Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin.

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol).

Large Incarcerated Inguinal Hernia: A Case Report.

A 90-year-old white male cadaver was found to have an incarcerated left inguinal hernia (IH). Although IHs are a very common pathology, the size and extent of this IH make it a unique case study. Upon gross dissection of the abdominal and pelvic cavities, 79 cm of small and large bowel was removed from the scrotal sac. The extent of the herniation had enlarged the scrotal sac to over 14 cm in both height and width and over 10 cm in depth. The herniation also caused the penis to become buried in the skin and not visible.

Proline synthesis through PYCR1 is required to support cancer cell proliferation and survival in oxygen-limiting conditions.

The demands of cancer cell proliferation alongside an inadequate angiogenic response lead to insufficient oxygen availability in the tumor microenvironment. Within the mitochondria, oxygen is the major electron acceptor for NADH, with the result that the reducing potential produced through tricarboxylic acid (TCA) cycle activity and mitochondrial respiration are functionally linked. As the oxidizing activity of the TCA cycle is required for efficient synthesis of anabolic precursors, tumoral hypoxia could lead to a cessation of proliferation without another means of correcting the redox imbalance. We show that in hypoxic conditions, mitochondrial pyrroline 5-carboxylate reductase 1 (PYCR1) activity is increased, oxidizing NADH with the synthesis of proline as a by-product. We further show that PYCR1 activity is required for the successful maintenance of hypoxic regions by permitting continued TCA cycle activity, and that its loss leads to significantly increased hypoxia in vivo and in 3D culture, resulting in widespread cell death.

Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin.

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol).

Dopamine in Auditory Nuclei and Lemniscal Projections is Poised to Influence Acoustic Integration in the Inferior Colliculus.

Dopamine (DA) modulates the activity of nuclei within the ascending and descending auditory pathway. Previous studies have identified neurons and fibers in the inferior colliculus (IC) which are positively labeled for tyrosine hydroxylase (TH), a key enzyme in the synthesis of dopamine. However, the origins of the tyrosine hydroxylase positive projections to the inferior colliculus have not been fully explored. The lateral lemniscus (LL) provides a robust inhibitory projection to the inferior colliculus and plays a role in the temporal processing of sound. In the present study, immunoreactivity for tyrosine hydroxylase was examined in animals with and without 6-hydroxydopamine (6-OHDA) lesions. Lesioning, with 6-OHDA placed in the inferior colliculus, led to a significant reduction in tyrosine hydroxylase immuno-positive labeling in the lateral lemniscus and inferior colliculus. Immunolabeling for dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), enzymes responsible for the synthesis of norepinephrine (NE) and epinephrine (E), respectively, were evaluated. Very little immunoreactivity for DBH and no immunoreactivity for PNMT was found within the cell bodies of the dorsal, intermediate, or ventral nucleus of the lateral lemniscus. The results indicate that catecholaminergic neurons of the lateral lemniscus are likely dopaminergic and not noradrenergic or adrenergic. Next, high-pressure liquid chromatography (HPLC) analysis was used to confirm that dopamine is present in the inferior colliculus and nuclei that send projections to the inferior colliculus, including the cochlear nucleus (CN), superior olivary complex (SOC), lateral lemniscus, and auditory cortex (AC). Finally, fluorogold, a retrograde tracer, was injected into the inferior colliculus of adult rats. Each subdivision of the lateral lemniscus contained fluorogold within the somata, with the dorsal nucleus of the lateral lemniscus showing the most robust projections to the inferior colliculus. Fluorogold-tyrosine hydroxylase colocalization within the lateral lemniscus was assessed. The dorsal and intermediate nuclei neurons exhibiting similar degrees of colocalization, while neurons of the ventral nucleus had significantly fewer colocalized fluorogold-tyrosine hydroxylase labeled neurons. These results suggest that several auditory nuclei that project to the inferior colliculus contain dopamine, dopaminergic neurons in the lateral lemniscus project to the inferior colliculus and that dopaminergic neurotransmission is poised to play a pivotal role in the function of the inferior colliculus.

A human pluripotent stem cell model for the analysis of metabolic dysfunction in hepatic steatosis.

Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term encompasses a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis, which have, to date, been challenging to model in the laboratory setting. Here, we present a human pluripotent stem cell (hPSC)-derived model of hepatic steatosis, which overcomes inherent challenges of current models and provides insights into the metabolic rewiring associated with steatosis. Following induction of macrovesicular steatosis in hepatocyte-like cells using lactate, pyruvate, and octanoate (LPO), respirometry and transcriptomic analyses revealed compromised electron transport chain activity. 13C isotopic tracing studies revealed enhanced TCA cycle anaplerosis, with concomitant development of a compensatory purine nucleotide cycle shunt leading to excess generation of fumarate. This model of hepatic steatosis is reproducible, scalable, and overcomes the challenges of studying mitochondrial metabolism in currently available models.

Total Synthesis of Kalimantacin A.

The kalimantacins make up a family of hybrid polyketide-nonribosomal peptide-derived natural products that display potent and selective antibiotic activity against multidrug resistant strains of Staphylococcus aureus. Herein, we report the first total synthesis of kalimantacin A, in which three fragments are prepared and then united via Sonogashira and amide couplings. The enantioselective synthetic approach is convergent, unlocking routes to further kalimantacins and analogues for structure-activity relationship studies and clinical evaluation.

Greyhound racing ideal trajectory path generation for straight to bend based on jerk rate minimization.

This paper presents methods for modelling and designing an ideal path trajectory between straight and bend track path segments for racing greyhounds. To do this, we numerically generate clothoid and algebraic curve segments for racing quadrupeds using a sequential vector transformation method as well as using a helper equation for approaching ideal clothoid segments that would respect greyhound kinematic parameters and boundary conditions of the track. Further, we look into the limitations of using a clothoid curve for racing dog track path design and propose a smooth composite curve for track transition design which roughly maintains G3 curvature continuity for smooth jerk to overcome limitations of a clothoid transition. Finally, we show results from race data modelling and past injury data, which provide a strong indication of clothoid curve segments improving the dynamics and safety of racing greyhounds while reducing injuries.

A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis.

Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens NCIMB 10586, and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml, respectively, remain unresolved. To define starter unit generation, the purified mupirocin proteins MupQ, MupS, and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonyl coenzyme A (CoA) and succinyl CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units, respectively, via the MupQ/TmlQ catalyzed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalyzed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA. MacpD/TacpD were interchangeable but alternate trans-acting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were nonfunctional. MupS and TmlS selectivity was more varied, and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titers of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.

Control of ß-Branching in Kalimantacin Biosynthesis: Application of 13 C†NMR to Polyketide Programming.

The presence of ß-branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature. Kalimantacin is a polyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential ß-branching modifications. We use purified single and multi-domain enzyme components of the kalimantacin biosynthetic machinery to address in vitro how the pattern of ß-branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of a generalisable assay that takes advantage of 13 C NMR of a single 13 C label incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report a previously unassigned modular enoyl-CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate each specific ß-branch.

Elucidation of the relative and absolute stereochemistry of the kalimantacin/batumin antibiotics.

Kalimantacin A and batumin exhibit potent and selective antibiotic activity against Staphylococcus species including MRSA. Both compounds are formed via a hybrid polyketide synthase/non-ribosomal peptide synthetase (PKS-NRPS) biosynthetic pathway and from comparison of the gene clusters it is apparent that batumin from Pseudomonas batumici and kalimantacin from P. fluorescens are the same compound. The linear structure of this unsaturated acid was assigned by spectroscopic methods, but the relative and absolute stereochemistry of the five stereocentres remained unknown. Herein we describe isolation of kalimantacin A and two further metabolites 17,19-diol 2 and 27-descarbomyl hydroxyketone 3 from cultures of P. fluorescens. Their absolute and relative stereochemistries are rigorously determined using a multidisciplinary approach combining natural product degradation and fragment synthesis with bioinformatics and NMR spectroscopy. Diol 2 has the 5R, 15S, 17S, 19R, 26R, 27R configuration and is the immediate biosynthetic precursor of the bioactive kalimantacin A formed by oxidation of the 17-alcohol to the ketone.

CREBH Maintains Circadian Glucose Homeostasis by Regulating Hepatic Glycogenolysis and Gluconeogenesis.

Cyclic AMP-responsive element binding protein, hepatocyte specific (CREBH), is a liver-enriched, endoplasmic reticulum-tethered transcription factor known to regulate the hepatic acute-phase response and lipid homeostasis. In this study, we demonstrate that CREBH functions as a circadian transcriptional regulator that plays major roles in maintaining glucose homeostasis. The proteolytic cleavage and posttranslational acetylation modification of CREBH are regulated by the circadian clock. Functionally, CREBH is required in order to maintain circadian homeostasis of hepatic glycogen storage and blood glucose levels. CREBH regulates the rhythmic expression of the genes encoding the rate-limiting enzymes for glycogenolysis and gluconeogenesis, including liver glycogen phosphorylase (PYGL), phosphoenolpyruvate carboxykinase 1 (PCK1), and the glucose-6-phosphatase catalytic subunit (G6PC). CREBH interacts with peroxisome proliferator-activated receptor α (PPARα) to synergize its transcriptional activities in hepatic gluconeogenesis. The acetylation of CREBH at lysine residue 294 controls CREBH-PPARα interaction and synergy in regulating hepatic glucose metabolism in mice. CREBH deficiency leads to reduced blood glucose levels but increases hepatic glycogen levels during the daytime or upon fasting. In summary, our studies revealed that CREBH functions as a key metabolic regulator that controls glucose homeostasis across the circadian cycle or under metabolic stress.

CREBH Couples Circadian Clock With Hepatic Lipid Metabolism.

The circadian clock orchestrates diverse physiological processes critical for health and disease. CREB, hepatocyte specific (CREBH) is a liver-enriched, endoplasmic reticulum (ER)-tethered transcription factor known to regulate the hepatic acute phase response and energy homeostasis under stress conditions. We demonstrate that CREBH is regulated by the circadian clock and functions as a circadian regulator of hepatic lipid metabolism. Proteolytic activation of CREBH in the liver exhibits typical circadian rhythmicity controlled by the core clock oscillator BMAL1 and AKT/glycogen synthase kinase 3ß (GSK3ß) signaling pathway. GSK3ß-mediated phosphorylation of CREBH modulates the association between CREBH and the coat protein complex II transport vesicle and thus controls the ER-to-Golgi transport and subsequent proteolytic cleavage of CREBH in a circadian manner. Functionally, CREBH regulates circadian expression of the key genes involved in triglyceride (TG) and fatty acid (FA) metabolism and is required to maintain circadian amplitudes of blood TG and FA in mice. During the circadian cycle, CREBH rhythmically regulates and interacts with the hepatic nuclear receptors peroxisome proliferator-activated receptor α and liver X receptor α as well as with the circadian oscillation activator DBP and the repressor E4BP4 to modulate CREBH transcriptional activities. In conclusion, these studies reveal that CREBH functions as a circadian-regulated liver transcriptional regulator that integrates energy metabolism with circadian rhythm.

Dopamine D1 receptor expression is bipolar cell type-specific in the mouse retina.

In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a-tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type-dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5-2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5-1, 9, and rod bipolar cells did not express Drd1a-tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion-coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type-dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059-2079, 2016. © 2015 Wiley Periodicals, Inc.

Effects of gender on locomotor sensitivity to amphetamine, body weight, and fat mass in regulator of G protein signaling 9 (RGS9) knockout mice.

Regulator of G-protein signaling (RGS) protein 9-2 is enriched in the striatum where it modulates dopamine and opioid receptor-mediated signaling. RGS9 knockout (KO) mice show increased psychostimulant-induced behavioral sensitization, as well as exhibit higher body weights and greater fat accumulation compared to wild-type (WT) littermates. In the present study, we found gender influences on each of these phenotypic characteristics. Female RGS9 KO mice exhibited greater locomotor sensitization to amphetamine (1.0mg/kg) treatment as compared to male RGS9 KO mice. Male RGS9 KO mice showed increased body weights as compared to male WT littermates, while no such differences were detected in female mice. Quantitative magnetic resonance showed that male RGS9 KO mice accumulated greater fat mass vs. WT littermates at 5months of age. Such observations could not be explained by increased caloric consumption since male and female RGS9 KO mice demonstrated equivalent daily food intake as compared to their respective WT littermates. Although indirect calorimetry methods found decreased oxygen consumption and carbon dioxide production during the 12-hour dark phase in male RGS9 KO vs. WT mice which are indicative of less energy expenditure, male RGS9 KO mice exhibited lower levels of locomotor activity during this period. Genotype had no effect on metabolic activities when KO and WT groups were compared under fasting vs. feeding treatments. In summary, these results highlight the importance of factoring gender into the experimental design since many studies conducted in RGS9 KO mice utilize locomotor activity as a measured outcome.

Silhouette estimation.

Silhouettes arise in a variety of imaging scenarios. Pristine silhouettes are often degraded via blurring, detector sampling, and detector noise. We present a maximum a posteriori estimator for the restoration of parameterized facial silhouettes. Extreme dealiasing and dramatic superresolution, well beyond the diffraction limit, are demonstrated through the use of strong prior knowledge.

A Tet-on system for DRD1-expressing cells.

Cells expressing the dopamine D1 receptor (DRD1) have significant functional roles in diverse physiological processes including locomotion and drug addiction. The present work presents a novel in vivo DRD1-Bacterial Artificial Chromosome (BAC) Tet-on system allowing for the inducible activation of tet-operated transgenes specifically within DRD1-expressing cells of transgenic mice. It is shown that the DRD1-rtTA BAC-driven expression of a tet-operated reporter is under tight regulation by doxycycline and is restricted to DRD1-expressing brain regions. The model will be a useful research tool in studies of movement and reward and associated pathologies such as Parkinson's disease and addiction.

Role of the primary motor cortex in L-Dopa-induced dyskinesia and its modulation by 5-HT1A receptor stimulation.

While serotonin 5-HT1A receptor (5-HT1AR) agonists reduce L-DOPA-induced dyskinesias (LID) by normalizing activity in the basal ganglia neurocircuitry, recent evidence suggests putative 5-HT1AR within the primary motor cortex (M1) may also contribute. To better characterize this possible mechanism, c-fos immunohistochemistry was first used to determine the effects of systemic administration of the full 5-HT1AR agonist ±8-OH-DPAT on L-Dopa-induced immediate early gene expression within M1 and the prefrontal cortex (PFC) of rats with unilateral medial forebrain bundle (MFB) dopamine (DA) lesions. Next, in order to determine if direct stimulation of 5-HT1AR within M1 attenuates the onset of LID, rats with MFB lesions were tested for L-Dopa-induced abnormal involuntary movements (AIMs) and rotations following M1 microinfusions of ±8-OH-DPAT with or without coadministration of the 5-HT1AR antagonist WAY100635. Finally, ±8-OH-DPAT was infused into M1 at peak dyskinesia to determine if 5-HT1AR stimulation attenuates established L-Dopa-induced AIMs and rotations. While no treatment effects were seen within the PFC, systemic ±8-OH-DPAT suppressed L-Dopa-induced c-fos within M1. Intra-M1 5-HT1AR stimulation diminished the onset of AIMs and this effect was reversed by WAY100635 indicating receptor specific effects. Finally, continuous infusion of ±8-OH-DPAT into M1 at peak dyskinesia alleviated L-Dopa-induced AIMs. Collectively, these findings support an integral role for M1 in LID and its modulation by local 5-HT1AR.

Contribution of the striatum to the effects of 5-HT1A receptor stimulation in L-DOPA-treated hemiparkinsonian rats.

Clinical and experimental studies implicate the use of serotonin (5-HT)1A receptor agonists for the reduction of L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID). Although raphe nuclei likely play a role in these antidyskinetic effects, an unexplored population of striatal 5-HT1A receptors (5-HT1AR) may also contribute. To better characterize this mechanism, L-DOPA-primed hemiparkinsonian rats received the 5-HT1AR agonist +/-8-OH-DPAT (0, 0.1, 1.0 mg/kg, i.p.) with or without cotreatment with the 5-HT1AR antagonist WAY100635 (0.5 mg/kg, i.p.) 5 min after L-DOPA, after which abnormal involuntary movements (AIMs), rotations, and forelimb akinesia were quantified. To establish the effects of 5-HT1AR stimulation on L-DOPA-induced c-fos and preprodynorphin (PPD) mRNA within the dopamine-depleted striatum, immunohistochemistry and real-time reverse transcription polymerase chain reaction, respectively, were used. Finally, to determine the contribution of striatal 5-HT1AR to these effects, L-DOPA-primed hemiparkinsonian rats received bilateral intrastriatal microinfusions of +/-8-OH-DPAT (0, 5, or 10 microg/side), WAY100635 (5 microg/side), or both (10 microg + 5 microg/side) 5 min after L-DOPA, after which AIMs and rotations were examined. Systemic +/-8-OH-DPAT dose- and receptor-dependently attenuated L-DOPA-mediated AIMs and improved forelimb akinesia. Striatal c-fos immunoreactivity and PPD mRNA ipsilateral to the lesion were strongly induced by L-DOPA, while +/-8-OH-DPAT suppressed these effects. Finally, intrastriatal infusions of +/-8-OH-DPAT reduced AIMs while coinfusion of WAY100635 reversed its antidyskinetic effect. Collectively, these results support the hypothesis that the cellular and behavioral properties of 5-HT1AR agonists are conveyed in part via a population of functional 5-HT1AR within the striatum.

Ontogeny of serotonin and serotonin2A receptors in rat auditory cortex.

Maturation of the mammalian cerebral cortex is, in part, dependent upon multiple coordinated afferent neurotransmitter systems and receptor-mediated cellular linkages during early postnatal development. Given that serotonin (5-HT) is one such system, the present study was designed to specifically evaluate 5-HT tissue content as well as 5-HT(2A) receptor protein levels within the developing auditory cortex (AC). Using high performance liquid chromatography (HPLC), 5-HT and the metabolite, 5-hydroxyindoleacetic acid (5-HIAA), was measured in isolated AC, which demonstrated a developmental dynamic, reaching young adult levels early during the second week of postnatal development. Radioligand binding of 5-HT(2A) receptors with the 5-HT(2A/2C) receptor agonist, (125)I-DOI ((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl; in the presence of SB206553, a selective 5-HT(2C) receptor antagonist, also demonstrated a developmental trend, whereby receptor protein levels reached young adult levels at the end of the first postnatal week (P8), significantly increased at P10 and at P17, and decreased back to levels not significantly different from P8 thereafter. Immunocytochemical labeling of 5-HT(2A) receptors and confocal microscopy revealed that 5-HT(2A) receptors are largely localized on layer II/III pyramidal cell bodies and apical dendrites within AC. When considered together, the results of the present study suggest that 5-HT, likely through 5-HT(2A) receptors, may play an important role in early postnatal AC development.