The Interfaces of Genetic Conflict Are Hot Spots for Innovation.

RNA-guided Cas9 endonucleases protect bacteria from viral infection and have been creatively repurposed as programmable molecular scalpels for surgical manipulation of DNA. Now, two papers in Cell (Pawluk et al. and Rauch et al.) identify viral proteins that suppress Cas9 and may function like molecular sheaths for the Cas9 scalpel.

Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex.

Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.

SnapShot: CRISPR-RNA-guided adaptive immune systems.

Bacteria and archaea have evolved sophisticated adaptive immune systems that reply on CRISPR loci and a diverse cassette of Cas genes that are classified into three main types and at least eleven subtypes. All CRISPR-Cas immune systems operate through three main stages: acquisition, biogenesis, and interference. This SnapShot summarizes our current knowledge of these fascinating immune systems.

Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.

Vaccine design via antigen reorientation.

A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an 'upside-down' configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines.

Incidence of Anterior Knee Pain and Functional Difficulty in a Prospectively Recruited Cohort Following Total Knee Arthroplasty With Selective Patellar Resurfacing.

BACKGROUND: The influence of patellar resurfacing on anterior knee pain, stair climbing, and functional activity outcomes following total knee arthroplasty (TKA) are incompletely understood. This study examined the influence of patellar resurfacing on patient-reported outcome measures (PROMs) relating to anterior knee pain and function. METHODS: The Knee Injury and Osteoarthritis Outcome Score of Joint Replacement (KOOS, JR.) patient PROMs were collected preoperatively and at 12 months follow-up for 950 TKAs performed over 5 years. Indications for patellar resurfacing included Grade IV patello-femoral (PFJ) changes or mechanical PFJ findings during patellar trialing. Patellar resurfacing was performed on 393 (41%) of the 950 TKAs performed. Multivariable binomial logistic regressions were performed, using KOOS, JR. questions assessing pain during stair climbing, standing upright, and function during rising from sitting as surrogates for anterior knee pain. Independent regression models were performed for each of the targeted KOOS, JR. questions, with adjustment for age at surgery, sex, and baseline pain and function. RESULTS: No association was observed between 12-month postoperative anterior knee pain or function with patellar resurfacing (P = .17, .97). Patients who had moderate or greater preoperative pain on stairs had an increased likelihood of postoperative pain and functional difficulty (odds ratio 2.3, P = .013), while males were 42% less likely to report postoperative anterior knee pain (odds ratio 0.58, P = .002). CONCLUSION: Selective patellar resurfacing based on PFJ degeneration and mechanical PFJ symptoms produces similar improvement in PROMs for resurfaced and unresurfaced knees.

What Role Does Patello-Femoral Joint Degeneration Have on Pain and Function After Unicompartmental Knee Arthroplasty? A Prospective Observational Cohort Study.

BACKGROUND: Influences on anterior knee pain, stair climbing limitations, and function such as rising from sitting are poorly understood in unicompartmental knee arthroplasty (UKA). Original indications for UKA excluded patients who had patello-femoral disease, while more recent studies have expanded the indications to include these patients. This study examined the influence of the patello-femoral joint degeneration on patient-reported outcome measures relating to anterior knee pain and function after UKA. METHODS: Between October 2017 and August 2021, Knee Injury and Osteoarthritis Outcome Score of Joint Replacement (KOOS, JR) patient-reported outcome measures were collected preoperatively and at 12 months of follow-up for 678 medial UKAs. Patello-femoral joint status was visually graded intraoperatively. Radiographic or intraoperative medial patellar facet and trochlear patello-femoral arthritis and preoperative anterior knee pain were not considered contraindications for UKA, while grade IV lateral patello-femoral arthritis was considered a contraindication for UKA. Multivariable ordinal logistic regressions were performed, using the KOOS, JR questionnaire assessing pain during stair climbing, standing upright, and function during rising from sitting. Independent regressions were performed for each targeted KOOS, JR question, with adjustments for age at surgery, sex, and baseline pain and function scores. RESULTS: No association was observed between 12-month postoperative anterior knee pain (P = .575) and function (P = .854) with patellar osteoarthritis grading after UKA. When comparing fixed and mobile-bearing UKA designs, no association was observed between bearing type and pain (P = .663) or functional outcomes (P = .758). CONCLUSION: Pain and function improved significantly following medial UKA and was independent of medial patellar and trochlear degenerative status.

Phylogenetic review of the comb-tooth blenny genus Hypleurochilus in the northwest Atlantic and Gulf of Mexico.

As some of the smallest vertebrates, yet largest producers of consumed reef biomass, cryptobenthic reef fishes serve a disproportionate role in reef ecosystems and are one of the most poorly understood groups of fish. The blenny genera Hypleurochilus and Parablennius are currently considered paraphyletic and the interrelationships of Parablennius have been the focus of recent phylogenetic studies. However, the interrelationships of Hypleurochilus remain understudied. This genus is transatlantically distributed and comprises 11 species with a convoluted taxonomic history. In this study, relationships for ten Hypleurochilus species are resolved using multi-locus nuclear and mtDNA sequence data, morphological data, and mined COI barcode data.  Mitochondrial and nuclear sequence data from 61 individuals collected from the western Atlantic and northern Gulf of Mexico (N. GoM) delimit seven species into a temperate clade, a tropical clade, and a third distinct lineage. This lineage, herein referred to as H. cf. aequipinnis, may represent a species of Hypleurochilus whose range has expanded into the N. GoM. Inclusion of publicly available COI sequence for an additional three species provides further phylogenetic resolution. H. bananensis forms a new eastern Atlantic clade with H. cf. aequipinnis, providing further evidence for a western Atlantic range expansion. Single marker COI delimitation was unable to elucidate the relationships between H. springeri/H. pseudoaequipinnis and between H. multifilis/H. caudovittatus due to incomplete lineage sorting. Mitochondrial data are also unable to accurately resolve the placement of H. bermudensis. However, a comprehensive approach using multi-locus phylogenetic and species delimitation methods was able to resolve these relationships. While mining publicly available sequence data allowed for the inclusion of an increased number of species in the analysis and a more comprehensive phylogeny, it was not without drawbacks, as a handful of sequences are potentially mis-identified. Overall, we find that the recent divergence of some species within this genus and potential introgression events confound the results of single locus delimitation methods, yet a combination of single and multi-locus analyses has allowed for insights into the biogeography of this genus and uncovered a potential transatlantic range expansion.

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity.

The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/32:Cas14) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1-2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.

Transiting circumbinary planets Kepler-34 b and Kepler-35 b.

Most Sun-like stars in the Galaxy reside in gravitationally bound pairs of stars (binaries). Although long anticipated, the existence of a 'circumbinary planet' orbiting such a pair of normal stars was not definitively established until the discovery of the planet transiting (that is, passing in front of) Kepler-16. Questions remained, however, about the prevalence of circumbinary planets and their range of orbital and physical properties. Here we report two additional transiting circumbinary planets: Kepler-34 (AB)b and Kepler-35 (AB)b, referred to here as Kepler-34 b and Kepler-35 b, respectively. Each is a low-density gas-giant planet on an orbit closely aligned with that of its parent stars. Kepler-34 b orbits two Sun-like stars every 289 days, whereas Kepler-35 b orbits a pair of smaller stars (89% and 81% of the Sun's mass) every 131 days. The planets experience large multi-periodic variations in incident stellar radiation arising from the orbital motion of the stars. The observed rate of circumbinary planets in our sample implies that more than ∼1% of close binary stars have giant planets in nearly coplanar orbits, yielding a Galactic population of at least several million.

Comparisons of Alcohol and Drug Dependence in Terms of Attachments and Clinical Issues.

BACKGROUND: Alcohol and drug dependencies are associated with different social stigmas, and some studies suggest they might exhibit different clinical presentations. Further, the treatments for each vary considerably. Alcohol versus drug use problems were compared in terms of attachment patterns and related clinical treatment issues in two interlocking studies using converging logic and designs. METHODS: In Study 1, alcohol versus drug dependence was defined in terms of a known groups design. Patients from an inpatient alcohol treatment center, patients receiving treatment for opiate dependence in a methadone maintenance clinic, and controls were compared on the 29 scales of the Attachment and Clinical Issues Questionnaire (ACIQ). Study 2 sampled a substantially different population (491 university students) and used different operational definitions of substance use problems, relying on screening instruments. RESULTS: Study 1 found that, although the drug and alcohol dependent patients differed substantially from the controls, they did not differ from one another on any of the 29 ACIQ scales measuring attachments and clinical issues. Study 2 converged on the known groups design of Study 1, showing convergent and concurrent rather than discriminate evidence for the alcohol and drug dependence screening instruments. CONCLUSION: Alcohol and drug dependencies were not found to differ significantly in terms of attachments or clinical issues. These studies aimed to provide clearer and more empirically grounded guidance to the clinician and researcher.

Outpatient-Focused Joint Arthroplasty Is the Future: The Midwest Center for Joint Replacement Experience.

BACKGROUND: Perhaps, the most significant developments in joint arthroplasty in the past decade have been in the area of multimodal perioperative management reducing pain, nausea, and length of stay leading to outpatient arthroplasty. METHODS: Over a 2-year period, we performed 1230 arthroplasty cases including partial knee, total hip, total knee, and selected revision cases. RESULTS: Patient satisfaction ranged from 98% to 100% great/good. Ninety-eight percent of patients were discharged the same day. There were no readmissions for pain control and an overall readmission rate of 2%. CONCLUSION: The outpatient program centers on the patient needs, family engagement, essentials of home recovery, preoperative education, efficient surgery, and a surgeon-controlled environment with a highly standardized care. This is a distinct shift in today's health-care environment, which has seen the expansion of regulatory demands; focus on Electronic Health Record, and distractions from real discussions of demonstrated value creation. The future is bright for both ambulatory surgery center and hospital development of successful outpatient joint arthroplasty program for patients and surgeons alike.

A closely packed system of low-mass, low-density planets transiting Kepler-11.

When an extrasolar planet passes in front of (transits) its star, its radius can be measured from the decrease in starlight and its orbital period from the time between transits. Multiple planets transiting the same star reveal much more: period ratios determine stability and dynamics, mutual gravitational interactions reflect planet masses and orbital shapes, and the fraction of transiting planets observed as multiples has implications for the planarity of planetary systems. But few stars have more than one known transiting planet, and none has more than three. Here we report Kepler spacecraft observations of a single Sun-like star, which we call Kepler-11, that reveal six transiting planets, five with orbital periods between 10 and 47 days and a sixth planet with a longer period. The five inner planets are among the smallest for which mass and size have both been measured, and these measurements imply substantial envelopes of light gases. The degree of coplanarity and proximity of the planetary orbits imply energy dissipation near the end of planet formation.

Cognitive dysfunction in patients with brain metastases: influences on caregiver resilience and coping.

PURPOSE: Neurologic deficits that may be manifested as cognitive impairment contribute to the challenges faced by caregivers of patients with brain metastases. To better address their needs, we examined how caregivers respond to these challenges and explore the relationship between the patient's cognitive impairment and caregiver resilience and coping. METHODS: We conducted a descriptive, cross-sectional study using self-reported data from 56 caregivers of patients with brain metastases. Study participants from a comprehensive cancer center were asked to complete a series of instruments that measured their perception of the patient's cognitive dysfunction (revised memory and behavior problems checklist, RMBC), their own personal resilience (Resilience Scale, RS), and their utilization of a broad range of coping responses (COPE inventory and Emotional-Approach Coping scale). RESULTS: Caregivers reported that memory-related problems occurred more frequently in the patients they cared for compared to depression and disruptive behavior (mean scores 3.52 vs 2.34 vs. 1.32, respectively). Coping strategies most frequently used by caregivers were acceptance (3.28), planning (3.08), and positive reinterpretation and growth (2.95). Most caregivers scored moderate to high on the RS (77%). The coping strategy acceptance correlated significantly with the memory and disruptive behavior subscales of the RMBC. CONCLUSIONS: Given the protective effect of problem-focused coping and the high rate of caregivers utilizing less effective coping strategies in instances of worsening cognitive dysfunction, healthcare professionals need to systematically assess the coping strategies of caregivers and deliver a more personalized approach to enhance effective coping among caregivers of patients with brain metastases.

Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli.

In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition.

Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.

Kepler constraints on planets near hot Jupiters.

We present the results of a search for planetary companions orbiting near hot Jupiter planet candidates (Jupiter-size candidates with orbital periods near 3 d) identified in the Kepler data through its sixth quarter of science operations. Special emphasis is given to companions between the 21 interior and exterior mean-motion resonances. A photometric transit search excludes companions with sizes ranging from roughly two-thirds to five times the size of the Earth, depending upon the noise properties of the target star. A search for dynamically induced deviations from a constant period (transit timing variations) also shows no significant signals. In contrast, comparison studies of warm Jupiters (with slightly larger orbits) and hot Neptune-size candidates do exhibit signatures of additional companions with these same tests. These differences between hot Jupiters and other planetary systems denote a distinctly different formation or dynamical history.

Cobalt to Chromium Ratio is Not a Key Marker for Adverse Local Tissue Reaction (ALTR) in Metal on Metal Hips.

The diagnosis of adverse local tissue reaction (ALTR) after metal-on-metal total hip arthroplasty (MoMTHA) presents a significant challenge. No single biomarker is specific for ALTR. The purpose of this study was to determine if the ratio of cobalt to chromium ions is useful for diagnosing ALTR in MoMTHA. In 89 bearing-related revision THAs, preoperative cobalt and chromium ion levels were compared to an intraoperative soft tissue damage grading scale. The average cobalt to chromium ratio was 2.96 (0-20). There was no correlation between the tissue scale and the cobalt to chromium ratio (R=0.095; P=0.41). Many variables affecting ion production/excretion mitigate the use of the ion ratio. The cobalt to chromium ratio is not a predictive biomarker for ALTR in MoMTHA.

Birth of common shovelnose rays (Glaucostegus typus) under captive conditions.

The common shovelnose ray (Glaucostegus typus) is a poorly studied species of the Rhinobatidae family that occurs throughout the Indo-West Pacific. Although common in aquariums throughout the United States, there are currently no records of captive birth events. In 2013, a female common shovelnose ray housed at the Downtown Aquarium in Houston, Texas, USA gave birth to eleven pups. Although all pups were stillborn, this event demonstrates that it is possible to breed common shovelnose rays in a controlled environment. The single female and two male common shovelnose rays at the aquarium are of sexually mature size (between 206 and 240 cm total length, TL), demonstrate mating behaviors, and provide an excellent opportunity to investigate the reproductive biology of this species. Captive environmental conditions of the birth enclosure may be useful in replicating the birthing event in order to develop a breeding program that could potentially relieve collection pressures on wild populations of guitarfish given their vulnerable status.

Deep-pelagic fishes: Demographic instability in a stable environment.

Demographic histories are frequently a product of the environment, as populations expand or contract in response to major environmental changes, often driven by changes in climate. Meso- and bathy-pelagic fishes inhabit some of the most temporally and spatially stable habitats on the planet. The stability of the deep-pelagic could make deep-pelagic fishes resistant to the demographic instability commonly reported in fish species inhabiting other marine habitats, however the demographic histories of deep-pelagic fishes are unknown. We reconstructed the historical demography of 11 species of deep-pelagic fishes using mitochondrial and nuclear DNA sequence data. We uncovered widespread evidence of population expansions in our study species, a counterintuitive result based on the nature of deep-pelagic ecosystems. Frequency-based methods detected potential demographic changes in nine species of fishes, while extended Bayesian skyline plots identified population expansions in four species. These results suggest that despite the relatively stable nature of the deep-pelagic environment, the fishes that reside here have likely been impacted by past changes in climate. Further investigation is necessary to better understand how deep-pelagic fishes, by far Earth's most abundant vertebrates, will respond to future climatic changes.