The RNA-binding protein, ZFP36L2, influences ovulation and oocyte maturation

ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004), suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3′UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest.Fil: Ball, Christopher B.. University of North Carolina; Estados UnidosFil: Rodriguez, Karina F.. National Institutes of Health; Estados UnidosFil: Stumpo, Deborah J.. National Institutes of Health; Estados UnidosFil: Ribeiro Neto, Fernando. National Institutes of Health; Estados UnidosFil: Korach, Kenneth S.. National Institutes of Health; Estados UnidosFil: Blackshear, Perry J.. University of Duke; Estados Unidos. National Institutes of Health; Estados UnidosFil: Birnbaumer, Lutz. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ramos, Silvia B. V.. University of North Carolina; Estados Unido

Paramedic educational program attrition accounts for significant loss of potential EMS workforce

OBJECTIVE: Recent concerns for the strength and stability of the emergency medical services (EMS) workforce have fueled interest in enhancing the entry of EMS clinicians into the workforce. However, the educational challenges associated with workforce entry remain unclear. Our objective was to evaluate the educational pathway of entry into the EMS workforce and to identify factors that lead to the loss of potential EMS clinicians. METHODS: This is a cross-sectional evaluation of all US paramedic educational programs, with enrolled students, in the 2019 Committee on Accreditation of Educational Programs for the EMS Professions annual report survey. This data set includes detailed program characteristics and metrics including program attrition rate (leaving before completion), and certifying exam pass rates. Descriptive statistics were calculated, and multivariable logistic regression analysis was conducted to evaluate the association between high program attrition rates (\u3e30%) and program specific characteristics. RESULTS: In 2019, 640 accredited programs met inclusion with 17,457 students enrolled in paramedic educational programs. Of these, 13,884 students successfully graduated (lost to attrition, 3,573/17,457 [21%]) and 12,002 passed the certifying exam on the third attempt (lost to unable to certify, 1,882/17,457 [11%]). High program attrition rates were associated with longer programs (\u3e12 months), small class sizes (\u3c12 \u3estudents), and regional locations. CONCLUSIONS: Nearly 1 in 3 paramedic students were lost from the potentially available workforce either owing to attrition during the educational program or failure to certify after course completion. Attrition represented the largest loss, providing an avenue for future targeted research and interventions to improve EMS workforce stability

Approaches for advancing scientific understanding of macrosystems

The emergence of macrosystems ecology (MSE), which focuses on regional- to continental-scale ecological patterns and processes, builds upon a history of long-term and broad-scale studies in ecology. Scientists face the difficulty of integrating the many elements that make up macrosystems, which consist of hierarchical processes at interacting spatial and temporal scales. Researchers must also identify the most relevant scales and variables to be considered, the required data resources, and the appropriate study design to provide the proper inferences. The large volumes of multi-thematic data often associated with macrosystem studies typically require validation, standardization, and assimilation. Finally, analytical approaches need to describe how cross-scale and hierarchical dynamics and interactions relate to macroscale phenomena. Here, we elaborate on some key methodological challenges of MSE research and discuss existing and novel approaches to meet them

LeMaRns: A length-based multi-species analysis by numerical simulation in R

Fish stocks interact through predation and competition for resources, yet stocks are typically managed independently on a stock-by-stock basis. The need to take account of multispecies interactions is widely acknowledged. However, examples of the application of multi-species models to support management decisions are limited as they are often seen as too complex and lacking transparency. Thus there is a need for simple and transparent methods to address stock interactions in a way that supports managers. Here we introduce LeMaRns, a new R-package of a general length-structured fish community model, LeMans, that characterises fishing using fleets that can have different gears and species catch preferences. We describe the model, package implementation, and give three examples of use: determination of multi-species reference points; modelling of mixed-fishery interactions; and examination of the response of community indicators to dynamical changes in fleet effort within a mixed-fishery. LeMaRns offers a diverse array of options for parameterisation. This, along with the speed, comprehensive documentation, and open source nature of the package makes LeMans newly accessible, transparent, and easy to use, which we hope will lead to increased uptake by the fisheries management community

Inducing circular RNA formation using the CRISPR endoribonuclease Csy4

Circular RNAs (circRNAs) are highly stable, covalently closed RNAs that are regulated in a spatiotemporal manner and whose functions are largely unknown. These molecules have the potential to be incorporated into engineered systems with broad technological implications. Here we describe a switch for inducing back-splicing of an engineered circRNA that relies on the CRISPR endoribonuclease, Csy4, as an activator of circularization. The endoribonuclease activity and 3' end-stabilizing properties of Csy4 are particularly suited for this task. Coexpression of Csy4 and the circRNA switch allows for the removal of downstream competitive splice sites and stabilization of the 5' cleavage product. This subsequently results in back-splicing of the 5' cleavage product into a circRNA that can translate a reporter protein from an internal ribosomal entry site (IRES). Our platform outlines a straightforward approach toward regulating splicing and could find potential applications in synthetic biology as well as in studying the properties of different circRNAs

4-Alkyloxyimino Derivatives of Uridine-5′-triphosphate: Distal Modification of Potent Agonists as a Strategy for Molecular Probes of P2Y 2 , P2Y 4 , and P2Y 6 Receptors

Extended N4-(3-arylpropyl)oxy derivatives of uridine-5′-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N4-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N4-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N4-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs

4-Alkyloxyimino-cytosine nucleotides: tethering approaches to molecular probes for the P2Y6 receptor

4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we probed two positions (N4 and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y6R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y6R expressed in 1321N1 human astrocytoma cells with an EC50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 (t1/2 of 18 min) and surface-bound fluorescence. Known P2Y6R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R

Approaches to advance scientific understanding of macrosystems ecology

The emergence of macrosystems ecology (MSE), which focuses on regional- to continental-scale ecological pat- terns and processes, builds upon a history of long-term and broad-scale studies in ecology. Scientists face the difficulty of integrating the many elements that make up macrosystems, which consist of hierarchical processes at interacting spatial and temporal scales. Researchers must also identify the most relevant scales and variables to be considered, the required data resources, and the appropriate study design to provide the proper inferences. The large volumes of multi-thematic data often associated with macrosystem studies typically require valida- tion, standardization, and assimilation. Finally, analytical approaches need to describe how cross-scale and hierarchical dynamics and interactions relate to macroscale phenomena. Here, we elaborate on some key methodological challenges of MSE research and discuss existing and novel approaches to meet them

The photosynthesis - leaf nitrogen relationship at ambient and elevated atmospheric carbon dioxide: a meta-analysis

Estimation of leaf photosynthetic rate (A) from leaf nitrogen content (N) is both conceptually and numerically important in models of plant, ecosystem and biosphere responses to global change. The relationship between A and N has been studied extensively at ambient CO{sub 2} but much less at elevated CO{sub 2}. This study was designed to (1) assess whether the A-N relationship was more similar for species within than between community and vegetation types, and (2) examine how growth at elevated CO{sub 2} affects the A-N relationship. Data were obtained for 39 C{sub 3} species grown at ambient CO{sub 2} and 10 C{sub 3} species grown at ambient and elevated CO{sub 2}. A regression model was applied to each species as well as to species pooled within different community and vegetation types. Cluster analysis of the regression coefficients indicated that species measured at ambient CO{sub 2} did not separate into distinct groups matching community or vegetation type. Instead, most community and vegetation types shared the same general parameter space for regression coefficients. Growth at elevated CO{sub 2} increased photosynthetic nitrogen use efficiency for pines and deciduous trees. When species were pooled by vegetation type, the A-N relationship for deciduous trees expressed on a leaf-mass bask was not altered by elevated CO{sub 2}, while the intercept increased for pines. When regression coefficients were averaged to give mean responses for different vegetation types, elevated CO{sub 2} increased the intercept and the slope for deciduous trees but increased only the intercept for pines. There were no statistical differences between the pines and deciduous trees for the effect of CO{sub 2}. Generalizations about the effect of elevated CO{sub 2} on the A-N relationship, and differences between pines and deciduous trees will be enhanced as more data become available