Pediatric Eosinophilia: A Review and Multiyear Investigation into Etiologies.

OBJECTIVES: To identify the etiology of peripheral eosinophilia in a large pediatric population and to develop a diagnostic algorithm to help guide diagnosis and management of peripheral eosinophilia in the outpatient pediatric population. STUDY DESIGN: We performed a retrospective chart review of children presenting to Texas Children's Hospital in Houston with peripheral eosinophilia between January 1, 2011 and December 31, 2019. Eosinophilia was classified as mild (absolute eosinophil count [AEC] >500 and <1500 cells/µL), moderate (AEC >1500 and <4500 cells/µL), or severe (AEC >4500 cells/µL). Demographic information and diagnostic workup data were collected. RESULTS: A total of 771 patients aged <18 years were evaluated. The most common cause of eosinophilia was allergy (n = 357; 46%), with atopy (n = 296) and drug reaction (n = 54) the most common subcauses. This was followed by unknown etiology (n = 274; 36%), infectious causes (n = 72; 9%), and eosinophilic disorders (n = 47; 6%). Many patients with an unknown cause (n = 202; 74%) had limited or no follow-up testing. CONCLUSIONS: More information on the etiology of pediatric eosinophilia and workup data could help identify the causes. This study provides important information on the evaluation of eosinophilia in the US pediatric population, including a diagnostic algorithm to guide primary care pediatricians.

Visualizing virus assembly intermediates inside marine cyanobacteria.

Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography.

Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer.

Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.

A tail-like assembly at the portal vertex in intact herpes simplex type-1 virions.

Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of the genome packaging and release machinery in the capsids of these large and complex double-stranded DNA viruses. However, such machinery is well characterized in tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In tailed bacteriophage, the genome exits from the virus particle through a portal and is transferred into the host cell by a complex apparatus (i.e. the tail) located at the portal vertex. Here we use electron cryo-tomography of human herpes simplex type-1 (HSV-1) virions to reveal a previously unsuspected feature at the portal vertex, which extends across the HSV-1 tegument layer to form a connection between the capsid and the viral membrane. The location of this assembly suggests that it plays a role in genome release into the nucleus and is also important for virion architecture.

Outcomes and Complications of Pediatric Eustachian Tube Dilation Surgery.

OBJECTIVE: To determine whether balloon dilation of Eustachian tube (BDET) improves postoperative audiology and quality of life scores in children with chronic Eustachian tube dysfunction. STUDY DESIGN: Retrospective study. SETTING: Tertiary care pediatric center. METHODS: Eligible participants were patients 8 years or older, with a history of 2 prior tubes placement. Group 1-patients completed pre-and post-Eustachian Tube Dysfunction Quality of Life Survey (ETDQ-7) survey scores, Group 2-patients had available pre- and postdilation tympanogram data (TD), and Group 3-patients had both ETDQ-7 survey and TD. The average time for the first and subsequent follow-ups was 3.8 and 12.9 months, respectively. RESULTS: A total of 43 patients (85 ears) underwent BDET. The mean age was 13.3 years (8-18 years). Twenty-four patients were male (55.8%) and over 80% were Caucasian. The average mean ETDQ-7 score before and after dilation was 3.9 and 2.5, respectively. Ninety-three percent experienced improvement of their postoperative ETDQ-7 scores and 53% had normal postdilation ETDQ-7 score (P < .0001). Thirty-seven ears in Group 2 (60.7%) had improvement in postdilation TD. A greater proportion of ears showed improvement of 62.3% with a 95% confidence interval (CI) [50.1%-74.5%] compared to 37.7% without improvement, 95% CI [25.5%-49.87%]. Ears with type A or B TD were more likely to show improvement than ears with type C, perforated, or with tubes (P < .0001). Eighteen out of 30 ears in Group 3 (60%) experienced an improvement in both ETDQ-7 and tympanogram. CONCLUSION: BDET is a safe, efficacious alternative to tubes in selected pediatric patients.

Utility of Dried Blood Spots for the Diagnosis of Congenital Cytomegaloviruses within the First 21 Days of Life in a Single Center.

In this retrospective study, we aimed to evaluate the performance of dried-blood-spot (DBS) testing as a diagnostic method for the congenital cytomegalovirus (cCMV). We reviewed the medical records and DBS test results of 89 patients who had also undergone diagnostic cCMV testing within the first 21 days of life. The DBS test had a sensitivity of 83.9%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 73%. Patients with a true-positive DBS had a higher median level of CMV in blood according to PCR than those with a false-negative result. Additionally, all patients with cCMV and hearing loss had a positive DBS test, with higher median viremia levels observed in those with hearing loss compared to those without a CMV PCR blood test. These results suggest that DBS-based testing is useful in the diagnosis of cCMV, and its performance may be related to levels of CMV viremia. DBS testing accurately identified those patients with congenital/early onset hearing loss and those at risk of developing late-onset hearing loss.

Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency.

One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron microscope. Direct electron detection using CMOS-based sensors for digitally recording images has the potential to alleviate this shortcoming. Here, we report a practical performance evaluation of a Direct Detection Device (DDD®) for biological cryo-EM at two different microscope voltages: 200 and 300 kV. Our DDD images of amorphous and graphitized carbon show strong per-pixel contrast with image resolution near the theoretical sampling limit of the data. Single-particle reconstructions of two frozen-hydrated bacteriophages, P22 and ε15, establish that the DDD is capable of recording usable signal for 3D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the performance of conventional imaging media. We anticipate the unparalleled performance of this digital recording device will dramatically benefit cryo-EM for routine tomographic and single-particle structural determination of biological specimens.

Reconstructing virus structures from nanometer to near-atomic resolutions with cryo-electron microscopy and tomography.

The past few decades have seen tremendous advances in single-particle electron -cryo-microscopy (cryo-EM). The field has matured to the point that near-atomic resolution density maps can be generated for icosahedral viruses without the need for crystallization. In parallel, substantial progress has been made in determining the structures of nonicosahedrally arranged proteins in viruses by employing either single-particle cryo-EM or cryo-electron tomography (cryo-ET). Implicit in this course have been the availability of a new generation of electron cryo-microscopes and the development of the computational tools that are essential for generating these maps and models. This methodology has enabled structural biologists to analyze structures in increasing detail for virus particles that are in different morphogenetic states. Furthermore, electron imaging of frozen, hydrated cells, in the process of being infected by viruses, has also opened up a new avenue for studying virus structures "in situ". Here we present the common techniques used to acquire and process cryo-EM and cryo-ET data and discuss their implications for structural virology both now and in the future.

From Telemedicine to the ICU-Fever and Rash in a 9-Year-Old Girl.

A 9-year-old girl presented to her primary care pediatrician via telemedicine during the initial months of the coronavirus disease 2019 pandemic because of 4 days of warmth perceived by her mother, decreased energy, and a new rash on her upper extremities. After 10 additional days of documented fever >38°C, worsening fatigue, and 1 day of nausea, vomiting, and diarrhea, she was allowed to schedule an in-person visit with her pediatrician after testing negative for severe acute respiratory syndrome coronavirus 2. She appeared ill on arrival to clinic, and her pediatrician recommended evaluation in an emergency department. Her initial laboratory testing revealed nonspecific elevation in several inflammatory markers and leukopenia, and she responded well to intravenous hydration. Over the next 2 weeks, her fever persisted, constitutional symptoms worsened, and she developed progressively painful cervical lymphadenopathy and pancytopenia. She was evaluated in clinic by several specialists and eventually was urged to present to the emergency department again, at which time she was admitted to the PICU. After consulting additional specialists and waiting for laboratory results, the team reached a definitive diagnosis and initiated therapy; however, she experienced rapid clinical decline shortly thereafter. The specialists who assisted with identification of the underlying etiology of her symptoms were able to work together to manage the subsequent complications.

Lessons learned from an enterprise-wide clinical datathon.

In 2020, Baylor College of Medicine held a datathon to inform potential users of a new data warehouse, allow users to address clinical questions, identify warehouse capabilities and limitations, foster collaborations, and engage trainees. Senior faculty selected proposals based on feasibility and impact. Selectees worked with Information Technology for 2 months and presented findings. A survey of participants showed diverse levels of experience, high perceived value of the datathon, high rates of collaboration, and significant increases in knowledge. A datathon can promote familiarity with a new data warehouse, guide data warehouse improvement, and promote collaboration.

Practical performance evaluation of a 10k × 10k CCD for electron cryo-microscopy.

Electron cryo-microscopy (cryo-EM) images are commonly collected using either charge-coupled devices (CCD) or photographic film. Both film and the current generation of 16 megapixel (4k × 4k) CCD cameras have yielded high-resolution structures. Yet, despite the many advantages of CCD cameras, more than two times as many structures of biological macromolecules have been published in recent years using photographic film. The continued preference to film, especially for subnanometer-resolution structures, may be partially influenced by the finer sampling and larger effective specimen imaging area offered by film. Large format digital cameras may finally allow them to overtake film as the preferred detector for cryo-EM. We have evaluated a 111-megapixel (10k × 10k) CCD camera with a 9 µm pixel size. The spectral signal-to-noise ratios of low dose images of carbon film indicate that this detector is capable of providing signal up to at least 2/5 Nyquist frequency potentially retrievable for 3D reconstructions of biological specimens, resulting in more than double the effective specimen imaging area of existing 4k × 4k CCD cameras. We verified our estimates using frozen-hydrated ε15 bacteriophage as a biological test specimen with previously determined structure, yielding a ∼7 Å resolution single particle reconstruction from only 80 CCD frames. Finally, we explored the limits of current CCD technology by comparing the performance of this detector to various CCD cameras used for recording data yielding subnanometer resolution cryo-EM structures submitted to the electron microscopy data bank (http://www.emdatabank.org/).

A matching algorithm to address imbalances in study populations: application to the National Institute of Neurological Diseases and Stroke Recombinant Tissue Plasminogen Activator acute stroke trial.

BACKGROUND AND PURPOSE: Outcome from stroke is highly dependent on baseline conditions. Patients with stroke have a wide range of severities, ages, and etiologies and it has proven difficult to achieve randomization of key variables in clinical trials. We present a new post hoc approach to achieve balance among selected variables. To illustrate the approach, we rebalanced the National Institute of Neurological Diseases and Stroke Recombinant Tissue Plasminogen Activator trial, in which the contribution of baseline imbalances continues to be debated. METHODS: We selected baseline stroke severity (National Institutes of Health Stroke Scale), age, and glucose as matching criteria. The closest matched placebo and treated subjects were identified based on nearness to each other in 3-dimensional Euclidean space. Matching was performed within the quintiles of National Institutes of Health Stroke Scale that have been previously used to assess balance. Subjects who could not be matched were eliminated. Outcomes were assessed using the original specified National Institute of Neurological Diseases and Stroke trial measures. RESULTS: We successfully matched the 2 arms resulting in nearly identical baseline characteristics and distribution among quintiles. Despite fewer subjects after outlier elimination, the primary outcome measures remained significantly improved. After rebalancing, the magnitude of benefit was reduced by 13% to 23%. Benefit was apparent mostly in the large vessel occlusion subtype. CONCLUSION: This study demonstrated the feasibility of rebalancing individual subjects within a randomized trial. After rebalancing and outlier elimination, recombinant tissue plasminogen activator continued to demonstrate improved outcome. That the apparent treatment effect was reduced suggests that imbalances contributed to the magnitude of the original National Institute of Neurological Diseases and Stroke outcomes. This method could in theory be applied to any data set to find matched subjects for outcome or other analyses.

Cryo-EM techniques to resolve the structure of HSV-1 capsid-associated components.

Electron cryo-microscopy has become a routine technique to determine the structure of biochemically purified herpes simplex virus capsid particles. This chapter describes the procedures of specimen preparation by cryopreservation; low dose and low temperature imaging in an electron cryo-microscope; and data processing for reconstruction. This methodology has yielded subnanometer resolution structures of the icosahedral capsid shell where α-helices and ß-sheets of individual subunits can be recognized. A relaxation of the symmetry in the reconstruction steps allows us to resolve the DNA packaging protein located at one of the 12 vertices in the capsid.