Directed evolution of hyperactive integrases for site specific insertion of transgenes.

The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.

Directed evolution of hyperactive integrases for site specific insertion of transgenes.

The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing Von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.

Evolution of avian heat tolerance: The role of atmospheric humidity.

The role of atmospheric humidity in the evolution of endotherms' thermoregulatory performance remains largely unexplored, despite the fact that elevated humidity is known to impede evaporative cooling capacity. Using a phylogenetically informed comparative framework, we tested the hypothesis that pronounced hyperthermia tolerance among birds occupying humid lowlands evolved to reduce the impact of humidity-impeded scope for evaporative heat dissipation by comparing heat tolerance limits (HTLs; maximum tolerable air temperature), maximum body temperatures (Tbmax), and associated thermoregulatory variables in humid (19.2 g H2O m-3) versus dry (1.1 g H2O m-3) air among 30 species from three climatically distinct sites (arid, mesic montane, and humid lowland). Humidity-associated decreases in evaporative water loss and resting metabolic rate were 27%-38% and 21%-27%, respectively, and did not differ significantly between sites. Decreases in HTLs were significantly larger among arid-zone (mean ± SD = 3.13 ± 1.12°C) and montane species (2.44 ± 1.0°C) compared to lowland species (1.23 ± 1.34°C), with more pronounced hyperthermia among lowland (Tbmax = 46.26 ± 0.48°C) and montane birds (Tbmax = 46.19 ± 0.92°C) compared to arid-zone species (45.23 ± 0.24°C). Our findings reveal a functional link between facultative hyperthermia and humidity-related constraints on evaporative cooling, providing novel insights into how hygric and thermal environments interact to constrain avian performance during hot weather. Moreover, the macrophysiological patterns we report provide further support for the concept of a continuum from thermal specialization to thermal generalization among endotherms, with adaptive variation in body temperature correlated with prevailing climatic conditions.

Respirometry protocols for avian thermoregulation at high air temperatures: stepped and steady-state profiles yield similar results.

Relationships between air temperature (Tair) and avian body temperature (Tb), resting metabolic rate (RMR) and evaporative water loss (EWL) during acute heat exposure can be quantified through respirometry using several approaches. One involves birds exposed to a stepped series of progressively increasing Tair setpoints for short periods (<20-30 min), whereas a second seeks to achieve steady-state conditions by exposing birds to a single Tair for longer periods (>1-2 h). To compare these two approaches, we measured Tb, RMR and EWL over Tair=28°C to 44°C in the dark-capped bulbul (Pycnonotus tricolor). The two protocols yielded indistinguishable values of Tb, RMR and EWL and related variables at most Tair values, revealing that both are appropriate for quantifying avian thermal physiology during heat exposure over the range of Tair in the present study. The stepped protocol, however, has several ethical and practical advantages.

Combating Health Care Fraud and Abuse: Conceptualization and Prototyping Study of a Blockchain Antifraud Framework.

BACKGROUND: An estimated US $2.6 billion loss is attributed to health care fraud and abuse. With traditional health care claims verification and reimbursement, the health care provider submits a claim after rendering services to a patient, which is then verified and reimbursed by the payer. However, this process leaves out a critical stakeholder: the patient for whom the services are actually rendered. This lack of patient participation introduces a risk of fraud and abuse. Blockchain technology enables secure data management with transparency, which could mitigate this risk of health care fraud and abuse. OBJECTIVE: The aim of this study is to develop a framework using blockchain to record claims data and transactions in an immutable format and to enable the patient to act as a validating node to help detect and prevent health care fraud and abuse. METHODS: We developed a health care fraud and abuse blockchain technical framework and prototype using key blockchain tools and application layers including consensus algorithms, smart contracts, tokens, and governance based on digital identity on the Ethereum platform (Ethereum Foundation). RESULTS: Our technical framework maps to the claims adjudication process and focuses on Medicare claims, with the US Centers for Medicare and Medicaid Services (CMS) as the central authority. A prototype of the framework system was developed using the blockchain platform Ethereum (Ethereum Foundation), with its design features, workflow, smart contract functions, system architecture, and software implementation outlined. The software stack used to build the system consisted of a front-end user interface framework, a back-end processing server, and a blockchain network. React was used for the user interface framework, and NodeJS and an Express server were used for the back-end processing server; Solidity was the smart contract language used to interact with a local Ethereum blockchain network. CONCLUSIONS: The proposed framework and the initial prototype have the potential to improve the health care claims process by using blockchain technology for secure data storage and consensus mechanisms, which make the claims adjudication process more patient-centric for the purposes of identifying and preventing health care fraud and abuse. Future work will focus on the use of synthetic or historic CMS claims data to assess the real-world viability of the framework.

Binding and entry of a non-enveloped T=4 insect RNA virus is triggered by alkaline pH.

Tetraviruses are small, non-enveloped, RNA viruses that exclusively infect lepidopteran insects. Their particles comprise 240 copies of a single capsid protein precursor (CP), which undergoes autoproteolytic cleavage during maturation. The molecular mechanisms of capsid assembly and maturation are well understood, but little is known about the viral infectious lifecycle due to a lack of tissue culture cell lines that are susceptible to tetravirus infection. We show here that binding and entry of the alphatetravirus, Helicoverpa armigera stunt virus (HaSV), is triggered by alkaline pH. At pH 9.0, wild-type HaSV virus particles undergo conformational changes that induce membrane-lytic activity and binding to Spodoptera frugiperda Sf9 cells. Binding is followed by entry and infection, with virus replication complexes detected by immunofluorescence microscopy within 2h post-infection and the CP after 12h. HaSV particles produced in S. frugiperda Sf9 cells are infectious. Helicoverpa armigera larval virus biofeed assays showed that pre-treatment with the V-ATPase inhibitor, Bafilomycin A1, resulted in a 50% decrease in larval mortality and stunting, while incubation of virus particles at pH 9.0 prior to infection restored infectivity. Together, these data show that HaSV, and likely other tetraviruses, requires the alkaline environment of the lepidopteran larval midgut for binding and entry into host cells.

Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication.

UNLABELLED: Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCE: It is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

Computed Tomography Artifact Created by Air in the X-ray Tube Oil.

A subtle artifact of patchy hypodensities in computed tomography images of the head mimicked acute or subacute cerebral infarct. The cause of the artifact was air in the oil of the x-ray tube. The artifact manifested only when the acquisition parameters included a rotation time of 0.5 second and a gantry tilt angle of 11 to 20 degrees. Routine quality control testing did not detect nonuniformities in the water phantom.

An encapsidated viral protein and its role in RNA packaging by a non-enveloped animal RNA virus.

Alphatetraviruses are small (+) ssRNA viruses with non-enveloped, icosahedral, T=4 particles that assemble from 240 copies of a single capsid protein precursor. This study is focused on the mechanisms underlying selection and packaging of genomic vRNAs by Helicoverpa armigera stunt virus. We demonstrate that the viral protein, p17, is packaged at low levels (between 4 and 8 copies per capsid) raising the possibility of icosahedral asymmetry in wild-type particles. p17 promotes packaging of vRNA2 by virus-like particles (VLPs) generated from plasmid-expressed vRNA2. The 5' and 3' UTRs of RNA2 are not required for encapsidation. VLPs produced by recombinant baculoviruses package vRNA2 at detectable levels even in the absence of p17 and apparently excluding baculoviral transcripts. This suggests a role for p17 in vRNA selectivity. This is one of few examples of the packaging of a minor non-structural protein by (+) ssRNA animal viruses.

Cytoplasmic granule formation and translational inhibition of nodaviral RNAs in the absence of the double-stranded RNA binding protein B2.

Flock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3' end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.

Providence virus (family: Carmotetraviridae) replicates vRNA in association with the Golgi apparatus and secretory vesicles.

Providence virus (PrV) is the sole member of the family Carmotetraviridae (formerly Tetraviridae) sharing the characteristic T=4 capsid architecture with other tetravirus families. Despite significant structural similarities, PrV differs from other tetraviruses in terms of genome organization, non-structural protein sequence and regulation of gene expression. In addition, it is the only tetravirus that infects tissue culture cells. Previous studies showed that in persistently infected Helicoverpa zea MG8 cells, the PrV replicase associates with detergent-resistant membranes in punctate cytosolic structures, which is similar to the distribution of an alpha-like tetravirus replicase (Helicoverpa armigera stunt virus). Here, we demonstrate that the site of PrV vRNA replication coincides with the presence of PrV p40/p104 proteins in infected cells and that these replication proteins associate with the Golgi apparatus and secretory vesicles in transfected cells.

Membrane targeting of an alpha-like tetravirus replicase is directed by a region within the RNA-dependent RNA polymerase domain.

The members of the family Tetraviridae are small positive-sense insect RNA viruses that exhibit stringent host specificity and a high degree of tissue tropism, suggesting that complex virus-host interactions are likely to occur during infection and viral replication. The alpha-like replicase of Helicoverpa armigera stunt virus (HaSV) (genus Omegatetravirus) has been proposed to associate with membranes of the endocytic pathway, which is similar to Semliki Forest virus, Sindbis virus and rubella virus. Here, we have used replicase-EGFP fusion proteins and recombinant baculovirus expression to demonstrate that the HaSV replicase associates strongly with cellular membranes, including detergent-resistant membranes, and that this association is maintained through a novel membrane targeting domain within the C-terminal region of the RNA-dependent RNA polymerase domain. We show a similar subcellular localization and strong association with detergent-resistant membranes for the carmo-like replicase of another tetravirus, Providence virus, in replicating cells, suggesting a common site of replication for these two tetraviruses.

Subcellular localization and live-cell imaging of the Helicoverpa armigera stunt virus replicase in mammalian and Spodoptera frugiperda cells.

Whilst their structure has been well studied, there is little information on the replication biology of tetraviruses because of the lack of suitable tissue-culture cell lines that support virus replication. In this study, the potential site of Helicoverpa armigera stunt virus replication was investigated by transient expression of the replicase protein fused to enhanced green fluorescent protein (EGFP) in mammalian and insect cells. When EGFP was present at the C terminus of the protein, fluorescence was located in punctate cytoplasmic structures that were distinct from the peripheral Golgi, endoplasmic reticulum, early endosomes, lysosomes and mitochondria, but overlapped partially with late endosomes. In experiments where targeting to endosomal compartments was examined further by using Cascade Blue-dextran in live cells, no overlap between the replicase and active endocytic organelles was apparent. Analysis of the punctate structures using time-lapse imaging in live cells revealed that they undergo fusion, fission and 'kiss-and-run' events. Whilst the source of the membranes used to form the punctate structures remains unclear, we propose that the replicase sequesters membranes from the late endosomes and actively excludes host proteins, either by normal recycling processes or by a replicase-dependent mechanism that may result in the destabilization of the associated membranes and a release of luminal contents into the cytosol. This is the first study describing the localization of a tetravirus.

The technique of pointing in the fabrication of a dental prosthesis.

This article describes a technique that makes it possible to copy the anatomy of teeth when fabricating a dental prosthesis. This technique, called pointing, has been used in sculpting since the time of the ancient Greeks. Pointing makes it possible to copy a predetermined model of the dentition accurately, saving time and aiding communication between dentist and laboratory technician.

Posterior reversible encephalopathy syndrome: incidence of atypical regions of involvement and imaging findings.

OBJECTIVE: Posterior reversible encephalopathy syndrome (PRES) is classically characterized as symmetric parietooccipital edema but may occur in other distributions with varying imaging appearances. This study determines the incidence of atypical and typical regions of involvement and unusual imaging manifestations. MATERIALS AND METHODS: Seventy-six patients were eventually included as having confirmed PRES from 111 initially suspected cases, per imaging and clinical follow-up. Two neuroradiologists retrospectively reviewed each MR image. Standard sequences were unenhanced FLAIR and T1- and T2-weighted images in all patients, with diffusion-weighted imaging (n = 75) and contrast-enhanced T1-weighted imaging (n = 69) in most. The regions involved were recorded on the basis of FLAIR findings, and the presence of atypical imaging findings (contrast enhancement, restricted diffusion, hemorrhage) was correlated with the severity (extent) of hyperintensity or mass effect on FLAIR. RESULTS: The incidence of regions of involvement was parietooccipital, 98.7%; posterior frontal, 78.9%; temporal, 68.4%; thalamus, 30.3%; cerebellum, 34.2%; brainstem, 18.4%; and basal ganglia, 11.8%. The incidence of less common manifestations was enhancement, 37.7%; restricted diffusion, 17.3%; hemorrhage, 17.1%; and a newly described unilateral variant, 2.6%. Poor correlation was found between edema severity and enhancement (r = 0.072), restricted diffusion (r = 0.271), hemorrhage (r = 0.267), blood pressure (systolic, r = 0.13; diastolic, r = 0.02). Potentially new PRES causes included contrast-related anaphylaxis and alcohol withdrawal. CONCLUSION: This large series of PRES cases shows that atypical distributions and imaging manifestations of PRES have a higher incidence than commonly perceived, and atypical manifestations do not correlate well with the edema severity.

Angiographic frequency of blunt cerebrovascular injury in patients with carotid canal or vertebral foramen fractures on multidetector CT.

PURPOSE: Blunt carotid injuries (BCI's) and blunt vertebral artery injuries (BVI's), known jointly as BCVI's, are common in "high risk" patients. The purpose is to evaluate the rate of occurrence of BCI/BVI in patients screened purely by the radiologic criteria of fracture through the carotid canal or vertebral transverse foramina, or significant cervical subluxation, noted by multidetector CT. METHODS: Seventy-one patients with 108 catheterized vessels were included over a 13-month interval. The angiographic examinations were prompted by current hospital protocol, solely by the presence of fractures involving/adjacent to the carotid canal, cervical fractures involving/adjacent to the foramen transversarium, or cervical fractures with significant subluxation. The incidence of each grade of blunt injury was calculated after review of the CT scans and catheter angiograms by two neuroradiologists. RESULTS: Two thousand and seventy-three total blunt trauma admissions occurred during the time period, with a BCVI rate of 0.92-1.0% (depending on the reviewer), similar to previous studies. Mean time to catheter angiography was 16.6 h. Of the 71 included patients, there were 11-12 BCI's and 10-12 BVI's, an overall rate of 27-30% of BCVI in the patients with foraminal fractures. Interobserver agreement in reviewing the catheter angiograms was excellent (Kappa 0.795). Of note, three internal carotid pseudoaneurysms resolved spontaneously after anticoagulation or aspirin. CONCLUSION: This study confirms that there is a high rate of BCVI in the presence of carotid canal or vertebral foramen fractures that are noted by multidetector CT. Utilization of purely radiologic criteria of foraminal involvement may be a significant screening tool in the decision of whether to evaluate these patients acutely by catheter or CT angiography, and for early detection of patients at risk for symptomatology, to initiate prompt, prophylactic treatment.

Utility of fat-suppressed FLAIR and subtraction imaging in detecting meningeal abnormalities.

We describe the use of a combination of fat-suppression SPIR (spectral inversion recovery) and subtraction FLAIR imaging to aid in detection of abnormal meningeal enhancement.

Transalar encephalocele associated with Wegener granulomatosis and meningeal enhancement: case report.

Transalar encephaloceles are rare lesions that do not fit the standard classification of basal encephaloceles. Typically, these lesions present in adulthood, with nonspecific symptoms. We report here a case of a patient with Wegener disease in whom a large transalar encephalocele posterior to the sinus was noted when he was preoperative for left maxillary sinus surgery. The encephalocele demonstrated irregular peripheral enhancement along the margin--a very uncommon finding--as well as contrast enhancement of the basal meninges, which can be seen occasionally with Wegener granulomatosis.