Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

On a sugar high: Role of O-GlcNAcylation in cancer.

Recent advances in the understanding of the molecular mechanisms underlying cancer progression have led to the development of novel therapeutic targeting strategies. Aberrant glycosylation patterns and their implication in cancer have gained increasing attention as potential targets due to the critical role of glycosylation in regulating tumor-specific pathways that contribute to cancer cell survival, proliferation, and progression. A special type of glycosylation that has been gaining momentum in cancer research is the modification of nuclear, cytoplasmic, and mitochondrial proteins, termed O-GlcNAcylation. This protein modification is catalyzed by an enzyme called O-GlcNAc transferase (OGT), which uses the final product of the Hexosamine Biosynthetic Pathway (HBP) to connect altered nutrient availability to changes in cellular signaling that contribute to multiple aspects of tumor progression. Both O-GlcNAc and its enzyme OGT are highly elevated in cancer and fulfill the crucial role in regulating many hallmarks of cancer. In this review, we present and discuss the latest findings elucidating the involvement of OGT and O-GlcNAc in cancer.

Correction: Recombinant Lloviu virus as a tool to study viral replication and host responses.

[This corrects the article DOI: 10.1371/journal.ppat.1010268.].

Recombinant Lloviu virus as a tool to study viral replication and host responses.

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.

Cerebrospinal fluid immunoglobulins in primary progressive multiple sclerosis are pathogenic.

Multiple sclerosis is clinically characterized by relapses and remissions (relapsing-remitting multiple sclerosis) that over time may evolve to a progressive course (secondary progressive multiple sclerosis) or as having a progressive course from disease onset (primary progressive multiple sclerosis). At present, it is not definitively known whether these clinical entities constitute a single pathological disease or whether these manifestations represent two distinct disease entities sharing inflammatory demyelination as a pathological feature. Here we show using a novel mouse model that CSF of primary progressive multiple sclerosis patients is unique in its capacity to induce motor disability and spinal cord pathology including demyelination, impaired remyelination, reactive astrogliosis and axonal damage. Notably, removal of immunoglobulin G from primary progressive multiple sclerosis CSF via filtration or immunodepletion attenuates its pathogenic capacity. Furthermore, injection of recombinant antibodies derived from primary progressive multiple sclerosis CSF recapitulates the pathology. Our findings suggest that the clinical and pathological features of primary progressive multiple sclerosis are antibody-mediated and pathogenically distinct from relapsing-remitting and secondary progressive multiple sclerosis. Our study has potentially important implications for the development of specific therapies for patients with primary progressive multiple sclerosis.

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes.

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Covalent docking and molecular dynamics simulations reveal the specificity-shifting mutations Ala237Arg and Ala237Lys in TEM beta-lactamase.

The rate of modern drug discovery using experimental screening methods still lags behind the rate at which pathogens mutate, underscoring the need for fast and accurate predictive simulations of protein evolution. Multidrug-resistant bacteria evade our defenses by expressing a series of proteins, the most famous of which is the 29-kilodalton enzyme, TEM ß-lactamase. Considering these challenges, we applied a covalent docking heuristic to measure the effects of all possible alanine 237 substitutions in TEM due to this codon's importance for catalysis and effects on the binding affinities of commercially-available ß-lactam compounds. In addition to the usual mutations that reduce substrate binding due to steric hindrance, we identified two distinctive specificity-shifting TEM mutations, Ala237Arg and Ala237Lys, and their respective modes of action. Notably, we discovered and verified through minimum inhibitory concentration assays that, while these mutations and their bulkier side chains lead to steric clashes that curtail ampicillin binding, these same groups foster salt bridges with the negatively-charged side-chain of the cephalosporin cefixime, widely used in the clinic to treat multi-resistant bacterial infections. To measure the stability of these unexpected interactions, we used molecular dynamics simulations and found the binding modes to be stable despite the application of biasing forces. Finally, we found that both TEM mutants also bind strongly to other drugs containing negatively-charged R-groups, such as carumonam and ceftibuten. As with cefixime, this increased binding affinity stems from a salt bridge between the compounds' negative moieties and the positively-charged side chain of the arginine or lysine, suggesting a shared mechanism. In addition to reaffirming the power of using simulations as molecular microscopes, our results can guide the rational design of next-generation ß-lactam antibiotics and bring the community closer to retaking the lead against the recurrent threat of multidrug-resistant pathogens.

The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR.

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.

Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection.

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

Heterogeneity in Human Induced Pluripotent Stem Cell-derived Alveolar Epithelial Type II Cells Revealed with ABCA3/SFTPC Reporters.

Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPCtdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPCtdTomato expression. Comparing ABCA3:GFP/SFTPCtdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.

Topical Review: Assessment of Binocular Sensory Processes in Low Vision.

SIGNIFICANCE: This article summarizes the evidence for a higher prevalence of binocular vision dysfunctions in individuals with vision impairment. Assessment for and identification of binocular vision dysfunctions can detect individuals experiencing difficulties in activities including reading, object placement tasks, and mobility.Comprehensive vision assessment in low vision populations is necessary to identify the extent of remaining vision and to enable directed rehabilitation efforts. In patients with vision impairment, little attention is typically paid to assessments of binocular vision, including ocular vergence, stereopsis, and binocular summation characteristics. In addition, binocular measurements of threshold automated visual fields are not routinely performed in clinical practice, leading to an incomplete understanding of individuals' binocular visual field and may affect rehabilitation outcomes.First, this review summarizes the prevalence of dysfunctions in ocular vergence, stereopsis, and binocular summation characteristics across a variety of ocular pathologies causing vision impairment. Second, this review examines the links between clinical measurements of binocular visual functions and outcome measures including quality of life and performance in functional tasks. There is an increased prevalence of dysfunctions in ocular alignment, stereopsis, and binocular summation across low vision cohorts compared with those with normal vision. The identification of binocular vision dysfunctions during routine low vision assessments is especially important in patients experiencing difficulties in activities of daily living, including but not limited to reading, object placement tasks, and mobility. However, further research is required to determine whether addressing the identified deficits in binocular vision in low vision rehabilitative efforts directly impacts patient outcomes.

Clinical Trial: Diurnal IOP Fluctuations in Glaucoma Using Latanoprost and Timolol with Self-Tonometry.

SIGNIFICANCE: Assessment of treatment efficacy via comparison with a target IOP is fundamental in monitoring patients with open-angle glaucoma and ocular hypertension. This article highlights that diurnal IOP fluctuations obtained using self-tonometry may more accurately reflect IOP responses to therapy. PURPOSE: This study aimed to investigate fluctuations in diurnal IOP measurements in patients with open-angle glaucoma and ocular hypertension treated with latanoprost 0.005% and timolol 0.25%. METHODS: In this crossover treatment trial, 14 participants performed self-tonometry with iCare HOME 4 times daily for (1) 1 week using latanoprost, (2) 4 weeks using no medications, and (3) 2 weeks using timolol. Daily peak IOPs, IOP fluctuations, and mean IOPs from different treatments were compared on an individual basis. Treatment efficacy between medications was assessed by comparing mean percentage IOP reductions with latanoprost and timolol across participants. In addition, effects of age, years since commencing latanoprost, sex, and diagnosis were investigated, and peak IOP times were compared with assess impacts on diurnal profiles. RESULTS: Between individuals, IOP responses ranged from reductions in peak IOPs, IOP fluctuations, and mean IOPs on both medications to no change in any parameter and medication. IOP fluctuations showed greater mean percentage reductions than did peak and mean IOPs (χ2 = 16.51, P = .002). There were significant associations between years since commencing latanoprost and peak and mean IOP responses on timolol (r = 0.69, P = .007), and sex and relative reductions in IOP fluctuations on both medications (P = .03). There were no differences in peak IOP times between treatment conditions. CONCLUSIONS: Despite variability in IOP responses to latanoprost and timolol, IOP fluctuation with self-tonometry was more consistent in evaluating target IOP, reflecting its importance in ascertaining true IOP response to topical therapies. These findings may impact clinical decision making based on target IOP criteria in patients on topical therapy.

Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells.

Global DNA hydroxymethylation mediated by the TET (ten-eleven translocation) enzyme was induced in allergen-induced airway hyperresponsiveness in mouse lung tissues and specifically in isolated airway smooth muscle (ASM) cells. TET is an α-ketoglutarate (α-KG)-dependent enzyme, and the production of α-KG is catalyzed by IDH (isocitrate dehydrogenase). However, the role of IDH in the regulation of DNA hydroxymethylation in ASM cells is unknown. In comparison with nonasthmatic cells, asthmatic ASM cells exhibited higher TET activity and IDH2 (but not IDH-1 or IDH-3) gene expression levels. We modified the expression of IDH2 in ASM cells from humans with asthma by siRNA and examined the α-KG levels, TET activity, global DNA hydroxymethylation, cell proliferation, and expression of ASM phenotypic genes. Inhibition of IDH2 in asthmatic ASM cells decreased the α-KG levels, TET activity, and global DNA hydroxymethylation, and reversed the aberrant ASM phenotypes (including decreased cell proliferation and ASM phenotypic gene expression). Specifically, asthmatic cells transfected with siRNA against IDH2 showed decreased 5hmC (5-hydroxymethylcytosine) levels at the TGFB2 (transforming growth factor-ß2) promoter determined by oxidative bisulfite sequencing. Taken together, our findings reveal that IDH2 plays an important role in the epigenetic regulation of ASM phenotypic changes in asthmatic ASM cells, suggesting that IDH2 is a potential therapeutic target for reversing the abnormal phenotypes seen in asthma.

Determining Significant Elevation of Intraocular Pressure Using Self-tonometry.

SIGNIFICANCE: Icare HOME rebound tonometry is increasingly adopted into clinical practice for IOP phasing of glaucoma patients and suspects. Because of measurement differences with applanation tonometry and diurnal fluctuations, interpretation of the IOP measured with Icare HOME phasing can be challenging. PURPOSE: The purpose of this study was to use a large patient cohort to develop a practical, analytical tool for interpreting Icare HOME measurements with respect to applanation pressure. METHODS: IOP measurements using the Icare HOME and an applanation tonometer were taken prospectively in 498 consecutive patients. Bland-Altman, frequency distribution, and linear regression analysis were applied to determine measurement differences. A novel criterion, Threshold Icare HOME IOP, was developed to assist identification of elevation above target applanation pressure, considering the expected diurnal variation and measurement variability. RESULTS: Icare HOME tended to underestimate applanation tonometry (mean bias, -1.7 mmHg; 95% limits of agreement, -7.0 to +3.6). Overall, differences were within ±3 mmHg in 71.5% and ±5 mmHg in 92% of patients. Based on the novel criterion developed, Icare HOME measurements that exceed target applanation pressure by 6 mmHg or greater are generally outside the 95% limit of expected observations. CONCLUSIONS: The Threshold Icare HOME IOP is a novel and practical criterion that can assist clinicians in their interpretation of Icare HOME phasing measurements with respect to target applanation pressures. Elevation above the expected thresholds may prompt closer monitoring or even modifications to glaucoma management.

Evaluation of a commercial deformable image registration algorithm for dual-energy CT processing.

PURPOSE: Several dual-energy computed tomography (DECT) techniques require a deformable image registration to correct for motion between the acquisition of low and high energy data. However, current DECT software does not provide tools to assess registration accuracy or allow the user to export deformed images, presenting a unique challenge for image registration quality assurance (QA). This work presents a methodology to evaluate the accuracy of DECT deformable registration and to quantify the impact of registration errors on end-product images. METHODS: The deformable algorithm implemented in Siemen Healthineers's Syngo was evaluated using a deformable abdomen phantom and a rigid phantom to mimic sliding motion in the thorax. Both phantoms were imaged using sequential 80 and 140 kVp scans with motion applied between the two scans. Since Syngo does not allow the export of the deformed images, this study focused on quantifying the accuracy of various end-product, dual-energy images resulting from processing of deformed images. RESULTS: The Syngo algorithm performed well for the abdomen phantom with a mean registration error of 0.4 mm for landmark analysis, Dice similarity coefficients (DSCs) > 0.90 for five organs contoured, and mean iodine concentrations within 0.2 mg/mL of values measured on static images. For rigid sliding motion, the algorithm performed poorer and resulted in noticeable registration errors toward the superior and inferior scan extents and DSCs as low as 0.41 for iodine rods imaged in the phantom. Additionally, local iodine concentration errors in areas of misregistration exceeded 3 mg/mL. CONCLUSIONS: This work represents the first methodology for DECT image registration QA using commercial software. Our data support the clinical use of the Syngo algorithm for abdominal sites with limited motion (i.e., pancreas and liver). However, dual-energy images generated with this algorithm should be used with caution for quantitative measurements in areas with sliding motion.

Investigating split-filter dual-energy CT for improving liver tumor visibility for radiation therapy.

PURPOSE: Accurate liver tumor delineation is crucial for radiation therapy, but liver tumor volumes are difficult to visualize with conventional single-energy CT. This work investigates the use of split-filter dual-energy CT (DECT) for liver tumor visibility by quantifying contrast and contrast-to-noise ratio (CNR). METHODS: Split-filter DECT contrast-enhanced scans of 20 liver tumors including cholangiocarcinomas, hepatocellular carcinomas, and liver metastases were acquired. Analysis was performed on the arterial and venous phases of mixed 120 kVp-equivalent images and VMIs at 57 keV and 40 keV gross target volume (GTV) contrast and CNR were calculated. RESULTS: For the arterial phase, liver GTV contrast was 12.1 ± 10.0 HU and 43.1 ± 32.3 HU (P < 0.001) for the mixed images and 40 keV VMIs. Image noise increased on average by 116% for the 40 keV VMIs compared to the mixed images. The average CNR did not change significantly (1.6 ± 1.5, 1.7 ± 1.4, 2.4 ± 1.7 for the mixed, 57 keV and 40 keV VMIs (P > 0.141)). For individual cases, however, CNR increases of up to 607% were measured for the 40 keV VMIs compared to the mixed image. Venous phase 40 keV VMIs demonstrated an average increase of 35.4 HU in GTV contrast and 121% increase in image noise. Average CNR values were also not statistically different, but for individual cases CNR increases of up to 554% were measured for the 40 keV VMIs compared to the mixed image. CONCLUSIONS: Liver tumor contrast was significantly improved using split-filter DECT 40 keV VMIs compared to mixed images. On average, there was no statistical difference in CNR between the mixed images and VMIs, but for individual cases, CNR was greatly increased for the 57 keV and 40 keV VMIs. Therefore, although not universally successful for our patient cohort, split-filter DECT VMIs may provide substantial gains in tumor visibility of certain liver cases for radiation therapy treatment planning.

Evaluation of a commercial Monte Carlo dose calculation algorithm for electron treatment planning.

The RayStation treatment planning system implements a Monte Carlo (MC) algorithm for electron dose calculations. For a TrueBeam accelerator, beam modeling was performed for four electron energies (6, 9, 12, and 15 MeV), and the dose calculation accuracy was tested for a range of geometries. The suite of validation tests included those tests recommended by AAPM's Medical Physics Practice Guideline 5.a, but extended beyond these tests in order to validate the MC algorithm in more challenging geometries. For MPPG 5.a testing, calculation accuracy was evaluated for square cutouts of various sizes, two custom cutout shapes, oblique incidence, and heterogenous media (cork). In general, agreement between ion chamber measurements and RayStation dose calculations was excellent and well within suggested tolerance limits. However, this testing did reveal calculation errors for the output of small cutouts. Of the 312 output factors evaluated for square cutouts, 20 (6.4%) were outside of 3% and 5 (1.6%) were outside of 5%, with these larger errors generally being for the smallest cutout sizes within a given applicator. Adjustment of beam modeling parameters did not fix these calculation errors, nor does the planning software allow the user to input correction factors as a function of field size. Additional validation tests included several complex phantom geometries (triangular nose phantom, lung phantom, curved breast phantom, and cortical bone phantom), designed to test the ability of the algorithm to handle high density heterogeneities and irregular surface contours. In comparison to measurements with radiochromic film, RayStation showed good agreement, with an average of 89.3% pixels passing for gamma analysis (3%/3mm) across four phantom geometries. The MC algorithm was able to accurately handle the presence of irregular surface contours (curved cylindrical phantom and a triangular nose phantom), as well as heterogeneities (cork and cortical bone).

Diurnal Intraocular Pressure Fluctuations with Self-tonometry in Glaucoma Patients and Suspects: A Clinical Trial.

SIGNIFICANCE: This article shows that self-tonometry can provide robust measures of diurnal intraocular pressure (IOP) and also detect changes to IOP in response to treatment within a short period of monitoring. These advances in IOP monitoring may contribute to improved management of glaucoma patients and suspects. PURPOSE: The aim of this study was to prospectively investigate the utility of rebound self-tonometry performed over several weeks for detecting diurnal IOP fluctuations in glaucoma patients and suspects and also initial response to topical treatment in glaucoma patients. METHODS: Forty patients were recruited following glaucoma-specific examination. Subsequent to successful training with the iCare HOME tonometer, patients were instructed to measure IOP, in a sitting position, four times a day over 4 to 6 weeks. Date, time, laterality, and IOP downloaded from the tonometer and clinical examination data, including applanation IOP and corneal thickness, were analyzed. A user satisfaction survey was also administered at study completion. t Test and analysis of variance were used to compare groups and IOP across days. Pearson correlation was used to compare measurements to Goldmann applanation tonometry and IOP measurements from the first day/s to the overall mean IOP. RESULTS: Twenty-seven patients (18 suspects and 9 glaucoma patients) completed data collection. Patients self-measured IOP on 118 (±29) occasions for 40 (±7.4) days. Two dominant patterns of fluctuation were revealed: peak IOP upon awakening (n = 11) and at midday (n = 13). Diurnal IOP measured in the first 7 days showed strong correlation to diurnal IOP across the entire study period (r = 0.82, P < .0001). Within 24 hours of treatment commencement (latanoprost 0.005% ophthalmic solution), IOP reduced from 23.9 (±5.2) to 16.1 (±2.6) mmHg. Overall, patients rated the instrument as easy to use, although difficulties with correct alignment were expressed. CONCLUSIONS: Rebound self-tonometry demonstrated utility for measuring diurnal IOP fluctuations in most patients, hence enhancing management of patient with or at risk of developing glaucoma.