Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messenger RNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

Molecular and structural basis of olfactory sensory neuron axon coalescence by Kirrel receptors.

Projections from sensory neurons of olfactory systems coalesce into glomeruli in the brain. The Kirrel receptors are believed to homodimerize via their ectodomains and help separate sensory neuron axons into Kirrel2- or Kirrel3-expressing glomeruli. Here, we present the crystal structures of homodimeric Kirrel receptors and show that the closely related Kirrel2 and Kirrel3 have evolved specific sets of polar and hydrophobic interactions, respectively, disallowing heterodimerization while preserving homodimerization, likely resulting in proper segregation and coalescence of Kirrel-expressing axons into glomeruli. We show that the dimerization interface at the N-terminal immunoglobulin (IG) domains is necessary and sufficient to create homodimers and fail to find evidence for a secondary interaction site in Kirrel ectodomains. Furthermore, we show that abolishing dimerization of Kirrel3 in vivo leads to improper formation of glomeruli in the mouse accessory olfactory bulb as observed in Kirrel3-/- animals. Our results provide evidence for Kirrel3 homodimerization controlling axonal coalescence.

The SARS-CoV-2 Programmed -1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography.

The programmed -1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses' genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1-10 and NSPs 1-16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the -1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the -1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.

Perceptions of Diabetes Technology Use in Cystic Fibrosis-Related Diabetes Management.

Background: Diabetes technologies are associated with improvements in glycemic control and health-related quality of life among people with type 1 diabetes (T1D). Use and perceptions of continuous glucose monitors (CGM) and insulin pumps within the cystic fibrosis (CF) community have not been well studied. Methods: A 30-item online survey addressing cystic fibrosis-related diabetes (CFRD) diagnosis, CGM and insulin pump use, and perceptions of diabetes technologies was sent to a CF community group, including people with CF (pwCF) and parents of children with CF (cwCF). Results: The response rate was 11% (n = 120; 83 pwCF, 35 cwCF). Sixty-one percent of pwCF and 34% of cwCF reported a diagnosis of CFRD. CGM use was reported by 75% (n = 47) of respondents with CFRD but was discontinued by 19% (n = 9), most commonly due to cost and increased worry about glycemia. Insulin pump therapy was reported by 29% (n = 18 of 62) of respondents with CFRD and was discontinued by 28% (n = 5), most commonly due to pain or skin irritation. Overall, 91% agreed or strongly agreed that CGM facilitated CFRD management. Eighty-one percent agreed with at least five of seven positive statements about CGM as compared with 22% for insulin pumps. Potential embarrassment over device wear, concerns about cost, and pain were commonly held negative perceptions of both technologies. Conclusions: As compared with T1D and despite perceived benefits, rates of sustained diabetes technology use are low in the CFRD community. Better insurance coverage to mitigate cost, better patient education, and confirmation that these technologies improve health and patient-reported outcomes may increase uptake.

Designing the GALAXY study: Partnering with the cystic fibrosis community to optimize assessment of gastrointestinal symptoms.

BACKGROUND: Gastrointestinal (GI) involvement among persons with cystic fibrosis (CF) is highly prevalent, representing a significant source of morbidity. Persons with CF have identified GI concerns as a top research priority, yet universal clinical outcome measures capturing many of the GI symptoms experienced in CF are lacking. The GALAXY study was envisioned to address this unmet need. METHODS: The GALAXY study team partnered with Community Voice, a community of patients with CF and their caregivers, to identify the patient reported outcome measures that most accurately reflected their experience with GI symptoms in CF. We also surveyed CF care teams to identify the comfort level of various team members (providers, nurses and dieticians) in managing a variety of GI conditions. RESULTS: Members of Community Voice identified the combination of PAC-SYM, PAGI-SYM, PAC-QOL and the Bristol Stool scale with three additional symptom-specific questions as patient-reported outcome measures that comprehensively captured the CF experience with GI disease. CF care team providers reported a high level of comfort in treating GI conditions including constipation (92%), GERD (93%), and gassiness (77%), however comfort level was limited to only first-line interventions. CONCLUSION: By partnering with persons with CF as well as their caregivers and medical providers, the GALAXY study is designed to uniquely capture the prevalence and severity of GI involvement among persons with CF in a manner that reflects the CF patient experience. The results of GALAXY will inform the development of future interventional trials and serve as a reproducible and objective study endpoint.

Hemoptysis from the perspective of people with cystic fibrosis.

INTRODUCTION: People with cystic fibrosis (CF) are living longer, thus complications associated with age, such as hemoptysis, are increasing. The Institute of Medicine has emphasized the importance of patient-centeredness. Although guidelines about hemoptysis in people with CF are available, these focus on management of the complication and not the patient perspective. OBJECTIVE: We sought to understand hemoptysis from the point of view of those who have experienced it. METHODS: We fielded an 11-question survey to adults with CF and asked those who had hemoptysis to respond. Four questions had open-ended options: (1) the person's first experience with hemoptysis, (2) how that experience affected the way they approach their CF, (3) how they deal with hemoptysis when it occurs outside the home and (4) a free text box for general comments. RESULTS: Thirty-one of 132 adults with CF who were sent a survey completed it (23% response rate); 63% F), indicated that they had experienced hemoptysis and described their triggers. In response to open questioning, 77% of respondents found their first experience with hemoptysis to be 'scary,' 'frightening,' 'worrying' or 'jarring.' Half of respondents reported quality of life being negatively affected by worsening stress or anxiety, fear of bleeding in public or other life impacts. CONCLUSIONS: Focusing on how to cope with future episodes of hemoptysis and the associated anxiety can be helpful to patients. Proactive communication and sensitivity to patient experience may deepen physician-patient rapport, increase self-efficacy to cope with future episodes and lead to more comprehensive care of hemoptysis.

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative.

Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA-binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-family replicative helicases, belongs to the pre-sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.

Alternate deacylating specificities of the archaeal sirtuins Sir2Af1 and Sir2Af2.

Sirtuins were originally shown to regulate a wide array of biological processes such as transcription, genomic stability, and metabolism by catalyzing the NAD(+) -dependent deacetylation of lysine residues. Recent proteomic studies have revealed a much wider array of lysine acyl modifications in vivo than was previously known, which has prompted a reevaluation of sirtuin substrate specificity. Several sirtuins have now been shown to preferentially remove propionyl, succinyl, and long-chain fatty acyl groups from lysines, which has changed our understanding of sirtuin biology. In light of these developments, we revisited the acyl specificity of several well-studied archaeal and bacterial sirtuins. We find that the Archaeoglobus fulgidus sirtuins, Sir2Af1 and Sir2Af2, preferentially remove succinyl and myristoyl groups, respectively. Crystal structures of Sir2Af1 bound to a succinylated peptide and Sir2Af2 bound to a myristoylated peptide show how the active site of each enzyme accommodates a noncanonical acyl chain. As compared to its structure in complex with an acetylated peptide, Sir2Af2 undergoes a conformational change that expands the active site to accommodate the myristoyl group. These findings point to both structural and biochemical plasticity in sirtuin active sites and provide further evidence that sirtuins from all three domains of life catalyze noncanonical deacylation.

A ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation.

Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.

The challenge of funding hospital employee retirement benefits.

Hospitals face a difficult challenge in meeting existing benefits obligations to employees while maintaining financial reserves to invest in electronic health records, quality improvement, and more effective integration of care. Although they may no longer be able to afford offering employees defined-benefit plans, many forward-looking healthcare organizations are finding ways to keep their commitments without sacrificing the balance sheet. One such organization is Scripps Health in San Diego, whose innovative benefits packages have contributed to its being ranked 56th in Fortune's "100 Best Companies to Work For" list in 2012.