Two RNA binding proteins, ADAD2 and RNF17, interact to form a heterogeneous population of novel meiotic germ cell granules with developmentally dependent organelle association.

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells. This work aimed to understand the role of ADAD2 granules in male germ cell differentiation by clearly defining their molecular composition and relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutants identified a rare post-meiotic chromatin defect, suggesting shared biological roles. ADAD2 and RNF17 were found to be dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs and organelle-specific markers, a subset of the ADAD2-RNF17 granules are found to be associated with the intermitochondrial cement and piRNA biogenesis. In contrast, a second, morphologically distinct population of ADAD2-RNF17 granules co-localized with the translation regulators NANOS1 and PUM1, along with the molecular chaperone PDI. These large granules form a unique funnel-shaped structure that displays distinct protein subdomains and is tightly associated with the endoplasmic reticulum. Developmental studies suggest the different granule populations represent different phases of a granule maturation process. Lastly, a double Adad2-Rnf17 mutant model suggests the interaction between ADAD2 and RNF17, as opposed to loss of either, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. These findings shed light on the relationship between germ cell granule pools and define new genetic approaches to their study.

ADAD2 regulates heterochromatin in meiotic and post-meiotic male germ cells via translation of MDC1.

Male germ cells establish a unique heterochromatin domain, the XY-body, early in meiosis. How this domain is maintained through the end of meiosis and into post-meiotic germ cell differentiation is poorly understood. ADAD2 is a late meiotic male germ cell-specific RNA-binding protein, loss of which leads to post-meiotic germ cell defects. Analysis of ribosome association in Adad2 mouse mutants revealed defective translation of Mdc1, a key regulator of XY-body formation, late in meiosis. As a result, Adad2 mutants show normal establishment but failed maintenance of the XY-body. Observed XY-body defects are concurrent with abnormal autosomal heterochromatin and ultimately lead to severely perturbed post-meiotic germ cell heterochromatin and cell death. These findings highlight the requirement of ADAD2 for Mdc1 translation, the role of MDC1 in maintaining meiotic male germ cell heterochromatin and the importance of late meiotic heterochromatin for normal post-meiotic germ cell differentiation.

A comprehensive assessment of the prolonged febrile neutropenia evaluation in pediatric oncology patients.

BACKGROUND: Pediatric oncology patients with prolonged (≥96 hours) febrile neutropenia (absolute neutrophil count < 500/µL) often undergo an evaluation for invasive fungal disease (IFD) and other infections. Current literature suggests that beta-D-glucan (BDG), galactomannan, bronchoalveolar lavage (BAL), and computed tomography (CT) scans (sinus, chest, and abdomen/pelvis) may help determine a diagnosis in this population. METHODS: In a retrospective cohort study of all cancer/stem cell transplant patients (diagnosed 2005-2019) from one pediatric hospital, all episodes with prolonged febrile neutropenia or IFD evaluations (defined as sending a fungal biomarker or performing a CT scan to assess for infection) were identified. RESULTS: In total, 503 episodes met inclusion criteria and 64% underwent IFD evaluations. In total, 36.4% of episodes documented an infection after initiation of prolonged febrile evaluation, most commonly Clostridioides difficile colitis (6.4%) followed by a true bacterial bloodstream infection (BSI) (5.2%), proven/probable IFD (4.8%), and positive respiratory pathogen panel (3.6%). There was no difference in sinus CTs showing sinusitis (74% vs 63%, p = 0.46), whereas 32% of abdomen/pelvis CTs led to a non-IFD diagnosis, and 25% of chest CTs showed possible pneumonia. On chest CT, the positive predictive value (PPV) for IFD was 19% for nodules and 14% for tree and bud lesions. BDG had a PPV of 25% for IFD and GM 50%. BAL diagnosed IFD once and pneumocystis jirovecii pneumonia twice. CONCLUSIONS: Chest CTs and abdomen/pelvis CTs provide clinically relevant information during the prolonged febrile neutropenia evaluation, whereas BDG, galactomannan, BAL, and sinus CTs have less certain utility.

Spontaneous splenic rupture in a neonate: a case report and literature review.

Splenic rupture in a neonate is a rare but potentially fatal condition that may trigger evaluation for child abuse. It is a diagnosis of exclusion that has been reported in the surgical literature but may be underrecognized by pediatric radiologists. We report a case of a newborn with an unremarkable prenatal, delivery, and nursery course who presented with anemia, abdominal distension, and lethargy. Abdominal ultrasound with Doppler and computed tomography (CT) of the head, cervical spine, chest, abdomen, and pelvis without contrast showed findings of splenic rupture and anoxic brain injury. An extensive workup for traumatic, infectious, coagulopathic, and congenital etiologies was unrevealing, leading to a presumptive diagnosis of spontaneous splenic rupture in a neonate.

Perinatal intermittent hypoxia increases early susceptibility to ANG II-induced hypertension in adult male but not in female Sprague-Dawley rats.

Prenatal, perinatal, and adulthood exposure to chronic intermittent hypoxia (IH) increases blood pressure in rodents. Males exposed to chronic IH have higher blood pressure versus females. However, it is unknown if this same-sex difference exists with acute perinatal IH. We tested the hypothesis that acute perinatal IH increases baseline blood pressure and enhances sensitivity to angiotensin II (ANG II)-induced hypertension in male Sprague-Dawley rats. Male and female pups were randomized to control (room air) or IH (10 min of ∼10% O2 for 3 times/day) for the first 8 days of life. IH decreased oxygen saturation, as confirmed via a pulse oximeter. Pups were weaned at postnatal day 21. Blood pressure was measured via telemetry beginning at 14 wk of age and analyzed separately into light and dark phases to assess circadian rhythm. Osmotic minipumps to deliver ANG II were implanted at 15 wk of age. Perinatal IH exposure did not alter baseline blood pressure. One week of ANG II treatment increased blood pressure in light and dark periods in males exposed to IH versus control; there was no effect in females. Blood pressure among the groups was comparable following 2 wk of ANG II infusion. Perinatal IH did not change the circadian rhythm. Following ANG II treatment, indexes of renal injury were measured. Perinatal IH did not alter kidney size, structure, nephron number, or creatinine clearance. These data indicate that acute perinatal IH enhances early ANG II-induced hypertension in males, independent of nephron loss or decreases in body weight or kidney function.NEW & NOTEWORTHY The impact of acute intermittent hypoxia (IH) in early life on blood pressure in adulthood is unknown. This study used a new model exposing female and male rat pups to acute IH in the first 8 days of life, without exposing the dam. Although baseline blood pressure was not altered in adulthood, IH increased susceptibility to angiotensin II hypertension only in males, supporting increased susceptibility of males exposed to IH to a second cardiovascular stressor.

ADAD1 is required for normal translation of nuclear pore and transport protein transcripts in spermatids of Mus musculus†.

ADAD1 is a testis-specific RNA-binding protein expressed in post-meiotic spermatids whose loss leads to defective sperm and male infertility. However, the drivers of the Adad1 phenotype remain unclear. Morphological and functional analysis of Adad1 mutant sperm showed defective DNA compaction, abnormal head shaping, and reduced motility. Mutant testes demonstrated minimal transcriptome changes; however, ribosome association of many transcripts was reduced, suggesting ADAD1 may be required for their translational activation. Further, immunofluorescence of proteins encoded by select transcripts showed delayed protein accumulation. Additional analyses demonstrated impaired subcellular localization of multiple proteins, suggesting protein transport is also abnormal in Adad1 mutants. To clarify the mechanism giving rise to this, the manchette, a protein transport microtubule network, and the LINC (linker of nucleoskeleton and cytoskeleton) complex, which connects the manchette to the nuclear lamin, were assessed across spermatid development. Proteins of both displayed delayed translation and/or localization in mutant spermatids implicating ADAD1 in their regulation, even in the absence of altered ribosome association. Finally, ADAD1's impact on the NPC (nuclear pore complex), a regulator of both the manchette and the LINC complex, was examined. Reduced ribosome association of NPC encoding transcripts and reduced NPC protein abundance along with abnormal localization in Adad1 mutants confirmed ADAD1's impact on translation is required for a NPC in post-meiotic germ cells. Together, these studies lead to a model whereby ADAD1's influence on nuclear transport leads to deregulation of the LINC complex and the manchette, ultimately generating the range of physiological defects observed in the Adad1 phenotype.

Complications of Cancer Therapy in Children: A Comprehensive Review of Neuroimaging Findings.

ABSTRACT: Complications of cancer therapy in children can result in a spectrum of neurologic toxicities that may occur at the initiation of therapy or months to years after treatment. Although childhood cancer remains rare, increasing survival rates mean that more children will be living longer after cancer treatment. Therefore, complications of cancer therapy will most likely occur with increasing frequency.At times, it is very difficult to differentiate between therapeutic complications and other entities such as tumor recurrence, development of secondary malignancy, and infection (among other conditions). Radiologists often play a key role in the diagnosis and evaluation of pediatric patients with malignancies, and thus, awareness of imaging findings of cancer complications and alternative diagnoses is essential in guiding management and avoiding misdiagnosis. The aim of this review article is to illustrate the typical neuroimaging findings of cancer therapy-related toxicities, including both early and late treatment effects, highlighting pearls that may aid in making the appropriate diagnosis.

Complications of Cancer Therapy in Children: A Comprehensive Review of Body Imaging Findings.

ABSTRACT: Complications of cancer therapy in children can result in a spectrum of toxicities that can affect any organ system and result in a range of morbidity. Complications may occur at the initiation of therapy or years following treatment. Although childhood cancer remains rare, increasing survival rates means more children are living longer following their treatment. Radiologists often play an important role in the diagnosis and evaluation of these complications, and thus, awareness of their imaging findings is essential to guide management and avoid misdiagnosis. This second part of a 2-part review aims to illustrate the typical body imaging findings of cancer therapy-related toxicities, including both early and late treatment effects. The article also discusses the differential diagnosis of imaging findings, highlighting pearls and pitfalls in making the appropriate diagnosis.

Making intussusception reductions easier: use of medical air in lieu of manual pump.

BACKGROUND: Pneumatic reduction of ileocolic intussusception is commonly performed with manual insufflators. The challenge of operating a handheld device while controlling the fluoroscope and monitoring the reduction could be obviated if the manual insufflation could be eliminated. OBJECTIVE: The aim in this retrospective study was to describe and evaluate the use of medical wall air in intussusception reduction. MATERIALS AND METHODS: We retrospectively reviewed all intussusception reductions over a period of years: from 2015 to 2018 using the manual insufflator and from 2018 to 2021 using medical air. We compared success rates, complication rates and time to reduction as documented on fluoroscopic image time stamps. Demographic data were obtained from the medical record. Attending radiologists and fluoroscopic technologists indicated their preference between methods, ease of use, perceived duration of reduction and perceived difference in success rates through an anonymous internal survey. RESULTS: There were 179 first reduction attempts in 167 patients (93 attempts during the period using the manual insufflator and 86 after converting to wall air). There was no difference in reduction duration (8:23 min for insufflation, 8:22 min for wall air, P=0.99) and no statistically significant difference in success rate (66.8% for insufflation and 79.1% for wall air, P=0.165). All survey respondents preferred the wall air method. The vast majority (93%) perceived that the wall air method was faster. CONCLUSION: Hospital wall air can be used to successfully reduce intussusceptions without incurring time burden or loss of effectiveness. The method leads to a perception of increased efficiency.

Prenatal Diagnosis of Skeletal Dysplasias: What Can CT Do for You?

Skeletal dysplasias (SDs) are a heterogeneous group of heritable disorders that affect development of bone and cartilage. Because each SD is individually rare and because of the heterogeneity within and among disorders, prenatal diagnosis of a specific SD remains challenging. Molecular genetic diagnosis involves invasive testing, which some patients are not amenable to. Further, genetic analysis is time consuming, and results may not become available in time to make pregnancy management decisions. Low-dose fetal CT can aid in the prenatal evaluation of SDs. The main downside is the low but true risk of fetal radiation exposure. As such, fetal CT should only be performed when there is concern for a severe skeletal dysplasia and the diagnosis is in question after a detailed ultrasound or if molecular genetic testing is unavailable and when prenatal diagnosis may affect management or counseling. Fetal CT should be obtained after consultation with geneticists, maternal-fetal medicine specialists, and fetal radiologists, and sometimes orthopedic surgeons or neonatologists. The purpose of this study was to review the technique of and indications for fetal CT, as well as discuss fetal radiation risk. Illustrative cases will demonstrate when and how CT may be helpful in the diagnosis of SDs.

Lymphatic Anomalies in Children: Update on Imaging Diagnosis, Genetics, and Treatment.

Lymphatic anomalies comprise a spectrum of disorders ranging from common localized microcystic and macrocystic lymphatic malformations (LMs) to rare complex lymphatic anomalies, including generalized lymphatic anomaly, Kaposiform lymph-angiomatosis, central conducting lymphatic anomaly, and Gorham-Stout disease. Imaging diagnosis of cystic LMs is generally straightforward, but complex lymphatic anomalies, particularly those with multiorgan involvement or diffuse disease, may be more challenging to diagnose. Complex lymphatic anomalies are rare but associated with high morbidity. Imaging plays an important role in their diagnosis, and radiologists may be the first clinicians to suggest the diagnosis. Furthermore, radiologists are regularly involved in management given the frequent need for image-guided interventions. For these reasons, it is crucial for radiologists to be familiar with the spectrum of entities comprising complex lymphatic anomalies and their typical imaging findings. In this article, we review the imaging findings of lymphatic anomalies, including LMs and complex lymphatic anomalies. We discuss characteristic imaging findings, multimodality imaging techniques used for evaluation, pearls and pitfalls in diagnosis, and potential complications. We also review recently discovered genetic changes underlying lymphatic anomaly development and the advent of new molecularly targeted therapies.

Characterization of germinal matrix hemorrhage in extremely premature infants: recognition of posterior location and diagnostic pitfalls.

BACKGROUND: Traditionally, descriptions of germinal matrix hemorrhage (GMH), derived from observations in preterm and very preterm infants, indicate its location at the caudothalamic grooves. However, before the germinal matrix begins to recede at approximately 28 weeks' gestational age (GA), it extends along the floor of the lateral ventricles far posterior to the caudothalamic grooves. Germinal matrix-intraventricular hemorrhage (GMH-IVH) can occur along any site from which the germinal matrix has not yet involuted. Therefore, as current advances in neonatology have allowed the routine survival of extremely preterm infants as young as 23 weeks' GA, postnatal GMH-IVH can occur in previously undescribed locations. Hemorrhage in the more posterior GMH on head ultrasound, if unrecognized, may lead to errors in diagnosis and mislocalization of this injury to the periventricular white matter or lateral walls of the lateral ventricles instead of to the subependyma, where it is in fact located. OBJECTIVE: Our aim is to describe posterior GMH in extremely premature infants, including its characteristic imaging appearance and potential pitfalls in diagnosis. MATERIALS AND METHODS: Over a 5-year period, all consecutive extremely preterm infants of 27 weeks' GA or less who developed GMH-IVH of any grade were included. A consecutive group of 100 very preterm infants of 31 weeks' GA with a GMH-IVH of any grade served as controls. RESULTS: In 106 extremely preterm neonates (mean GA: 25 weeks, range: 23.1-26.6 weeks) with 212 potential lateral ventricular germinal matrix bleeding sites, 159 sites had bleeds. In 70/159 (44%), the GMH-IVH was located posterior to the caudothalamic grooves and the foramina of Monro, 52 (32.7%) were both anterior and posterior and 21 (13.2%) were exclusively anterior. In 16 ventricles with intraventricular hemorrhage, an origin site in the germinal matrix could not be determined. In the control population of very preterm infants, all hemorrhages were at the anterior caudothalamic grooves and 95% were grade I. CONCLUSION: Unlike the older very preterm and moderately preterm infants that form the basis of our GMH-IVH description and classification, the extremely preterm infants now routinely surviving have a more fetal pattern of germinal matrix distribution, which is reflected in a different distribution and size of germinal matrix injury. We report the postnatal occurrence of subependymal GMH-IVH in extremely preterm infants in these more primitive, posterior locations, its potential imaging pitfalls and sonographic findings.

Early pulmonary complications related to cancer treatment in children.

In this review, we summarize early pulmonary complications related to cancer therapy in children and highlight characteristic findings on imaging that should be familiar to a radiologist reviewing imaging from pediatric cancer patients.

Telehealth Billing for Nurse Practitioners During COVID-19: Policy Updates.

Telehealth is a growing valuable strategy to assist patients accessing needed care when unable to get to a health care setting for one of several reasons. During the coronavirus disease 2019 (COVID-19) pandemic of 2020, many health care practices were forced to implement telehealth services to meet patient and practice needs. In 2020, several temporary waivers, exceptions, and telehealth policy changes emerged across the nation. Many telehealth policies are state or federal specific. This report provides a general overview of essential telehealth policies and legislative updates along with resources and websites to guide and support nurse practitioners with contemporary regulations regarding telehealth billing.

Hypertensive female Sprague-Dawley rats require an intact nitric oxide synthase system for compensatory increases in renal regulatory T cells.

We have previously shown that hypertensive female rats have more regulatory T cells (Tregs), which contribute more to blood pressure (BP) control in female versus male rats. Based on known protective properties of Tregs, the goal of the present study was to investigate the mechanisms by which female rats maintain Tregs. The present study was designed to 1) compare the impact of three hypertension models on the percentage of renal Tregs and 2) test the hypothesis that nitric oxide synthase (NOS) inhibition prevents increases in renal Tregs and exacerbates renal damage in female Sprague-Dawley rats. Rats (11-14 wk old) were randomized to one of the following four groups: control, norepinephrine (NE) infusion, angiotensin II infusion, or the NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) in drinking water. BP was measured via tail cuff. After 2 wk of treatment, kidneys were isolated and processed to measure Tregs via flow cytometric analysis and renal injury via urinary albumin excretion, plasma creatinine, and histological analyses. Hypertensive treatments increased BP in all experimental animals. Increases in BP in norepinephrine-and angiotensin II-treated rats were associated with increases in renal Tregs versus control. In contrast, l-NAME treatment decreased Tregs compared with all groups. l-NAME treatment modestly increased albumin excretion. However, plasma creatinine was comparable among the groups, and there was no histological evidence of glomerular or tubular injury. This study provides insights into the mechanisms regulating renal Tregs and supports that an intact NOS system is crucial for female rats to have BP-related increases in renal Tregs.

An expanded mouse testis transcriptome and mass spectrometry defines novel proteins.

The testis transcriptome is exceptionally complex. Despite its complexity, previous testis transcriptome analyses relied on a reductive method for transcript identification, thus underestimating transcriptome complexity. We describe here a more complete testis transcriptome generated by combining Tuxedo, a reductive method, and spliced-RUM, a combinatorial transcript-building approach. Forty-two percent of the expanded testis transcriptome is composed of unannotated RNAs with novel isoforms of known genes and novel genes constituting 78 and 9.8% of the newly discovered transcripts, respectively. Across tissues, novel transcripts were predominantly expressed in the testis with the exception of novel isoforms which were also highly expressed in the adult ovary. Within the testis, novel isoform expression was distributed equally across all cell types while novel genes were predominantly expressed in meiotic and post-meiotic germ cells. The majority of novel isoforms retained their protein-coding potential while most novel genes had low protein-coding potential. However, a subset of novel genes had protein-coding potentials equivalent to known protein-coding genes. Shotgun mass spectrometry of round spermatid total protein identified unique peptides from four novel genes along with seven annotated non-coding RNAs. These analyses demonstrate the testis expresses a wide range of novel transcripts that give rise to novel proteins.

Head Ultrasound Resistive Indices Are Associated With Brain Injury on Diffusion Tensor Imaging Magnetic Resonance Imaging in Neonates With Hypoxic-Ischemic Encephalopathy.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) is associated with dysfunctional cerebral autoregulation. Resistive index (RI) measured in the anterior cerebral artery on transfontanellar head ultrasound is a noninvasive measure of blood flow and may indicate autoregulation dysfunction. We tested whether RI was associated with brain injury on diffusion tensor imaging magnetic resonance imaging (MRI). MATERIALS AND METHODS: Seventy-five neonates who underwent therapeutic hypothermia for HIE were enrolled. Resistive index values were obtained from head ultrasound performed at the end of therapeutic hypothermia. Apparent diffusion coefficient scalars were measured on MRIs performed before day of life 10. RESULTS: Lower RI was associated with lower apparent diffusion coefficient in the centrum semiovale, basal ganglia, thalamus, and posterior limb of the internal capsule. Combining RI and Apgar scores improved the ability to distinguish injury severity on MRI relative to either metric alone. CONCLUSIONS: Low RI correlated with worse brain injury on diffusion tensor imaging and may serve as an early marker of brain injury in cooled HIE neonates.

APOBEC1 complementation factor (A1CF) is dispensable for C-to-U RNA editing in vivo.

Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney.