Extracorporeal life support without systemic anticoagulation: a nitric oxide-based non-thrombogenic circuit for the artificial placenta in an ovine model.

BACKGROUND: Clinical translation of the extracorporeal artificial placenta (AP) is impeded by the high risk for intracranial hemorrhage in extremely premature newborns. The Nitric Oxide Surface Anticoagulation (NOSA) system is a novel non-thrombogenic extracorporeal circuit. This study aims to test the NOSA system in the AP without systemic anticoagulation. METHODS: Ten extremely premature lambs were delivered and connected to the AP. For the NOSA group, the circuit was coated with DBHD-N2O2/argatroban, 100 ppm nitric oxide was blended into the sweep gas, and no systemic anticoagulation was given. For the Heparin control group, a non-coated circuit was used and systemic anticoagulation was administered. RESULTS: Animals survived 6.8 ± 0.6 days with normal hemodynamics and gas exchange. Neither group had any hemorrhagic or thrombotic complications. ACT (194 ± 53 vs. 261 ± 86 s; p < 0.001) and aPTT (39 ± 7 vs. 69 ± 23 s; p < 0.001) were significantly lower in the NOSA group than the Heparin group. Platelet and leukocyte activation did not differ significantly from baseline in the NOSA group. Methemoglobin was 3.2 ± 1.1% in the NOSA group compared to 1.6 ± 0.6% in the Heparin group (p < 0.001). CONCLUSIONS: The AP with the NOSA system successfully supported extremely premature lambs for 7 days without significant bleeding or thrombosis. IMPACT: The Nitric Oxide Surface Anticoagulation (NOSA) system provides effective circuit-based anticoagulation in a fetal sheep model of the extracorporeal artificial placenta (AP) for 7 days. The NOSA system is the first non-thrombogenic circuit to consistently obviate the need for systemic anticoagulation in an extracorporeal circuit for up to 7 days. The NOSA system may allow the AP to be implemented clinically without systemic anticoagulation, thus greatly reducing the intracranial hemorrhage risk for extremely low gestational age newborns. The NOSA system could potentially be applied to any form of extracorporeal life support to reduce or avoid systemic anticoagulation.

The entropic force generated by intrinsically disordered segments tunes protein function.

Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (such as ligand binding, catalysis and regulation)3-5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood3,4. One of the constraints on the evolution of proteins is the stability of the folded 'native' state. Despite this, 44% of the human proteome contains intrinsically disordered peptide segments greater than 30 residues in length6, the majority of which have no known function7-9. Here we show that the entropic force produced by an intrinsically disordered carboxy terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (UGDH) towards a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the intrinsically disordered segment, and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10-13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of UGDH to favour inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of intrinsic disorder in the proteome.

Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence.

Many viruses employ ATP-powered motors during assembly to translocate DNA into procapsid shells. Previous reports raise the question if motor function is modulated by substrate DNA sequence: (i) the phage T4 motor exhibits large translocation rate fluctuations and pauses and slips; (ii) evidence suggests that the phage phi29 motor contacts DNA bases during translocation; and (iii) one theoretical model, the 'B-A scrunchworm', predicts that 'A-philic' sequences that transition more easily to A-form would alter motor function. Here, we use single-molecule optical tweezers measurements to compare translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor. We observed no significant differences in motor velocities, even with A-philic sequences predicted to show higher translocation rate at high applied force. We also observed no significant changes in motor pausing and only modest changes in slipping. To more generally test for sequence dependence, we conducted correlation analyses across pairs of packaging events. No significant correlations in packaging rate, pausing or slipping versus sequence position were detected across repeated measurements with several different DNA sequences. These studies suggest that viral genome packaging is insensitive to DNA sequence and fluctuations in packaging motor velocity, pausing and slipping are primarily stochastic temporal events.

Enhanced Hemocompatibility and In Vivo Analytical Accuracy of Intravascular Potentiometric Carbon Dioxide Sensors via Nitric Oxide Release.

In this letter, the innate ability of nitric oxide (NO) to inhibit platelet activation/adhesion/thrombus formation is employed to improve the hemocompatibility and in vivo accuracy of an intravascular (IV) potentiometric PCO2 (partial pressure of carbon dioxide) sensor. The catheter-type sensor is fabricated by impregnating a segment of dual lumen silicone tubing with a proton ionophore, plasticizer, and lipophilic cation-exchanger. Subsequent filling of bicarbonate and strong buffer solutions and placement of Ag/AgCl reference electrode wires within each lumen, respectively, enables measurement of the membrane potential difference across the inner wall of the tube, with this potential changing as a function of the logarithm of sample PCO2. The dual lumen device is further encapsulated within a S-nitroso-N-acetyl-DL-penicillamine (SNAP)-doped silicone tube that releases physiological levels of NO. The NO releasing sensor exhibits near-Nernstian sensitivity toward PCO2 (slope = 59.31 ± 0.78 mV/decade) and low drift rates (<2 mV/24 h after initial equilibration). In vivo evaluation of the NO releasing sensors, performed in the arteries and veins of anesthetized pigs for 20 h, shows enhanced accuracy (vs non-NO releasing sensors) when benchmarked to measurements of discrete blood samples made with a commercial blood gas analyzer. The accurate, continuous monitoring of blood PCO2 levels achieved with this new IV NO releasing PCO2 sensor configuration could help better manage hospitalized patients in critical care units.

PD-L1/B7-H1 Inhibits Viral Clearance by Macrophages in HSV-1-Infected Corneas.

Immune privilege helps protect the cornea from damaging inflammation but can also impair pathogen clearance from this mucosal surface. Programmed death-ligand 1 (PD-L1 or B7-H1) contributes to corneal immune privilege by inhibiting the function of a variety of immune cells. We asked whether programmed death-1 (PD-1)/PD-L1 interaction regulates HSV-1 clearance from infected corneas. We show that PD-L1 is constitutively expressed in the corneal epithelium and is upregulated upon HSV-1 corneal infection, with peak expression on CD45+ cells NK cells, dendritic cells, neutrophils, and macrophages and CD45- corneal epithelial cells at 4 d postinfection (dpi). As early as 1 dpi, HSV-1-infected corneas of B7-H1-/- mice as compared with wild-type mice showed increased chemokine expression and this correlated with increased migration of inflammatory cells into the viral lesions and decreased HSV-1 corneal titers. Local PD-L1 blockade caused a similar increase in viral clearance, suggesting a local effect of PD-1/PD-L1 in the cornea. The enhanced HSV-1 clearance at 2 dpi resulting from PD-1/PD-L1 blockade is mediated primarily by a monocyte/macrophage population. Studies in bone marrow chimeras demonstrated enhanced viral clearance when PD-L1 was absent only from nonhematopoietic cells. We conclude that PD-L1 expression on corneal cells negatively impacts the ability of the innate immune system to clear HSV-1 from infected corneas.

DNA Conformational Changes Play a Force-Generating Role during Bacteriophage Genome Packaging.

Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens as well as dsDNA bacteriophages-viruses that infect bacteria. We previously proposed that DNA is not a passive substrate of bacteriophage packaging motors but is instead an active component of the machinery. We carried out computational studies on dsDNA in the channels of viral portal proteins, and they reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer ("stretched") in regions of high negative electrostatic potential and shorter ("scrunched") in regions of high positive potential. These results suggest a mechanism that electrostatically couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis, and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA as the phosphate groups respond to the protein's electrostatic potential. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal, and dsDNA work synergistically to promote genome packaging.

Enhancing Next-Generation Sequencing-Guided Cancer Care Through Cognitive Computing.

BACKGROUND: Using next-generation sequencing (NGS) to guide cancer therapy has created challenges in analyzing and reporting large volumes of genomic data to patients and caregivers. Specifically, providing current, accurate information on newly approved therapies and open clinical trials requires considerable manual curation performed mainly by human "molecular tumor boards" (MTBs). The purpose of this study was to determine the utility of cognitive computing as performed by Watson for Genomics (WfG) compared with a human MTB. MATERIALS AND METHODS: One thousand eighteen patient cases that previously underwent targeted exon sequencing at the University of North Carolina (UNC) and subsequent analysis by the UNCseq informatics pipeline and the UNC MTB between November 7, 2011, and May 12, 2015, were analyzed with WfG, a cognitive computing technology for genomic analysis. RESULTS: Using a WfG-curated actionable gene list, we identified additional genomic events of potential significance (not discovered by traditional MTB curation) in 323 (32%) patients. The majority of these additional genomic events were considered actionable based upon their ability to qualify patients for biomarker-selected clinical trials. Indeed, the opening of a relevant clinical trial within 1 month prior to WfG analysis provided the rationale for identification of a new actionable event in nearly a quarter of the 323 patients. This automated analysis took <3 minutes per case. CONCLUSION: These results demonstrate that the interpretation and actionability of somatic NGS results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing could potentially improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of up-to-date availability of clinical trials. IMPLICATIONS FOR PRACTICE: The results of this study demonstrate that the interpretation and actionability of somatic next-generation sequencing results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing can significantly improve patient care by providing a fast, cost-effective, and comprehensive approach for data analysis in the delivery of precision medicine. Patients and physicians who are considering enrollment in clinical trials may benefit from the support of such tools applied to genomic data.

History of the ribosome and the origin of translation.

We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.

Associations Between Bicycling and Reduced Fall-Related Physical Performance in Older Adults.

Falls among older adults remain a significant public health issue. Bicycling positively influences falls risk factors including reduced balance, muscle weakness, and low self-perceived confidence in maintaining balance. However, this association has not been systematically examined. We recruited 107 community-dwelling participants aged 65 years and older in the Netherlands to determine the relationship between bicycling and falls risk factors. Participants completed three questionnaires on cycling behavior and balance confidence, and also undertook five falls-related physical performance tasks encompassing tests of balance, strength, gait, and endurance. On average, current bicyclists showed significantly better scores in all physical tasks and confidence compared with nonriders ranging from a 10% difference in 6-m walk time to a 141% difference in single-leg balance time (all ps = .01). Type of bike used and duration of bicycling displayed varied associations (.01 < ps < .79). Our findings suggest that bicycle riding warrants further prospective investigation for fall prevention and active aging.

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy.

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.

Evolution of the ribosome at atomic resolution.

The origins and evolution of the ribosome, 3-4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be "observed" by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call "insertion fingerprints." Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

A review of the use of a systematic observation method in coaching research between 1997 and 2016.

A systematic observation method has been one of the most popularly employed methods in coaching research. Kahan's review of this method conducted between 1975 and 1997 highlighted the key trends in this research, and offered methodological guidance for researchers wishing to use this method in their research. The purpose of this review was to provide an update of the use of a systematic observation method in coaching research and assess the extent to which the calls made by Kahan have been addressed. While in some respect this field of study has progressed (i.e., the introduction of qualitative methods), researchers adopting this method have failed to attend to many of the issues Kahan raised. For this method to continue to make a positive contribution towards the coaching research literature, researchers need to more critically reflect on how and why they are employing this method. At present, some of the decisions made by researchers who have conducted work in this area are not justified with a rationale. It is our intention that this review will serve as guidance for researchers and practitioners, and editors and reviewers of journals when attempting to assess the quality of this type of work.

Exposing athletes to playing form activity: outcomes of a randomised control trial among community netball teams using a game-centred approach.

This study evaluated whether exposing junior netball players to greater amounts of competition relevant activity (playing form activity) had an effect on game play outcomes and session involvement. A group-randomised controlled trial in one junior netball club in the Hunter Region, NSW, Australia. Ninety female athletes (mean age = 9.04 years, SD 1.53) were randomised by team (n = 11) into the intervention (n = 41) or 9-week wait-list control (n = 49) condition. The Professional Learning for Understanding Games Education into Sport (PLUNGE into Sport) programme was undertaken in the first half of nine training sessions (9 × 30 min). The intervention exposed athletes to playing form activity through a coach development programme within training sessions. Athletes' decision-making, support and skill outcomes during a small-sided invasion game, and session involvement (pedometer step/min), were measured at baseline and 9-week follow-up. Linear mixed models revealed significant group-by-time intervention effects (P < 0.05) for decision-making (d = 0.4) and support (d = 0.5) during game play, and in-session activity (d = 1.2). An intervention exposing athletes to greater levels of playing form activity, delivered via a coach education programme, was efficacious in improving athlete decision-making and support skills in game play and increasing athlete involvement during sessions.

Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Augmenter of liver regeneration (ALR, encoded by GFER) is a widely distributed pleiotropic protein originally identified as a hepatic growth factor. However, little is known about its roles in hepatic physiology and pathology. We created mice with liver-specific deletion of ALR to study its function. METHODS: We developed mice with liver-specific deletion of ALR (ALR-L-KO) using the albumin-Cre/LoxP system. Liver tissues were collected from ALR-L-KO mice and ALR(floxed/floxed) mice (controls) and analyzed by histology, reverse-transcription polymerase chain reaction, immunohistochemistry, electron microscopy, and techniques to measure fibrosis and lipids. Liver tissues from patients with and without advanced liver disease were determined by immunoblot analysis. RESULTS: Two weeks after birth, livers of ALR-L-KO mice contained low levels of ALR and adenosine triphosphate (ATP); they had reduced mitochondrial respiratory function and increased oxidative stress, compared with livers from control mice, and had excessive steatosis, and hepatocyte apoptosis. Levels of carbamyl-palmitoyl transferase 1a and ATP synthase subunit ATP5G1 were reduced in livers of ALR-L-KO mice, indicating defects in mitochondrial fatty acid transport and ATP synthesis. Electron microscopy showed mitochondrial swelling with abnormalities in shapes and numbers of cristae. From weeks 2-4 after birth, levels of steatosis and apoptosis decreased in ALR-L-KO mice, and numbers of ALR-expressing cells increased, along with ATP levels. However, at weeks 4-8 after birth, livers became inflamed, with hepatocellular necrosis, ductular proliferation, and fibrosis; hepatocellular carcinoma developed by 1 year after birth in nearly 60% of the mice. Hepatic levels of ALR were also low in ob/ob mice and alcohol-fed mice with liver steatosis, compared with controls. Levels of ALR were lower in liver tissues from patients with advanced alcoholic liver disease and nonalcoholic steatohepatitis than in control liver tissues. CONCLUSIONS: We developed mice with liver-specific deletion of ALR, and showed that it is required for mitochondrial function and lipid homeostasis in the liver. ALR-L-KO mice provide a useful model for investigating the pathogenesis of steatohepatitis and its complications.

The scrunchworm hypothesis: transitions between A-DNA and B-DNA provide the driving force for genome packaging in double-stranded DNA bacteriophages.

Double-stranded DNA bacteriophages have motors that drive the genome into preformed capsids, using the energy released by hydrolysis of ATP to overcome the forces opposing DNA packaging. Viral packaging motors are the strongest of all biological motors, but it is not known how they generate these forces. Several models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active force-generating core of the motor, not simply a substrate on which the motor operates. The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening-lengthening motions of the B→A→B cycle ("scrunching"), so that DNA is pulled into the motor and pushed forward into the capsid. This DNA-centric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions.

Reversible nerve damage and corneal pathology in murine herpes simplex stromal keratitis.

Herpes simplex virus type 1 (HSV-1) shedding from sensory neurons can trigger recurrent bouts of herpes stromal keratitis (HSK), an inflammatory response that leads to progressive corneal scarring and blindness. A mouse model of HSK is often used to delineate immunopathogenic mechanisms and bears many of the characteristics of human disease, but it tends to be more chronic and severe than human HSK. Loss of blink reflex (BR) in human HSK is common and due to a dramatic retraction of corneal sensory nerve termini in the epithelium and the nerve plexus at the epithelial/stromal interface. However, the relationship between loss of BR due to nerve damage and corneal pathology associated with HSK remains largely unexplored. Here, we show a similar retraction of corneal nerves in mice with HSK. Indeed, we show that much of the HSK-associated corneal inflammation in mice is actually attributable to damage to the corneal nerves and accompanying loss of BR and can be prevented or ameliorated by tarsorrhaphy (suturing eyelids closed), a clinical procedure commonly used to prevent corneal exposure and desiccation. In addition, we show that HSK-associated nerve retraction, loss of BR, and severe pathology all are reversible and regulated by CD4(+) T cells. Thus, defining immunopathogenic mechanisms of HSK in the mouse model will necessitate distinguishing mechanisms associated with the immunopathologic response to the virus from those associated with loss of corneal sensation. Based on our findings, investigation of a possible contribution of nerve damage and BR loss to human HSK also appears warranted. Importance: HSK in humans is a potentially blinding disease characterized by recurrent inflammation and progressive scarring triggered by viral release from corneal nerves. Corneal nerve damage is a known component of HSK, but the causes and consequences of HSK-associated nerve damage remain obscure. We show that desiccation of the corneal surface due to nerve damage and associated loss of BR severely exacerbates and prolongs inflammation-induced pathology in mice. Preventing corneal desiccation results in a milder and more transient HSK with variable scarring that mirrors HSK seen in most humans. We further show that nerve damage is reversible and regulated by CD4(+) T cells. Thus, we provide a mouse model that more closely resembles typical human HSK and suggest nerve damage is an important but largely overlooked factor in human disease.

Secondary structure and domain architecture of the 23S and 5S rRNAs.

We present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23S and 5S rRNAs, using 3D structures, determined by X-ray diffraction, as input. In the traditional 2° structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure. Unlike the traditional 2° structure, the revised 2° structure of the 23S rRNA shows architectural similarity with the 16S rRNA. The revised 2° structure also reveals a clear relationship with the 3D structure and is generalizable to rRNAs of other species from all three domains of life. The 2° structure revision required us to reconsider the domain architecture. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities and compactness. The best domain model for the 23S rRNA contains seven domains, not six as previously ascribed. Domain 0 forms the core of the 23S rRNA, to which the other six domains are rooted. Editable 2° structures mapped with various data are provided (http://apollo.chemistry.gatech.edu/RibosomeGallery).

Molecular paleontology: a biochemical model of the ancestral ribosome.

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

Comment on the letter by A. Ben-Shaul: "entropy, energy, and bending of DNA in viral capsids".

The conformational entropic penalty associated with packaging double-stranded DNA into viral capsids remains an issue of contention. So far, models based on a continuum approximation for DNA have either left the question unexamined, or they have assumed that the entropic penalty is negligible, following an early analysis by Riemer and Bloomfield. In contrast, molecular-dynamics (MD) simulations using bead-and-spring models consistently show a large penalty. A recent letter from Ben-Shaul attempts to reconcile the differences. While the letter makes some valid points, the issue of how to include conformational entropy in the continuum models remains unresolved. In this Comment, I show that the free energy decomposition from continuum models could be brought into line with the decomposition from the MD simulations with two adjustments. First, the entropy from Flory-Huggins theory should be replaced by the estimate of the entropic penalty given in Ben-Shaul's letter, which corresponds closely to that from the MD simulations. Second, the DNA-DNA repulsions are well described by the empirical relationship given by the Cal Tech group, but the strength of these should be reduced by about half, using parameters based on the Rau-Parsegian experiments, rather than treating them as "fitting parameters (tuned) to fit the data from (single molecule pulling) experiments."