Although the role of Hedgehog (Hh) signalling in embryonic pattern formation is well established 1 , its functions in adult tissue renewal and maintenance remain unclear, and the relationship of these functions to cancer development has not been determined. Here we show that the lossof Smoothened (Smo), an essential component of the Hh pathway 2 , impairs haematopoietic stem cell renewal and decreases induction of chronic myelogenous leukaemia (CML) by the BCR-ABL1 oncoprotein 3 . Loss of Smo causes depletion of CML stem cells-the cells that propagate the leukaemia-whereas constitutively active Smo augments CML stem cell number and accelerates disease. As a possible mechanism for Smo action, we show that the cell fate determinant Numb, which depletes CML stem cells, is increased in the absence of Smo activity. Furthermore, pharmacological inhibition of Hh signalling impairs not only the propagation of CML driven by wildtype BCR-ABL1, but also the growth of imatinib-resistant mouse and human CML. These data indicate that Hh pathway activity is required for maintenance of normal and neoplastic stem cells of the haematopoietic system and raise the possibility that the drug resistance and disease recurrence associated with imatinib treatment of CML 4,5 might be avoided by targeting this essential stem cell maintenance pathway.

A key characteristic of stem cells and cancer cells is their ability to self-renew. To test if Wnt signaling can regulate the self-renewal of both stem cells and cancer cells in the hematopoietic system, we developed mice that lack beta-catenin in their hematopoietic cells. Here we show that beta-catenin-deficient mice can form HSCs, but that these cells are deficient in long-term growth and maintenance. Moreover, beta-catenin deletion causes a profound reduction in the ability of mice to develop BCR-ABL-induced chronic myelogenous leukemia (CML), while allowing progression of acute lymphocytic leukemia (ALL). These studies demonstrate that Wnt signaling is required for the self-renewal of normal and neoplastic stem cells in the hematopoietic system.

We report the de novo folding of three hyperstable RNA tetraloops to 1-3 Å rmsd from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. RNA tetraloops with loop sequences UUCG, GCAA, or CUUG are hyperstable because of the formation of noncanonical loop-stabilizing interactions, and they are all faithfully reproduced to angstrom-level accuracy in replica exchange molecular dynamics simulations, including explicit solvent and ion molecules. This accuracy is accomplished using unique RNA parameters, in which biases that favor rigid, highly stacked conformations are corrected to accurately capture the inherent flexibility of ssRNA loops, accurate base stacking energetics, and purine syn-anti interconversions. In a departure from traditional quantum chemistrycentric approaches to force field optimization, our parameters are calibrated directly from thermodynamic and kinetic measurements of intra-and internucleotide structural transitions. The ability to recapitulate the signature noncanonical interactions of the three most abundant hyperstable stem loop motifs represents a significant milestone to the accurate prediction of RNA tertiary structure using unbiased all-atom molecular dynamics simulations.RNA folding | molecular simulations S tructured RNAs exhibit a distinct preference for loops of precisely 4 nt, which was originally noted by Woese et al. (1) using comparative sequence analysis of ribosomes. Approximately 70% of these tetraloops are comprised of just three specific loop sequences: UUCG, GCAA, or CUUG. The abundance of these sequences is thermodynamic in origin, because each motif forms a unique network of noncanonical interactions within their loops that stabilizes the folded state. The abundance of high-resolution structural and thermodynamic data available for these motifs coupled with their characteristic noncanonical signatures make them ideal for adjudicating the accuracy of RNA folding simulations.RNA folding is understood to be hierarchical in nature, with secondary and tertiary folds stabilized by distinct thermodynamic driving forces (2). Secondary structure (the formation of canonical helices stabilized by Watson-Crick base pairs) can be accurately predicted from the nucleotide sequence alone using simple nearest neighbor thermodynamic models (3). In contrast, tertiary structure formation is a subtle competition between intrinsic flexibility of single-stranded segments, rigidity imparted from base-stacking interactions, stabilization of noncanonical hydrogen bonding patterns, and site-specific ion binding. In principle, a molecular dynamics simulation using a properly calibrated force field should capture all of the physicochemical properties of ribonucleotides relevant to the RNA folding process. Up until now, however, even small, fast-folding tetraloops cannot be accurately and reversibly folded from the unfolded state (4-6). In contrast, numerous documented successes have been reported using de novo protein folding with all-ato...

No abstract

Aqueous solutions of 1:1 strong electrolytes are considered to be the prototype for complete ionic dissociation. Nonetheless, clustering of strong 1:1 electrolytes has been widely reported in all atom molecular dynamics simulations, and their presence is indirectly implicated in a diverse range of experimental results. Is there a physical basis for nonidealities such as ion pairing and cluster formation in aqueous solutions of strong 1:1 electrolytes? We attempt to answer this question by direct comparison of results from detailed molecular dynamics simulations to experimentally observed properties of 1:1 electrolytes. We report the analysis of a series of lengthy molecular dynamics simulations of alkali-halide solutions carried out over a wide range of physiologically relevant concentrations using explicit representations of water molecules. We find evidence for pronounced nonideal behavior of ions at all concentrations in the form of ion pairs and clusters which are in rapid equilibrium with dissociated ions. The phenomenology for ion pairing seen in these simulations is congruent with the multistep scheme proposed by Eigen and Tamm based on data from ultrasonic absorption experiments. For a given electrolyte, we show that the dependence of cluster populations on concentration can be described through a single set of equilibrium constants. We assess the accuracy of calculated ion pairing constants by favorable comparison to estimates obtained by Fuoss and co-workers and based on conductometric experiments. Ion pairs and clusters form on length scales where the size of individual water molecules is as important as the hard core radius of ions. Ion pairing results as a balance between the favorable Coulomb interactions and the unfavorable partial desolvation of ions needed to form a pair.

The monovalent ion parameters used by the AMBER-99 forcefield are shown to exhibit physically inaccurate behavior in molecular dynamics simulations of strong 1:1 electrolytes. These errors arise from an ad hoc adaptation of Aqvist's cation parameters. The result is the rapid formation of large, unphysical clusters at concentrations that are well below solubility limits. The observed unphysical behavior poses a serious challenge for simulating ions around highly charged polymers such as nucleic acids. In this communication, we explain the source of this unphysical behavior. To facilitate the continued use of the popular AMBER parameters, we prescribe a simple fix whereby Aqvist's cations and anions are used in conjunction with the AMBER forcefield for nucleic acids. A preliminary test of this strategy suggests that the proposed fix is reasonable and is likely to be generalizable for simulating diffuse and specific ion binding to nucleic acids.

Objective To characterize the hospitalization and death rates among patients with inflammatory arthritis (IA) affected by coronavirus disease 2019 (COVID‐19) and to analyze the associations of comorbidities and immunomodulatory medications with infection outcomes. Methods Data on clinical and demographic features, maintenance treatment, disease course, and outcomes in individuals with IA (rheumatoid arthritis and spondyloarthritis) with symptomatic COVID‐19 infection were prospectively assessed via web‐based questionnaire followed by individual phone calls and electronic medical record review. Baseline characteristics and medication use were summarized for hospitalized and ambulatory patients, and outcomes with the different medication classes were compared using multivariable logistic regression. Results A total of 103 patients with IA were included in the study (80 with confirmed COVID‐19 and 23 with high suspicion of COVID‐19). Hospitalization was required in 26% of the participants, and 4% died. Patients who were hospitalized were significantly more likely to be older (P < 0.001) and have comorbid hypertension (P = 0.001) and chronic obstructive pulmonary disease (P = 0.02). IA patients taking oral glucocorticoids had an increased likelihood of being admitted for COVID‐19 (P < 0.001), while those receiving maintenance anticytokine biologic therapies did not. Conclusion Among patients with underlying IA, COVID‐19 outcomes were worse in those receiving glucocorticoids but not in patients receiving maintenance anticytokine therapy. Further work is needed to understand whether immunomodulatory therapies affect COVID‐19 incidence.

The worldwide pandemic of COVID-19, caused by the virus SARS-CoV,-2 has continued to progress, and increasing information is becoming available about the incidence of digestive symptoms as well as abnormal liver-associated enzymes in patients who are infected. These are postulated to be related to the virus's use of ACE-2 receptors located on certain intestinal cells, cholangiocytes, and hepatocytes. This brief review summarizes the available limited data on digestive manifestations of COVID-19. A significant proportion of COVID-19 patients can present initially with only digestive complaints. The most common digestive symptoms are anorexia, nausea, vomiting, and diarrhea. Liver-related transaminases are elevated in a substantial proportion of patients, although generally only mildly elevated. Currently there is no firm evidence to suggest that severity of digestive symptoms corresponds to severity of COVID-19 clinical course, however, more severe alterations in liver enzymes may correlate with worse clinical course. Given use of antiviral and antibacterial agents in sicker patients, drug-induced liver injury cannot be ruled out either in these cases. Although viral RNA can be detected in stool, it is unclear whether fecal-oral transmission can be achieved by the virus. As further data becomes available, our understanding of the digestive manifestations of COVID-19 will continue to evolve.