ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling.

Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-ß-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/ß-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46,XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

Parent Experiences and Perceptions of Safety When Transporting Children With Autism Spectrum Disorder.

IMPORTANCE: Motor vehicle accidents are the leading cause of unintentional deaths of children ages 1 and older, particularly children with health care needs. OBJECTIVE: To explore family caregivers' experiences and current practices while transporting children diagnosed with autism spectrum disorder (ASD). DESIGN: A concurrent nested mixed-methods approach was used to gain insight into caregiver experiences. SETTING: Participants completed an online Qualtrics survey. PARTICIPANTS: A convenience sample of 54 caregivers of children with ASD from 17 U.S. states. RESULTS: Themes that emerged include sensory behaviors of children, education related to child passenger safety, and participation in the community. Statistically significant findings suggest a correlation between caregivers (n = 39) being less likely to participate in activities away from home if the child had attempted to elope (p = .013), displayed aggressive behaviors (p = .005), or demonstrated self-injurious behaviors (p = .001). CONCLUSIONS AND RELEVANCE: The findings suggest a correlation between caregivers limiting the distance traveled during vehicular transportation and behavioral safety concerns. If caregivers must limit travel, they may refrain from accessing the community and engaging in leisure pursuits. What This Article Adds: The findings suggest that therapists who treat children with ASD should be familiar with child passenger safety restraints, particularly for elopement.

CHTF18 ensures the quantity and quality of the ovarian reserve†.

The number and quality of oocytes, as well as the decline in both of these parameters with age, determines reproductive potential in women. However, the underlying mechanisms of this diminution are incompletely understood. Previously, we identified novel roles for CHTF18 (Chromosome Transmission Fidelity Factor 18), a component of the conserved Replication Factor C-like complex, in male fertility and gametogenesis. Currently, we reveal crucial roles for CHTF18 in female meiosis and oocyte development. Chtf18-/- female mice are subfertile and have fewer offspring beginning at 6 months of age. Consistent with age-dependent subfertility, Chtf18-/- ovaries contain fewer follicles at all stages of folliculogenesis than wild type ovaries, but the decreases are more significant at 3 and 6 months of age. By 6 months of age, both primordial and growing ovarian follicle pools are markedly reduced to near depletion. Chromosomal synapsis in Chtf18-/- oocytes is complete, but meiotic recombination is impaired resulting in persistent DNA double-strand breaks, fewer crossovers, and early homolog disjunction during meiosis I. Consistent with poor oocyte quality, the majority of Chtf18-/- oocytes fail to progress to metaphase II following meiotic resumption and a significant percentage of those that do progress are aneuploid. Collectively, our findings indicate critical functions for CHTF18 in ensuring both the quantity and quality of the mammalian oocyte pool.

Reduced expression of ribosomal proteins relieves microRNA-mediated repression.

MicroRNAs (miRNAs) regulate physiological and pathological processes by inducing posttranscriptional repression of target messenger RNAs (mRNAs) via incompletely understood mechanisms. To discover factors required for human miRNA activity, we performed an RNAi screen using a reporter cell line of miRNA-mediated repression of translation initiation. We report that reduced expression of ribosomal protein genes (RPGs) dissociated miRNA complexes from target mRNAs, leading to increased polysome association, translation, and stability of miRNA-targeted mRNAs relative to untargeted mRNAs. RNA sequencing of polysomes indicated substantial overlap in sets of genes exhibiting increased or decreased polysomal association after Argonaute or RPG knockdowns, suggesting similarity in affected pathways. miRNA profiling of monosomes and polysomes demonstrated that miRNAs cosediment with ribosomes. RPG knockdowns decreased miRNAs in monosomes and increased their target mRNAs in polysomes. Our data show that most miRNAs repress translation and that the levels of RPGs modulate miRNA-mediated repression of translation initiation.

Response of protein S100B to playing American football, lifting weights, and treadmill running.

OBJECTIVE: To determine if serum S100B increases similarly as a result of playing American football compared to exercise alone. METHODS: Serum S100B was measured in division III collegiate football players before and after every home game during a single football season. Serum S100B was also measured before and after subjects walked on a treadmill for 30 minutes at a leisurely pace, ran on a treadmill while wearing and not wearing a football helmet at 6 mph for 8 minutes, and performed low-, moderate-, or high-intensity resistance exercise. RESULTS: Serum S100B increased significantly (P < 0.05) when subjects played in a football game, ran on a treadmill, or performed moderate-intensity resistance exercise. Pre-game serum S100B did not accumulate throughout the football season in any of the players (P > 0.05). The increase in serum S100B during football games was moderately and significantly correlated with both the number of hits (R2  = 0.407) and the number of plays (R2  = 0.484) that each player experienced (P < 0.001). Post-game serum S100B was greater in football players who played more than 50 plays compared to those players who played <50 plays, subjects who exercised on a treadmill, or subjects performing resistance exercise (P < 0.05). CONCLUSION: It is unclear if the higher S100B concentration in football players playing at least 50 plays was caused by exercise or hits. Therefore, if serum S100B is to be used as a biomarker of impacts, and possible brain injury in sport, exercise time and intensity should be taken into account as confounding variables.

Alternative RISC assembly: binding and repression of microRNA-mRNA duplexes by human Ago proteins.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate protein output from the majority of human mRNAs. In contrast to the consensus view that all miRNAs are associated with Argonaute (Ago) proteins, we determine that miRNAs are expressed in a 13-fold excess relative to Agos in HeLa cells and that miRNAs are bound to mRNAs in a sevenfold excess relative to Agos, implying the existence of miRNA-mRNA duplexes not stoichiometrically bound by Agos. We show that all four human Agos can repress miRNA-mRNA duplexes, but only Ago2 can cleave small interfering RNA-mRNA duplexes in vitro. We visualize direct Ago binding to miRNA-mRNA duplexes in live cells using fluorescence lifetime imaging microscopy. In contrast to the consensus view that Agos bind miRNA duplexes, these data demonstrate that Agos can bind and repress miRNA-mRNA duplexes and support a model of catalytic Ago function in translational repression.

A standardized reporting system for assessment of diverse public health programs.

State public health agencies face challenges when monitoring the efforts and effects of public health programs that use disparate strategies and address various diseases, locations, and populations. The external evaluators of a complex portfolio of grant funding sought a standardized reporting framework and tool that could be used for all grants in the portfolio, without having to redesign it for each disease or intervention approach. Evaluators iteratively reviewed grant-funded projects to identify common project delivery strategies, then developed and implemented a common reporting framework and spreadsheet-based data capture tool. Evaluators provided training, technical assistance, and ongoing data reviews. During 2 fiscal years, 103 public health programs throughout Colorado submitted quarterly reports; agencies funded to implement these programs ranged from small community-based organizations to university- and hospital-affiliated groups in urban and rural settings. Aggregated reports supported estimates of program reach by strategy and by disease area, and the system supported production of summary descriptions of program implementation. Standardized language and expectations for reporting helped to align grant applications and work plans with reporting tools. A common language and standardized reporting tool can be used for diverse projects in a comprehensive evaluation framework. Decentralized data collection using common spreadsheet software enabled the aggregation of common data elements across multiple programs and projects. Further refinements could enable wider dissemination of common reporting criteria and expectations.

The experiences of active duty military spouses with advanced degrees in maintaining and advancing their careers.

BACKGROUND: Individuals married to active duty military members experience a significantly higher rate of un/underemployment than their civilian counterparts; those with advanced degrees are most impacted. Occupation is central to individual identity and adaptation; therefore, individual occupational struggles can impact family systems. Evidence shows military spouse career concerns impact service member retention, making spouse employment related to national security; however there is limited research on active duty spouses, particularly those with advanced degrees. OBJECTIVE: This phenomenological study explored the experiences of activity duty military spouses with advanced degrees focusing on maintaining and advancing their careers. METHODS: Semi-structured face-to-face interviews were used to explore experiences of military spouses with advanced degrees actively seeking to work in their profession. Inductive content analysis was used to derive overarching themes to respond to the research questions. RESULTS: Four major themes emerged: uniqueness of military life impacts; professional adaptation: flexibility, creativity and reinvention of the professional self; enhanced sense of duty/professional integrity; and need for enhanced forms of career support. CONCLUSIONS: Military spouses with advanced degrees value and have a right to occupation, including work. Military life presents unique barriers to this. Results have implications for healthcare providers and policy-makers.

Unrequited love: The role of prior commitment, motivation to remain friends, and friendship maintenance.

This study tested a moderated mediation model that commitment prior to an unrequited love episode will be related to higher levels of friendship maintenance behaviors after the episode and that this relationship will be mediated by the individual's motivations to remain friends with the rejecter. We predicted that rejection distress would weaken the mediational model among those reporting high distress. Participants wrote about an unrequited love experience as a pursuer and completed measures of pre-unrequited love commitment, rejection distress, motivations to remain friends, and friendship maintenance behaviors. Our results confirmed the moderated mediation model when the motivations to remain friends measure overall score, the interpersonal connection motivation, or the social connections motivation was the mediator. When covariates were added, only the model with the overall motivations score as mediator was supported. These data can help laypersons and mental health professionals understand and potentially repair friendships following an unrequited love episode.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Metabolic and regulatory engineering of Serratia marcescens: mimicking phage-mediated horizontal acquisition of antibiotic biosynthesis and quorum-sensing capacities.

Serratia marcescens is an important cause of opportunistic human infections. Many, but not all, strains produce prodigiosin, a secondary metabolic, red-pigment antibiotic, the biosynthesis of which is directed by the pig gene cluster. Quorum sensing (QS) involves the production and detection of chemical signal molecules as a means to regulate gene expression in response to population cell density. Several strains of S. marcescens have previously been shown to possess an N-acyl-L-homoserine lactone (aHSL) QS system. This study aimed to determine the impact of introducing, by phage-mediated horizontal gene transfer, a biosynthetic gene cluster (pig) and a regulatory locus (aHSL QS) into strains lacking the respective trait. The pig cluster from S. marcescens ATCC 274 (Sma 274) was transferred to the non-pigmented strain, S. marcescens strain 12 (Sma 12). In the engineered strain, pigment was expressed and brought under the control of the recipient's native regulatory systems (aHSL QS and luxS). Moreover, transfer of the aHSL locus from Sma 12 to the non-QS Sma 274 resulted in the imposition of aHSL control onto a variety of native traits, including pigment production. In addition, during this study, the QS regulon of the clinical strain, Sma 12, was characterized, and some novel QS-regulated traits in S. marcescens were identified. The results have implications for the evolution and dissemination of biosynthetic and QS loci, illustrating the genetic modularity and ease of acquisition of these traits and the capacity of phages to act as vectors for horizontal gene transfer.

The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation.

The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respective pig clusters revealed 14 ORFs in S. marcescens ATCC 274 and 15 ORFs in Serratia sp. ATCC 39006. In each Serratia species, predicted gene products showed similarity to polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs) and the Red proteins of Streptomyces coelicolor A3(2). Comparisons between the two Serratia pig clusters and the red cluster from Str. coelicolor A3(2) revealed some important differences. A modified scheme for the biosynthesis of prodigiosin, based on the pathway recently suggested for the synthesis of undecylprodigiosin, is proposed. The distribution of the pig cluster within several Serratia sp. isolates is demonstrated and the presence of cryptic clusters in some strains shown. The pig cluster of Serratia marcescens ATCC 274 is flanked by cueR and copA homologues and this configuration is demonstrated in several S. marcescens strains, whilst these genes are contiguous in strains lacking the pig cluster.

The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations.

Erwinia carotovora is a Gram-negative bacterial phytopathogen that causes soft-rot disease and potato blackleg. The organism is environmentally widespread and exhibits an opportunistic plant pathogenesis. The ability to secrete multiple plant cell wall-degrading enzymes is a key virulence trait and exoenzyme production is responsive to multiple environmental and physiological cues. One important cue is the cell population density of the pathogen. Cell density is monitored via an acylated homoserine lactone (acyl HSL) signalling molecule, which is thought to diffuse between Erwinia cells in a process now commonly known as 'quorum sensing'. This molecule also acts as the chemical communication signal controlling production of a broad-spectrum beta-lactam antibiotic (1-carbapen-2-em-3-carboxylic acid; carbapenem) synthesised in concert with exoenzyme elaboration, possibly for niche defence. In antibiotic production control, quorum sensing acts at the level of transcriptional activation of the antibiotic biosynthetic cluster. This is achieved via a dedicated LuxR-type protein, CarR that is bound to the signalling molecule. The molecular relay connecting acyl HSL production and exoenzyme induction is not clear, despite the identification of a multitude of global regulatory genes, including those of the RsmA/rsmB system, impinging on enzyme synthesis. Quorum sensing control mediated by acyl HSLs is widespread in Gram-negative bacteria and is responsible for the regulation of diverse phenotypes. Although there is still a paucity of meaningful information on acyl HSL availability and in-situ biological function, there is growing evidence that such molecules play significant roles in microbial ecology.