Impact assessment of metabolite instability in the development and validation of LC-MS/MS bioanalytical assays for measurement of rosuvastatin in human plasma and urine samples.

During bioanalytical assay development and validation, maintaining the stability of the parent drug and metabolites of interest is critical. While stability of the parent drug has been thoroughly investigated, the stability of unanalyzed metabolites is often overlooked. When an unstable metabolite is known or suspected to interfere with measurement of the parent drug or other metabolites of interest through back-conversion or other routes, additional tests with these unstable metabolites should be conducted. Here, the development and validation of two assays for quantification of rosuvastatin, one in human plasma and one in human urine, was reported. To this end, additional sets of quality control samples were added during assay validation to ensure the reliability of the assays. Acid treatment of samples is shown to be necessary for rosuvastatin quantification. In this regard, stability issues caused by the metabolite, rosuvastatin lactone, may have been overlooked if assay development and validation had only considered the parent drug, rosuvastatin. These assays represent a case study for how to develop and validate assays with unstable metabolites. Taken together, unstable metabolites should be included in all applicable stability tests.

Validation of LC-MS/MS methods for quantitative analysis of kynurenine pathway metabolites in human plasma and cerebrospinal fluid.

Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

Genetic Diversity in the mtDNA of Physarum polycephalum.

The mtDNA of the myxomycete Physarum polycephalum can contain as many as 81 genes. These genes can be grouped in three different categories. The first category includes 46 genes that are classically found on the mtDNA of many organisms. However, 43 of these genes are cryptogenes that require a unique type of RNA editing (MICOTREM). A second category of gene is putative protein-coding genes represented by 26 significant open reading frames. However, these genes do not appear to be transcribed during the growth of the plasmodium and are currently unassigned since they do not have any apparent similarity to other classical mitochondrial protein-coding genes. The third category of gene is found in the mtDNA of some strains of P. polycephalum. These genes derive from a linear mitochondrial plasmid with nine significant, but unassigned, open reading frames which can integrate into the mitochondrial DNA by recombination. Here, we review the mechanism and evolution of the RNA editing necessary for cryptogene expression, discuss possible origins for the 26 unassigned open reading frames based on tentative identification of their protein product, and discuss the implications to mtDNA structure and replication of the integration of the linear mitochondrial plasmid.

Iron from Co-Encapsulation of Defatted Nannochloropsis Oceanica with Inulin Is Highly Bioavailable and Does Not Impact Wheat Flour Shelf Life or Sensorial Attributes.

Defatted green microalgae Nannochloropsis oceanica (DGM) is a rich source of bioavailable iron. However, its use in foods results in unacceptable color and taste development. Therefore, the purpose of this study was to investigate strategies to enhance the use of DGM in foods. DGM and inulin were encapsulated (EC) in an oil-in-water emulsion using high-pressure homogenization. To confirm iron bioavailability, C57BL/6 mice were fed an iron-deficient diet (ID) for 2 weeks. The mice were then fed one of the four diets: ID, ID + DGM (DGM), ID + EC (EC50 or EC100) for 4 weeks. To test the stability of DGM as an iron fortificant at two different fortification rates of 17.5 mg Fe/kg (50%) or 35 mg Fe/kg (100%), whole (DGM50/DGM100), encapsulated (EC50/EC100) and color-masked (CM50/CM100) DGM were added to wheat flour (WF) at two different temperatures: 20 °C and 45 °C and were examined for 30 days. Acceptability studies were conducted to determine sensory differences between rotis (Indian flat bread) prepared from WF/EC50/CM50/EC100. The mice consuming EC50/EC100 diets showed comparable iron status to DGM-fed mice, suggesting that encapsulation did not negatively impact iron bioavailability. Addition of EC to wheat flour resulted in the lowest Fe2+ oxidation and color change amongst treatments, when stored for 30 days. There were no differences in the overall liking and product acceptance of rotis amongst treatments at both day 0 and day 21 samples. Our results suggest that EC50 can be effectively used as an iron fortificant in WF to deliver highly bioavailable iron without experiencing any stability or sensory defects, at least until 30 days of storage.

A first-in-human study of BLZ-100 (tozuleristide) demonstrates tolerability and safety in skin cancer patients.

BLZ-100 (tozuleristide) is an intraoperative fluorescent imaging agent that selectively detects malignant tissue and can be used in real time to guide tumor resection. The purpose of this study was to assess the safety, tolerability, and pharmacokinetics of BLZ-100 and to explore the pharmacodynamics of fluorescence imaging of skin tumors. In this first-in-human study, BLZ-100 was administered intravenously to 21 adult patients 2 days before excising known or suspected skin cancers. Doses were 1, 3, 6, 12, and 18 mg, with 3-6 patients/cohort. Fluorescence imaging was conducted before and up to 48 h after dosing. BLZ-100 was well tolerated. There were no serious adverse events, deaths, or discontinuations due to adverse events, and no maximum tolerated dose (MTD) was identified. Headache (n = 2) and nausea (n = 2) were the only BLZ-100 treatment-related adverse events reported for >1 patient. Median time to maximal serum concentration was <0.5 h. Exposure based on maximal serum concentrations increased in a greater than dose-proportional manner. For intermediate dose-levels (3-12 mg), 4 of 5 basal cell carcinomas and 4 of 4 melanomas were considered positive for BLZ-100 fluorescence. BLZ-100 was well tolerated at all dose levels tested and these results support further clinical testing of this imaging agent in surgical oncology settings. Clinicaltrials.gov: NCT02097875.

RNA-sequencing and behavioral testing reveals inherited physical inactivity co-selects for anxiogenic behavior without altering depressive-like behavior in Wistar rats.

Physical inactivity is positively associated with anxiety and depression. Considering physical inactivity, anxiety, and depression each have a genetic basis for inheritance, our lab used artificial selectively bred low-voluntary running (LVR) and wild type (WT) female Wistar rats to test if physical inactivity genes selected over multiple generations would lead to an anxiety or depressive-like phenotype. We performed next generation RNA sequencing and immunoblotting on the dentate gyrus to reveal key biological functions from heritable physical inactivity. LVR rats did not display depressive-like behavior. However, LVR rats did display anxiogenic behavior with gene networks associated with reduced neuronal development, proliferation, and function compared to WT counterparts. Additionally, immunoblotting revealed LVR deficits in neuronal development and function. To our knowledge, this is the first study to show that by selectively breeding for physical inactivity genes, anxiety-like genes were co-selected. The study also reveals molecular insights to the genetic influences that physical inactivity has on anxiety-like behavior.

Gas hydrate dissociation linked to contemporary ocean warming in the southern hemisphere.

Ocean warming related to climate change has been proposed to cause the dissociation of gas hydrate deposits and methane leakage on the seafloor. This process occurs in places where the edge of the gas hydrate stability zone in sediments meets the overlying warmer oceans in upper slope settings. Here we present new evidence based on the analysis of a large multi-disciplinary and multi-scale dataset from such a location in the western South Atlantic, which records massive gas release to the ocean. The results provide a unique opportunity to examine ocean-hydrate interactions over millennial and decadal scales, and the first evidence from the southern hemisphere for the effects of contemporary ocean warming on gas hydrate stability. Widespread hydrate dissociation results in a highly focused advective methane flux that is not fully accessible to anaerobic oxidation, challenging the assumption that it is mostly consumed by sulfate reduction before reaching the seafloor.

Supplemental Microalgal Iron Helps Replete Blood Hemoglobin in Moderately Anemic Mice Fed a Rice-Based Diet.

Iron deficiency anemia affects 1.2 billion people globally. Our objectives were to determine if (1) supplemental iron extracted from defatted microalgae (Nannochloropsis oceanica, DGM) and (2) a combination of minute amount of plant phytase and inulin could help replete hemoglobin in anemic mice. Mice (7 weeks old) were fed a control diet (6 mg Fe/kg). After 10 weeks, the mice were assigned to three treatments: control, control + DGM iron (Fe-DGM, 39 mg Fe/kg), or control + 1% inulin + 250 units of phytase/kg (INU-PHY, 6 mg Fe/kg). The mice had free access to diets and water for 6 weeks. The Fe-DGM group had elevated blood hemoglobin (p < 0.01) and a two-fold greater (p < 0.0001) liver non-heme iron over the control. Strikingly, the INU-PHY group had 34% greater non-heme iron than the control, despite the same concentrations of iron in their diets. Fe-DGM group had altered (p < 0.05) mRNA levels of hepcidin, divalent metal transporter 1, transferrin and transferrin receptor 1. Iron extracted from defatted microalgae seemed to be effective in alleviating moderate anemia, and INU-PHY enhanced utilization of intrinsic iron present in the rice diet. Our findings may lead to a novel formulation of these ingredients to develop safer and bioavailable iron supplements for iron-deficient populations.

A potent peptide-steroid conjugate accumulates in cartilage and reverses arthritis without evidence of systemic corticosteroid exposure.

On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads.

Harpagophytum procumbens Extract Ameliorates Allodynia and Modulates Oxidative and Antioxidant Stress Pathways in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) is a deliberating disorder with impairments in locomotor deficits and incapacitating sensory abnormalities. Harpagophytum procumbens (Hp) is a botanical widely used for treating inflammation and pain related to various inflammatory and musculoskeletal conditions. Using a modified rodent contusion model of SCI, we explored the effects of this botanical on locomotor function and responses to mechanical stimuli, and examined possible neurochemical changes associated with SCI-induced allodynia. Following spinal cord contusion at T10 level, Hp (300 mg/kg, p.o.) or vehicle (water) was administered daily starting 24 h post-surgery, and behavioral measurements made every-other day until sacrifice (Day 21). Hp treatment markedly ameliorated the contusion-induced decrease in locomotor function and increased sensitivity to mechanical stimuli. Determination of Iba1 expression in spinal cord tissues indicated microglial infiltration starting 3 days post-injury. SCI results in increased levels of 4-hydroxynonenal, an oxidative stress product and proalgesic, which was diminished at 7 days by treatment with Hp. SCI also enhanced antioxidant heme oxygenase-1 (HO-1) expression. Concurrent studies of cultured murine BV-2 microglial cells revealed that Hp suppressed oxidative/nitrosative stress and inflammatory responses, including production of nitric oxide and reactive oxygen species, phosphorylation of cytosolic phospholipases A2, and upregulation of the antioxidative stress pathway involving the nuclear factor erythroid 2-related factor 2 and HO-1. These results support the use of Hp for management of allodynia by providing resilience against the neuroinflammation and pain associated with SCI and other neuropathological conditions.

The sigma receptor ligand N-phenylpropyl-N'-(4-methoxyphenethyl)3piperazine (YZ-067) enhances the cocaine conditioned-rewarding properties while inhibiting the development of sensitization of cocaine in mice.

RATIONALE: The N-phenylpropyl-N'-substituted piperazines SA-4503 (N-phenylpropyl-N'-(3,4-dimethoxyphenethyl)piperazine) and YZ-185 (N-phenylpropyl-N'-(3-methoxyphenethyl)piperazine) bind to sigma (σ) receptors and block the development of cocaine-induced conditioned place preference at concentrations that inhibit cocaine-induced hyperactivity. YZ-067 (N-phenylpropyl-N'-(4-methoxyphenethyl)piperazine) also binds to sigma receptors and attenuates cocaine-induced hyperactivity in mice. OBJECTIVES: The present study determined the effect of YZ-067 on the development and expression of cocaine (66 µmol/kg or 33 µmol/kg) conditioned place preference (CPP) and locomotor sensitization in mice. RESULTS: YZ-067 (10 or 31.6 µmol/kg) did not have intrinsic effects on place preference or place aversion. Interestingly, the 31.6 µmol/kg YZ-067 dose enhanced the development of cocaine place preference, while 10 µmol/kg YZ-067 attenuated the development of cocaine-induced locomotor sensitization. However, YZ-067 did not alter the expression of cocaine place preference nor cocaine-induced locomotor sensitization. In follow-up studies, YZ-067 did not affect performance in the zero maze or rotarod, indicating that sigma receptors probed by this ligand do not regulate anxiety-like or coordinated motor skill behaviors, respectively. CONCLUSION: Overall, these results are consistent with previous studies demonstrating a role for sigma receptors in the behavioral effects of cocaine. However, the present findings also indicate that N-phenylpropyl-N'-substituted piperazines do not strictly block cocaine's behavioral effects and that sigma receptor may differentially mediate cocaine-induced hyperactivity and place conditioning.

Promoter Length Affects the Initiation of T7 RNA Polymerase In Vitro: New Insights into Promoter/Polymerase Co-evolution.

Polymerases are integral factors of gene expression and are essential for the maintenance and transmission of genetic information. RNA polymerases (RNAPs) differ from other polymerases in that they can bind promoter sequences and initiate transcription de novo and this promoter recognition requires the presence of specific DNA binding domains in the polymerase. Bacteriophage T7 RNA polymerase (T7RNAP) is the prototype for single subunit RNA polymerases which include bacteriophage and mitochondrial RNAPs, and the structure and mechanistic aspects of transcription by T7 RNAP are well characterized. Here, we describe experiments to determine whether the prototype T7 RNAP is able to recognize and initiate at truncated promoters similar to mitochondrial promoters. Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP. These assays have not only defined the limits of conventional de novo initiation on truncated promoters, but have identified novel activities of initiation of RNA synthesis. We propose that these novel activities may be vestigial activities surviving from the transition of single subunit polymerase initiation using primers to de novo initiation using promoters.

Neurosteroid and benzodiazepine combination therapy reduces status epilepticus and long-term effects of whole-body sarin exposure in rats.

OBJECTIVE: Our objective was to evaluate the protective efficacy of the neurosteroid pregnanolone (3α-hydroxy-5ß pregnan-20-one), a GABAA receptor-positive allosteric modulator, as an adjunct to benzodiazepine therapy against the chemical warfare nerve agent (CWNA) sarin (GB), using whole-body exposure, an operationally relevant route of exposure to volatile GB. METHODS: Rats implanted with telemetry transmitters for the continuous measurement of cortical electroencephalographic (EEG) activity were exposed for 60 minutes to 3.0 LCt50 of GB via whole-body exposure. At the onset of toxic signs, rats were administered an intramuscular injection of atropine sulfate (2 mg/kg) and the oxime HI-6 (93.6 mg/kg) to increase survival rate and, 30 minutes after seizure onset, treated subcutaneously with diazepam (10 mg/kg) and intravenously with pregnanolone (4 mg/kg) or vehicle. Animals were evaluated for GB-induced status epilepticus (SE), spontaneous recurrent seizures (SRS), impairment in spatial memory acquisition, and brain pathology, and treatment groups were compared. RESULTS: Delayed dual therapy with pregnanolone and diazepam reduced time in SE in GB-exposed rats compared to those treated with delayed diazepam monotherapy. The combination therapy of pregnanolone with diazepam also prevented impairment in the Morris water maze and reduced the neuronal loss and neuronal degeneration, evaluated at one and three months after exposure. SIGNIFICANCE: Neurosteroid administration as an adjunct to benzodiazepine therapy offers an effective means to treat benzodiazepine-refractory SE, such as occurs following delayed treatment of GB exposure. This study is the first to present data on the efficacy of delayed pregnanolone and diazepam dual therapy in reducing seizure activity, performance deficits and brain pathology following an operationally relevant route of exposure to GB and supports the use of a neurosteroid as an adjunct to standard anticonvulsant therapy for the treatment of CWNA-induced SE.

Sigma-1 receptor antagonist, PD144418, selectively reduces female motivation for food during negative energy balance.

Sigma-1 (σ1) receptors have been investigated for their involvement in learning, rewarding and motivational processes. PD144418, a σ1 receptor antagonist, has been found to produce a dose-dependent attenuation of locomotor activity induced by cocaine, and by itself, does not suppress basal locomotor activity in mice. Moreover, PD144418 decreases the motivational effort of a food-reinforced behavior in male rats, without altering appetite or food palatability. It remains unknown whether the PD144418 can alter the motivational effort of a food-reinforced behavior in response to altered energy homeostasis, as is the case under 24 -h food deprivation. Additionally, while the previous experiments indicate effects in male rats, there has been no research examining the effects of PD144418, or any other σ1 receptor antagonist, on motivational aspects of feeding in females. The present study examined the effects of PD144418 on motivational aspects of feeding in male and female rats using an operant task under sated or food deprived conditions. Results indicated that when animals are sated, at the highest dose (10 µmol/kg), under a progressive ratio (PR) reinforcement schedule, PD144418 significantly attenuated the breakpoint and the number of active lever responses for sucrose pellets in both males and females. When animals are in a state of energy deficit, as is the case following 24-hr food deprivation, PD144418 does not alter motivationally driven operant responding as measured by the breakpoint in either sex but does alter the number of earned reinforcers responses in females.

Phase 1 Safety, Pharmacokinetics, and Fluorescence Imaging Study of Tozuleristide (BLZ-100) in Adults With Newly Diagnosed or Recurrent Gliomas.

BACKGROUND: Fluorescence-guided surgery (FGS) can improve extent of resection in gliomas. Tozuleristide (BLZ-100), a near-infrared imaging agent composed of the peptide chlorotoxin and a near-infrared fluorophore indocyanine green, is a candidate molecule for FGS of glioma and other tumor types. OBJECTIVE: To perform a phase 1 dose-escalation study to characterize the safety, pharmacokinetics, and fluorescence imaging of tozuleristide in adults with suspected glioma. METHODS: Patients received a single intravenous dose of tozuleristide 3 to 29 h before surgery. Fluorescence images of tumor and cavity in Situ before and after resection and of excised tissue ex Vivo were acquired, along with safety and pharmacokinetic measures. RESULTS: A total of 17 subjects received doses between 3 and 30 mg. No dose-limiting toxicity was observed, and no reported adverse events were considered related to tozuleristide. At doses of 9 mg and above, the terminal serum half-life for tozuleristide was approximately 30 min. Fluorescence signal was detected in both high- and low-grade glial tumors, with high-grade tumors generally showing greater fluorescence intensity compared to lower grade tumors. In high-grade tumors, signal intensity increased with increased dose levels of tozuleristide, regardless of the time of dosing relative to surgery. CONCLUSION: These results support the safety of tozuleristide at doses up to 30 mg and suggest that tozuleristide imaging may be useful for FGS of gliomas.

Sigma-1 receptor ligand PD144418 and sigma-2 receptor ligand YUN-252 attenuate the stimulant effects of methamphetamine in mice.

RATIONALE: Previous research indicates that the selective sigma-1 receptor ligand PD144418 and the selective sigma-2 ligands YUN-252 can inhibit cocaine-induced hyperactivity. The effects of these ligands on other stimulants, such as methamphetamine, have not been reported. OBJECTIVES: The present study examined the effects of PD144418 and YUN-252 pretreatment on methamphetamine-induced hyperactivity after acute treatment. METHODS: Mice (n = 8-14/group) were injected with PD144418 (3.16, 10, or 31.6 µmol/kg), YUN-252 (0.316, 3.16, 31.6 µmol/kg), or saline. After 15 min, mice injected with 2.69 µmol/kg methamphetamine or saline vehicle, where distance traveled during a 60-min period was recorded. Additionally, the effect of PD144418 on the initiation and expression of methamphetamine sensitization was determined by treating mice (n = 8-14/group) with PD144418, methamphetamine or saline repeatedly over a 5-day period, and testing said mice with a challenge dose after a 7-day withdrawal period. RESULTS: Results indicate that both PD144418 and YUN-252, in a dose-dependent manner, attenuated hyperactivity induced by an acute methamphetamine injection. Specifically, 10 µmol/kg or 31.6 µmol/kg of PD144418 and 31 µmol/kg of YUN-252 suppressed methamphetamine-induced hyperactivity. In regard to methamphetamine sensitization, while 10 µmol/kg PD144418 prevented the initiation of methamphetamine sensitization, it did not have an effect on the expression. CONCLUSIONS: Overall, the current results suggest an intriguing potential for this novel sigma receptor ligand as a treatment for the addictive properties of methamphetamine. Future analysis of this novel sigma receptor ligand in assays directly measuring reinforcement properties will be critical.

Sigma-1 receptor antagonist PD144418 suppresses food reinforced operant responding in rats.

Sigma-1 (σ1) receptors have been investigated for their involvement in learning, rewarding and motivational processes, particularly as it relates to substances of abuse. Few studies have examined the effects of σ1 receptor agonists and antagonists on the rewarding and motivational properties of natural reinforcers, such as food. Studies that have investigated σ1 receptor agonists and antagonists has produced conflicting results. σ1 receptor antagonist PD144418 has been found to produce a dose-dependent attenuation of locomotor activity induced by cocaine, and by itself, does not suppress basal locomotor activity in mice. However, its effects on reward and motivation as it relates to food are unknown. The present study examined the involvement of σ1 receptors in mediating the rewarding and motivational properties of food using an operant task. The results indicated that at the highest dose (10 µmol/kg), PD144418 significantly attenuated the number of active lever responses for chow pellets but did not decrease the number of active lever responses for sucrose pellets under a fixed ratio (FR2) schedule of reinforcement. However, under a progressive ratio (PR) reinforcement schedule, 10 µmol/kg of PD14418 significantly reduced the breakpoint, a measure indicative of effort or motivation, for both chow and sucrose pellets. When ad libitum chow or sucrose pellets were made freely available (i.e. no lever press required) inside the operant chamber, 10 µmol/kg, PD144418 did not have an effect on number of pellets consumed. These findings indicate that PD144418 reduces the motivational effort of a food reinforced behavior.

Real-time Visualization of Breast Carcinoma in Pathology Specimens From Patients Receiving Fluorescent Tumor-Marking Agent Tozuleristide.

CONTEXT.­: Resection of breast carcinoma with adequate margins reduces the risk of local recurrence and reoperation. Tozuleristide (BLZ-100) is an investigational peptide-fluorophore agent that may aid in intraoperative tumor detection and margin assessment. In this study, fluorescence imaging was conducted ex vivo on gross breast pathology specimens. OBJECTIVES.­: To determine the potential of tozuleristide to detect breast carcinoma in fresh pathology specimens and the feasibility of fluorescence-guided intraoperative pathology assessment of surgical margins. DESIGN.­: Twenty-three patients received an intravenous bolus dose of 6 or 12 mg of tozuleristide at least 1 hour before surgery. Fifteen lumpectomy and 12 mastectomy specimens were evaluated for fluorescence by the site's clinical pathology staff using the SIRIS, an investigational near-infrared imaging device. The breast tissue was then processed per usual procedures. Fluorescent patterns were correlated with the corresponding hematoxylin-eosin-stained sections. Clinical pathology reports were used to correlate fluorescent signal to grade, histotype, prognostic marker status, and margin measurements. RESULTS.­: Tozuleristide fluorescence was readily observed in invasive and in situ breast carcinoma specimens. Most invasive carcinomas were bright and focal, whereas in situ lesions demonstrated a less intense, more diffuse pattern. Tozuleristide was detected in ductal and lobular carcinomas with a similar fluorescent pattern. Fluorescence was detected in high- and low-grade lesions, and molecular marker/hormone receptor status did not affect signal. Fluorescence could be used to identify the relationship of carcinoma to margins intraoperatively. CONCLUSIONS.­: Tumor targeting with tozuleristide allowed visual real-time distinction between pathologically confirmed breast carcinoma and normal tissue.

A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases.

Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3' ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3' end of T7 RNAP transcripts in vitro. Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3' ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3' ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3' ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3' end. Optimization of this activity is obtained through alteration of the lengths of the 5'-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.

A validated surrogate analyte LC-MS/MS assay for quantitation of endogenous kynurenine and tryptophan in human plasma.

AIM: Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) catalyze the initial and rate-controlling step of tryptophan metabolism through the kynurenine pathway, which plays an important role in mediating immune response. Accurate measurement of tryptophan and kynurenine is critical for monitoring the activity of IDO/TDO. Experimental: Surrogate analytes ([15N2]-Tryptophan and [13C6]-Kynurenine) were used for preparation of calibration standard and quality control. A fit-for-purpose validation using an approach of surrogate analyte and authentic matrix was carried out. RESULTS: Acid precipitation was used in sample preparation, which yielded good recovery without significant matrix effect. Precision and accuracy results were well within the acceptance criteria. The assay demonstrated successful application to a clinical study to confirm a transient depletion of kynurenine upon IDO inhibition. CONCLUSION: A robust, specific and simple LC-MS/MS method was developed and validated with a fit-for-purpose style for measuring tryptophan and kynurenine in human plasma samples.