Engineered Ribonucleoprotein Granules Inhibit Translation in Protocells.

Liquid granules rich in intrinsically disordered proteins and RNA play key roles in critical cellular functions such as RNA processing and translation. Many details of the mechanism via which this occurs remain to be elucidated. Motivated by the lacuna in the field and by the prospects of developing de novo artificial granules that provide extrinsic control of translation, we report a bottom-up approach to engineer ribonucleoprotein granules composed of a recombinant RNA-binding IDP that exhibits phase behavior in water. We developed a kinetic model to illustrate that these granules inhibit translation through reversible or irreversible sequestration of mRNA. Within monodisperse droplets capable of transcription and translation, we experimentally demonstrate temporal inhibition of translation by using designer IDPs that exhibit tunable phase behavior. This work lays the foundation for developing artificial granules that promise to further our mechanistic understanding of their naturally occurring counterparts.

Constraints on the Dynamical Environments of Supermassive Black-Hole Binaries Using Pulsar-Timing Arrays.

We introduce a technique for gravitational-wave analysis, where Gaussian process regression is used to emulate the strain spectrum of a stochastic background by training on population-synthesis simulations. This leads to direct Bayesian inference on astrophysical parameters. For pulsar timing arrays specifically, we interpolate over the parameter space of supermassive black-hole binary environments, including three-body stellar scattering, and evolving orbital eccentricity. We illustrate our approach on mock data, and assess the prospects for inference with data similar to the NANOGrav 9-yr data release.

Stimulating social interest: The translational value of basic investigations into frontal cortex function.

Fan et al. use electrical stimulation during a novel social interaction paradigm to demonstrate a role for the orbitofrontal cortex in directing social attention. Their results shed new light on the basic functions of the orbitofrontal cortex and have translational value in understanding circuit modulation for psychiatric disorders.

Conversion of an outpatient pharmacy to a mail-order pharmacy within a health system.

DISCLAIMER: In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. PURPOSE: The establishment of a new outpatient pharmacy provided a strategic opportunity to repurpose and convert an existing outpatient pharmacy into a closed-door mail-order pharmacy within a health system. This article describes the steps taken to successfully make this change and evaluates the impact. SUMMARY: The mail-order pharmacy conversion project was divided into 3 phases: phase 1 (before conversion) from July through August 2022, phase 2 (conversion) from October through November 2022, and phase 3 (after conversion) from December 2022 through February 2023. Phase 1 included standardizing workflows with standard operating procedure (SOP) development, improving automation, determining staffing ratios, gathering baseline staff engagement data, and identifying primary and secondary outcomes of interest. Phase 2 encompassed SOP implementation and training of mail-order team members. Phase 3 involved evaluating available pharmacy floorspace, marketing mail-order services, and the second distribution of the staff engagement survey. The measured outcomes of this project were total prescription volumes, increase in total revenue, and staff engagement. Data collection was completed in phase 3. CONCLUSION: The existing outpatient pharmacy was successfully converted to a closed-door pharmacy, and the associated prescription volume increased. Developing a strategic action plan to establish SOPs, calculate staffing performance metrics, and identify opportunities for growth and engaging frontline team members were essential to the success of this project.

Activation of UTP-sensitive P2Y2 receptor induces the expression of cholinergic genes in cultured cortical neurons: a signaling cascade triggered by Ca2+ mobilization and extracellular regulated kinase phosphorylation.

ATP functions as an extracellular signaling molecule that is costored and coreleased with neurotransmitters at central and peripheral neuronal synapses. Stimulation by ATP upregulates the expression of synaptic genes in muscle-including the genes for nicotine acetylcholine receptor (α-, δ-, and ε-subunits) and acetylcholinesterase (AChE)-via the P2Y receptor (P2YR), but the trophic response of neurons to the activation of P2YRs is less well understood. We reported that cultured cortical neurons and the developing rat brain expressed different types of P2YRs, and among these the UTP-sensitive P2Y2R was the most abundant. P2Y2R was found to exist in membrane rafts and it colocalized with the postsynaptic protein PSD-95 in cortical neurons. Notably, agonist-dependent stimulation of P2Y2R elevated the neuronal expression of cholinergic genes encoding AChE, PRiMA (an anchor for the globular form AChE), and choline acetyltransferase, and this induction was mediated by a signaling cascade that involved Ca(2+) mobilization and extracellular regulated kinases 1/2 activation. The importance of P2Y2R action was further shown by the receptor's synergistic effect with P2Y1R in enhancing cholinergic gene expression via the robust stimulation of Ca(2+) influx. Taken together our results revealed a developmental function of P2Y2R in promoting synaptic gene expression and demonstrated the influence of costimulation of P2Y1R and P2Y2R in neurons.

The scaffold protein NHERF2 determines the coupling of P2Y1 nucleotide and mGluR5 glutamate receptor to different ion channels in neurons.

Expressed metabotropic group 1 glutamate mGluR5 receptors and nucleotide P2Y1 receptors (P2Y1Rs) show promiscuous ion channel coupling in sympathetic neurons: their stimulation inhibits M-type [Kv7, K(M)] potassium currents and N-type (Ca(V)2.2) calcium currents (Kammermeier and Ikeda, 1999; Brown et al., 2000). These effects are mediated by G(q) and G(i/o) G-proteins, respectively. Via their C-terminal tetrapeptide, these receptors also bind to the PDZ domain of the scaffold protein NHERF2, which enhances their coupling to G(q)-mediated Ca(2+) signaling (Fam et al., 2005; Paquet et al., 2006b). We investigated whether NHERF2 could modulate coupling to neuronal ion channels. We find that coexpression of NHERF2 in sympathetic neurons (by intranuclear cDNA injections) does not affect the extent of M-type potassium current inhibition produced by either receptor but strongly reduced Ca(V)2.2 inhibition by both P2Y1R and mGluR5 activation. NHERF2 expression had no significant effect on Ca(V)2.2 inhibition by norepinephrine (via alpha(2)-adrenoceptors, which do not bind NHERF2), nor on Ca(V)2.2 inhibition produced by an expressed P2Y1R lacking the NHERF2-binding DTSL motif. Thus, NHERF2 selectively restricts downstream coupling of mGluR5 and P2Y1Rs in neurons to G(q)-mediated responses such as M-current inhibition. Differential distribution of NHERF2 in neurons may therefore determine coupling of mGluR5 receptors and P2Y1 receptors to calcium channels.

From affective to cognitive processing: Functional organization of the medial frontal cortex.

The medial wall of the primate frontal lobe encompasses multiple anatomical subregions. Based on distinct neurophysiological correlates and effects of lesions, individual areas are thought to play unique roles in behavior. Further, evidence suggests that dysfunction localized to specific subregions is commonly found in different neuropsychiatric disorders. The neurobiological underpinnings of these disorders, however, remain far from clear. Here, to better understand the functions of medial frontal cortex (MFC) and its role in psychiatric disease, we focus on its functional organization. We describe the emerging pattern in which more dorsal regions subserve temporally extended cognitive functions and more ventral regions predominantly subserve affective functions. We focus on two specific domains, decision-making and social cognition, that require integration across emotion and cognition. In each case, we discuss the current understanding of the functions believed to depend on subregions of MFC as a stepping-stone to speculate on how they might work in unison. We conclude with an overview of how symptoms of certain psychiatric disorders relate to our understanding of MFC functional organization and how further discovery could fuel advances in circuit-based therapies.

Typifications of some species names in Anthurium section Pachyneurium (Araceae).

During a taxonomic study of Anthurium sect. Pachyneurium, it was found that the names of four species required typification. Verification of the protologues and cited collections is discussed and typifications are proposed as follows: the illustration Schott Icones Aroideae No. 465 is designated as the neotype of A. affine Schott. A lectotype is designated for A. bonplandii G.S.Bunting since the holotype, cited in the protologue at MY, was not found there. An epitype is selected for A. solitarium Schott because the lectotype illustration of J.M.C. Vellozo (Flora Fluminensis t. 123) lacks sufficient detail to determine it unambiguously to species in A. sect. Pachyneurium. A lectotype is selected for A. glaziovii Hook.f., a synonym of A. solitarium.

ATP induces synaptic gene expressions in cortical neurons: transduction and transcription control via P2Y1 receptors.

Studies in vertebrate neuromuscular synapses have revealed previously that ATP, via P2Y receptors, plays a critical role in regulating postsynaptic gene expressions. An equivalent regulatory role of ATP and its P2Y receptors would not necessarily be expected for the very different situation of the brain synapses, but we provide evidence here for a brain version of that role. In cultured cortical neurons, the expression of P2Y(1) receptors increased sharply during neuronal differentiation. Those receptors were found mainly colocalized with the postsynaptic scaffold postsynaptic density protein 95 (PSD-95). This arises through a direct interaction of a PDZ domain of PSD-95 with the C-terminal PDZ-binding motif, D-T-S-L of the P2Y(1) receptor, confirmed by the full suppression of the colocalization upon mutation of two amino acids therein. This interaction is effective in recruiting PSD-95 to the membrane. Specific activation of P2Y(1) (G-protein-coupled) receptors induced the elevation of intracellular Ca(2+) and activation of a mitogen-activated protein kinase/Raf-1 signaling cascade. This led to distinct up-regulation of the genes encoding acetylcholinesterase (AChE(T) variant), choline acetyltransferase, and the N-methyl-d-aspartate receptor subunit NR2A. This was confirmed, in the example of AChE, to arise from P2Y(1)-dependent stimulation of a human ACHE gene promoter. That involved activation of the transcription factor Elk-1; mutagenesis of the ACHE promoter revealed that Elk-1 binding at its specific responsive elements in that promoter was induced by P2Y(1) receptor activation. The combined findings reveal that ATP, via its P2Y(1) receptor, can act trophically in brain neurons to regulate the gene expression of direct effectors of synaptic transmission.

Complex microparticle architectures from stimuli-responsive intrinsically disordered proteins.

The controllable production of microparticles with complex geometries is useful for a variety of applications in materials science and bioengineering. The formation of intricate microarchitectures typically requires sophisticated fabrication techniques such as flow lithography or multiple-emulsion microfluidics. By harnessing the molecular interactions of a set of artificial intrinsically disordered proteins (IDPs), we have created complex microparticle geometries, including porous particles, core-shell and hollow shell structures, and a unique 'fruits-on-a-vine' arrangement, by exploiting the metastable region of the phase diagram of thermally responsive IDPs within microdroplets. Through multi-site unnatural amino acid (UAA) incorporation, these protein microparticles can also be photo-crosslinked and stably extracted to an all-aqueous environment. This work expands the functional utility of artificial IDPs as well as the available microarchitectures of this class of biocompatible IDPs, with potential applications in drug delivery and tissue engineering.

Ileal-anal pouches: A review of its history, indications, and complications.

The ileal pouch anal anastomosis (IPAA) has revolutionised the surgical management of ulcerative colitis (UC) and familial adenomatous polyposis (FAP). Despite refinement in surgical technique(s) and patient selection, IPAA can be associated with significant morbidity. As the IPAA celebrated its 40th anniversary in 2018, this review provides a timely outline of its history, indications, and complications. IPAA has undergone significant modification since 1978. For both UC and FAP, IPAA surgery aims to definitively cure disease and prevent malignant degeneration, while providing adequate continence and avoiding a permanent stoma. The majority of patients experience long-term success, but "early" and "late" complications are recognised. Pelvic sepsis is a common early complication with far-reaching consequences of long-term pouch dysfunction, but prompt intervention (either radiological or surgical) reduces the risk of pouch failure. Even in the absence of sepsis, pouch dysfunction is a long-term complication that may have a myriad of causes. Pouchitis is a common cause that remains incompletely understood and difficult to manage at times. 10% of patients succumb to the diagnosis of pouch failure, which is traditionally associated with the need for pouch excision. This review provides a timely outline of the history, indications, and complications associated with IPAA. Patient selection remains key, and contraindications exist for this surgery. A structured management plan is vital to the successful management of complications following pouch surgery.

Bidirectional scaling of vocal variability by an avian cortico-basal ganglia circuit.

Behavioral variability is thought to be critical for trial and error learning, but where such motor exploration is generated in the central nervous system is unclear. The zebra finch songbird species offers a highly appropriate model in which to address this question. The male song is amenable to detailed measurements of variability, while the brain contains an identified cortico-basal ganglia loop that underlies this behavior. We used pharmacogenetic interventions to separately interrogate cortical and basal ganglia nodes of zebra finch song control circuitry. We show that bidirectional manipulations of each node produce near mirror image changes in vocal control: Cortical activity promotes song variability, whereas basal ganglia activity promotes song stability. Furthermore, female conspecifics can detect these pharmacogenetically elicited changes in song quality. Our results indicate that cortex and striatopallidum can jointly and reciprocally affect behaviorally relevant levels of vocal variability, and point to endogenous mechanisms for its control.

Photo-Crosslinkable Unnatural Amino Acids Enable Facile Synthesis of Thermoresponsive Nano- to Microgels of Intrinsically Disordered Polypeptides.

Hydrogel particles are versatile materials that provide exquisite, tunable control over the sequestration and delivery of materials in pharmaceutics, tissue engineering, and photonics. The favorable properties of hydrogel particles depend largely on their size, and particles ranging from nanometers to micrometers are used in different applications. Previous studies have only successfully fabricated these particles in one specific size regime and required a variety of materials and fabrication methods. A simple yet powerful system is developed to easily tune the size of polypeptide-based, thermoresponsive hydrogel particles, from the nano- to microscale, using a single starting material. Particle size is controlled by the self-assembly and unique phase transition behavior of elastin-like polypeptides in bulk and within microfluidic-generated droplets. These particles are then stabilized through ultraviolet irradiation of a photo-crosslinkable unnatural amino acid (UAA) cotranslationally incorporated into the parent polypeptide. The thermoresponsive property of these particles provides an active mechanism for actuation and a dynamic responsive to the environment. This work represents a fundamental advance in the generation of crosslinked biomaterials, especially in the form of soft matter colloids, and is one of the first demonstrations of successful use of UAAs in generating a novel material.

Exposing migratory sparrows to Plasmodium suggests costs of resistance, not necessarily of infection itself.

Migratory birds move through multiple habitats and encounter a diverse suite of parasites. This raises concern over migrants' role in transporting infectious disease between breeding and wintering grounds, and along migratory flyways. Trade-offs between flight and immune defenses could interfere with infected individuals' migratory timing and success, potentially affecting infection dynamics. However, experimental evidence that parasitic infection affects migratory preparation or timing remains scant. We hypothesized that birds encountering hematozoan parasites shortly before migration incur physical costs (reduced body condition) and behavioral costs (delayed migration), due to the infection itself and/or to the demands of mounting an immune response. We experimentally inoculated song sparrows (Melospiza melodia) with Plasmodium shortly before fall migration. We monitored infection and body composition for 2 weeks after inoculation, and used radiotelemetry to track timing of migratory departure for another 7 weeks after release. Inoculated individuals that resisted infection had lower lean mass 12 days post exposure, relative to controls and infected individuals. This suggests trade-offs between body composition and immune defenses that might reduce migration success of resistant individuals. Despite group differences in body composition prior to release, we did not detect significant differences in timing of migration departure several weeks later. Thus, malarial infection did not appear to incur detectable costs to body composition or to migratory timing, at least when exposure occurs several weeks before migration. This study is novel considering not only the costs of infection, but also the costs of resisting infection, in an experimental context.

Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature-triggered hierarchical self-assembly.

Post-translational modification of proteins is a strategy widely used in biological systems. It expands the diversity of the proteome and allows for tailoring of both the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the potential of post-translational modifications in biomaterials synthesis has remained largely untapped. As a proof of concept to demonstrate the feasibility of creating a genetically encoded biohybrid material through post-translational modification, we report here the generation of a family of three stimulus-responsive hybrid materials-fatty-acid-modified elastin-like polypeptides-using a one-pot recombinant expression and post-translational lipidation methodology. These hybrid biomaterials contain an amphiphilic domain, composed of a ß-sheet-forming peptide that is post-translationally functionalized with a C14 alkyl chain, fused to a thermally responsive elastin-like polypeptide. They exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be controlled at the sequence level.

Recognition of the genus Thaumatophyllum Schott - formerly Philodendron subg. Meconostigma (Araceae) - based on molecular and morphological evidence.

Philodendron subgenus Meconostigma has been a well-circumscribed group since 1829. Members of this group are easily distinguished by diagnostic morphological characters as well as by a distinct ecology and geographical distribution. Based on molecular, morphological and cytological evidence, we propose the recognition of P. subg. Meconostigma as a distinct genus, Thaumatophyllum Schott. We also present the necessary new combinations, an emended key and some nomenclatural and taxonomic corrections regarding 21 names of Thaumatophyllum.

Activation of P2Y1 nucleotide receptors induces inhibition of the M-type K+ current in rat hippocampal pyramidal neurons.

We have shown previously that stimulation of heterologously expressed P2Y1 nucleotide receptors inhibits M-type K+ currents in sympathetic neurons. We now report that activation of endogenous P2Y1 receptors induces inhibition of the M-current in rat CA1/CA3 hippocampal pyramidal cells in primary neuron cultures. The P2Y1 agonist adenosine 5'-[beta-thio]diphosphate trilithium salt (ADPbetaS) inhibited M-current by up to 52% with an IC50 of 84 nM. The hydrolyzable agonist ADP (10 microM) produced 32% inhibition, whereas the metabotropic glutamate receptor 1/5 agonist DHPG [(S)-3,5-dihydroxyphenylglycine] (10 microM) inhibited M-current by 44%. The M-channel blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride] produced 73% inhibition at 3 microM; neither ADPbetaS nor ADP produced additional inhibition in the presence of XE991. The effect of ADPbetaS was prevented by a specific P2Y1 antagonist, MRS 2179 (2'-deoxy-N'-methyladenosine-3',5'-bisphosphate tetra-ammonium salt) (30 microM). Inhibition of the M-current by ADPbetaS was accompanied by increased neuronal firing in response to injected current pulses. The neurons responding to ADPbetaS were judged to be pyramidal cells on the basis of (1) morphology, (2) firing characteristics, and (3) their distinctive staining for the pyramidal cell marker neurogranin. Strong immunostaining for P2Y1 receptors was shown in most cells in these cultures: 74% of the cells were positive for both P2Y1 and neurogranin, whereas 16% were only P2Y1 positive. These results show the presence of functional M-current-inhibitory P2Y1 receptors on hippocampal pyramidal neurons, as predicted from their effects when expressed in sympathetic neurons. However, the mechanism of inhibition in the two cell types seems to differ because, unlike nucleotide-mediated M-current inhibition in sympathetic neurons, that in hippocampal neurons did not appear to result from raised intracellular calcium.

Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells.

Pathological cellular hallmarks of Duchenne muscular dystrophy (DMD) include, among others, abnormal calcium homeostasis. Changes in the expression of specific receptors for extracellular ATP in dystrophic muscle have been recently documented: here, we demonstrate that at the earliest, myoblast stage of developing dystrophic muscle a purinergic dystrophic phenotype arises. In myoblasts of a dystrophin-negative muscle cell line established from the mdx mouse model of DMD but not in normal myoblasts, exposure to extracellular ATP triggered a strong increase in cytoplasmic Ca2+ concentrations. Influx of extracellular Ca2+ was stimulated by ATP and BzATP and inhibited by zinc, Coomassie Brilliant Blue-G, and KN-62, demonstrating activation of P2X7 receptors. Significant expression of P2X4 and P2X7 proteins was immunodetected in dystrophic myoblasts. Therefore, full-length dystrophin appears, surprisingly, to play an important role in myoblasts in controlling responses to ATP. Our results suggest that altered function of P2X receptors may be an important contributor to pathogenic Ca2+ entry in dystrophic mouse muscle and may have implications for the pathogenesis of muscular dystrophies. Treatments aiming at inhibition of specific ATP receptors could be of a potential therapeutic benefit.