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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.

Adenosine-to-inosine RNA editing, a fundamental RNA modification, is regulated by adenosine deaminase (AD) domain containing proteins. Within the testis, RNA editing is catalyzed by ADARB1 and is regulated in a cell-type dependent manner. This study examined the role of two testis-specific AD domain proteins, ADAD1 and ADAD2, on testis RNA editing and male germ cell differentiation. ADAD1, previously shown to localize to round spermatids, and ADAD2 had distinct localization patterns with ADAD2 expressed predominantly in mid- to late-pachytene spermatocytes suggesting a role for both in meiotic and post-meiotic germ cell RNA editing. AD domain analysis showed the AD domain of both ADADs was likely catalytically inactive, similar to known negative regulators of RNA editing. To assess the impact of Adad mutation on male germ cell RNA editing, CRISPR-induced alleles of each were generated in mouse. Mutation of either Adad resulted in complete male sterility with Adad1 mutants displaying severe teratospermia and Adad2 mutant germ cells unable to progress beyond round spermatid. However, mutation of neither Adad1 nor Adad2 impacted RNA editing efficiency or site selection. Taken together, these results demonstrate ADAD1 and ADAD2 are essential regulators of male germ cell differentiation with molecular functions unrelated to A-to-I RNA editing.

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. Summary Sentence: ADAD1 is a post-meiotic spermatid RNA-binding protein that is required for normal translation of mRNAs important for post-meiotic differentiation and mRNAs associated with nuclear and intracellular transport. Graphical Abstract

Connexin43 and pannexin1 are found in immune cells. While gap junctional communication has been demonstrated between immune cells, hemichannels have been implicated in many cellular functions. Among the functions involved as being connexin dependent and pannexin dependent are cell migration, phagocytosis, antigen presentation, T-cell reactivity and B-cell responses. Surprisingly, many of these connexin-related and pannexin-related functions are not recapitulated in in vivo models. This is leading to a reevaluation of the role of these proteins in immune function.

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.

BackgroundTraditionally, 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.ObjectiveOur aim is to describe posterior GMH in extremely premature infants, including its characteristic imaging appearance and potential pitfalls in diagnosis.Materials and methodsOver 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.ResultsIn 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.ConclusionUnlike 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.

Abstract Ruminants are major producers of meat and milk, thus managing their reproductive potential is a key element in cost-effective, safe, and efficient food production. Of particular concern, defects in male germ cells and female germ cells may lead to significantly reduced live births relative to fertilization. However, the underlying molecular drivers of these defects are unclear. Small noncoding RNAs, such as piRNAs and miRNAs, are known to be important regulators of germ-cell physiology in mouse (the best-studied mammalian model organism) and emerging evidence suggests that this is also the case in a range of ruminant species, in particular bovine. Similarities exist between mouse and bovids, especially in the case of meiotic and postmeiotic male germ cells. However, fundamental differences in small RNA abundance and metabolism between these species have been observed in the female germ cell, differences that likely have profound impacts on their physiology. Further, parentally derived small noncoding RNAs are known to influence early embryos and significant species-specific differences in germ-cell born small noncoding RNAs have been observed. These findings demonstrate the mouse to be an imperfect model for understanding germ-cell small noncoding RNA biology in ruminants and highlight the need to increase research efforts in this underappreciated aspect of animal reproduction.

Background: An essential component of advanced practice nursing (APRN) education includes how to facilitate and adopt telehealth into practice, which includes becoming familiar with what is needed to practice telehealth and how to effectively bill for this service. This article describes the integration of telehealth credentialing, licensing, and billing into a preexisting APRN Role Practicum course. Method: Participants consisted of two cohorts (n = 129) of APRN students enrolled in a role preparation course within a Doctor of Nursing Practice curriculum. Students' knowledge and perceptions regarding the content of a prerecorded lecture were assessed using a pretest/posttest design. The posttest also included optional module assessment questions. Results: Student knowledge regarding the information presented increased, and overall student feedback was positive. Conclusion: Telehealth content was successfully integrated into a preexisting APRN Role Practicum course. Students found the information relevant to future practice and became knowledgeable regarding telehealth laws and regulations. [J Nurs Educ. 2022;61(7):413–416.]

Effective management of behavioral mental health crises in pediatric emergency departments is critical for patient and staff safety. This quality improvement initiative evaluated a structured crisis management algorithm combined with targeted staff training in a southeastern United States pediatric emergency department. Restraint use was compared in the 3 months before and after implementation of crisis management training and the use of the algorithm. Implementation of the algorithm showed a significant impact on restraint use, the use of the algorithm, and staff confidence in crisis management. These findings support the integration of standardized protocols and education to minimize physical restraints. •Pediatric restraint use is influenced by modifiable system factors.•Lack of clear criteria increases restraint use in emergency care.•Crisis algorithms plus education reduce restraint events.•Long-term sustainability requires scalable implementation.

Hypertension remains the leading risk factor for cardiovascular disease. Young females tend to be protected from hypertension compared with age-matched males. Although it has become increasingly clear that the immune system plays a key role in the development of hypertension in both sexes, few studies have examined how cytokines mediate hypertension in males versus females. We previously published that there are sex differences in the levels of the cytokine tumor necrosis factor (TNF)-α in spontaneously hypertensive rats (SHR). The goal of this study was to test the hypothesis that TNF-α inhibition with etanercept will lower BP in male and female SHR. However, as male SHR have a more pro-inflammatory status than female SHR, we further hypothesize that males will have a greater decrease in BP with TNF-α inhibition than females. Young adult male and female SHR were administered increasing doses of the TNF-α inhibitor etanercept or vehicle twice weekly for 31 days and BP was continuously measured via telemetry. Following treatment, kidneys and urine were collected and analyzed for markers of inflammation and injury. Despite significantly decreasing renal TNF-α levels, renal phospho-NFκB and urinary MCP-1 excretion, etanercept did not alter BP in either male or female SHR. Interestingly, treatment with etanercept increased urinary excretion of protein, creatinine and KIM-1 in both sexes. These results indicate that TNF-α does not contribute to sex differences in BP in SHR but may be vital in the maintenance of renal health. Highlights Etanercept treatment successfully decreased renal TNF-α levels in male and female SHR. Inhibition of TNF-α did not alter BP in SHR of either sex. Inhibition of TNF-α significantly elevated creatinine and KIM-1 levels in both male and female SHR, indicating TNF-α may be necessary for maintaining renal health.