Pediatric Cervical Spine Injury Following Blunt Trauma in Children Younger Than 3 Years: The PEDSPINE II Study.

Importance: There is variability in practice and imaging usage to diagnose cervical spine injury (CSI) following blunt trauma in pediatric patients. Objective: To develop a prediction model to guide imaging usage and to identify trends in imaging and to evaluate the PEDSPINE model. Design, Setting, and Participants: This cohort study included pediatric patients (<3 years years) following blunt trauma between January 2007 and July 2017. Of 22 centers in PEDSPINE, 15 centers, comprising level 1 and 2 stand-alone pediatric hospitals, level 1 and 2 pediatric hospitals within an adult hospital, and level 1 adult hospitals, were included. Patients who died prior to obtaining cervical spine imaging were excluded. Descriptive analysis was performed to describe the population, use of imaging, and injury patterns. PEDSPINE model validation was performed. A new algorithm was derived using clinical criteria and formulation of a multiclass classification problem. Analysis took place from January to October 2022. Exposure: Blunt trauma. Main Outcomes and Measures: Primary outcome was CSI. The primary and secondary objectives were predetermined. Results: The current study, PEDSPINE II, included 9389 patients, of which 128 (1.36%) had CSI, twice the rate in PEDSPINE (0.66%). The mean (SD) age was 1.3 (0.9) years; and 70 patients (54.7%) were male. Overall, 7113 children (80%) underwent cervical spine imaging, compared with 7882 (63%) in PEDSPINE. Several candidate models were fitted for the multiclass classification problem. After comparative analysis, the multinomial regression model was chosen with one-vs-rest area under the curve (AUC) of 0.903 (95% CI, 0.836-0.943) and was able to discriminate between bony and ligamentous injury. PEDSPINE and PEDSPINE II models' ability to identify CSI were compared. In predicting the presence of any injury, PEDSPINE II obtained a one-vs-rest AUC of 0.885 (95% CI, 0.804-0.934), outperforming the PEDSPINE score (AUC, 0.845; 95% CI, 0.769-0.915). Conclusion and Relevance: This study found wide clinical variability in the evaluation of pediatric trauma patients with increased use of cervical spine imaging. This has implications of increased cost, increased radiation exposure, and a potential for overdiagnosis. This prediction tool could help to decrease the use of imaging, aid in clinical decision-making, and decrease hospital resource use and cost.

Efficient manufacturing and engraftment of CCR5 gene-edited HSPCs following busulfan conditioning in nonhuman primates.

Hematopoietic stem cell gene therapy has been successfully used for a number of genetic diseases and is also being explored for HIV. However, toxicity of the conditioning regimens has been a major concern. Here we compared current conditioning approaches in a clinically relevant nonhuman primate model. We first customized various aspects of the therapeutic approach, including mobilization and cell collection protocols, conditioning regimens that support engraftment with minimal collateral damage, and cell manufacturing and infusing schema that reflect and build on current clinical approaches. Through a series of iterative in vivo experiments in two macaque species, we show that busulfan conditioning significantly spares lymphocytes and maintains a superior immune response to mucosal challenge with simian/human immunodeficiency virus, compared to total body irradiation and melphalan regimens. Comparative mobilization experiments demonstrate higher cell yield relative to our historical standard, primed bone marrow and engraftment of CRISPR-edited hematopoietic stem and progenitor cells (HSPCs) after busulfan conditioning. Our findings establish a detailed workflow for preclinical HSPC gene therapy studies in the nonhuman primate model, which in turn will support testing of novel conditioning regimens and more advanced HSPC gene editing techniques tailored to any disease of interest.

Implementation of Bedaquiline, Pretomanid, and Linezolid in the United States: Experience Using a Novel All-Oral Treatment Regimen for Treatment of Rifampin-Resistant or Rifampin-Intolerant Tuberculosis Disease.

BACKGROUND: Rifampin-resistant tuberculosis is a leading cause of morbidity worldwide; only one-third of persons start treatment, and outcomes are often inadequate. Several trials demonstrate 90% efficacy using an all-oral, 6-month regimen of bedaquiline, pretomanid, and linezolid (BPaL), but significant toxicity occurred using 1200-mg linezolid. After US Food and Drug Administration approval in 2019, some US clinicians rapidly implemented BPaL using an initial 600-mg linezolid dose adjusted by serum drug concentrations and clinical monitoring. METHODS: Data from US patients treated with BPaL between 14 October 2019 and 30 April 2022 were compiled and analyzed by the BPaL Implementation Group (BIG), including baseline examination and laboratory, electrocardiographic, and clinical monitoring throughout treatment and follow-up. Linezolid dosing and clinical management was provider driven, and most patients had linezolid adjusted by therapeutic drug monitoring. RESULTS: Of 70 patients starting BPaL, 2 changed to rifampin-based therapy, 68 (97.1%) completed BPaL, and 2 of the 68 (2.9%) experienced relapse after completion. Using an initial 600-mg linezolid dose daily adjusted by therapeutic drug monitoring and careful clinical and laboratory monitoring for adverse effects, supportive care, and expert consultation throughout BPaL treatment, 3 patients (4.4%) with hematologic toxicity and 4 (5.9%) with neurotoxicity required a change in linezolid dose or frequency. The median BPaL duration was 6 months. CONCLUSIONS: BPaL has transformed treatment for rifampin-resistant or intolerant tuberculosis. In this cohort, effective treatment required less than half the duration recommended in 2019 US guidelines for drug-resistant tuberculosis. Use of individualized linezolid dosing and monitoring likely enhanced safety and treatment completion. The BIG cohort demonstrates that early implementation of new tuberculosis treatments in the United States is feasible.

Dual Disseminated Aspergillosis and Mucormycosis Diagnosed at Autopsy: A Report of Two Cases of Coinfection and a Review of the Literature.

Coinfection with invasive aspergillosis and mucormycosis in immunocompromised patients has been reported but is rarely confirmed by tissue histology or autopsy. Serum fungal biomarkers and culture are the primary diagnostic tools but are suboptimal for detecting fungal coinfection. Here, we present the cases of two patients who were immunocompromised due to hematologic malignancy where disseminated aspergillosis and mucormycosis coinfection was only diagnosed upon autopsy despite extensive fungal diagnostic workup, and also review recent literature of such instances of coinfection.

Extended stability of vasopressin 0.2 unit/mL in PVC containers.

PURPOSE: Vasopressin is used to maintain blood pressure in vasodilatory shock. Vasopressin is diluted from concentrated vials prior to administration as a continuous infusion. This study evaluates the physical and chemical stability changes of vasopressin diluted to 0.2 units/mL with 0.9% sodium chloride injection in polyvinyl chloride (PVC) bags stored under refrigeration. METHODS: Vasopressin Injection, USP, 20 unit/mL solution was diluted to 0.2 unit/mL with 0.9% sodium chloride injection, and stability changes were evaluated over 10 days via mass spectrometry on days 0, 7, and 10. RESULTS: Solutions of vasopressin 0.2 unit/mL in 0.9% sodium chloride injection in PVC bags were physically stable and showed less than 10% degradation over 10 days of refrigerated storage. CONCLUSION: Vasopressin 0.2 unit/mL may be given a beyond-use date (BUD) of 10 days based on United States Pharmacopeia BUD recommendations, with this study showing less than 10% degradation over 10 days of refrigerated storage.

dATP elevation induces myocardial metabolic remodeling to support improved cardiac function.

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ∼1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs.

Engineered red blood cells (activating antigen carriers) drive potent T cell responses and tumor regression in mice.

Activation of T cell responses is essential for effective tumor clearance; however, inducing targeted, potent antigen presentation to stimulate T cell responses remains challenging. We generated Activating Antigen Carriers (AACs) by engineering red blood cells (RBCs) to encapsulate relevant tumor antigens and the adjuvant polyinosinic-polycytidylic acid (poly I:C), for use as a tumor-specific cancer vaccine. The processing method and conditions used to create the AACs promote phosphatidylserine exposure on RBCs and thus harness the natural process of aged RBC clearance to enable targeting of the AACs to endogenous professional antigen presenting cells (APCs) without the use of chemicals or viral vectors. AAC uptake, antigen processing, and presentation by APCs drive antigen-specific activation of T cells, both in mouse in vivo and human in vitro systems, promoting polyfunctionality of CD8+ T cells and, in a tumor model, driving high levels of antigen-specific CD8+ T cell infiltration and tumor killing. The efficacy of AAC therapy was further enhanced by combination with the chemotherapeutic agent Cisplatin. In summary, these findings support AACs as a potential vector-free immunotherapy strategy to enable potent antigen presentation and T cell stimulation by endogenous APCs with broad therapeutic potential.

Tendon Healing in a Mouse Model of Loeys-Dietz Syndrome: Controlled Study Using a Patellar Tendon Transection Model.

BACKGROUND: Loeys-Dietz syndrome (LDS) is a rare autosomal-dominant connective tissue disorder caused by genetic mutations in the transforming growth factor-ß (TGFß) signaling pathway. In addition to vascular malformations, patients with LDS commonly present with bone and tendon abnormalities, including joint laxity. While TGFß signaling dysregulation has been implicated in many of these clinical manifestations, the degree to which it influences the tendinopathy and tendon healing issues in LDS has not been determined. METHODS: Wound healing after patellar tendon transection was compared between wild-type (WT) and Tgfbr2-mutant (LDS) mice (7 mice per group). In all mice, the right patellar tendon was transected at midsubstance, while the left was untouched to serve as a control. Mice were euthanized 6 weeks after surgery. Tendon specimens were harvested for histopathologic grading according to a previously validated scoring metric, and gene expression levels of Mmp2, Tgfb2, and other TGFß-signaling genes were assayed. Between-group comparisons were made using 1-way analysis of variance with post hoc Tukey honestly significant difference testing. RESULTS: Expression levels of assayed genes were similar between LDS and WT tendons at baseline; however, at 6 weeks after patellar tendon transection, LDS tendons showed sustained elevations in Mmp2 and Tgfb2 compared with baseline values; these elevations were not seen in normal tendons undergoing the same treatments. Histologically, untreated LDS tendons had significantly greater cellularity and cell rounding compared with untreated WT tendons, and both WT and LDS tendons had significantly worse histologic scores after surgery. CONCLUSION: We present the first mechanistic insight into the effect of LDS on tendons and tendon healing. The morphologic differences between LDS and WT tendons at baseline may help explain the increased risk of tendon/ligament dysfunction in patients with LDS, and the differential healing response to injury in LDS may account for the delayed healing and weaker repair tissue. LEVEL OF EVIDENCE: Level V.

Discovery of C13-Aminobenzoyl Cycloheximide Derivatives that Potently Inhibit Translation Elongation.

Employed for over half a century to study protein synthesis, cycloheximide (CHX, 1) is a small molecule natural product that reversibly inhibits translation elongation. More recently, CHX has been applied to ribosome profiling, a method for mapping ribosome positions on mRNA genome-wide. Despite CHX's extensive use, CHX treatment often results in incomplete translation inhibition due to its rapid reversibility, prompting the need for improved reagents. Here, we report the concise synthesis of C13-amide-functionalized CHX derivatives with increased potencies toward protein synthesis inhibition. Cryogenic electron microscopy (cryo-EM) revealed that C13-aminobenzoyl CHX (8) occupies the same site as CHX, competing with the 3' end of E-site tRNA. We demonstrate that 8 is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.

Impact of etiology on force and kinetics of left ventricular end-stage failing human myocardium.

BACKGROUND: Heart failure (HF) is associated with highly significant morbidity, mortality, and health care costs. Despite the significant advances in therapies and prevention, HF remains associated with poor clinical outcomes. Understanding the contractile force and kinetic changes at the level of cardiac muscle during end-stage HF in consideration of underlying etiology would be beneficial in developing targeted therapies that can help improve cardiac performance. OBJECTIVE: Investigate the impact of the primary etiology of HF (ischemic or non-ischemic) on left ventricular (LV) human myocardium force and kinetics of contraction and relaxation under near-physiological conditions. METHODS AND RESULTS: Contractile and kinetic parameters were assessed in LV intact trabeculae isolated from control non-failing (NF; n = 58) and end-stage failing ischemic (FI; n = 16) and non-ischemic (FNI; n = 38) human myocardium under baseline conditions, length-dependent activation, frequency-dependent activation, and response to the ß-adrenergic stimulation. At baseline, there were no significant differences in contractile force between the three groups; however, kinetics were impaired in failing myocardium with significant slowing down of relaxation kinetics in FNI compared to NF myocardium. Length-dependent activation was preserved and virtually identical in all groups. Frequency-dependent activation was clearly seen in NF myocardium (positive force frequency relationship [FFR]), while significantly impaired in both FI and FNI myocardium (negative FFR). Likewise, ß-adrenergic regulation of contraction was significantly impaired in both HF groups. CONCLUSIONS: End-stage failing myocardium exhibited impaired kinetics under baseline conditions as well as with the three contractile regulatory mechanisms. The pattern of these kinetic impairments in relation to NF myocardium was mainly impacted by etiology with a marked slowing down of kinetics in FNI myocardium. These findings suggest that not only force development, but also kinetics should be considered as a therapeutic target for improving cardiac performance and thus treatment of HF.

Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2'-deoxy-ATP.

KEY POINTS: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation. The naturally occurring nucleotide 2-deoxy-ATP (dATP) is a myosin activator that enhances cross-bridge binding and kinetics. X-ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin-binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation. This study of the dATP-induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin-targeted molecular compounds to treat muscle diseases. ABSTRACT: Here we used time-resolved small-angle X-ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first-order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6 ) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2-deoxy-ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X-ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin-binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions.

Microscopic Investigation of the Combined Use of Antibiotics and Biosurfactants on Methicillin Resistant Staphylococcus aureus.

One current strategy to deal with the serious issue of antibiotic resistance is to use biosurfactants, weak antimicrobials in their own right, with antibiotics in order to extend the efficacy of antibiotics. Although an adjuvant effect has been observed, the underlying mechanisms are poorly understood. To investigate the nature of the antibiotic and biosurfactant interaction, we undertook a scanning electron microscopy (SEM) and atomic force microscopy (AFM) microscopic study of the effects of the tetracycline antibiotic, combined with sophorolipid and rhamnolipid biosurfactants, on Methicillin-resistant Staphylococcus aureus using tetracycline concentrations below and above the minimum inhibitory concentration (MIC). Control and treated bacterial samples were prepared with an immersion technique by adsorbing the bacteria onto glass substrates grafted with the poly-cationic polymer polyethyleneimine. Bacterial surface morphology, hydrophobic and hydrophilic surface characters as well as the local bacterial cell stiffness were measured following combined antibiotic and biosurfactant treatment. The sophorolipid biosurfactant stands alone insofar as, when used with the antibiotic at sub-MIC concentration, it resulted in bacterial morphological changes, larger diameters (from 758 ± 75 to 1276 ± 220 nm, p-value = 10-4) as well as increased bacterial core stiffness (from 205 ± 46 to 396 ± 66 mN/m, p-value = 5 × 10-5). This investigation demonstrates that such combination of microscopic analysis can give useful information which could complement biological assays to understand the mechanisms of synergy between antibiotics and bioactive molecules such as biosurfactants.

Restoration Scaling Approaches to Addressing Ecological Injury: The Habitat-Based Resource Equivalency Method.

Natural resource trustee agencies must determine how much, and what type of environmental restoration will compensate for injuries to natural resources that result from releases of hazardous substances or oil spills. To fulfill this need, trustees, and other natural resource damage assessment (NRDA) practitioners have relied on a variety of approaches, including habitat equivalency analysis (HEA) and resource equivalency analysis (REA). The purpose of this paper is to introduce the Habitat-Based Resource Equivalency Method (HaBREM), which integrates REA's reproducible injury metrics and population modeling with HEA's comprehensive habitat approach to restoration. HaBREM is intended to evaluate injury and restoration using organisms that use the habitat to represent ecological habitat functions. This paper seeks to expand and refine the use of organism-based metrics (biomass-based REA), providing an opportunity to integrate sublethal injuries to multiple species, as well as the potential to include error rates for injury and restoration parameters. Applied by NRDA practitioners in the appropriate context, this methodology can establish the relationship between benefits of compensatory restoration projects and injuries to plant or animal species within an affected habitat. HaBREM may be most effective where there are appropriate data supporting the linkage between habitat and species gains (particularly regionally specific habitat information), as well as species-specific monitoring data and predictions on the growth, density, productivity (i.e., rate of generation of biomass or individuals), and age distributions of indicator species.

Cardiac myosin activation with 2-deoxy-ATP via increased electrostatic interactions with actin.

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

Structures of translationally inactive mammalian ribosomes.

The cellular levels and activities of ribosomes directly regulate gene expression during numerous physiological processes. The mechanisms that globally repress translation are incompletely understood. Here, we use electron cryomicroscopy to analyze inactive ribosomes isolated from mammalian reticulocytes, the penultimate stage of red blood cell differentiation. We identify two types of ribosomes that are translationally repressed by protein interactions. The first comprises ribosomes sequestered with elongation factor 2 (eEF2) by SERPINE mRNA binding protein 1 (SERBP1) occupying the ribosomal mRNA entrance channel. The second type are translationally repressed by a novel ribosome-binding protein, interferon-related developmental regulator 2 (IFRD2), which spans the P and E sites and inserts a C-terminal helix into the mRNA exit channel to preclude translation. IFRD2 binds ribosomes with a tRNA occupying a noncanonical binding site, the 'Z site', on the ribosome. These structures provide functional insights into how ribosomal interactions may suppress translation to regulate gene expression.

Etiology-dependent impairment of relaxation kinetics in right ventricular end-stage failing human myocardium.

BACKGROUND: In patients with end-stage heart failure, the primary etiology often originates in the left ventricle, and eventually the contractile function of the right ventricle (RV) also becomes compromised. RV tissue-level deficits in contractile force and/or kinetics need quantification to understand involvement in ischemic and non-ischemic failing human myocardium. METHODS AND RESULTS: The human population suffering from heart failure is diverse, requiring many subjects to be studied in order to perform an adequately powered statistical analysis. From 2009-present we assessed live tissue-level contractile force and kinetics in isolated myocardial RV trabeculae from 44 non-failing and 41 failing human hearts. At 1 Hz stimulation rate (in vivo resting state) the developed active force was not different in non-failing compared to failing ischemic nor non-ischemic failing trabeculae. In sharp contrast, the kinetics of relaxation were significantly impacted by disease, with 50% relaxation time being significantly shorter in non-failing vs. non-ischemic failing, while the latter was still significantly shorter than ischemic failing. Gender did not significantly impact kinetics. Length-dependent activation was not impacted. Although baseline force was not impacted, contractile reserve was critically blunted. The force-frequency relation was positive in non-failing myocardium, but negative in both ischemic and non-ischemic myocardium, while the ß-adrenergic response to isoproterenol was depressed in both pathologies. CONCLUSIONS: Force development at resting heart rate is not impacted by cardiac pathology, but kinetics are impaired and the magnitude of the impairment depends on the underlying etiology. Focusing on restoration of myocardial kinetics will likely have greater therapeutic potential than targeting force of contraction.

Fast and sensitive HPLC-MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells.

Deoxyribonucleoside triphosphates (dNTPs) are used in DNA synthesis and repair. Even slight imbalances can have adverse biological effects. This study validates a fast and sensitive HPLC-MS/MS method for direct quantification of intracellular dNTPs from tissue. Equal volumes of methanol and water were used for nucleotide extraction from mouse heart and gastrocnemius muscle and isolated cardiomyocytes followed by centrifugation to remove particulates. The resulting supernatant was analyzed on a porous graphitic carbon chromatography column using an elution gradient of ammonium acetate in water and ammonium hydroxide in acetonitrile with a run time of just 10min. Calibration curves of all dNTPs ranged from 62.5 to 2500fmol injections and demonstrated excellent linearity (r2>0.99). The within day and between day precision, as measured by the coefficient of variation (CV (%)), was <25% for all points, including the lower limit of quantification (LLOQ). The inter-day accuracy was within 12% of expected concentration for the LLOQ and within 7% for all other points on the calibration curve. The intra-day accuracy was within 22% for the LLOQ and within 11% for all points on the curve. Compared to existing methods, this study presents a faster and more sensitive method for dNTP quantification.

Evaluation of bactericidal and anti-biofilm properties of a novel surface-active organosilane biocide against healthcare associated pathogens and Pseudomonas aeruginosa biolfilm.

Healthcare acquired infections (HAI) pose a great threat in hospital settings and environmental contamination can be attributed to the spread of these. De-contamination and, significantly, prevention of re-contamination of the environment could help in preventing/reducing this threat. Goldshield (GS5) is a novel organosilane biocide marketed as a single application product with residual biocidal activity. We tested the hypothesis that GS5 could provide longer-term residual antimicrobial activity than existing disinfectants once applied to surfaces. Thus, the residual bactericidal properties of GS5, Actichlor and Distel against repeated challenge with Staphylococcus aureus ATCC43300 were tested, and showed that GS5 alone exhibited longer-term bactericidal activity for up to 6 days on 316I stainless steel surfaces. Having established efficacy against S. aureus, we tested GS5 against common healthcare acquired pathogens, and demonstrated that, on average, a 1 log10 bactericidal effect was exhibited by GS5 treated surfaces, although biocidal activity varied depending upon the surface type and the species of bacteria. The ability of GS5 to prevent Pseudomonas aeruginosa biofilm formation was measured in standard microtitre plate assays, where it had no significant effect on either biofilm formation or development. Taken together the data suggests that GS5 treatment of surfaces may be a useful means to reducing bacterial contamination in the context of infection control practices.

Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes.

In eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factor⋅GTPase complexes representing intermediates of translation elongation (aminoacyl-tRNA⋅eEF1A), termination (eRF1⋅eRF3), and ribosome rescue (Pelota⋅Hbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase. Additional structural snapshots of the translation termination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework for how different states of the mammalian ribosome are selectively recognized by the appropriate decoding factor⋅GTPase complex to ensure translational fidelity.

Structural characterization of ribosome recruitment and translocation by type IV IRES.

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts tvhe otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.