The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.

Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

Estradiol Regulation of the Prelimbic Cortex and the Reinstatement of Cocaine Seeking in Female Rats.

Relapse susceptibility in women with substance use disorders (SUDs) has been linked to the estrogen, 17ß-estradiol (E2). Our previous findings in female rats suggest that the influence of E2 on cocaine seeking can be localized to the prelimbic prefrontal cortex (PrL-PFC). Here, we investigated the receptor mechanisms through which E2 regulates the reinstatement of extinguished cocaine seeking. Sexually mature female rats underwent intravenous cocaine self-administration (0.5 mg/inf; 14 × 2 h daily) and extinction, and then were ovariectomized before reinstatement testing. E2 (10 µg/kg, i.p.) alone did not reinstate cocaine seeking, but it potentiated reinstatement when combined with an otherwise subthreshold priming dose of cocaine. A similar effect was observed following intra-PrL-PFC microinfusions of E2 and by systemic or intra-PrL-PFC administration of the estrogen receptor (ER)ß agonist, DPN, but not agonists at ERα or the G-protein-coupled ER1 (GPER1). By contrast, E2-potentiated reinstatement was prevented by intra-PrL-PFC microinfusions of the ERß antagonist, MPP, or the GPER1 antagonist, G15, but not an ERα antagonist. Whole-cell recordings in PrL-PFC layer (L)5/6 pyramidal neurons revealed that E2 decreases the frequency, but not amplitude, of GABAA-dependent miniature IPSCs (mIPSC). As was the case with E2-potentiated reinstatement, E2 reductions in mIPSC frequency were prevented by ERß and GPER1, but not ERα, antagonists and mimicked by ERß, but not GPER1, agonists. Altogether, the findings suggest that E2 activates ERß and GPER1 in the PrL-PFC to attenuate the GABA-mediated constraint of key outputs that mediate cocaine seeking.

Covalent Cross-Linking of Diketopyrrolopyrrole-Based Organogels with Polydiacetylenes.

A new strategy toward functional materials with novel properties and well-defined structures has been developed through the topochemical polymerization of diacetylene-containing diketopyrrolopyrrole (DPP) derivatives. In order to enable the efficient photopolymerization and cross-linking of the materials, a rational design of DPP-based derivatives has been performed to incorporate amide moieties, thus enabling the formation of intermolecular hydrogen bonds and the formation of an organogel. The new materials showed good gelation properties in aromatic solvents, resulting in the formation of a dense fibrous network in the gel state. Upon UV irradiation, the supramolecular self-assemblies obtained were shown to be efficiently cross-linked through the conversion of diacetylene into polydiacetylene. A detailed investigation of new resulting materials was performed by a combination of morphological characterization tools, including X-ray diffraction, Raman spectroscopy, and atomic force microscopy. Our results demonstrate that the topochemical polymerization of diacetylene-containing DPP-based compounds is a promising strategy toward new electroactive and well-defined materials, without the use of catalysts or additives, thus creating new opportunities for the preparation and processing of π-conjugated materials.

Purification and characterization of a Rieske oxygenase and its NADH-regenerating partner proteins.

The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins.

Molecular Structure and Conformational Design of Donor-Acceptor Conjugated Polymers to Enable Predictable Optoelectronic Property.

Tuning the optoelectronic properties of donor-acceptor conjugated polymers (D-A CPs) is of great importance in designing various organic optoelectronic devices. However, there remains a critical challenge in precise control of bandgap through synthetic approach, since the chain conformation also affects molecular orbital energy levels. Here, D-A CPs with different acceptor units are explored that show an opposite trend in energy band gaps with the increasing length of oligothiophene donor units. By investigating their chain conformation and molecular orbital energy, it is found that the molecular orbital energy alignment between donor and acceptor units plays a crucial role in dictating the final optical bandgap of D-A CPs. For polymers with staggered orbital energy alignment, the higher HOMO with increasing oligothiophene length leads to a narrowing of the optical bandgap despite decreased chain rigidity. On the other hand, for polymers with sandwiched orbital energy alignment, the increased band gap with increasing oligothiophene length originates from the reduction of bandwidth due to more localized charge density distribution. Thus, this work provides a molecular understanding of the role of backbone building blocks on the chain conformation and bandgaps of D-A CPs for organic optoelectronic devices through the conformation design and segment orbital energy alignment.

Investigation of the Effect of Turn Residues on Tetrapeptide Aldol Catalysts with ß-Turn Propensity.

Peptide catalysts for a wide diversity of reaction types contain a common motif-residues that bias the sequence toward ß-turn secondary structure. In this work, we explore what role that secondary structure plays in the catalysis of aldol reactions for primary amine tetrapeptide aldol catalysts. Using a lead tetrapeptide ß-turn catalytic sequence, we varied the i + 1 and i + 2 residues to amino acids that would affect the ß-turn propensity. We then studied the correlation between secondary structure, aldol rate enhancement, and stereoselectivity of the reaction between hydroxyacetone and 4-nitrobenzaldehyde. Using the i + 3 amide chemical shift as a measure of ß-turn character, we found a rough correlation between the peptide structure and reaction kinetics but minimal effect on stereoselectivity. These trends may help aid the design of future catalytic sequences.

Impairing proliferation of glioblastoma multiforme with CD44+ selective conjugated polymer nanoparticles.

Glioblastoma is one of the most aggressive types of cancer with success of therapy being hampered by the existence of treatment resistant populations of stem-like Tumour Initiating Cells (TICs) and poor blood-brain barrier drug penetration. Therapies capable of effectively targeting the TIC population are in high demand. Here, we synthesize spherical diketopyrrolopyrrole-based Conjugated Polymer Nanoparticles (CPNs) with an average diameter of 109 nm. CPNs were designed to include fluorescein-conjugated Hyaluronic Acid (HA), a ligand for the CD44 receptor present on one population of TICs. We demonstrate blood-brain barrier permeability of this system and concentration and cell cycle phase-dependent selective uptake of HA-CPNs in CD44 positive GBM-patient derived cultures. Interestingly, we found that uptake alone regulated the levels and signaling activity of the CD44 receptor, decreasing stemness, invasive properties and proliferation of the CD44-TIC populations in vitro and in a patient-derived xenograft zebrafish model. This work proposes a novel, CPN- based, and surface moiety-driven selective way of targeting of TIC populations in brain cancer.

The role of ß-hairpin conformation in ester hydrolysis peptide catalysts based on a TrpZip scaffold.

To explore the role of peptide conformation on catalytic activity in the context of ester hydrolysis catalysts, pairs of sequences were designed that contained or lacked ß-hairpin character. For the hydrolysis of para-nitrophenylacetate in aqueous media, we found small but consistent trends wherein His-containing sequences based on a TrpZip scaffold showed higher catalytic activity without ß-hairpin character.

Sex, stress, and prefrontal cortex: influence of biological sex on stress-promoted cocaine seeking.

Clinical reports suggest that females diagnosed with substance use disorder experience enhanced relapse vulnerability compared with males, particularly during stress. We previously demonstrated that a stressor (footshock) can potentiate cocaine seeking in male rats via glucocorticoid-dependent cannabinoid type-1 receptor (CB1R)-mediated actions in the prelimbic prefrontal cortex (PrL-PFC). Here, we investigated the influence of biological sex on stress-potentiated cocaine seeking. Despite comparable self-administration and extinction, females displayed a lower threshold for cocaine-primed reinstatement than males. Unlike males, footshock, tested across a range of intensities, failed to potentiate cocaine-primed reinstatement in females. However, restraint potentiated reinstatement in both sexes. While sex differences in stressor-induced plasma corticosterone (CORT) elevations and defensive behaviors were not observed, differences were evident in footshock-elicited ultrasonic vocalizations. CORT administration, at a dose which recapitulates stressor-induced plasma levels, reproduced stress-potentiated cocaine-primed reinstatement in both sexes. In females, CORT effects varied across the estrous cycle; CORT-potentiated reinstatement was only observed during diestrus and proestrus. As in males, CORT-potentiated cocaine seeking in females was localized to the PrL-PFC and both CORT- and restraint-potentiated cocaine seeking required PrL-PFC CB1R activation. In addition, ex vivo whole-cell electrophysiological recordings from female layer V PrL-PFC pyramidal neurons revealed CB1R-dependent CORT-induced suppression of inhibitory synaptic activity, as previously observed in males. These findings demonstrate that, while stress potentiates cocaine seeking via PrL-PFC CB1R in both sexes, sensitivity to cocaine priming injections is greater in females, CORT-potentiating effects vary with the estrous cycle, and whether reactivity to specific stressors may manifest as drug seeking depends on biological sex.

Photophysical and Optical Properties of Semiconducting Polymer Nanoparticles Prepared from Hyaluronic Acid and Polysorbate 80.

A nanoprecipitation procedure was utilized to prepare novel diketopyrrolopyrrole-based semiconducting polymer nanoparticles (SPNs) with hyaluronic acid (HA) and polysorbate 80. The nanoprecipitation led to the formation of spherical nanoparticles with average diameters ranging from 100 to 200 nm, and a careful control over the structure of the parent conjugated polymers was performed to probe the influence of π-conjugation on the final photophysical and thermal stability of the resulting SPNs. Upon generation of a series of novel SPNs, the optical and photophysical properties of the new nanomaterials were probed in solution using various techniques including transmission electron microscopy, dynamic light scattering, small-angle neutron scattering, transient absorption, and UV-vis spectroscopy. A careful comparison was performed between the different SPNs to evaluate their excited-state dynamics and photophysical properties, both before and after nanoprecipitation. Interestingly, although soluble in organic solution, the nanoparticles were found to exhibit aggregative behavior, resulting in SPNs that exhibit excited-state behaviors that are very similar to aggregated polymer solutions. Based on these findings, the formation of HA- and polysorbate 80-based nanoparticles does not influence the photophysical properties of the conjugated polymers, thus opening new opportunities for the design of bioimaging agents and nanomaterials for health-related applications.

17ß-Estradiol Potentiates the Reinstatement of Cocaine Seeking in Female Rats: Role of the Prelimbic Prefrontal Cortex and Cannabinoid Type-1 Receptors.

Clinical observations imply that female cocaine addicts experience enhanced relapse vulnerability compared with males, an effect tied to elevated estrogen phases of the ovarian hormone cycle. Although estrogens can enhance drug-seeking behavior, they do not directly induce reinstatement on their own. To model this phenomenon, we tested whether an estrogen could augment drug-seeking behavior in response to an ordinarily subthreshold reinstatement trigger. Following cocaine self-administration and extinction, female rats were ovariectomized to isolate estrogen effects on reinstatement. Although neither peak proestrus levels of the primary estrogen 17ß-estradiol (E2; 10 µg/kg, i.p., 1-h pretreatment) nor a subthreshold cocaine dose (1.25 mg/kg, i.p.) alone were sufficient to reinstate drug-seeking behavior, pretreatment with E2 potentiated reinstatement to the ordinarily subthreshold cocaine dose. Furthermore, E2 microinfusions revealed that E2 (5 µg/0.3 µl, 15-min pretreatment) acts directly within the prelimbic prefrontal cortex (PrL-PFC) to potentiate reinstatement. As E2 has been implicated in endocannabinoid mobilization, which can disinhibit PrL-PFC projection neurons, we investigated whether cannabinoid type-1 receptor (CB1R) activation is necessary for E2 to potentiate reinstatement. The CB1R antagonist AM251 (1 or 3 mg/kg, i.p., 30-min pretreatment) administered prior to E2 and cocaine suppressed reinstatement in a dose-dependent manner. Finally, PrL-PFC AM251 microinfusions (300 ng/side, 15-min pretreatment) also suppressed E2-potentiated reinstatement. Together, these results suggest that E2 can augment reactivity to an ordinarily subthreshold relapse trigger in a PrL-PFC CB1R activation-dependent manner.

Anti-human cytomegalovirus activity and toxicity of sulfonated anthraquinones and anthraquinone derivatives.

Sulfonated anthraquinones and other anthraquinone derivatives were evaluated for anti-human cytomegalovirus (HCMV) activity, cytotoxicity and genotoxicity. Acid blues 40 and 129, acid black 48, alizarin violet R and reactive blue 2 were the most active compounds having selective indices of greater than 30 and EC50 values of 4-30 microM. When tested against a clinical isolate, the 4 compounds were 2- to 5-fold less active. The antiviral activity was distinctly separate from the virucidal activity (> 1000 microM). The compounds were weakly toxic to either log phase or stationary cells in most of the following cytotoxicity assays: neutral red uptake assay, lactic acid dehydrogenase assay, trypan blue exclusion assay and radiolabeled macromolecular precursor uptake assays. Using a genotoxicity assay, the comet assay, only reactive blue 2 and acid black 48 were found to cause DNA strand breakage. This occurred at concentrations of 30 and 170 microM, respectively. These results suggest that these compounds could be a prototype for synthesizing even more effective HCMV-inhibitory anthraquinone derivatives.

Potent inhibition of respiratory syncytial virus by polyoxometalates of several structural classes.

A series of polyoxometalates (POM) were synthesized and evaluated for anti-respiratory syncytial virus (RSV) activity. POM containing zirconium, tungsten, silicon, platinum, niobium or germanium of a variety of structural types have been evaluated. Sixteen of the compounds had very striking anti-RSV activity against a clinical isolate, Utah 89, with median effective concentration (EC50) values < or = 3 microM and selective indices > 80 as determined by viral cytopathic inhibition effect, neutral red uptake and virus yield reduction assays. The EC50 values for all three assays correlated very well (Pearson correlation coefficients > 0.90). POM containing sodium cations were totally inactive. Germanium-, niobium-, tin- and zirconium-containing compounds were found to be highly potent and selective. The antiviral activity was not cell line-dependent. The median cytotoxic concentration (IC50) values were generally greater than 100 microM. The compounds were also comparably active against a known laboratory RSV strain, A2, as well as other RSV strains. To detect any virus strain-specific inhibitory activity, seven POM were tested against other RSV strains; all were nearly equally inhibitory to the human virus strains, suggesting no strain specificity. Timing studies suggested that these compounds were most inhibitory during virus adsorption and penetration, although RSV was still significantly inhibited when the compound was added 3 h post-infection; which is considered well into the eclipse period. These data suggest that these potent, non-toxic compounds should be further studied as potential chemotherapeutic agents for treating RSV infections.

Nonuniform irradiation of the canine intestine. I. Effects.

To investigate the effects of nonuniform irradiation on the small intestine, we prepared 24 dogs for continent isoperistaltic ileostomies under aseptic surgical conditions and general anesthesia. After a 3-week recovery period, the ileum was catheterized with a fiberoptic endoscope to observe the intestinal mucosa and to harvest mucosal biopsies. The baseline macroscopic and microscopic appearance of the intestinal mucosa was determined. Two weeks later, the ileum was catheterized with a 100-cm soft tube containing 40 groups of three thermoluminescent dosimeters placed at equally spaced intervals, and a dose of either 4.5, 8, 10, 11, or 15 Gy 60Co gamma rays was delivered to the right abdomen (nonuniform exposure). This method allowed a direct and precise assessment of the dose received at 40 sites located in the 100-cm intestinal segment. The intestinal mucosa was again evaluated 1, 4, and 6 days after irradiation. All animals exposed to 4.5 and 8 Gy survived, whereas none survived after 11 and 15 Gy. After exposure to 10 Gy, 60% of the animals died within 4-6 days and 40% survived with symptoms associated with both the intestinal and the hematopoietic syndromes. Crypt cell necrosis, blunting of villi, and reduction of the mucosal lining increased between 1 and 4 days after irradiation, and mucosal damage was correlated with intraintestinal dosimetry at Day 6. The granulocyte counts at Day 4 were significantly lower than baseline level in animals that died within 4-6 days but not in survivors. The present model appears to be realistic and clinically relevant, allowing the concurrent study of the intestinal and hematopoietic effects of high-dose nonuniform irradiation similar to that received by patients during radiation therapy as well as by radiation accident victims.

Effect of various growth-promoting factors on preimplantation bovine embryo development in vitro.

One-cell bovine embryos produced by in vitro oocyte maturation (IVM) and in vitro fertilization (IVF) were cultured in chemically defined medium (CDM) to which the following growth-promoting factors were added separately: transferrin (Tf, 10 ug/ml), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), transforming growth factor-betal (TGF-beta1), insulin-like growth factor-one (IGF-I), insulin-like growth factor-two (IGF-II), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), all at 10 ng/ml. The embryos were cultured for a total of 10 days and were assessed. The culture medium was changed every 2 days. There were no beneficial effects of growth factors on embryo development to morula or hatched blastocyst stages of development. However, supplementation with EGF approached significance (P<0.08) when compared with that of CDM alone at the blastocyst stage of development. The results suggest that supplementation with growth factors does not improve bovine IVM-IVF embryo development when cultured under chemically-defined conditions.

Patient medication costs as a therapeutic consideration.

Don't neglect therapeutic considerations in selecting drugs, but take their cost into account. Sometimes, selecting an older, established drug or generic equivalent can represent a significant savings for the patient.

Adverse drug interactions in dental practice. Professional and educational implications.

BACKGROUND: Rapid progress in dental pharmacotherapeutics requires that clinicians constantly update their knowledge of new drugs, drug interactions and useful therapeutic trends. This article is the first in a five-part series based on a 1998 International Association for Dental Research symposium entitled "Adverse Drug Interactions in Dentistry: Separating the Myths From the Facts." The goal of the series is to identify specific adverse drug interactions that are relevant to the therapeutic agents commonly used in general dental practice: analgesics, antibiotics, sedatives, local anesthetics and vasoconstrictors. METHODS: A group of dentist/clinical pharmacologists, with documented expertise in specific areas of dental therapeutics, reviewed the current literature regarding adverse drug interactions in dentistry. This expert panel evaluated the quality of information used to document these drug interactions and assess the severity of these drug reactions with respect to the drugs' use in dental practice. RESULTS: On the basis of the quality and severity of each reported interaction, the authors summarized the clinical importance of these drug interactions using a Significance Rating for Dental Drug Interactions. The participants presented their recommendations at the above-mentioned IADR symposium. CONCLUSIONS: Although thousands of drug interactions are described in the literature, the authors found many to be poorly documented or of minor importance to dental practitioners. For interactions that they determined to be relevant, the participants provided recommendations and precautions for preventing these potential complications. This article discusses the professional impact of drug interactions on dental practice; the classification and documentation of drug interactions; the determination of causality between drug interactions and adverse effects; risk factors; and unique characteristics of dental therapeutics. Subsequent articles will present specific summary recommendations for drug interactions associated with the use of antibiotics, analgesics, sedatives, and local anesthetics and vasoconstrictors. CLINICAL IMPLICATIONS: Although thousands of drug interactions have been reported in the literature, only a few are significantly associated with dental therapeutic agents. Avoiding these drug interactions will prevent potentially severe reactions in dental practice.

Prevention of bacterial endocarditis: recommendations by the American Heart Association.

OBJECTIVE: To update recommendations issued by the American Heart Association last published in 1990 for the prevention of bacterial endocarditis in individuals at risk for this disease. PARTICIPANTS: An ad hoc writing group appointed by the American Heart Association for their expertise in endocarditis and treatment with liaison members representing the American Dental Association, the infectious Diseases Society of America, the American Academy of Pediatrics and the American Society for Gastrointestinal Endoscopy. EVIDENCE: The recommendations in this article reflect analyses of relevant literature regarding procedure-related endocarditis, in vitro susceptibility data of pathogens causing endocarditis, results of prophylactic studies in animal models of endocarditis and retrospective analyses of human endocarditis cases in terms of antibiotic prophylaxis usage patterns and apparent prophylaxis failures. MEDLINE database searches from 1936 through 1996 were done using root words endocarditis, bacteremia and antibiotic prophylaxis. Recommendations in this document fall into evidence level III of the U.S. Preventive Services Task Force categories of evidence. CONSENSUS PROCESS: The recommendations were formulated by the writing group after specific therapeutic regimens were discussed. The consensus statement was subsequently reviewed by outside experts not affiliated with the writing group and by the Science Advisory and Coordinating Committee of the American Heart Association. These guidelines are meant to aid practitioners but are not intended as the standard of care or as a substitute for clinical judgment. CONCLUSIONS: Major changes in the updated recommendations include the following: (1) emphasis that most cases of endocarditis are not attributable to an invasive procedure; (2) cardiac conditions are stratified into high-, moderate- and negligible-risk categories based on potential outcome if endocarditis develops; (3) procedures that may cause bacteremia and for which prophylaxis is recommended are more clearly specified; (4) an algorithm was developed to more clearly define when prophylaxis is recommended for patients with mitral valve prolapse; (5) for oral or dental procedures the initial amoxicillin dose is reduced to 2 g, a follow-up antibiotic dose is no longer recommended, erythromycin is no longer recommended for penicillin-allergic individuals, but clindamycin and other alternatives are offered.

Anxiolytic therapy. Oral and intravenous sedation.

Anxiety associated with dental treatment is a well recognized problem that has a negative impact on patients' willingness to seek care. Today, this anxiety can be controlled by the practitioner. This article describes those techniques for both oral and parenteral administration as well as the pharmacokinetics of the drugs most commonly used for anxiety control.