified differentially methylated regions (DMRs) between a reference database of cancer tissue biopsy samples and cfDNA from individuals without cancer. Then, without utilizing matched tissue biopsy, counts of fragments matching the cancer-indicative hyper/hypo-methylated patterns within DMRs were used to determine a tumor methylated fraction (TMeF; a methylation-based quantification of the circulating tumor allele fraction and estimate of ctDNA abundance) for plasma samples. TMeF and small variant allele fraction (SVAF) estimates of the same cancer plasma samples were correlated (Spearman's correlation coefficient: 0.73), and synthetic dilutions to expected TMeF of 10-3 and 10-4 had estimated TMeF within two-fold for 95% and 77% of samples, respectively. TMeF increased with cancer stage and tumor size and inversely correlated with survival probability. Therefore, tumor-derived fragments in the cfDNA of patients with cancer can be leveraged to estimate ctDNA abundance without the need for a tumor biopsy, which may provide non-invasive clinical approximations of tumor burden.

Structure of the PAPP-ABP5 complex reveals mechanism of substrate recognition.

Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-ABP5) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.

High-throughput screening for improved microbial cell factories, perspective and promise.

High-throughput screening is a critical part of any industrial strain engineering effort, helping ensure the lowest cost product is produced in the shortest amount of time. Small-scale testing that correlates to manufacturing scale allows rapid strain development with confidence that engineering changes are relevant at-scale. In this review, the current state of high-throughput screening, the technological advances for the next generation strain screening pipeline, and options for implementation are reviewed. New technologies in cell culture (optofluidics) and measurement (acoustic mist ionization mass spectrometry) are highlighted, and special considerations (i.e. cost models, correlation between scales, data quality) are discussed.

The population dynamics of bacteria in physically structured habitats and the adaptive virtue of random motility.

Why is motility so common in bacteria? An obvious answer to this ecological and evolutionary question is that in almost all habitats, bacteria need to go someplace and particularly in the direction of food. Although the machinery required for motility and chemotaxis (acquiring and processing the information needed to direct movement toward nutrients) are functionally coupled in contemporary bacteria, they are coded for by different sets of genes. Moreover, information that resources are more abundant elsewhere in a habitat would be of no value to a bacterium unless it already had the means to get there. Thus, motility must have evolved before chemotaxis, and bacteria with flagella and other machinery for propulsion in random directions must have an advantage over bacteria relegated to moving at the whim of external forces alone. However, what are the selection pressures responsible for the evolution and maintenance of undirected motility in bacteria? Here we use a combination of mathematical modeling and experiments with Escherichia coli to generate and test a parsimonious and ecologically general hypothesis for the existence of undirected motility in bacteria: it enables bacteria to move away from each other and thereby obtain greater individual shares of resources in physically structured environments. The results of our experiments not only support this hypothesis, but are quantitatively and qualitatively consistent with the predictions of our model.

Discovering functional novelty in metagenomes: examples from light-mediated processes.

The emerging coverage of diverse habitats by metagenomic shotgun data opens new avenues of discovering functional novelty using computational tools. Here, we apply three different concepts for predicting novel functions within light-mediated microbial pathways in five diverse environments. Using phylogenetic approaches, we discovered two novel deep-branching subfamilies of photolyases (involved in light-mediated repair) distributed abundantly in high-UV environments. Using neighborhood approaches, we were able to assign seven novel functional partners in luciferase synthesis, nitrogen metabolism, and quorum sensing to BLUF domain-containing proteins (involved in light sensing). Finally, by domain analysis, for RcaE proteins (involved in chromatic adaptation), we predict 16 novel domain architectures that indicate novel functionalities in habitats with little or no light. Quantification of protein abundance in the various environments supports our findings that bacteria utilize light for sensing, repair, and adaptation far more widely than previously thought. While the discoveries illustrate the opportunities in function discovery, we also discuss the immense conceptual and practical challenges that come along with this new type of data.

Modularity of stress response evolution.

Responses to extracellular stress directly confer survival fitness by means of complex regulatory networks. Despite their complexity, the networks must be evolvable because of changing ecological and environmental pressures. Although the regulatory networks underlying stress responses are characterized extensively, their mechanism of evolution remains poorly understood. Here, we examine the evolution of three candidate stress response networks (chemotaxis, competence for DNA uptake, and endospore formation) by analyzing their phylogenetic distribution across several hundred diverse bacterial and archaeal lineages. We report that genes in the chemotaxis and sporulation networks group into well defined evolutionary modules with distinct functions, phenotypes, and substitution rates as compared with control sets of randomly chosen genes. The evolutionary modules vary in both number and cohesiveness among the three pathways. Chemotaxis has five coherent modules whose distribution among species shows a clear pattern of interdependence and rewiring. Sporulation, by contrast, is nearly monolithic and seems to be inherited vertically, with three weak modules constituting early and late stages of the pathway. Competence does not seem to exhibit well defined modules either at or below the pathway level. Many of the detected modules are better understood in engineering terms than in protein functional terms, as we demonstrate using a control-based ontology that classifies gene function according to roles such as "sensor," "regulator," and "actuator." Moreover, we show that combinations of the modules predict phenotype, yet surprisingly do not necessarily correlate with phylogenetic inheritance. The architectures of these three pathways are therefore emblematic of different modes and constraints on evolution.

Early adoption of new drug treatments: the role of continuing medical education and physician adaptivity.

The influence of continuing medical education (CME) on the adoption of new treatments is widely regarded as self-evident. Less well understood is how the dynamics of dissemination of new healthcare practices are influenced by the intersection of education with the adaptive characteristics of providers. We developed and validated a 43-item online instrument (eSAIL) for measuring adaptive style and used it to investigate the interplay of physician adaptivity with key components of effective medical education. Satisfactory Cronbach alpha and test-retest reliability coefficients were observed for all primary psychometric scales and a composite adaptivity scale. Discriminant, convergent, and predictive validities of eSAIL scales were consistent across all cohorts. Using an online medical education program for which data on physician behavioral change are available, we show that the rate of adoption of new drugs is driven by both psychological (adaptivity) and environmental (educational) inputs. We show for the first time that topic eminence, length of reinforcement period and physician adaptive score in the eSAIL are each proportional to early-adoption-related behavioral change. Using a simple forced-choice question, a cohort of 208 physicians was segmented into "A" (adaptive) and "C" (conservative) segments based on their eSAIL adaptivity z scores (+0.170 versus -0.234 respectively; P < 0.01). Early adoption of new drug treatments by similarly segmented physician cohorts was driven almost entirely by A-segment physicians, but only when those physicians were additionally exposed to effective medical education.