Ion binding with charge inversion combined with screening modulates DEAD box helicase phase transitions.

Membraneless organelles, or biomolecular condensates, enable cells to compartmentalize material and processes into unique biochemical environments. While specific, attractive molecular interactions are known to stabilize biomolecular condensates, repulsive interactions, and the balance between these opposing forces, are largely unexplored. Here, we demonstrate that repulsive and attractive electrostatic interactions regulate condensate stability, internal mobility, interfaces, and selective partitioning of molecules both in vitro and in cells. We find that signaling ions, such as calcium, alter repulsions between model Ddx3 and Ddx4 condensate proteins by directly binding to negatively charged amino acid sidechains and effectively inverting their charge, in a manner fundamentally dissimilar to electrostatic screening. Using a polymerization model combined with generalized stickers and spacers, we accurately quantify and predict condensate stability over a wide range of pH, salt concentrations, and amino acid sequences. Our model provides a general quantitative treatment for understanding how charge and ions reversibly control condensate stability.

Surface tension measurement and calculation of model biomolecular condensates.

The surface tension of liquid-like protein-rich biomolecular condensates is an emerging physical principle governing the mesoscopic interior organisation of biological cells. In this study, we present a method to evaluate the surface tension of model biomolecular condensates, through straighforward sessile drop measurements of capillary lengths and condensate densities. Our approach bypasses the need for characterizing condensate viscosities, which was required in previously reported techniques. We demonstrate this method using model condensates comprising two mutants of the intrinsically disordered protein Ddx4N. Notably, we uncover a detrimental impact of increased protein net charge on the surface tension of Ddx4N condensates. Furthermore, we explore the application of Scheutjens-Fleer theory, calculating condensate surface tensions through a self-consistent mean-field framework using Flory-Huggins interaction parameters. This relatively simple theory provides semi-quantitative accuracy in predicting Ddx4N condensate surface tensions and enables the evaluation of molecular organisation at condensate surfaces. Our findings shed light on the molecular details of fluid-fluid interfaces in biomolecular condensates.

Structure-conditioned amino-acid couplings: How contact geometry affects pairwise sequence preferences.

Relating a protein's sequence to its conformation is a central challenge for both structure prediction and sequence design. Statistical contact potentials, as well as their more descriptive versions that account for side-chain orientation and other geometric descriptors, have served as simplistic but useful means of representing second-order contributions in sequence-structure relationships. Here we ask what happens when a pairwise potential is conditioned on the fully defined geometry of interacting backbones fragments. We show that the resulting structure-conditioned coupling energies more accurately reflect pair preferences as a function of structural contexts. These structure-conditioned energies more reliably encode native sequence information and more highly correlate with experimentally determined coupling energies. Clustering a database of interaction motifs by structure results in ensembles of similar energies and clustering them by energy results in ensembles of similar structures. By comparing many pairs of interaction motifs and showing that structural similarity and energetic similarity go hand-in-hand, we provide a tangible link between modular sequence and structure elements. This link is applicable to structural modeling, and we show that scoring CASP models with structured-conditioned energies results in substantially higher correlation with structural quality than scoring the same models with a contact potential. We conclude that structure-conditioned coupling energies are a good way to model the impact of interaction geometry on second-order sequence preferences.

In Vitro Transition Temperature Measurement of Phase-Separating Proteins by Microscopy.

Intracellular compartmentalization through liquid-liquid phase separation is an emerging organizing principle of cell biology. These compartments, such as the nucleolus and stress granules, are collectively known as membraneless organelles or biomolecular condensates. In vitro studies of many protein components of biomolecular condensates, such as the intrinsically disordered regions of Ddx4, FUS, and Laf-1 proteins, have revealed much about the driving forces of the phase separation process. A common approach is to investigate how the temperature at which a protein solution forms condensates-the transition temperature-responds to changes in the solution composition. We describe a method to measure the in vitro transition temperature of a sub-10 µL sample of a phase-separating solution using transmitted light microscopy. Through monitoring changes in transition temperature with solution conditions, this approach allows the impact of additional biomolecules and additives to be quantitatively assessed and permits the construction of phase diagrams.

Contact prediction is hardest for the most informative contacts, but improves with the incorporation of contact potentials.

Co-evolution between pairs of residues in a multiple sequence alignment (MSA) of homologous proteins has long been proposed as an indicator of structural contacts. Recently, several methods, such as direct-coupling analysis (DCA) and MetaPSICOV, have been shown to achieve impressive rates of contact prediction by taking advantage of considerable sequence data. In this paper, we show that prediction success rates are highly sensitive to the structural definition of a contact, with more permissive definitions (i.e., those classifying more pairs as true contacts) naturally leading to higher positive predictive rates, but at the expense of the amount of structural information contributed by each contact. Thus, the remaining limitations of contact prediction algorithms are most noticeable in conjunction with geometrically restrictive contacts-precisely those that contribute more information in structure prediction. We suggest that to improve prediction rates for such "informative" contacts one could combine co-evolution scores with additional indicators of contact likelihood. Specifically, we find that when a pair of co-varying positions in an MSA is occupied by residue pairs with favorable statistical contact energies, that pair is more likely to represent a true contact. We show that combining a contact potential metric with DCA or MetaPSICOV performs considerably better than DCA or MetaPSICOV alone, respectively. This is true regardless of contact definition, but especially true for stricter and more informative contact definitions. In summary, this work outlines some remaining challenges to be addressed in contact prediction and proposes and validates a promising direction towards improvement.

Spatial gradients of protein-level time delays set the pace of the traveling segmentation clock waves.

The vertebrate segmentation clock is a gene expression oscillator controlling rhythmic segmentation of the vertebral column during embryonic development. The period of oscillations becomes longer as cells are displaced along the posterior to anterior axis, which results in traveling waves of clock gene expression sweeping in the unsegmented tissue. Although various hypotheses necessitating the inclusion of additional regulatory genes into the core clock network at different spatial locations have been proposed, the mechanism underlying traveling waves has remained elusive. Here, we combined molecular-level computational modeling and quantitative experimentation to solve this puzzle. Our model predicts the existence of an increasing gradient of gene expression time delays along the posterior to anterior direction to recapitulate spatiotemporal profiles of the traveling segmentation clock waves in different genetic backgrounds in zebrafish. We validated this prediction by measuring an increased time delay of oscillatory Her1 protein production along the unsegmented tissue. Our results refuted the need for spatial expansion of the core feedback loop to explain the occurrence of traveling waves. Spatial regulation of gene expression time delays is a novel way of creating dynamic patterns; this is the first report demonstrating such a control mechanism in any tissue and future investigations will explore the presence of analogous examples in other biological systems.

Genomic testing in cancer: patient knowledge, attitudes, and expectations.

BACKGROUND: Genomic testing in cancer (GTC) characterizes genes that play an important role in the development and growth of a patient's cancer. This form of DNA testing is currently being studied for its ability to guide cancer therapy. The objective of the current study was to describe patients' knowledge, attitudes, and expectations toward GTC. METHODS: A 42-item self-administered GTC questionnaire was developed by a multidisciplinary group and patient pretesting. The questionnaire was distributed to patients with advanced cancer who were referred to the Princess Margaret Cancer Center for a phase 1 clinical trial or GTC testing. RESULTS: Results were reported from 98 patients with advanced cancer, representing 66% of the patients surveyed. Seventy-six percent of patients were interested in learning more about GTC, and 64% reported that GTC would significantly improve their cancer care. The median score on a 12-item questionnaire to assess knowledge of cancer genomics was 8 of 12 items correct (67%; interquartile range, 7-9 of 12 items correct [58%-75%]). Scores were associated significantly with patients' education level (P < .0001). Sixty-six percent of patients would consent to a needle biopsy, and 39% would consent to an invasive surgical biopsy if required for GTC. Only 48% of patients reported having sufficient knowledge to make an informed decision to pursue GTC whereas 34% of patients indicated a need for formal genetic counseling. CONCLUSIONS: Patients with advanced cancer are motivated to participate in GTC. Patients require further education to understand the difference between somatic and germline mutations in the context of GTC. Educational programs are needed to support patients interested in pursuing GTC.

Short-lived Her proteins drive robust synchronized oscillations in the zebrafish segmentation clock.

Oscillations are prevalent in natural systems. A gene expression oscillator, called the segmentation clock, controls segmentation of precursors of the vertebral column. Genes belonging to the Hes/her family encode the only conserved oscillating genes in all analyzed vertebrate species. Hes/Her proteins form dimers and negatively autoregulate their own transcription. Here, we developed a stochastic two-dimensional multicellular computational model to elucidate how the dynamics, i.e. period, amplitude and synchronization, of the segmentation clock are regulated. We performed parameter searches to demonstrate that autoregulatory negative-feedback loops of the redundant repressor Her dimers can generate synchronized gene expression oscillations in wild-type embryos and reproduce the dynamics of the segmentation oscillator in different mutant conditions. Our model also predicts that synchronized oscillations can be robustly generated as long as the half-lives of the repressor dimers are shorter than 6 minutes. We validated this prediction by measuring, for the first time, the half-life of Her7 protein as 3.5 minutes. These results demonstrate the importance of building biologically realistic stochastic models to test biological models more stringently and make predictions for future experimental studies.

Consensus statement on the use of gonadotropin-releasing hormone analogs in children.

OBJECTIVE: Gonadotropin-releasing hormone analogs revolutionized the treatment of central precocious puberty. However, questions remain regarding their optimal use in central precocious puberty and other conditions. The Lawson Wilkins Pediatric Endocrine Society and the European Society for Pediatric Endocrinology convened a consensus conference to review the clinical use of gonadotropin-releasing hormone analogs in children and adolescents. PARTICIPANTS: When selecting the 30 participants, consideration was given to equal representation from North America (United States and Canada) and Europe, an equal male/female ratio, and a balanced spectrum of professional seniority and expertise. EVIDENCE: Preference was given to articles written in English with long-term outcome data. The US Public Health grading system was used to grade evidence and rate the strength of conclusions. When evidence was insufficient, conclusions were based on expert opinion. CONSENSUS PROCESS: Participants were put into working groups with assigned topics and specific questions. Written materials were prepared and distributed before the conference, revised on the basis of input during the meeting, and presented to the full assembly for final review. If consensus could not be reached, conclusions were based on majority vote. All participants approved the final statement. CONCLUSIONS: The efficacy of gonadotropin-releasing hormone analogs in increasing adult height is undisputed only in early-onset (girls <6 years old) central precocious puberty. Other key areas, such as the psychosocial effects of central precocious puberty and their alteration by gonadotropin-releasing hormone analogs, need additional study. Few controlled prospective studies have been performed with gonadotropin-releasing hormone analogs in children, and many conclusions rely in part on collective expert opinion. The conference did not endorse commonly voiced concerns regarding the use of gonadotropin-releasing hormone analogs, such as promotion of weight gain or long-term diminution of bone mineral density. Use of gonadotropin-releasing hormone analogs for conditions other than central precocious puberty requires additional investigation and cannot be suggested routinely.

The impact of privacy and confidentiality laws on the conduct of clinical trials.

Justifiable concerns about the use of personal data in many aspects of daily life have led to the recent introduction in many countries of laws intended to regulate data use. Although participation in randomized clinical trials is generally with informed consent, recruitment procedures, complete follow-up, and the efficient conduct of trials may be substantially affected by such national or local privacy legislation. The relevant laws often have exceptions that allow the use of patient information in the public interest - including the use of data collected to improve or monitor public health or as part of medical research. However, regulatory bodies often give conflicting interpretations of the law, and this affects the conduct of large-scale trials. In particular, unnecessarily restrictive interpretation of the law may be a serious impediment to identification of potential participants for a trial, access to records to confirm events, continued follow-up of patients after the trial has been concluded, and secondary use of the trial data for purposes not directly related to the original purpose of the study. These obstacles could be overcome by better informing patients of the uses of records for medical research purposes, by using informed consent procedures that explain the nature of the research and the uses of the data, and by the use of identifiers, such as social security numbers that allow central follow-up. The clinical trial research community needs to ensure that the substantial benefits of large-scale randomized trials are explained both to the public and to those responsible for introducing legislation. The negative impact of privacy legislation on the use of personal health information and on conducting large studies needs to be understood and minimized.