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RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free electron laser sources to reveal the formation and ready identification of Å scale features in structured and unstructured RNAs. New structural signatures of RNA secondary and tertiary structures are identified through wide angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base paired intermediate to assume a triple helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. In addition to understanding how RNA triplexes form and thereby function as dynamic signaling elements, this new method can vastly increase the rate of structure determination for these biologically essential, but mostly uncharacterized macromolecules.

Academic investigators have used behavioural economics, a method developed originally to study consumers and their sentiments towards products, to study matters of public policy. A recent article in the European Journal of International Law – ‘What Is Wrong with Investment Arbitration? Evidence from a Set of Behavioural Experiments’ – gives a detailed summary of a series of experiments performed in order to study public sentiment towards investment arbitration. The investigators, Maria Laura Marceddu and Pietro Ortolani observe that public sentiment improves towards the outcome of a dispute settlement procedure when survey respondents are told that the procedure was a ‘court’ with tenured judges, and it worsens when they are told that it was ‘arbitration’ with temporary appointees. From their observations, Marceddu and Ortolani conclude that an international investment court, such as that which the European Union promotes, is a good idea. We suggest, however, that a further inquiry should investigate in greater detail public understanding of what qualities the individuals who serve as judges or arbitrators ought to display, as distinct from the institutional format in which dispute settlement takes place.

No account of international law in the modern era would be intelligible without an account of the non-State actor in international law. However, States in no small part continue to set the terms by which others now share the stage. How States set the terms—and how to address the problems arising from the terms they set—are central questions for international law and the organs that apply it. The roles that non-state actors play have been routinized, for example in treaty arbitration. As a result, we sometimes forget that the starting point for what James Crawford called international law’s ‘open system’ is still the State. Non-State actors enter the system more readily than before, but, if we are to understand the continuing limitations on their rights, duties and functions in international law, then we should keep sight of how it is they enter: States are the gatekeepers of the open system, and, even if the terms of access are more liberal and more parties seek access than ever, States still decide who gets in.

Nature Communications 11(1), 4511 (2020). doi:10.1038/s41467-020-18156-7, Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported., Published by Nature Publishing Group UK, [London]

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Dissociation of transforming growth factor beta-1 (TGFβ-1) from the inhibitory protein latency-associated peptide (LAP) can occur from low doses of X-ray irradiation of the LAP–TGFβ-1 complex, resulting in the activation of TGFβ-1, and can have health-related consequences. Using the tools and knowledge developed in the study of radiation damage in the crystallographic setting, small-angle X-ray scattering (SAXS) and complementary techniques suggest an activation process that is initiated but not driven by the initial X-ray exposure. LAP is revealed to be extended when not bound to TGFβ-1 and has a different structural conformation compared to the bound state. These studies pave the way for the structural understanding of systems impacted at therapeutic X-ray doses and show the potential impact of radiation damage studies beyond their original intent.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Cytochrome c oxidase (C c O) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine C c O. It is assigned to the P R -intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a 3 iron atom is in a ferryl (Fe 4+ = O 2− ) configuration, and heme a and Cu B are oxidized while Cu A is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

Small-angle scattering (SAS) probes the size and shape of particles at low resolution through the analysis of the scattering of X-rays or neutrons passing through a solution of particles. One approach to extracting structural information from SAS data is the indirect Fourier transform (IFT). The IFT approach parameterizes the real-space pair distribution function [P(r)] of a particle using a set of basis functions, which simultaneously determines the scattering profile [I(q)] using corresponding reciprocal-space basis functions. This article presents an extension of an IFT algorithm proposed by Moore [J. Appl. Cryst. (1980), 13, 168–175] which used a trigonometric series to describe the basis functions, where the real-space and reciprocal-space basis functions are Fourier mates. An equation is presented relating the Moore coefficients to the intensities of the SAS profile at specific positions, as well as a series of new equations that describe the size and shape parameters of a particle from this distinct set of intensity values. An analytical real-space regularizer is derived to smooth the P(r) curve and ameliorate systematic deviations caused by series termination. Regularization is commonly used in IFT methods though not described in Moore's original approach, which is particularly susceptible to such effects. The algorithm is provided as a script, denss.fit_data.py, as part of the DENSS software package for SAS, which includes both command line and interactive graphical interfaces. Results of the program using experimental data show that it is as accurate as, and often more accurate than, existing tools.

This chapter traces the origins of arbitration. Arbitration initially developed in the absence of courts and later in opposition to courts, or at least as an alternative to them. The chapter then looks at the three episodes which illustrate the rise of modern arbitration. While arbitration takes many forms, all its forms share a core set of characteristics. Ultimately, arbitration is a procedure in which certain parties choose a decision-maker and grant the decision-maker the exclusive power to render a decision on a dispute between them—usually referred to as an arbitral award—following a procedure that complies with some standard of fairness also agreed by the parties.

This chapter reflects on the future directions that arbitration might take. The privatization of justice through arbitration no doubt has advantages. However, privatized justice all too readily looks like justice for hire. So is privatized justice to be promoted, or should it be restrained? A fair reply is, it depends. The answers one gives in the debate over arbitration, as in so many debates over institutions that affect the public interest, are often shaped by one’s ideological starting point. Ultimately, arbitrators and the parties who call upon them should remain conscious of both arbitration’s promise and its limits, if they mean this noble institution well.

This chapter focuses on the most politically charged type of arbitration: arbitration between an investor and a foreign government. Investment arbitration marks a great step forward for rule of law in international affairs. A government does not escape responsibility by saying that its own law or courts have declared a given abuse against an investor to be lawful. Investment treaties set out substantive protections, such as fair and equitable treatment, full protection and security, and a guarantee against discriminatory expropriation. They give investors legal rights independent of domestic rules that an unfriendly government might have manipulated at the investor’s cost. The chapter then considers the emerging critique of investment arbitration.

This chapter describes some of the main types of cases in which parties use arbitration. First, there are disputes that pit two countries one against the other, which can be referred to as ‘interstate’ or ‘international’ or sometimes ‘inter-governmental’ disputes. Then there are disputes between private parties: individuals in dispute with one another; individuals in dispute with companies; or companies in dispute with other companies. Legal scholars call these ‘private’ disputes. And, third, there are disputes between a private party, such as an individual or a company, and a government. In the field of dispute settlement, these types of disputes are known as ‘mixed’ disputes.

This chapter explores how arbitration relates to public courts, the law, and legal systems. Not all disputes are permitted to be arbitrated. The word lawyers use to describe disputes that the law permits parties to submit to arbitration is ‘arbitrability’. Disputes that the law does not permit parties to submit to arbitration are said to be ‘non-arbitrable’. The chapter then considers how parties can choose the law that applies in their arbitration. The typical arrangement is that the seat of arbitration is also the physical location where the proceedings take place—meaning that the law of the physical location is the procedural law of the arbitration.

This chapter explains how arbitration works in practice. Arbitration takes place if and only if parties have consented to it. Their consent needs to make clear how, specifically, arbitration is going to work for them. Parties in some situations may use courts and through them the executive apparatus to assist in arbitral proceedings. Under most arbitration agreements, once the arbitrator or arbitral tribunal has given the final arbitral award, arbitral jurisdiction comes to an end, and so there is no place for a party to turn to appeal against an adverse award. However, a losing party nevertheless may have opportunities to challenge an award, including in proceedings in national courts.

Holmes’s enduring interest was in the development of the law, as indicated by the title The Path of the Law. He drew on the philosophy of science and the role of induction in forming scientific theories, and he added a new ingredient: social induction. Social induction has two parts. First, the law develops through the accumulation of cases, which arise through the actions of agents who are embedded within society and who necessarily adapt their actions to the law. This is analogous to a subfield of machine learning called reinforcement learning, the basis for DeepMind’s AlphaGo, in which the machine is trained on a dataset of its own adaptive actions. The second part of social induction is the process whereby settled legal doctrine arises out of contested judicial decisions. We argue that this second part can be formulated in terms of prediction (law is constituted, after all, by prophecies of what courts will do), and that it is therefore a suitable topic for machine learning. This suggests new ways of thinking about explainability of machine learning decisions.

Holmes thought that the law is drawn from much wider sources than legal formalists of the day maintained: not just the law codes, but experience at large. “The life of the law has not been logic; it has been experience.” The algorithmists, analogous to legal formalists, see the source code as what matters. In machine learning there is still source code, but it is the training dataset that matters. The typical setup is this: the training dataset is assembled beforehand, then an algorithm is run by which the machine “learns” its parameters from the training data, then the trained machine is put to use to process new cases. The training data is what is given, the ground truth to be learnt from. The collected experience of society may be likened to a training dataset, and the learnt experience of a jurist may be likened to the learnt parameters of a machine learning system.

The output of a machine learning system is nothing more than statements of the following form: “such and such a new case is likely to behave similarly to other similar cases that belong to the training dataset that was used to train this machine.” It has been described as “just curve fitting”, in the sense of drawing a curve through the datapoints in the training dataset, perhaps smoothing out some irregularities; the “learning” in machine learning is nothing more than tuning parameter values to make a well-fitting curve. This curve is then used to make predictions for new cases. The patterns found by machine learning are not laws of nature like Newton’s laws, and they are not stipulative rules like those laid down in statutes, they are simply fitted curves. Analogously, Holmes said that law emerges as people and their institutions find patterns in human experience. He wrote that a page of history is worth a volume of logic. When lawmakers ask for “the logic involved” in machine learning, or refer to “inferring rules from data”, they should really be asking for “a story about the training dataset”.

There is an unavoidable tension between prediction and explanation: between the idea that predictive accuracy is what matters, and the requirement for explanation. In the law, Holmes drew a distinction between explanation as articulated in a judicial decision using the language of logic, and the real explanation that can be found by looking at the judge’s body of experience, what he called the “inarticulate major premise”; but he went beyond both of these and instead invoked prophecy as the fundamental constituent of law. Holmes prefigured a debate that machine learning has brought to the fore: the debate between machine learning’s “prediction culture” which values case-by-case predictive accuracy, and the scientific “explanation culture” which looks for explanations in terms of rules. The difference between these two cultures was missed by an earlier generation of philosophers of science, who it seems did not conceive of machines whose function is sufficiently advanced that it seems like magic. The prediction culture has made striking advances … and yet, explainability remains a policy priority, because of the impact that machine learning is having on society.

Machine learning gives rise to concerns about “algorithmic bias,” arising from bias in the training dataset. A dataset necessarily reflects a past state of affairs, but we may anticipate that the future will be different, or desire it to be so. Law has struggled with an analogous problem: how to deal with “bad evidence” and prejudice. Holmes’s broad view of experience suggests that once undesirable data has been revealed, its potential for mischief is there, and it is futile to pretend it does not exist. Instead, law has developed several strategies to deal with these negative influences. Under the doctrine of the “fruit of the poisonous tree,” in American jurisprudence, evidence that stems from improper actions by public authorities is inadmissible in court. Or, a judge can give instructions to the jury to guard against improper inferences. Or, a jury verdict may be struck down on appeal. Strategies analogous to these might help to make machine learning a tool for better outcomes, rather than a trap that entrenches past mistakes and prejudice.

Holmes’s famous epigram, that “prophecies of what the courts will do” is what constitutes the law, applies equally well to machine learning. In machine learning, prediction is the be-all and end-all. Machine learning requires that its tasks be formulated as prediction problems—not just forecasting, but prediction in the broader sense of “give an answer for cases where the true answer is not known to the machine”, for example naming the animal shown in a photo. A machine learning system is trained so as to maximize the accuracy of its predictions, and prediction accuracy is the benchmark by which machine learning systems are compared and evaluated. In the law, according to Holmes, prediction is everything, from the humble plaintiff who simply wants to guess how a case might turn out, to the appeals court judge whose decisions are written in anticipation of how they will be received by other judges and by the rest of society.

To the legal formalist, the law consists in applying rules to facts using logic, similar to working out a mathematical proof. It is a common view of computers that they should be thought of in the same way: that they run algorithms, which should be described using rules of logic. This view of computing is appealing to formalist lawyers, who see the link between statutory law and algorithmic source code. But just as Holmes argued against the formalist view of law, we must let go of algorithmic thinking if we are to make sense of machine learning. The essential parts of machine learning—the training dataset, and the learnt parameters—cannot be understood by looking at source code. True, it does use algorithms to learn those parameter values, most notably the Gradient Descent algorithm. But to focus on algorithms and logic is to miss what is distinctive about machine learning. It leads to misguided views about regulation and control.

Holmes saw juries as a conduit into the courtroom of popular feelings and prejudices, ensuring that the law is kept in accord with the will of the community. Thus juries are analogous to the training dataset: they provide the givens, the ground truth. A judge who is faced with an intractable question about which she would rather not make a decision will look for a way to send that question to the jury. Likewise, with the successes of machine learning it can be tempting to hand off decisions from humans such as sentencing judges or bank loan officers, to machines; and as the machine is nothing more than pattern-matching to its training data, this is really handing off to the dataset. Because juries have this authoritative role, Holmes treated their decisions with special caution; he placed the corrective elsewhere, namely in the hands of the court of appeal. His caution, and his approach, have relevance for the machine learning age.

This work demonstrates the use of polymer thin films and graphene to support and maintain the hydration of protein microcrystals on fixed targets for serial femtosecond crystallography at X-ray free-electron lasers. Rapid encystment protein (REP24) provides a benchmark for this encapsulation approach., For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.