Hem25p is required for mitochondrial IPP transport in fungi.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor composed of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for haem biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p failed to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enabled robust IPP uptake and incorporation into the CoQ biosynthetic pathway. HEM25 orthologues from diverse fungi, but not from metazoans, were able to rescue hem25∆ CoQ deficiency. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in fungi.

Hem25p is a mitochondrial IPP transporter.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor comprised of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing, and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for heme biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p fail to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enables robust IPP uptake demonstrating that Hem25p is sufficient for IPP transport. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in yeast.

Inquiry-Based Activities and Games That Engage Students in Learning Atomic Orbitals.

Atomic orbitals represent an essential construct used to develop chemical bonding models, upon which other more advanced chemistry topics are built. In this article, we share a series of active-learning activities and a gamified approach to develop students' representational competence about atomic orbitals and to engage students in learning the properties of atomic orbitals. These properties are essential for understanding an array of fundamental concepts such as penetration and shielding, relationships such as periodic trends, and models used to describe chemical bonding. The activities employ an inquiry-based approach to engage students in exploring the relationship between atomic orbitals' spatial properties and quantum numbers. The activities guide students to collect data to verify periodic trends and construct electronic configurations. The activities utilize Orbital Explorer Web site for visualization, comparison and analysis of atomic orbitals. The Orbital Explorer Web site is described in a related Technology Report. The activities and the game are suitable to be conducted in both in-person and remote-teaching settings.

Online Orbital Explorer and BingOrbital Game for Inquiry-Based Activities.

We report a new online suite of tools that enables inquiry-based active-learning activities to develop students' representational competence about atomic orbitals. Orbital Explorer is Web site hub for the visualization and interactive investigation of atomic orbital properties. Orbital Explorer contains two integrated tools, namely, Atomic Orbital Explorer, which enables one to visualize and interrogate individual atomic orbitals, and Orbital RDF Comparison, which enables one to make a more detailed quantitative comparison of orbital energies and properties of orbital radial distribution functions (RDFs). In addition, we present an original chemistry educational gamification design, BingOrbital, constructed in a format resembling Bingo (American version). The game aims to reinforce the recognition of atomic orbitals based on the RDF and 3D isosurface and has been applied as an engaging retrieval practice tool. A companion set of example activities that use the Orbital Explorer and BingOrbital game have been presented in another article.

Acceptance of Preventive Dental Services for Children at a Retail-Based Clinic: A Pilot Study.

Purpose: Low-income children with high caries risk are disproportionately affected by poor access to dental care. Retail-based clinics (RBCs) can provide accessible ancillary oral health care. The purposes of this study were: (1) to measure caregivers' acceptance rate of an oral health screening, fluoride varnish (FV) application, and caries risk assessment offered to children on a walk-in basis in an RBC; and (2) to categorize the caries risk and demographics among the participants.
Methods: Screenings and FV applications were provided to children younger than 18 years at a Walgreens Health Care Clinic in Baltimore, Md., USA, from October 2016 to October 2017. The acceptance rate and caries risk using the American Dental Association caries risk assessment form were documented. Descriptive statistics and Fisher's exact test were used to analyze the data.
Results: Eighty-five children and their caretakers were approached and 32 (38 percent) agreed to participate. Most children had high caries risk (84.3 percent) and a dental home (81.2 percent), but only 50 percent reported visiting their dentist in the last year.
Conclusion: Our results demonstrate modest acceptance of FV application for children on a convenience basis. This population had predominantly high caries risk, with poor adherence to follow-up with their dental home. Retail-based dental care should not replace the dental home but could support it by increasing access to preventive dental care in children. (J Dent Child 2019;86(1):40-6)
Received July 2, 2018; Last Revision August 13, 2018; Accepted August 13, 2018.

Genome-wide Methyl-Seq analysis of blood-brain targets of glucocorticoid exposure.

Chronic exposure to glucocorticoids (GCs) can lead to psychiatric complications through epigenetic mechanisms such as DNA methylation (DNAm). We sought to determine whether epigenetic changes in a peripheral tissue can serve as a surrogate for those in a relatively inaccessible tissue such as the brain. DNA extracted from the hippocampus and blood of mice treated with GCs or vehicle solution was assayed using a genome-wide DNAm platform (Methyl-Seq) to identify differentially methylated regions (DMRs) induced by GC treatment. We observed that ∼70% of the DMRs in both tissues lost methylation following GC treatment. Of the 3,095 DMRs that mapped to the same genes in both tissues, 1,853 DMRs underwent DNAm changes in the same direction. Interestingly, only 209 DMRs (<7%) overlapped in genomic coordinates between the 2 tissues, suggesting tissue-specific differences in GC-targeted loci. Pathway analysis showed that the DMR-associated genes were members of pathways involved in metabolism, immune function, and neurodevelopment. Also, changes in cell type composition of blood and brain were examined by fluorescence-activated cell sorting. Separation of the cortex into neuronal and non-neuronal fractions and the leukocytes into T-cells, B-cells, and neutrophils showed that GC-induced methylation changes primarily occurred in neurons and T-cells, with the blood tissue also undergoing a shift in the proportion of constituent cell types while the proportion of neurons and glia in the brain remained stable. From the current pilot study, we found that despite tissue-specific epigenetic changes and cellular heterogeneity, blood can serve as a surrogate for GC-induced changes in the brain.

Basal forebrain projections to the lateral habenula modulate aggression reward.

Maladaptive aggressive behaviour is associated with a number of neuropsychiatric disorders and is thought to result partly from the inappropriate activation of brain reward systems in response to aggressive or violent social stimuli. Nuclei within the ventromedial hypothalamus, extended amygdala and limbic circuits are known to encode initiation of aggression; however, little is known about the neural mechanisms that directly modulate the motivational component of aggressive behaviour. Here we established a mouse model to measure the valence of aggressive inter-male social interaction with a smaller subordinate intruder as reinforcement for the development of conditioned place preference (CPP). Aggressors develop a CPP, whereas non-aggressors develop a conditioned place aversion to the intruder-paired context. Furthermore, we identify a functional GABAergic projection from the basal forebrain (BF) to the lateral habenula (lHb) that bi-directionally controls the valence of aggressive interactions. Circuit-specific silencing of GABAergic BF-lHb terminals of aggressors with halorhodopsin (NpHR3.0) increases lHb neuronal firing and abolishes CPP to the intruder-paired context. Activation of GABAergic BF-lHb terminals of non-aggressors with channelrhodopsin (ChR2) decreases lHb neuronal firing and promotes CPP to the intruder-paired context. Finally, we show that altering inhibitory transmission at BF-lHb terminals does not control the initiation of aggressive behaviour. These results demonstrate that the BF-lHb circuit has a critical role in regulating the valence of inter-male aggressive behaviour and provide novel mechanistic insight into the neural circuits modulating aggression reward processing.

Subcellular modulation of protein VlsE stability and folding kinetics.

The interior of a cell interacts differently with proteins than a dilute buffer because of a wide variety of macromolecules, chaperones, and osmolytes that crowd and interact with polypeptide chains. We compare folding of fluorescent constructs of protein VlsE among three environments inside cells. The nucleus increases the stability of VlsE relative to the cytoplasm, but slows down folding kinetics. VlsE is also more stable in the endoplasmic reticulum, but unlike PGK, tends to aggregate there. Although fluorescent-tagged VlsE and PGK show opposite stability trends from in vitro to the cytoplasm, their trends from cytoplasm to nucleus are similar.

Total synthesis and antiplasmodial activity of pohlianin C and analogues.

The first synthesis of the glycine-rich cyclic octapeptide pohlianin C is reported, confirming the structure of this natural product. Screening against Plasmodium falciparum reveals moderate antiplasmodial activity, consistent with data obtained from the natural sample. In addition, the synthesis of three analogues reveals that the antiplasmodial activity of pohlianin C can be preserved or increased with simplified structures.

Targeting NAD+ metabolism in the human malaria parasite Plasmodium falciparum.

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite utilized as a redox cofactor and enzyme substrate in numerous cellular processes. Elevated NAD+ levels have been observed in red blood cells infected with the malaria parasite Plasmodium falciparum, but little is known regarding how the parasite generates NAD+. Here, we employed a mass spectrometry-based metabolomic approach to confirm that P. falciparum lacks the ability to synthesize NAD+ de novo and is reliant on the uptake of exogenous niacin. We characterized several enzymes in the NAD+ pathway and demonstrate cytoplasmic localization for all except the parasite nicotinamidase, which concentrates in the nucleus. One of these enzymes, the P. falciparum nicotinate mononucleotide adenylyltransferase (PfNMNAT), is essential for NAD+ metabolism and is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. Our results demonstrate the enzymatic activity of PfNMNAT in vitro and demonstrate its ability to genetically complement the closely related Escherichia coli NMNAT. Due to the similarity of PfNMNAT to the bacterial enzyme, we tested a panel of previously identified bacterial NMNAT inhibitors and synthesized and screened twenty new derivatives, which demonstrate a range of potency against live parasite culture. These results highlight the importance of the parasite NAD+ metabolic pathway and provide both novel therapeutic targets and promising lead antimalarial compounds.

Kinetic control in protein folding for light chain amyloidosis and the differential effects of somatic mutations.

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis-trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans-cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH2; reactions become slower and more stochastic as the pH increases up to pH7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.

The extracellular protein VlsE is destabilized inside cells.

We use U2OS cells as in vivo "test tubes" to study how the same cytoplasmic environment has opposite effects on the stability of two different proteins. Protein folding stability and kinetics were compared by fast relaxation imaging, which combines a temperature jump with fluorescence microscopy of FRET (Förster resonance energy transfer)-labeled proteins. While the stability of the cytoplasmic enzyme PGK (phosphoglycerate kinase) increases in cells, the stability of the cell surface antigen VlsE, which presumably did not evolve for stability inside cells, decreases. VlsE folding also slows down more than PGK folding in cells, relative to their respective aqueous buffer kinetics. Our FRET measurements provide evidence that VlsE is more compact inside cells than in aqueous buffer. Two kinetically distinct protein populations exist inside cells, making a connection with previous in vitro crowding studies. In addition, we confirm previous studies showing that VlsE is stabilized by 150mg/mL of the carbohydrate crowder Ficoll, even though it is destabilized in the cytoplasm relative to aqueous buffer. We propose two mechanisms for the observed destabilization of VlsE in U2OS cells: long-range interactions competing with crowding or shape-dependent crowding favoring more compact states inside the cell over the elongated aqueous buffer native state.