An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis.

Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5' 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

Factors Associated With Desaturation in Prehospital Rapid Sequence Intubation in a Helicopter Emergency Medical Service.

OBJECTIVE: Desaturation during prehospital rapid sequence intubation (RSI) is common and is associated with patient morbidity. Past studies have identified oxygen saturations at induction, the grade of laryngoscopy, and multiple attempts to intubate as being associated with desaturation. This study aimed to investigate whether there are other factors, identifiable before RSI, associated with desaturation. METHODS: This was a study of a physician-paramedic critical care team operating as Aeromedical Operations, NSW Ambulance. Prehospital RSIs (using paralysis) were studied retrospectively via patient case notes, monitor data, and an airway database. The review occurred between April 1, 2016, and December 31, 2018. Desaturation was defined as monitor recordings of saturations ≤ 92%. Logistic regression was performed for factors likely to be associated with desaturation. RESULTS: Desaturation occurred in 67 of 350 (19.1%) RSIs. Factors significantly associated with desaturation included male sex, a chest injury, increased weight, and lower saturations pre-RSI. CONCLUSION: Increased weight, chest injuries, and lower oxygen saturations are associated with desaturation at RSI. The variable male sex may be a surrogate for other as-yet unidentified factors.

Optimizing human α-galactosidase for treatment of Fabry disease.

Fabry disease is caused by a deficiency of α-galactosidase A (GLA) leading to the lysosomal accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids. Fabry patients experience significant damage to the heart, kidney, and blood vessels that can be fatal. Here we apply directed evolution to generate more stable GLA variants as potential next generation treatments for Fabry disease. GLAv05 and GLAv09 were identified after screening more than 12,000 GLA variants through 8 rounds of directed evolution. Both GLAv05 and GLAv09 exhibit increased stability at both lysosomal and blood pH, stability to serum, and elevated enzyme activity in treated Fabry fibroblasts (19-fold) and GLA-/- podocytes (10-fold). GLAv05 and GLAv09 show improved pharmacokinetics in mouse and non-human primates. In a Fabry mouse model, the optimized variants showed prolonged half-lives in serum and relevant tissues, and a decrease of accumulated Gb3 in heart and kidney. To explore the possibility of diminishing the immunogenic potential of rhGLA, amino acid residues in sequences predicted to bind MHC II were targeted in late rounds of GLAv09 directed evolution. An MHC II-associated peptide proteomics assay confirmed a reduction in displayed peptides for GLAv09. Collectively, our findings highlight the promise of using directed evolution to generate enzyme variants for more effective treatment of lysosomal storage diseases.

Real-world, Multicenter Experience With Meropenem-Vaborbactam for Gram-Negative Bacterial Infections Including Carbapenem-Resistant Enterobacterales and Pseudomonas aeruginosa.

BACKGROUND: We aimed to describe the clinical characteristics and outcomes of patients treated with meropenem-vaborbactam (MEV) for a variety of gram-negative infections (GNIs), primarily including carbapenem-resistant Enterobacterales (CRE). METHODS: This is a real-world, multicenter, retrospective cohort within the United States between 2017 and 2020. Adult patients who received MEV for ≥72 hours were eligible for inclusion. The primary outcome was 30-day mortality. Classification and regression tree analysis (CART) was used to identify the time breakpoint (BP) that delineated the risk of negative clinical outcomes (NCOs) and was examined by multivariable logistic regression analysis (MLR). RESULTS: Overall, 126 patients were evaluated from 13 medical centers in 10 states. The most common infection sources were respiratory tract (38.1%) and intra-abdominal (19.0%) origin, while the most common isolated pathogens were CRE (78.6%). Thirty-day mortality and recurrence occurred in 18.3% and 11.9%, respectively. Adverse events occurred in 4 patients: nephrotoxicity (n = 2), hepatoxicity (n = 1), and rash (n = 1). CART-BP between early and delayed treatment was 48 hours (P = .04). MEV initiation within 48 hours was independently associated with reduced NCO following analysis by MLR (adusted odds ratio, 0.277; 95% CI, 0.081-0.941). CONCLUSIONS: Our results support current evidence establishing positive clinical and safety outcomes of MEV in GNIs, including CRE. We suggest that delaying appropriate therapy for CRE significantly increases the risk of NCOs.

Surgical repair for canine tricuspid valve dysplasia: Technique and case report.

Tricuspid valve dysplasia (TVD) is a congenital malformation of the right atrioventricular valve characterized by restricted leaflet motion, annular dilation, and tricuspid regurgitation (TR). Severe cases typically exhibit progressive right-sided congestive heart failure, affecting the quality of life and survival. This article describes a technique for surgical repair of TVD and a case report with long-term follow-up. A 1.5-year-old intact male Labrador retriever with severe TR underwent surgical repair for TVD. Valve repair was performed under cardiopulmonary bypass and consisted of neochord mobilization of the valve leaflets and partial band annuloplasty. Transthoracic echocardiogram performed 5 days after surgery showed mild TR, a 93% decrease in anatomic regurgitant orifice area, and decreased right chamber dimensions. Forty-eight months after repair, the patient was free of clinical signs, did not have a heart murmur, and was receiving no cardiac medications. Based on this case, surgical repair of TVD is feasible with long-term durability, and the outcome suggests that the described technique may be a viable treatment option for patients with severe TVD.

Mendelian genes in primary open angle glaucoma.

Mutations in each of three genes, myocilin (MYOC), optineurin (OPTN), and TANK binding kinase 1 (TBK1), may cause primary open-angle glaucoma (POAG) that is inherited as a Mendelian trait. MYOC mutations cause 3-4% of POAG cases with IOP >21 mmHg, while mutations in OPTN, TBK1, and MYOC each cause ∼1% of POAG with IOP ≤21 mmHg, i.e. normal tension glaucoma. Identification of these disease-causing genes has provided insights into glaucoma pathogenesis. Mutations in MYOC cause a cascade of abnormalities in the trabecular meshwork including intracellular retention of MYOC protein, decreased aqueous outflow, higher intraocular pressure, and glaucoma. Investigation of MYOC mutations demonstrated that abnormal retention of intracellular MYOC and stimulation of endoplasmic reticular (ER) stress may be important steps in the development of MYOC-associated glaucoma. Mutations in OPTN and TBK1 cause a dysregulation of autophagy which may directly cause retinal ganglion cell damage and normal tension glaucoma. Discovery of these Mendelian causes of glaucoma has also provided a new set of potential therapeutic targets that may ultimately lead to novel, gene-directed glaucoma treatments.

Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum.

Genetic manipulation of malaria parasites remains an inefficient, time-consuming and resource-intensive process. Presented here is a set of methods for 96-well plate-based transfection and culture that improve the efficiency of genetic manipulation of Plasmodium falciparum. Compared to standard protocols plate-based transfection requires 20-fold less DNA, transient transfection efficiency achieved is approximately seven-fold higher, whilst stable transfection success rate is above 90%. Furthermore the utility of this set of protocols to generate a knockout of the PfRH3 pseudogene, screened by whole-cell PCR, is demonstrated. The methods and tools presented here will facilitate genome-scale genetic manipulation of P. falciparum.

Firearm restriction as suicide prevention: variation in belief and practice among providers in an urban emergency department.

Providers in an urban emergency department (ED) responded to a confidential, online survey assessing their beliefs and practices with respect to reducing a suicidal person's access to lethal means of suicide. 26% of respondents (37% of ED nurses, 23% of ED physicians, and 9% of psychiatrists) believed that all of the jumpers from the Golden Gate Bridge would have found a lethal suicide alternative had a barrier been present, and an additional 38% thought that most would have. Two-thirds of respondents believed that providers in the ED should always ask suicidal patients about access to firearms, yet 52% (67% of nurses, 54% of ED physicians, and 13% of psychiatrists) reported rarely or never doing so. Psychiatrists were more likely than ED staff to report always asking. Further understanding these attitudes and behaviours could enhance suicide prevention activities in the ED.

Characterizing dissolved organic matter using PARAFAC modeling of fluorescence spectroscopy: a comparison of two models.

We evaluated whether fitting fluorescence excitation-emission matrices (EEMs) to a previously validated PARAFAC model is an acceptable alternative to building an original model. To do this, we built a 10-component model using 307 EEMs collected from southeast Alaskan soil and streamwater. All 307 EEMs were then fit to the existing model (CM) presented in Cory and McKnight (Environ. Sci. Technol. 2005, 39, 8142-8149). The first approach for evaluating whether the EEMs were fit well to the CM model was an evaluation of the residual EEMs, and we found 22 EEMs were fit poorly by the CM model. Our second measure for verifying whether EEMs were fit well to the CM model was a comparison of correlations between the percent contribution of PARAFAC components and DOM measurements (e.g., dissolved nutrient concentrations), and we found no significant difference Ip > 0.05) between the two models. These results support the approach of fitting EEMs to an existing model when DOM is collected from similar environments, which can potentially reduce some of the problems when building an original PARAFAC model. However, it is important to recognize that some of the sensitivity or ecological interpretative power may be lost when fitting EEMs to an existing model.

Balance improvements in female high school basketball players after a 6-week neuromuscular-training program.

CONTEXT: Poor balance has been associated with increased injury risk among athletes. Neuromuscular-training programs have been advocated as a means of injury prevention, but little is known about the benefits of these programs on balance in high school athletes. OBJECTIVE: To determine whether there are balance gains after participation in a neuromuscular-training program in high school athletes. DESIGN: Nonrandomized controlled trial. SETTING: All data were collected at each participating high school before and after a 6-wk intervention or control period. PARTICIPANTS: 62 female high school basketball players recruited from the local high school community and assigned to a training (n = 37) or control (n = 25) group. INTERVENTION: Training-group subjects participated in a 6-wk neuromuscular-training program that included plyometric, functional-strengthening, balance, and stability-ball exercises. MAIN OUTCOME MEASURES: Data were collected for the Balance Error Scoring System (BESS) and Star Excursion Balance Test (SEBT) before and after the 6-wk intervention or control period. RESULTS: The authors found a significant decrease in total BESS errors in the trained group at the posttest compared with their pretest and the control group (P = .003). Trained subjects also scored significantly fewer BESS errors on the single-foam and tandem-foam conditions at the posttest than the control group and demonstrated improvements on the single-foam compared with their pretest (P = .033). The authors found improvements in reach in the lateral, anteromedial, medial, and posterior directions in the trained group at the posttest compared with the control group (P < .05) using the SEBT. CONCLUSION: The study demonstrates that a neuromuscular-training program can increase the balance and proprioceptive capabilities of female high school basketball players and that clinical balance measures are sensitive to detect these differences.

Injury prevention and control: reflections on the state and the direction of the field.

OBJECTIVE: To provide an assessment of the contours of the injury field today and to raise questions about our future direction. MATERIAL AND METHODS: We classified the self-reported activities of injury centers, assessed trends in injury-related publications in peer review journals, and compared data on current funding levels. RESULTS: The 47 identified centers are more likely to focus on unintentional injuries and on prevention than on intentional injuries, biomechanics, acute care or rehabilitation. Injury-related publications have doubled over the past decade, yet remained dwarfed by those on other diseases. Funding for injury prevention remains incommensurate with the burden of injury. Within the injury field itself, publications and funding are not commensurate with the burden imposed by particular injuries. CONCLUSION: Our responsibilities as injury prevention professionals will increase not only because of the projected increase in the global burden of injury but also because of our expanded conceptualization of what the scope of injury prevention should be. The lack of clarity we project about the substantive areas of our expertise and the incommensurate funding for our efforts relative to their toll on global health represent challenges to our field's coherence and ultimate effectiveness.

Injury prevention and control: reflections on the state and the direction of the field: [review]

OBJECTIVE: To provide an assessment of the contours of the injury field today and to raise questions about our future direction. MATERIAL AND METHODS: We classified the self-reported activities of injury centers, assessed trends in injury-related publications in peer review journals, and compared data on current funding levels. RESULTS: The 47 identified centers are more likely to focus on unintentional injuries and on prevention than on intentional injuries, biomechanics, acute care or rehabilitation. Injury-related publications have doubled over the past decade, yet remained dwarfed by those on other diseases. Funding for injury prevention remains incommensurate with the burden of injury. Within the injury field itself, publications and funding are not commensurate with the burden imposed by particular injuries. CONCLUSION: Our responsibilities as injury prevention professionals will increase not only because of the projected increase in the global burden of injury but also because of our expanded conceptualization of what the scope of injury prevention should be. The lack of clarity we project about the substantive areas of our expertise and the incommensurate funding for our efforts relative to their toll on global health represent challenges to our field's coherence and ultimate effectiveness.

OBJETIVO: Valorar el estado actual del área de la prevención de lesiones y plantear el futuro de la misma. MATERIAL Y MÉTODOS: Clasificamos las actividades llevadas a cabo por diferentes centros de prevención de lesiones; recabamos información sobre la tendencia en las publicaciones en revistas científicas, y comparamos los datos sobre financiación. RESULTADOS: Evaluamos las actividades de 47 instituciones. En general, los centros realizan actividades relacionadas con las lesiones no intencionadas y en la prevención en detrimento de las lesiones intencionales, la biomecánica, asistencia sanitaria o la rehabilitación. Las publicaciones relacionadas con el área de las lesiones se han duplicado en la pasada década, aunque siguen muy por debajo de las publicaciones de otras enfermedades. La financiación destinada a la prevención de lesiones sigue siendo desproporcionada con respecto a la carga de enfermedad. Dentro del propio ámbito de la prevención de lesiones, tanto la financiación como las publicaciones no se adecuan a la carga impuesta por diferentes tipos de lesiones. CONCLUSIÓN: Nuestra responsabilidad aumenta en tanto que aumenta la carga de las lesiones sobre la población mundial y al mismo tiempo se amplía nuestra visión sobre lo que constituye una lesión. En vista de las discrepancias sobre la percepción, necesidades y escasez de financiación, hemos de estudiar con detenimiento las estrategias de futuro.

Interlocking transcriptional feedback loops control white-opaque switching in Candida albicans.

The human pathogen Candida albicans can assume either of two distinct cell types, designated "white" and "opaque." Each cell type is maintained for many generations; switching between them is rare and stochastic, and occurs without any known changes in the nucleotide sequence of the genome. The two cell types differ dramatically in cell shape, colony appearance, mating competence, and virulence properties. In this work, we investigate the transcriptional circuitry that specifies the two cell types and controls the switching between them. First, we identify two new transcriptional regulators of white-opaque switching, Czf1 and white-opaque regulator 2 (Wor2). Analysis of a large set of double mutants and ectopic expression strains revealed genetic relationships between CZF1, WOR2, and two previously identified regulators of white-opaque switching, WOR1 and EFG1. Using chromatin immunoprecipitation, we show that Wor1 binds the intergenic regions upstream of the genes encoding three additional transcriptional regulators of white-opaque switching (CZF1, EFG1, and WOR2), and also occupies the promoters of numerous white- and opaque-enriched genes. Based on these interactions, we have placed these four genes in a circuit controlling white-opaque switching whose topology is a network of positive feedback loops, with the master regulator gene WOR1 occupying a central position. Our observations indicate that a key role of the interlocking feedback loop network is to stably maintain each epigenetic state through many cell divisions.

Nuclear fusion occurs during mating in Candida albicans and is dependent on the KAR3 gene.

It is now well established that mating can occur between diploid a and alpha cells of Candida albicans. There is, however, controversy over when, and with what efficiency, nuclear fusion follows cell fusion to create stable tetraploid a/alpha cells. In this study, we have analysed the mating process between C. albicans strains using both cytological and genetic approaches. Using strains derived from SC5314, we used a number of techniques, including time-lapse microscopy, to demonstrate that efficient nuclear fusion occurs in the zygote before formation of the first daughter cell. Consistent with these observations, zygotes micromanipulated from mating mixes gave rise to mononuclear tetraploid cells, even when no selection for successful mating was applied to them. Mating between different clinical isolates of C. albicans revealed that while all isolates could undergo nuclear fusion, the efficiency of nuclear fusion varied in different crosses. We also show that nuclear fusion in C. albicans requires the Kar3 microtubule motor protein. Deletion of the CaKAR3 gene from both mating partners had little or no effect on zygote formation but reduced the formation of stable tetraploids more than 600-fold, as determined by quantitative mating assays. These findings demonstrate that nuclear fusion is an active process that can occur in C. albicans at high frequency to produce stable, mononucleate mating products.

A collaborative approach to the development of psychiatric rehabilitation services in a public behavioral health managed care environment.

This article summarizes the collaborative effort that took place between providers, managed care administrators, and state and county officials in Pennsylvania to create a fluid system in the development of psychiatric rehabilitation services. The stakeholders involved solved the numerous logistical problems that are inherent in the start-up of new services and were able to cooperatively create procedures for service delivery, service coordination, and reimbursement across systems and funding mechanisms. The article reviews that process and highlights the services of Welcome House, a psychiatric rehabilitation program exemplifying the products of their collaboration.

Mechanism-based enzyme inactivators of phytosterol biosynthesis.

Current progress on the mechanism and substrate recognition by sterol methyl transferase (SMT), the role of mechanism-based inactivators, other inhibitors of SMT action to probe catalysis and phytosterol synthesis is reported. SMT is a membrane-bound enzyme which catalyzes the coupled C-methylation-deprotonation reaction of sterol acceptor molecules generating the 24-alkyl sterol side chains of fungal ergosterol and plant sitosterol. This C-methylation step can be rate-limiting in the post-lanosterol (fungal) or post-cycloartenol (plant) pathways. A series of sterol analogs designed to impair SMT activity irreversibly have provided deep insight into the C-methylation reaction and topography of the SMT active site and as reviewed provide leads for the development of antifungal agents.

Evolution of a combinatorial transcriptional circuit: a case study in yeasts.

Developing new regulation of existing genes is likely a key mechanism by which organismal complexity arises in evolution. To examine plasticity of gene regulation over evolutionary timescales, we have determined the transcriptional circuit regulating mating type in the human fungal pathogen Candida albicans, and compared it to that of Saccharomyces cerevisiae. Since the two yeasts last shared an ancestor 100-800 million years ago, several major differences in circuitry have arisen. For example, a positive regulator of mating type was retained in C. albicans but lost in S. cerevisiae; this circuit branch was replaced by the modification of an existing negative regulator, thereby conserving the circuit output. We also characterize a tier of mating type transcriptional regulation that is present only in C. albicans, and likely results from the vastly different environmental selections imposed on the two yeasts--in this case, the pressure on C. albicans to survive in a mammalian host.

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation.

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11-19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3beta are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3beta by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3beta is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.

Identification and characterization of a Candida albicans mating pheromone.

Candida albicans, the most prevalent fungal pathogen of humans, has recently been shown to undergo mating. Here we describe a mating pheromone produced by C. albicans alpha cells and show that the gene which encodes it (MFalpha) is required for alpha cells, but not a cells, to mate. We also identify the receptor for this mating pheromone as the product of the STE2 gene and show that this gene is required for the mating of a cells, but not alpha cells. Cells of the a mating type respond to the alpha mating pheromone by producing long polarized projections, similar to those observed in bona fide mating mixtures of C. albicans a and alpha cells. During this process, transcription of approximately 62 genes is induced. Although some of these genes correspond to those induced in Saccharomyces cerevisiae by S. cerevisiae alpha-factor, most are specific to the C. albicans pheromone response. The most surprising class encode cell surface and secreted proteins previously implicated in virulence of C. albicans in a mouse model of disseminated candidiasis. This observation suggests that aspects of cell-cell communication in mating may have been evolutionarily adopted for host-pathogen interactions in C. albicans.

Assessment of pharmacodynamic vascular response in a phase I trial of combretastatin A4 phosphate.

PURPOSE: Clinical evaluation of novel agents that target tumor blood vessels requires pharmacodynamic end points that measure vascular damage. Positron emission tomography (PET) was used to measure the effects of the vascular targeting agent combretastatin A4 phosphate (CA4P) on tumor and normal tissue perfusion and blood volume. PATIENTS AND METHODS: Patients with advanced solid tumors were enrolled onto part of a phase I, accelerated-titration, dose-escalation study. The effects of 5 to 114 mg/m2 CA4P on tumor, spleen, and kidney were investigated. Tissue perfusion was measured using oxygen-15 (15O)-labeled water and blood volume was measured using 15O-labeled carbon monoxide (C15O). Scans were performed immediately before, and 30 minutes and 24 hours after the first infusion of each dose level of CA4P. All statistical tests were two sided. RESULTS: PET data were obtained for 13 patients with intrapatient dose escalation. Significant dose-dependent reductions were seen in tumor perfusion 30 minutes after CA4P administration (mean change, -49% at >or= 52 mg/m2; P =.0010). Significant reductions were also seen in tumor blood volume (mean change, -15% at >or= 52 mg/m2; P =.0070). Although by 24 hours there was tumor vascular recovery, for doses >or= 52 mg/m2 the reduction in perfusion remained significant (P =.013). Thirty minutes after CA4P administration borderline significant changes were seen in spleen perfusion (mean change, -35%; P =.018), spleen blood volume (mean change, -18%; P =.022), kidney perfusion (mean change, -6%; P =.026), and kidney blood volume (mean change, -6%; P =.014). No significant changes were seen at 24 hours in spleen or kidney. CONCLUSION: CA4P produces rapid changes in the vasculature of human tumors that can be assessed using PET measurements of tumor perfusion.