Lesion Sterilization Tissue Repair (LSTR) Approach Of Non-Vital Primary Molars With A Chloramphenicol-Tetracycline-ZOE Antibiotic Paste: A Scoping Review.

The study aimed to perform a systematic scoping review with the need of exploring the actual clinical applications of the chloramphenicol-tetracycline-ZOE antibiotic paste (CTZ) as a lesion sterilization tissue repair (LSTR) therapy agent. Following a scoping framework suggested by Arksey and O'Malley, relevant articles (randomized controlled trials, literature reviews, observational studies, in vitro studies, and clinical case-series reports) published over the last 15 years (in English, Spanish, or Portuguese languages) were identified and retrieved from five internet databases: PubMed, Embase/Ovid, Cochrane Library, Google Scholar, and EBSCO. By title and abstract screening and after removing duplicates, 11 articles were finally included in the present scoping review: five randomized/non-randomized clinical trials, five in vitro studies, and one case-series report. According to the collected information, there were no differences between CTZ paste and conventional pulpectomy, antibiotic pastes, and intracanal filling materials, considering the clinical, radiographic, antimicrobial activity, and periapical tissue biocompatibility outcomes. CTZ has shown excellent rates of clinical success and good radiographic results, with adequate antimicrobial effects; however, its biocompatibility has been put into doubt. Contemporary pediatric dentists should carefully consider the CTZ paste as an alternative endodontic approach for pulpally involved primary molars, with the advantages of being simple and fast, and taking into account the limitations of instrumental pulpectomy such as the microbiological and morphological complexity of primary root canals.

Defining parameters of specificity for bioluminescent optogenetic activation of neurons using in vitro multi electrode arrays (MEA).

In Bioluminescent Optogenetics (BL-OG) a biological, rather than a physical, light source is used to activate light-sensing opsins, such as channelrhodopsins or pumps. This is commonly achieved by utilizing a luminopsin (LMO), a fusion protein of a light-emitting luciferase tethered to a light-sensing opsin. Light of the wavelength matching the activation peak of the opsin is emitted by the luciferase upon application of its small molecule luciferin, resulting in activation of the fused opsin and subsequent effects on membrane potential. Using optimized protocols for culturing, transforming, and testing primary neurons in multi electrode arrays, we systematically defined parameters under which changes in neuronal activity are specific to bioluminescent activation of opsins, rather than due to off-target effects of either the luciferin or its solvent on neurons directly, or on opsins directly. We further tested if there is a direct effect of bioluminescence on neurons. Critical for assuring specific BL-OG effects are testing the concentration and formulation of the luciferin against proper controls, including testing effects of vehicle on LMO expressing and of luciferin on nonLMO expressing targets.

Cannabidiol inhibits febrile seizure by modulating AMPA receptor kinetics through its interaction with the N-terminal domain of GluA1/GluA2.

Cannabidiol (CBD) is a major phytocannabinoid in Cannabis sativa. CBD is being increasingly reported as a clinical treatment for neurological diseases. Febrile seizure is one of the most common diseases in children with limited therapeutic options. We investigated possible therapeutic effects of CBD on febrile seizures and the underlying mechanism. Use of a hyperthermia-induced seizures model revealed that CBD significantly prolonged seizure latency and reduced the severity of thermally-induced seizures. Hippocampal neuronal excitability was significantly decreased by CBD. Further, CBD significantly reduced the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) mediated evoked excitatory postsynaptic currents (eEPSCs) and the amplitude and frequency of miniature EPSCs (mEPSCs). Furthermore, CBD significantly accelerated deactivation in GluA1 and GluA2 subunits. Interestingly, CBD slowed receptor recovery from desensitization of GluA1, but not GluA2. These effects on kinetics were even more prominent when AMPAR was co-expressed with γ-8, the high expression isoform 8 of transmembrane AMPAR regulated protein (TARPγ8) in the hippocampus. The inhibitory effects of CBD on AMPAR depended on its interaction with the distal N-terminal domain of GluA1/GluA2. CBD inhibited AMPAR activity and reduced hippocampal neuronal excitability, thereby improving the symptoms of febrile seizure in mice. The putative binding site of CBD in the N-terminal domain of GluA1/GluA2 may be a drug target for allosteric gating modulation of AMPAR.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.

BACKGROUND: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10µM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

C6-Deoxy coelenterazine analogues as an efficient substrate for glow luminescence reaction of nanoKAZ: the mutated catalytic 19 kDa component of Oplophorus luciferase.

The codon-optimized gene for the mutated 19 kDa protein (nanoKAZ), which is the catalytic component of Oplophorus luciferase, was expressed in Escherichia coli cells and the recombinant protein was highly purified. The secretory expression of nanoKAZ from CHO-K1 cells was performed by fusing the secretory signal peptide sequence of Gaussia luciferase to the amino-terminus of nanoKAZ. The substrate specificity for the purified nanoKAZ and the nanoKAZ secreted into the cultured medium was determined, indicating that bis-coelenterazine (bis-CTZ) and newly synthesized 6h-f-coelenterazine (6h-f-CTZ) are an efficient substrate for the glow luminescence reaction of nanoKAZ.

Propylene Glycol Potentiates the Inhibitory Action of CTZ Paste on Antibiotic-Resistant Enterococcus faecalis Isolated from the Root Canal: An In Vitro Study.

This study aimed to evaluate if the change of vehicle for CTZ (Chloramphenicol, Tetracycline, zinc oxide, and Eugenol) paste improves the inhibition of Enterococcus faecalis in vitro. The vehicles evaluated alone and mixed with CTZ were Eugenol, propylene glycol (PG), super-oxidized solution (SOS), grapefruit-seed extract (GSE), and 0.9% saline solution as a negative control. A clinical isolate of E. faecalis was morphologically and biochemically characterized, and its antimicrobial susceptibility was tested using 20 antimicrobial agents. Once characterized, the clinical isolate was cultivated to perform the Kirby-Bauer disc diffusion method with paper discs embedded with the different vehicles mixed or used alone, and incubated at 37 °C for 24 h. Data were analyzed using one-way ANOVA, and the means were compared using Tukey test with a significance level of p < 0.05. For vehicles used alone, GSE presented the greatest inhibition showing a statistically significant difference with the rest of the vehicles. When vehicles were mixed with the CTZ paste, PG showed a greater inhibition with a statistically significant difference from the rest of the vehicles. In conclusion, the vehicle used to mix the CTZ paste plays an important role in the inhibition of E. faecalis in vitro; therefore, we consider that this can be an important factor to achieve success in the use of this technique.

Characterization of a S1PR2 specific 11C-labeled radiotracer in streptozotocin-induced diabetic murine model.

BACKGROUND: Diabetes mellitus is a chronic progressive metabolic disorder that affects millions of people worldwide. Emerging evidence suggests the important roles of sphingolipid metabolism in diabetes. In particular, sphingosine-1-phosphate (S1P) and S1P receptor 2 (S1PR2) have important metabolic functions and are involved in several metabolic diseases. In diabetes, S1PR2 can effectively preserve ß cells and improve glucose/insulin tolerance in high-fat diet induced and streptozotocin (STZ)-induced diabetic mouse models. We previously developed a group of potent and selective S1PR2 ligands and radioligands. METHODS: In this study, we continued our efforts and characterized our leading S1PR2 radioligand, [11C]TZ34125, in a STZ-induced diabetic mouse model. [11C]TZ34125 was radiosynthesized in an automated synthesis module and in vitro saturation binding assay was performed using recombinant human S1PR2 membrane. In vitro saturation autoradiography analysis was also performed to determine the binding affinity of [11C]TZ34125 against mouse tissues. Type-1 diabetic mouse model was developed following a single high dose of STZ in C57BL/6 mice. Ex vivo biodistribution was performed to evaluate the distribution and amount of [11C]TZ34125 in tissues. In vitro autoradiography analysis was performed to compare the uptake of [11C]TZ34125 between diabetic and control animals in mouse spleen and pancreas. RESULTS: Our in vitro saturation binding assay using [11C]TZ34125 confirmed [11C]TZ34125 is a potent radioligand to recombinant human S1PR2 membrane with a Kd value of 0.9 nM. Saturation autoradiographic analysis showed [11C]TZ34125 has a Kd of 67.5, 45.9, and 25.0 nM to mouse kidney, spleen, and liver tissues respectively. Biodistribution study in STZ-induced diabetic mice showed the uptake of [11C]TZ34125 was significantly elevated in the spleen (~2 fold higher) and pancreas (~1.4 fold higher) compared to normal controls. The increased uptake of [11C]TZ34125 was further confirmed using autoradiographic analysis in the spleen and pancreases of STZ-induced diabetic mice, indicating S1PR2 can potentially act as a biomarker of diabetes in pancreases and inflammation in spleen. Future mechanistic analysis and in vivo quantitative assessment using non-invasive PET imaging in large animal model of diabetes is worthwhile. CONCLUSIONS: Overall, our data showed an increased uptake of our lead S1PR2-specific radioligand, [11C]TZ34125, in the spleen and pancreases of STZ-induced diabetic mice, and demonstrated [11C]TZ34125 has a great potential for preclinical and clinical usage for assessment of S1PR2 in diabetes and inflammation.

Synthetic Coelenterazine Derivatives and Their Application for Bioluminescence Imaging.

Bioluminescence (BL), the emission light resulting from the enzyme-catalyzed oxidative reaction, is a powerful imaging modality for monitoring biological phenomena both in vitro and in vivo. Coelenterazine (CTZ), the known widespread luciferin found in bioluminescent organisms, develops bioluminescence imaging (BLI). Here, we describe an approach to synthesize a series of novel CTZ derivatives for diversifying the toolbox of the BL substrates. Furthermore, we exemplify some of them display excellent BL signals in vitro and in vivo, and thus should be noted as one of the ideal substrates for in vivo BLI compared with a well-known conventional substrate, DeepBlueC.

Applications of Bioluminescence-Optogenetics in Rodent Models.

In the preceding chapter, we introduced bioluminescence-optogenetics (BL-OG) and luminopsin fusion proteins (LMOs), an emerging method of molecular neuromodulation. In addition to reviewing the fundamental principles of BL-OG, we provided a discussion of its application in vitro, including with cell lines and primary cells in culture in vitro. BL-OG is mediated by an easily diffusible molecule, luciferin, and when applied systemically in rodents, the substrate can spread throughout the body, including the brain, achieving powerful molecular neuromodulation with convenience even in awake and behaving animals. In this chapter, we provide a practical guide for BL-OG and LMO applications in rodent models of the nervous system, both ex vivo and in vivo.

LC-MS/MS method for simultaneous quantification of ten antibiotics in human plasma for routine therapeutic drug monitoring.

Background: Optimizing antimicrobial therapy to attain drug exposure that limits the emergence of resistance, effectively treats the infection, and reduces the risk of side effects is of a particular importance in critically ill patients, in whom normal functions are augmented or/and are infected with pathogens less sensitive to treatment. Achievement of these goals can be enhanced by therapeutic drug monitoring (TDM) for many antibiotics. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is presented here for simultaneous quantification of ten antimicrobials: cefazolin (CZO), cefepime (CEP), cefotaxime (CTA), ceftazidime (CTZ), ciprofloxacin (CIP), flucloxacillin (FLU), linezolid (LIN), meropenem (MER), piperacillin (PIP) and tazobactam (TAZ) in human plasma. Methods: Plasma samples were precipitated with acetonitrile and injected into the LC-MS/MS. Chromatographic separation was on a Waters Acquity BEH C18 column. Compounds were eluted with water and acetonitrile containing 0.1 % formic acid, using a gradient (0.5-65 % B), in 3.8 min. The flow rate was 0.4 mL/min, and the run time was 5.8 min. Results: The calibration curves were linear across the tested concentration ranges (0.5-250, CZO, CEP, CTA, CTZ and FLU; 0.2-100, MER and TAZ; 0.1-50, CIP and LIN and 1-500 mg/L, PIP). The intra and inter-day imprecision was < 11 %. Accuracy ranged from 95 to 114 %. CTZ and MER showed ionization suppression while CIP showed ionization enhancement, which was normalized with the use of the internal standard. Conclusion: An LC-MS/MS method for simultaneous quantification of ten antimicrobials in human plasma was developed for routine TDM.

Bioluminescence Imaging of Neuronal Network Dynamics Using Aequorin-Based Calcium Sensors.

Optogenetic calcium sensors enable the imaging in real-time of the activities of single or multiple neurons in brain slices and in vivo. Bioluminescent probes engineered from the natural calcium sensor aequorin do not require illumination, are virtually devoid of background signal, and exhibit wide dynamic range and low cytotoxicity. These probes are thus well suited for long-duration, whole-field recordings of multiple neurons simultaneously. Here, we describe a protocol for monitoring and analyzing the dynamics of neuronal ensembles using whole-field bioluminescence imaging of an aequorin-based sensor in brain slice.

On the rapid in situ oxidation of two-dimensional V2CTz MXene in culture cell media and their cytotoxicity.

The plethora of emerging two-dimensional (2D) materials exhibit wide potential application in novel technologies and advanced devices. However, their stability in environmental conditions could be an issue, affecting their application possibilities and posing health risks. Moreover, their decomposed leftovers can also induce a negative influence on human health. In particular, transition metal carbides commonly referred to as MXenes are susceptible to environmental oxidation being decomposed toward transition metal oxides and carbide-derived carbon. In this study we focused on the oxidation-state-related in vitro cytotoxicity of delaminated V2CTz onto immortalized keratinocytes (HaCaT) and malignant melanoma (A375) human cell lines. Due to the fact, that the V2CTx MXenes are least stable from all known obtained MXenes up to date, the vanadium ones were a practical choice to visualize the oxidation-cytotoxic correlation keeping the standards of 24-48 h of cell culturing. We found that the oxidation of V2CTz highly increases their cytotoxicity toward human cells, which is also time and dose dependent. The identified mode of action relates to the cell cycle as well as cellular membrane disintegration through direct physicochemical interactions.

Near-Infrared Bioluminescence Imaging of Animal Cells with Through-Bond Energy Transfer Cassette.

Coelenterazine (CTZ) is the most general substrate for marine luciferases. The present protocol introduces a near-infrared (NIR ) bioluminescence (BL) imaging of mammalian cells with a cyanine-5 (Cy5) dye-conjugated CTZ . This unique Cy5-conjugated CTZ, named Cy5-CTZ , can act as a dual optical readout emitting both fluorescence (FL) and BL. The Cy5-CTZ exerts through-bond energy transfer (TBET)-based imaging modalities for mammalian cells. This novel derivative, Cy5-CTZ , is intrinsically fluorescent and emits NIR-shifted BL when reacting with an appropriate luciferase , such as Renilla luciferase (RLuc). The protocol exemplifies a unique live-cell imaging with Cy5-CTZ that is optically stable in physiological samples and rapidly permeabilize through plasma membrane and emit NIR-BL in live mammalian cells.

Azide- and Dye-Conjugated Coelenterazine Analogues for Imaging Mammalian Cells.

Coelenterazine (CTZ) is a common substrate to most marine luciferases and photoproteins. The present protocol introduces mammalian cell imaging with nine novel dye- and azide-conjugated CTZ analogues, which were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of CTZ backbone. The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly selective bioluminescence (BL) to artificial luciferases (ALucs) and ca. 130 nm blue-shifted BL with Renilla luciferase variant 8.6 (RLuc8.6) in mammalian cells. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than CTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). The present protocol shows that the minimal spectral overlap occurs among the pairs of [Furimazine/NanoLuc], [6-N3-CTZ/ALuc26], [6-pi-OH-CTZ/RLuc8.6], and [6-N3-CTZ/RLuc8.6] because of the substrate-driven luciferase specificity or color shifts, convincing a cross talk-free multiplex bioassay platform. The present protocol introduces a new toolbox to bioassays and multiplex molecular imaging platforms for mammalian cells.

Comparative evaluation of clinical and radiographic success of three different lesion sterilization and tissue repair techniques as treatment options in primary molars requiring pulpectomy: An in vivo study.

INTRODUCTION: Paediatric endodontics is part of paediatric dental practice. Teeth with infected root canals, particularly those in which the infection has spread around the apical foramen and furcation area, is a common problem in primary dentition for such conditions pulpectomy is the procedure. Pulpectomy procedure proves to be long and complicated and has remained controversial for a number of reasons. Lesion sterilization and tissue repair therapy (LSTR) is a relatively new biologic approach for carious lesions with or without pulpal and periapical involvement using a mixture of antibiotics. OBJECTIVES: The aim of this study was to evaluate clinical and radiographic success of three different LSTR techniques as treatment options in primary molars requiring pulpectomy. METHODS: Sixty-three primary molars of fifty children aged between 4 and 8 years with primary molars requiring pulpectomy were treated with modified 3Mix-MP antibiotic paste without removal of accessible radicular pulp (Group I), modified 3Mix-MP with removal of accessible radicular pulp (Group II) and Chloramphenicol, tetracycline and zinc oxide eugeno (CTZ) paste (Group III). The subjects were followed up clinically at one, six, and twelve months whereas radiographically at six and twelve months, respectively. RESULTS: The results showed that clinical success rates of Group I, Group II and Group III were 90%, 90.5% and 81.8% respectively and radiographical success rates were 75%, 76.2% and 63.6% respectively after twelve months observation. CONCLUSION: On the basis of the overall success rates of all the three LSTR techniques, following order of performance can be inferred clinical success and radiographical success: - 3Mix-MP without removal of radicular pulp = 3Mix-MP with removal of radicular pulp >CTZ paste.

Upregulated Sphingosine 1-Phosphate Receptor 1 Expression in Human and Murine Atherosclerotic Plaques.

PURPOSE: Dysregulation of sphingosine 1-phosphate receptor 1 (S1PR1) signaling contributes to inflammation-related pathophysiological changes in cardiovascular diseases including atherosclerosis (AS). S1PR1-targeting compounds significantly reduce lesion size in murine models of AS. Therefore, characterization of S1PR1 expression in vitro and in vivo in atherosclerotic plaque could enable mechanistic studies and inform S1PR1 targeted therapies. PROCEDURES: H&E staining and immunostaining studies were performed on variably diseased human femoral endarterectomy plaque specimens, as well as mouse aortic sections from ApoE-/- mice maintained on a high-fat diet (AS mice). In vitro autoradiography study in human femoral plaques was used to confirm the tracer specificity. Micro positron emission tomography (PET) and ex vivo autoradiography studies were conducted in AS mice and their controls using a S1PR1-specific radioligand [11C]TZ3321 for in vivo and ex vivo quantification of S1PR1 expression in mouse aortic plaques. RESULTS: Increased S1PR1 expression was observed in areas of human femoral endarterectomy plaque specimens with foam cell accumulation compared with control tissue; in vitro autoradiography study indicated that SEW2781, a S1PR1 compound was able to reduce the uptake of [11C]TZ3321 by 56 %. S1PR1 levels were also upregulated in AS mouse aortic plaques. MicroPET data showed the aorta-to-blood tracer uptake ratio in AS mice was approximately 20 % higher than that in controls. Autoradiographic study also revealed elevated tracer accumulation in AS mouse aorta. CONCLUSIONS: Upregulated S1PR1 expression in human and mouse atherosclerotic plaques was successfully identified by immunostaining and radioligand-based methods. This data demonstrates that [11C]TZ3321 PET provides great promise in imaging S1PR1 expression in atherosclerotic plaques.

Simultaneous determination of cetirizine, phenyl propanolamine and nimesulide using third derivative spectrophotometry and high performance liquid chromatography in pharmaceutical preparations.

BACKGROUND: The combination between cetirizine (CET), phenylpropanolamine (PPA) and nimesulide (NMS) under trade name Nemeriv Cp tablet is prescribed for nasal congestion, cold, sneezing, and allergy. Among all published methods for the three drugs; there is no reported method concerning estimation of CTZ, PPA and NMS simultaneously and this motivates us to develop new and simple methods for their assay in pure form and tablet preparations. RESULTS: Two new methodologies were described for the simultaneous quantification of cetirizine (CTZ), PPA and NMS. Spectrophotometric procedures relies on measuring the amplitudes of the third derivative curves at 238 nm for CTZ, 218 nm for PPA and 305 nm for NMS. The calibration graphs were rectilinear over the ranges of 8-90 µg/mL for CTZ, 20-100 µg/mL for PPA and 20-200 µg/mL for NMS respectively. Regarding the HPLC method; monolithic column (100 mm × 4.6 mm i.d) was used for the separation. The used mobile phase composed of 0.1 M phosphate buffer and methanol in the ratio of 40:60, v/v at pH 7.0. The analysis was performed using UV detector at 215 nm. Calibration curves showed the linearity over concentration ranges of 5-40, 10-100 and 10-120 µg/mL for CTZ, PPA and NMS. CONCLUSION: Application of the proposed methods to the laboratory prepared tablets was carried out successfully. The results were compared with those obtained from previously published methods and they were satisfactory. Graphical abstract Graphical abstract represents the chemical structures, representative chromatogram for the HPLC separation of a PPA, b NMS and c CTZ and third derivative absorption spectra of a PPA, b NMS and c CTZ for the spectrophotometric method.

Synthetic Bioluminescent Coelenterazine Derivatives.

The development of coelenterazine (CTZ) derivatives resulting in superior optical characteristics is an efficient method to extend the range of its possible applications. Here, we describe the synthesis of three C-6 substituted CTZ derivatives retaining the recognition by Renilla luciferase (RLuc) and its derivatives. The novel derivatives are useful as bright blue-shifted CTZ derivatives, which can be used as an alternative to hitherto reported compound DeepBlueC™.

Kinetics modeling and occupancy studies of a novel C-11 PET tracer for VAChT in nonhuman primates.

INTRODUCTION: Deficits in cholinergic function have been found in the aged brain and in neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for the cholinergic system. We previously reported the initial in vitro and ex vivo characterization of (-)-[(11)C]TZ659 as a VAChT specific ligand. Here, we report the in vivo specificity, tracer kinetics, and dose-occupancy studies in the nonhuman primate brain. METHODS: MicroPET brain imaging of (-)-[(11)C]TZ659 was performed under baseline conditions in two male macaques. Tracer kinetic modeling was carried out using a two-tissue compartment model (2TCM) and Logan plot with arterial blood input function and using a simplified reference tissue model (SRTM) and Logan plot (LoganREF) without blood input. Specificity for VAChT was demonstrated by pretreatment with (+)-pentazocine, (-)-vesamicol, or S-(-)-eticlopride. Target occupancy (Occ) was calculated following pretreatment with escalating doses of (-)-vesamicol. RESULTS: Baseline PET imaging revealed selective retention in the striatum with rapid clearance from the cerebellar hemispheres as a reference region. Total volume of distribution (VT) values derived from both 2TCM and Logan analysis with blood input revealed ~3-fold higher levels of (-)-[(11)C]TZ659 in the striatum than the cerebellar hemispheres. Injection of (-)-vesamicol either as a blocking or displacing agent significantly reduced striatal uptake of (-)-[(11)C]TZ659. In contrast, pretreatment with the sigma-1 ligand (+)-pentazocine had no impact. Pretreatment with the S-(-)-eticlopride, a dopamine D2-like receptor antagonist, increased striatal uptake of (-)-[(11)C]TZ659. Striatal binding potential (BPND, range of 0.33-1.6 with cerebellar hemispheres as the reference region) showed good correlation (r(2)=0.97) between SRTM and LoganREF. Occupancy studies found that ~0.0057 mg/kg of (-)-vesamicol produced 50% VAChT occupancy in the striatum. CONCLUSION: (-)-[(11)C]TZ659 demonstrated specific and reversible VAChT binding and favorable pharmacokinetic properties for assessing the density of VAChT in the living brain.

Role of hexokinase-1 in the survival of HIV-1-infected macrophages.

Viruses have developed various strategies to protect infected cells from apoptosis. HIV-1 infected macrophages are long-lived and considered reservoirs for HIV-1. One significant deciding factor between cell survival and cell death is glucose metabolism. We hypothesized that HIV-1 protects infected macrophages from apoptosis in part by modulating the host glycolytic pathway specifically by regulating hexokinase-1 (HK-1) an enzyme that converts glucose to glucose-6-phosphate. Therefore, we analyzed the regulation of HK-1 in HIV-1 infected PBMCs, and in a chronically HIV-1 infected monocyte-like cell line, U1. Our results demonstrate that HIV-1 induces a robust increase in HK-1 expression. Surprisingly, hexokinase enzymatic activity was significantly inhibited in HIV-1 infected PBMCs and in PMA differentiated U1 cells. Interestingly, we observed increased levels of mitochondria-bound HK-1 in PMA induced U1 cells and in the HIV-1 accessory protein, viral protein R (Vpr) transduced U937 cell derived macrophages. Dissociation of HK-1 from mitochondria in U1 cells using a pharmacological agent, clotrimazole (CTZ) induced mitochondrial membrane depolarization and caspase-3/7 mediated apoptosis. Dissociation of HK-1 from mitochondria in Vpr transduced U937 also activated caspase-3/7 activity. These observations indicate that HK-1 plays a non-metabolic role in HIV-1 infected macrophages by binding to mitochondria thereby maintaining mitochondrial integrity. These results suggest that targeting the interaction of HK-1 with the mitochondria to induce apoptosis in persistently infected macrophages may prove beneficial in purging the macrophage HIV reservoir.