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Low dopamine D2 receptor (D2R) availability in the striatum can predispose for cocaine abuse; though how low striatal D2Rs facilitate cocaine reward is unclear. Overexpression of D2Rs in striatal neurons or activation of D2Rs by acute cocaine suppresses striatal Penk mRNA. Conversely, low D2Rs in D2-striatal neurons increases striatal Penk mRNA and enkephalin peptide tone, an endogenous mu-opioid agonist. In brain slices, met-enkephalin and inhibition of enkephalin catabolism suppresses intra-striatal GABA transmission. Pairing cocaine with intra-accumbens met-enkephalin during place conditioning facilitates acquisition of preference, while mu-opioid receptor antagonist blocks preference in wild-type mice. We propose that heightened striatal enkephalin potentiates cocaine reward by suppressing intra-striatal GABA to enhance striatal output. Surprisingly, a mu-opioid receptor antagonist does not block cocaine preference in mice with low striatal D2Rs, implicating other opioid receptors. The bidirectional regulation of enkephalin by D2R activity and cocaine offers insights into mechanisms underlying the vulnerability for cocaine abuse., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

3-Alkylpyridine alkaloids (3-APAs) isolated from the arctic sponge Haliclona viscosa are a promising group of bioactive marine alkaloids. However, due to limited bioavailability, investigations of their bioactivity have been hampered. Additionally, synthesis of a common intermediate requires the use of protecting groups and harsh conditions. In this work, we developed a simple and concise two-step route to nine different natural and synthetic haliclocyclins. These compounds displayed modest antibiotic activity against several Gram-positive bacterial strains., (© 2021 Wiley-VCH GmbH.)

Iron is an essential nutrient to nearly all living beings. However, its acquisition poses a significant challenge to many organisms, including most bacteria. One of the main iron uptake strategies employed by bacteria is the uptake of siderophores, small molecules that chelate extracellular iron. The pathogenic species Pseudomonas aeruginosa produces two different siderophores, pyochelin and pyoverdine. P. aeruginosa senses the amount of bioavailable extracellular iron in order to regulate the production levels of each of these two siderophores. In previous work, we found that a series of pyochelin biosynthetic shunt products enhanced the growth of P. aeruginosa in iron-depleted conditions when prechelated with iron. Thus, on the basis of these results, we investigated the physiochemical and biological properties of a series of non-native oxygen counterparts to these metabolites in the current study., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest

Covering: 2000 to 2020. trans-Bicyclo[4.4.0]decane/decene (such as trans-decalin and trans-octalin)-containing natural products display a wide range of structural diversity and frequently exhibit potent and selective antibacterial activities. With one of the major factors in combatting antibiotic resistance being the discovery of novel scaffolds, the efficient construction of these natural products is an attractive pursuit in the development of novel antibiotics. This highlight aims to provide a critical analysis on how the presence of dense architectural and stereochemical complexity necessitated special strategies in the synthetic pursuits of these natural trans-bicyclo[4.4.0]decane/decene antibiotics.

The C-terminal domain of Bacillus cereus hemolysin II (HlyIIC), stabilizes the trans-membrane-pore formed by the HlyII toxin and may aid in target cell recognition. Initial efforts to determine the NMR structure of HlyIIC were hampered by cis/trans isomerization about the single proline at position 405 that leads to doubling of NMR resonances. We used the mutant P405M-HlyIIC that eliminates the cis proline to determine the NMR structure of the domain, which revealed a novel fold. Here, we extend earlier studies to the NMR structure determination of the cis and trans states of WT-HlyIIC that exist simultaneously in solution. The primary structural differences between the cis and trans states are in the loop that contains P405, and structurally adjacent loops. Thermodynamic linkage analysis shows that at 25 C the cis proline, which already has a large fraction of 20% in the unfolded protein, increases to 50% in the folded state due to coupling with the global stability of the domain. The P405M or P405A substitutions eliminate heterogeneity due to proline isomerization but lead to the formation of a new dimeric species. The NMR structure of the dimer shows that it is formed through domain-swapping of strand β5, the last segment of secondary structure following P405. The presence of P405 in WT-HlyIIC strongly disfavors the dimer compared to the P405M-HlyIIC or P405A-HlyIIC mutants. The WT proline may thus act as a "gatekeeper," warding off aggregative misfolding., (© 2021 The Protein Society.)

Pseudomonads employ several strategies to sequester iron vital for their survival including the use of siderophores such as pyoverdine and pyochelin. Similar in structure but significantly less studied are pyochelin biosynthetic byproducts, dihydroaeruginoic acid, aeruginoic acid, aeruginaldehyde (IQS), and aeruginol, along with two other structurally related molecules, aerugine and pyonitrins A-D, which have all been isolated from numerous Pseudomonad extracts. Because of the analogous substructure of these compounds to pyochelin, we hypothesized that they may play a role in iron homeostasis or have a biological effect on other bacterial species. Herein, we discuss the physiochemical evaluation of these molecules and disclose, for the first time, their ability to bind iron and promote growth in Pseudomonads .

The delivery of cancer therapeutics can be limited by pharmacological issues such as poor bioavailability and high toxicity to healthy tissue. pH-low insertion peptides (pHLIPs) represent a promising tool to overcome these limitations. pHLIPs allow for the selective delivery of agents to tumors on the basis of pH, taking advantage of the acidity of the hypoxic tumor microenvironment. This review article highlights the various applications in which pHLIPs have been utilized for targeting and treating diseases in hypoxic environments, including delivery of small molecule inhibitors, toxins, nucleic acid analogs, fluorescent dyes, and nanoparticles.

Promysalin is a small-molecule natural product that specifically inhibits growth of the Gram-negative pathogen Pseudomonas aeruginosa ( PA ). This activity holds promise in the treatment of multidrug resistant infections found in immunocompromised patients with chronic illnesses, such as cystic fibrosis. In 2015, our lab completed the first total synthesis; subsequent analogue design and SAR investigation enabled identification of succinate dehydrogenase (Sdh) as the biological target in PA . Herein, we report the target-guided design of new promysalin analogues with varying alkyl chains, one of which is on par with our most potent analogue to date. Computational docking revealed that some analogues have a different orientation in the Sdh binding pocket, placing the terminal carbon proximal to a tryptophan residue. This inspired the design of an extended side chain analogue bearing a terminal phenyl moiety, providing a basis for the design of future analogues.

The development of therapeutic agents that specifically target cancer cells while sparing healthy tissue could be used to enhance the efficacy of cancer therapy without increasing its toxicity. Specific targeting of cancer cells can be achieved through the use of pH-low insertion peptides (pHLIP), which take advantage of the acidity of the tumor microenvironment to deliver cargoes selectively to tumor cells. We developed a pHLIP-peptide nucleic acid (PNA) conjugate as an antisense reagent to reduce expression of the otherwise undruggable DNA double-strand break repair factor, KU80, and thereby radiosensitize tumor cells. Increased antisense activity of the pHLIP-PNA conjugate was achieved by partial mini-PEG sidechain substitution of the PNA at the gamma position, designated pHLIP-αKu80(γ). We evaluated selective effects of pHLIP-αKu80(γ) in cancer cells in acidic culture conditions as well as in two subcutaneous mouse tumor models. Fluorescently labeled pHLIP-αKu80(γ) delivers specifically to acidic cancer cells and accumulates preferentially in tumors when injected i.v. in mice. Furthermore, pHLIP-αKu80(γ) selectively reduced KU80 expression in cells under acidic conditions and in tumors in vivo . When pHLIP-αKu80(γ) was administered to mice prior to local tumor irradiation, tumor growth was substantially reduced compared with radiation treatment alone. Furthermore, there was no evidence of acute toxicity associated with pHLIP-αKu80(γ) administration to the mice. These results establish pHLIP-αKu80(γ) as a tumor-selective radiosensitizing agent. IMPLICATIONS: This study describes a novel agent, pHLIP-αKu80(γ), which combines PNA antisense and pHLIP technologies to selectively reduce the expression of the DNA repair factor KU80 in tumors and confer tumor-selective radiosensitization., (©2020 American Association for Cancer Research.)

Hypoxia is a hallmark of the tumour microenvironment with profound effects on tumour biology, influencing cancer progression, the development of metastasis and patient outcome. Hypoxia also contributes to genomic instability and mutation frequency by inhibiting DNA repair pathways. This review summarises the diverse mechanisms by which hypoxia affects DNA repair, including suppression of homology-directed repair, mismatch repair and base excision repair. We also discuss the effects of hypoxia mimetics and agents that induce hypoxia on DNA repair, and we highlight areas of potential clinical relevance as well as future directions., (© The Author(s) 2019. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There is much interest in targeting DNA repair pathways for use in cancer therapy, as the effectiveness of many therapeutic agents relies on their ability to cause damage to DNA, and deficiencies in DSB repair pathways can make cells more sensitive to specific cancer therapies. For example, defects in the double-strand break (DSB) pathways, non-homologous end joining (NHEJ) and homology-directed repair (HDR), induce sensitivity to radiation therapy and poly(ADP)-ribose polymerase (PARP) inhibitors, respectively. However, traditional approaches to inhibit DNA repair through small molecule inhibitors have often been limited by toxicity and poor bioavailability. This review identifies several pharmacologic manipulations that modulate DSB repair by reducing expression of DNA repair factors. A number of pathways have been identified that modulate activity of NHEJ and HDR through this mechanism, including growth and hormonal receptor signaling pathways as well as epigenetic modifiers. We also discuss the effects of anti-angiogenic therapy on DSB repair. Preclinically, these pharmacological manipulations of DNA repair factor expression have been shown to increase sensitivity to specific cancer therapies, including ionizing radiation and PARP inhibitors. When applicable, relevant clinical trials are discussed and areas for future study are identified., (© 2020 Elsevier Inc. All rights reserved.)

Introduction: Opioid overdose deaths quintupled in Massachusetts between 2000 and 2016. Potentially inappropriate opioid prescribing practices (PIP) are associated with increases in overdoses. The purpose of this study was to conduct spatial epidemiological analyses of novel comprehensively linked data to identify overdose and PIP hotspots., Methods: Sixteen administrative datasets, including prescription monitoring, medical claims, vital statistics, and medical examiner data, covering >98% of Massachusetts residents between 2011-2015, were linked in 2017 to better investigate the opioid epidemic. PIP was defined by six measures: ≥100 morphine milligram equivalents (MMEs), co-prescription of benzodiazepines and opioids, cash purchases of opioid prescriptions, opioid prescriptions without a recorded pain diagnosis, and opioid prescriptions through multiple prescribers or pharmacies. Using spatial autocorrelation and cluster analyses, overdose and PIP hotspots were identified among 538 ZIP codes., Results: More than half of the adult population (n = 3,143,817, ages 18 and older) were prescribed opioids. Nearly all ZIP codes showed increasing rates of overdose over time. Overdose clusters were identified in Worcester, Northampton, Lee/Tyringham, Wareham/Bourne, Lynn, and Revere/Chelsea (Getis-Ord Gi*; p < 0.05). Large PIP clusters for ≥100 MMEs and prescription without pain diagnosis were identified in Western Massachusetts; and smaller clusters for multiple prescribers in Nantucket, Berkshire, and Hampden Counties (p < 0.05). Co-prescriptions and cash payment clusters were localized and nearly identical (p < 0.05). Overlap in PIP and overdose clusters was identified in Cape Cod and Berkshire County. However, we also found contradictory patterns in overdose and PIP hotspots., Conclusions: Overdose and PIP hotspots were identified, as well as regions where the two overlapped, and where they diverged. Results indicate that PIP clustering alone does not explain overdose clustering patterns. Our findings can inform public health policy decisions at the local level, which include a focus on PIP and misuse of heroin and fentanyl that aim to curb opioid overdoses., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Combining the anti-angiogenic agent cediranib with the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib improves progression-free survival compared to olaparib alone in ovarian cancer patients through an unknown mechanism. PARP inhibitors are used primarily in the treatment of patients with DNA repair-associated (BRCA1/2) mutated cancers because these mutations cause a deficit in homology-directed DNA repair (HDR) that confers sensitivity to these agents. However, the combination of cediranib and olaparib was effective in patients without BRCA1/2 mutations. We report here that cediranib confers sensitivity to olaparib by down-regulating HDR in tumor cells. This occurs partially as a result of cediranib inducing hypoxia, which suppresses expression of the HDR factors BRCA1/2 and RAD51 recombinase (RAD51). However, we also observed that cediranib has a direct effect on HDR independent of its ability to induce tumor hypoxia. This direct effect occurs through platelet-derived growth factor receptor (PDGFR) inhibition, activation of protein phosphatase 2A (PP2A), and E2F transcription factor 4 (E2F4)/RB transcriptional corepressor like 2 (RB2/p130)-mediated repression of BRCA1/2 and RAD51 gene expression. This down-regulation was seen in mouse tumor xenografts but not in mouse bone marrow, providing a therapeutic window for combining cediranib and olaparib in cancer therapy. Our work reveals a treatment strategy by which DNA repair can be manipulated in human tumors to induce synthetic lethality, broadening the potential therapeutic scope of cediranib based on its activity as a DNA repair inhibitor., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Despite very low sequence homology, the major capsid proteins of double-stranded DNA (dsDNA) bacteriophages, some archaeal viruses, and the herpesviruses share a structural motif, the HK97 fold. Bacteriophage P22, a paradigm for this class of viruses, belongs to a phage gene cluster that contains three homology groups: P22-like, CUS-3-like, and Sf6-like. The coat protein of each phage has an inserted domain (I-domain) that is more conserved than the rest of the coat protein. In P22, loops in the I-domain are critical for stabilizing intra- and intersubunit contacts that guide proper capsid assembly. The nuclear magnetic resonance (NMR) structures of the P22, CUS-3, and Sf6 I-domains reveal that they are all six-stranded, anti-parallel β-barrels. Nevertheless, significant structural differences occur in loops connecting the β-strands, in surface electrostatics used to dock the I-domains with their respective coat protein core partners, and in sequence motifs displayed on the capsid surfaces. Our data highlight the structural diversity of I-domains that could lead to variations in capsid assembly mechanisms and capsid surfaces adapted for specific phage functions. IMPORTANCE Comparative studies of protein structures often provide insights into their evolution. The HK97 fold is a structural motif used to form the coat protein shells that encapsidate the genomes of many dsDNA phages and viruses. The structure and function of coat proteins based on the HK97 fold are often embellished by the incorporation of I-domains. In the present work we compare I-domains from three phages representative of highly divergent P22-like homology groups. While the three I-domains share a six-stranded β-barrel skeleton, there are differences (i) in structure elements at the periphery of the conserved fold, (ii) in the locations of disordered loops important in capsid assembly and conformational transitions, (iii) in surfaces charges, and (iv) in sequence motifs that are potential ligand-binding sites. These structural modifications on the rudimentary I-domain fold suggest that considerable structural adaptability was needed to fulfill the versatile range of functional requirements for distinct phages., (Copyright © 2019 American Society for Microbiology.)

Vulnerability for cocaine abuse in humans is associated with low dopamine D2 receptor (D2R) availability in the striatum. The mechanisms driving this vulnerability are poorly understood. In this study, we found that downregulating D2R expression selectively in striatal indirect-pathway neurons triggers a multitude of changes in D1 receptor (D1R)-expressing direct-pathway neurons, which comprise the other main subpopulation of striatal projection neurons. These changes include a leftward shift in the dose-response to a D1-like agonist that indicates a behavioral D1R hypersensitivity, a shift from PKA to ERK intracellular signaling cascades upon D1R activation, and a reduction in the density of bridging collaterals from D1R-expressing neurons to pallidal areas. We hypothesize that the D1R hypersensitivity underlies abuse vulnerability by facilitating the behavioral responses to repeated cocaine, such as locomotor sensitization and drug self-administration. We found evidence that littermate control mice develop D1R hypersensitivity after they are sensitized to cocaine. Indeed, D1-like agonist and cocaine cross-sensitize in control littermates and this effect was potentiated in mice lacking striatal D2Rs from indirect-pathway neurons. To our surprise, mice with low striatal D2Rs acquired cocaine self-administration similarly to littermate controls and showed no significant change in motivation to take cocaine but lower seeking. These findings indicate that downregulation of striatal D2Rs triggers D1R hypersensitivity to facilitate cocaine locomotor sensitization, which by itself was not associated with greater cocaine taking or seeking under the conditions tested.

Twenty-one mono- and biscationic quaternary ammonium amphiphiles (monoQACs and bisQACs) were rapidly prepared in order to investigate the effects of rigidity of a diamine core structure on antiseptic activity. As anticipated, the bioactivity against a panel of six bacteria including methicillin-resistant Staphylococcus aureus (MRSA) strains was strong for bisQAC structures, and is clearly correlated with the length of non-polar side chains. Modest advantages were noted for amide-containing side chains, as compared with straight-chained alkyl substituents. Surprisingly, antiseptics with more rigidly disposed side chains, such as those in DABCO-12,12, showed the highest level of antimicrobial activity, with single-digit MIC values or better against the entire bacterial panel, including sub-micromolar activity against an MRSA strain., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The spread of antimicrobial resistance is a major threat to human health, and patients requiring prolonged antibiotic exposure are in desperate need of new therapeutic strategies. It has been hypothesized that tailoring our antibiotics to inhibit molecular targets specific to pathogens might stem the spread of resistance. A prime candidate for such a strategy is Pseudomonas aeruginosa, which can be found in the lungs of nearly all adult cystic fibrosis patients and, due to chronic exposure to antibiotics, has a high rate of multidrug-resistant strains. Although much research has been done on P. aeruginosa virulence factors as narrow-spectrum targets, less attention has been paid to primary carbon metabolism being leveraged for pathogen-specific mechanisms. However, early studies show that primary metabolic pathways, although shared amongst all organisms, contain intricacies specific to Pseudomonas species that have potential for antibiotic exploitation. Here we lay out some of this work in the hopes that it inspires researchers to continue developing a knowledge base for future antibiotic discovery to build upon and include a case study of a Pseudomonas primary metabolic pathway that has been targeted by small molecules in a species-specific manner., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study investigates the safety and efficacy of a large-dose, needle-based epidural technique in obstetric anesthesia. The technique differs from a standard, catheter-based approach in that the anesthetic dose is administered through an epidural needle prior to insertion of the epidural catheter. Using a data-driven informatics and machine learning approach, our findings show that the needle-based technique is faster and more dose-effective in achieving sensory level. We also find that injecting large doses in the epidural space through the epidural needle is safe, with complication rates similar to those reported in published literature for catheter-based technique. Further, machine learning reveals that if the needle dose is kept under 18 ml, the resulting hypotension rate will be significantly lower than published results. The machine learning framework can predict the incidence of hypotension with 85% accuracy. The findings from this investigation facilitate delivery improvement and establish an improved clinical practice guideline for training and for dissemination of safe practice.

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

In addition to multiple virulence factors, Bacillus cereus a pathogen that causes food poisoning and life-threatening wound infections, secretes the pore-forming toxin hemolysin II (HlyII). The HlyII toxin has a unique 94 amino acid C-terminal domain (HlyIIC). HlyIIC exhibits splitting of NMR resonances due to cis/trans isomerization of a single proline near the C-terminus. To overcome heterogeneity, we solved the structure of P405M-HlyIIC, a mutant that exclusively stabilizes the trans state. The NMR structure of HlyIIC reveals a novel fold, consisting of two subdomains αA-β1-β2 and β3-β4-αB-β5, that come together in a barrel-like structure. The barrel core is fastened by three layers of hydrophobic residues. The barrel end opposite the HlyIIC-core has a positively charged surface, that by binding negatively charged moieties on cellular membranes, may play a role in target-cell surface recognition or stabilization of the heptameric pore complex. In the WT domain, dynamic flexibility occurs at the N-terminus and the first α-helix that connects the HlyIIC domain to the HlyII-core structure. In the destabilizing P405M mutant, increased flexibility is evident throughout the first subdomain, suggesting that the HlyIIC structure may have arisen through gene fusion.

To understand the roles ion pairs play in stabilizing coiled coils, we determined nuclear magnetic resonance structures of GCN4p at three pH values. At pH 6.6, all acidic residues are fully charged; at pH 4.4, they are half-charged, and at pH 1.5, they are protonated and uncharged. The α-helix monomer and coiled coil structures of GCN4p are largely conserved, except for a loosening of the coiled coil quaternary structure with a decrease in pH. Differences going from neutral to acidic pH include (i) an unwinding of the coiled coil superhelix caused by the loss of interchain ion pair contacts, (ii) a small increase in the separation of the monomers in the dimer, (iii) a loosening of the knobs-into-holes packing motifs, and (iv) an increased separation between oppositely charged residues that participate in ion pairs at neutral pH. Chemical shifts (HN, N, C', Cα, and Cβ) of GCN4p display a seven-residue periodicity that is consistent with α-helical structure and is invariant with pH. By contrast, periodicity in hydrogen exchange rates at neutral pH is lost at acidic pH as the exchange mechanism moves into the EX1 regime. On the basis of 1 H- 15 N nuclear Overhauser effect relaxation measurements, the α-helix monomers experience only small increases in picosecond to nanosecond backbone dynamics at acidic pH. By contrast, 13 C rotating frame T 1 relaxation (T 1ρ ) data evince an increase in picosecond to nanosecond side-chain dynamics at lower pH, particularly for residues that stabilize the coiled coil dimerization interface through ion pairs. The results on the structure and dynamics of GCNp4 over a range of pH values help rationalize why a single structure at neutral pH poorly predicts the pH dependence of the unfolding stability of the coiled coil.

Obesity is associated with physical inactivity, which exacerbates the health consequences of weight gain. However, the mechanisms that mediate this association are unknown. We hypothesized that deficits in dopamine signaling contribute to physical inactivity in obesity. To investigate this, we quantified multiple aspects of dopamine signaling in lean and obese mice. We found that D2-type receptor (D2R) binding in the striatum, but not D1-type receptor binding or dopamine levels, was reduced in obese mice. Genetically removing D2Rs from striatal medium spiny neurons was sufficient to reduce motor activity in lean mice, whereas restoring G i signaling in these neurons increased activity in obese mice. Surprisingly, although mice with low D2Rs were less active, they were not more vulnerable to diet-induced weight gain than control mice. We conclude that deficits in striatal D2R signaling contribute to physical inactivity in obesity, but inactivity is more a consequence than a cause of obesity., (Published by Elsevier Inc.)

Striatal medium spiny neurons (MSNs) form inhibitory synapses on neighboring striatal neurons through axon collaterals. The functional relevance of this lateral inhibition and its regulation by dopamine remains elusive. We show that synchronized stimulation of collateral transmission from multiple indirect-pathway MSNs (iMSNs) potently inhibits action potentials in direct-pathway MSNs (dMSNs) in the nucleus accumbens. Dopamine D2 receptors (D2Rs) suppress lateral inhibition from iMSNs to disinhibit dMSNs, which are known to facilitate locomotion. Surprisingly, D2R inhibition of synaptic transmission was larger at axon collaterals from iMSNs than their projections to the ventral pallidum. Targeted deletion of D2Rs from iMSNs impaired cocaine's ability to suppress lateral inhibition and increase locomotion. These impairments were rescued by chemogenetic activation of Gi-signaling in iMSNs. These findings shed light on the functional significance of lateral inhibition between MSNs and offer a novel synaptic mechanism by which dopamine gates locomotion and cocaine exerts its canonical stimulant response. VIDEO ABSTRACT., (Published by Elsevier Inc.)

Bradykinesia is a prominent phenotype of Parkinson's disease, depression, and other neurological conditions. Disruption of dopamine (DA) transmission plays an important role, but progress in understanding the exact mechanisms driving slowness of movement has been impeded due to the heterogeneity of DA receptor distribution on multiple cell types within the striatum. Here we show that selective deletion of DA D2 receptors (D2Rs) from indirect-pathway medium spiny neurons (iMSNs) is sufficient to impair locomotor activity, phenocopying DA depletion models of Parkinson's disease, despite this mouse model having intact DA transmission. There was a robust enhancement of GABAergic transmission and a reduction of in vivo firing in striatal and pallidal neurons. Mimicking D2R signaling in iMSNs with Gi-DREADDs restored the level of tonic GABAergic transmission and rescued the motor deficit. These findings indicate that DA, through D2R activation in iMSNs, regulates motor output by constraining the strength of GABAergic transmission., (Published by Elsevier Inc.)

A prominent aspect of drug addiction is the ability of drug-associated cues to elicit craving and facilitate relapse. Understanding the factors that regulate cue reactivity will be vital for improving treatment of addictive disorders. Low availability of dopamine (DA) D2 receptors (D2Rs) in the striatum is associated with high cocaine intake and compulsive use. However, the role of D2Rs of nonstriatal origin in cocaine seeking and taking behavior and cue reactivity is less understood and possibly underestimated. D2Rs expressed by midbrain DA neurons function as autoreceptors, exerting inhibitory feedback on DA synthesis and release. Here, we show that selective loss of D2 autoreceptors impairs the feedback inhibition of DA release and amplifies the effect of cocaine on DA transmission in the nucleus accumbens (NAc) in vitro. Mice lacking D2 autoreceptors acquire a cued-operant self-administration task for cocaine faster than littermate control mice but acquire similarly for a natural reward. Furthermore, although mice lacking D2 autoreceptors were able to extinguish self-administration behavior in the absence of cocaine and paired cues, they exhibited perseverative responding when cocaine-paired cues were present. This enhanced cue reactivity was selective for cocaine and was not seen during extinction of sucrose self-administration. We conclude that low levels of D2 autoreceptors enhance the salience of cocaine-paired cues and can contribute to the vulnerability for cocaine use and relapse.

Pathogenic bacteria secrete pore-forming toxins (PFTs) to selectively defend against immune cells and to break through cellular barriers in the host. Understanding how PFTs attack cell membranes is not only essential for therapeutic intervention but for designing agents to deliver drugs to specific human cell subtypes, for example in anti-cancer or anti-viral therapies. Many toxins contain accessory domains that help recognize specific molecular epitopes on the membranes of target cells, including proteins, carbohydrates, and lipids. Here we report NMR assignments for the 94-residue 10 kDa C-terminal accessory domain of Bacillus cereus hemolysin II, HlyIIC, that has no known structural or functional homologues. The HlyIIC domain exists in a dynamic equilibrium due to cis/trans isomerization of its Gly86-Pro87 peptide bond. The cis and trans forms are about equally populated and are in slow exchange on the NMR timescale, giving rise to separate signals for approximately half of the residues in the domain. Assignments for the cis and trans forms were achieved with the aid of a P87M mutant that stabilizes the trans form, and separate sequential walks for the two forms in 3D NMR spectra of the wild-type HlyIIC. Based on backbone chemical shifts, the domain has a α1-α2-β1-β2-β3-β4-α3-β5 order of secondary structure elements. The last strand in the trans form and in the P87M mutant is shortened near Pro87 compared to the cis form. Both cis/trans isomerization and the P87M mutation cause large chemical shift changes throughout HlyIIC, suggesting that the proline is important in stabilizing the structure of the domain. The NMR assignments pave the way for solving the structures of the multiple conformational forms of HlyIIC and establishing their mechanism of interconversion.

A hallmark of addiction is the loss of control over drug intake, which is seen in only a fraction of those exposed to stimulant drugs such as cocaine. The cellular mechanisms underlying vulnerability or resistance to compulsive drug use remain unknown. We found that individual variability in the development of highly motivated and perseverative behavior toward cocaine is associated with synaptic plasticity in medium spiny neurons expressing dopamine D2 receptors (D2-MSNs) in the nucleus accumbens (NAc) of mice. Potentiation of glutamatergic inputs onto indirect pathway D2-MSNs was associated with resilience toward compulsive cocaine seeking. Inhibition of D2-MSNs using a chemicogenetic approach enhanced the motivation to obtain cocaine, whereas optogenetic activation of D2-MSNs suppressed cocaine self-administration. These results indicate that recruitment of D2-MSNs in NAc functions to restrain cocaine self-administration and serves as a natural protective mechanism in drug-exposed individuals.

DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.

Responses to vestibular stimulation can, under well-controlled experimental conditions, provide a measure of brain-stem function. Autistic children had significantly longer time constants during the primary nystagmus response and significantly fewer beats during the secondary response than normal children when stimulated with constant angular acceleration in complete darkness. These findings could not be attributed to gross differences in arousal, to developmental retardation, to associated clinical conditions, or to either the influence of vision or habituation. Rather, they are suggestive of a neurophysiologic dysfunction, perhaps involving the brain stem, and may be an expression of the process that underlies those autistic behaviors that suggest faulty modulation of sensory input and motor output. Brain-stem centers modulate both general sensory input and motor excitation and may play a role in the elaboration of the more complex adaptive and motivated behaviors that are also disturbed in autism.

Actual HL-A typing has been performed on 115 members of cancer family N, a large kindred (over 1000 members ascertained) showing the findings consistent with the cancer family syndrome. In the cancer-prone line (branches C and D) of the family, 20 of 21 members with cancer had one HL-A haplotype, HL-A2-HL-A12 (relative odds = 6.30), including some decreased family members who had haplotypes assigned. Eleven of 12 family members with cancer in branches C and D, actually typed, had HL-A2-HL-A12 (relative odds = 6.06). The single exception showing cancer and another haplotype in branch D is a child of a family member with haplotype HL-A2-HL-A12.

The significant differences in biochemical and chromosomal characteristics, and familial history observed between group of mothers who gave birth to children affected with G-trisomy (Down's syndrome) and their age-matched controls, indicate that these three maternal variables, in addition to the well known variable of maternal age, are associated with etiology of the aneuploidy. Since attempts to find statistical correlation between these chromosomal and biochemical variables failed, it is believed that these three are unrelated, but very possible etiological factors.

The time constants and gains of the vestibulo-ocular reflex were computed from the primary nystagmus evoked by constant angular acceleration in 79 normal infants and children, ranging in age from 2 months to 11 years old, and 10 normal adults. There were significant changes in both time constant and gain in respect to development. The time constants increased while the gains decreased as a function of the logarithm of age. The time constants of the youngest infants were close to the cupular time constant. The lengthening of the time constant with increasing age was discussed in respect to maturation of inhibitory brainstem reticular formation mechanisms.

The displacements, durations, and velocities of the slow and fast components of both the primary and secondary nystagmus induced by constant angular acceleration were measured in 46 normal children 1 month to 11 years old. There were significant changes in nystagmus parameters in respect to maturation. The young infant had larger amplitude, higher velocity beats than the older child during both the primary and the secondary nystagmus. Parameters describing both the primary and the secondary nystagmus reached their peak values and terminated earlier in the infant than in the older child. Although the slow component velocity during secondary nystagmus was much slower than during the primary nystagmus at all ages, the secondary nystagmus/primary nystagmus ratio was significantly greater in early infancy. Thus, in infancy, as compared with later childhood, the vigor of the secondary nystagmus was disproportionately greater than the primary nystagmus. These results were discussed in relation to the maturation both of vestibular responsiveness and of vestibular adaptation.