Qualitative analysis of 7- and 8-hydroxyzolpidem and discovery of novel zolpidem metabolites in postmortem urine using liquid chromatography–tandem mass spectrometry

Purpose Zolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography–triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QqTOFMS). Methods 7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS. Results The presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems. Conclusions The presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

Depicting the DNA Binding and Cytotoxicity Studies against Human Colorectal Cancer of Aquabis (1-Formyl-2-Naphtholato-k2O,O′) Copper(II): A Biophysical and Molecular Docking Perspective

In this study, we attempted to examine the biological activity of the copper(II)–based small molecule aquabis (1-formyl-2-naphtholato-k2O,O′)copper(II) (1) against colon cancer. The characterization of complex 1 was established by analytical and spectral methods in accordance with the single-crystal X-ray results. A monomeric unit of complex 1 exists in an O4 (H2O) coordination environment with slightly distorted square pyramidal geometry (τ = ~0.1). The interaction of complex 1 with calf thymus DNA (ctDNA) was determined by employing various biophysical techniques, which revealed that complex 1 binds to ctDNA at the minor groove with a binding constant of 2.38 × 105 M–1. The cytotoxicity of complex 1 towards human colorectal cell line (HCT116) was evaluated by the MTT assay, which showed an IC50 value of 11.6 μM after treatment with complex 1 for 24 h. Furthermore, the apoptotic effect induced by complex 1 was validated by DNA fragmentation pattern, which clarified that apoptosis might be regulated through the mitochondrial-mediated production of reactive oxygen species (ROS) causing DNA damage pathway. Additionally, molecular docking was also carried out to confirm the recognition of complex 1 at the minor groove.

Hemp Chemotype Definition by Cannabinoids Characterization Using LC-ESI(+)-LTQ-FTICR MS and Infrared Multiphoton Dissociation

The development and application of advanced analytical methods for a comprehensive analysis of Cannabis sativa L. extracts plays a pivotal role in order to have a reliable evaluation of their chemotype definition to guarantee the efficacy and safety in pharmaceutical use. This paper deals with the qualitative and quantitative determination of cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), and cannabigerol (CBG) based on a liquid chromategraphy-mass spectrometry (LC-MS) method using electrospray ionization in positive mode (ESI+), coupled with a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). For the first time, structural information of phytocannabinoids is available upon precursor ions’ isolation within the FTICR trapping cell and subsequent fragmentation induced by infrared multiphoton dissociation (IRMPD). Such fragmentation and accurate mass measurement of product ions, alongside collision-induced dissociation (CID) within LTQ, was advantageous to propose a reliable fragmentation pattern for each compound. Then, the proposed LC-ESI(+)-LTQ-FTICR MS method was successfully applied to the hemp chemotype definition of three registered Italian accessions of hemp C. sativa plants (Carmagnola C.S., Carmagnola, and Eletta Campana), thus resulting in the Eletta Campana accession being the best one for cannabis product manufacturing.

Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia

Taxadiene synthase (TDS) is the rate-limiting enzyme of Taxol biosynthesis that cyclizes the geranylgeranyl pyrophosphate into taxadiene. Attenuating Taxol productivity by fungi is the main challenge impeding its industrial application; it is possible that silencing the expression of TDS is the most noticeable genomic feature associated with Taxol-biosynthetic abolishing in fungi. As such, the characterization of TDS with unique biochemical properties and autonomous expression that is independent of transcriptional factors from the host is the main challenge. Thus, the objective of this study was to kinetically characterize TDS from endophytic bacteria isolated from different plants harboring Taxol-producing endophytic fungi. Among the recovered 23 isolates, Bacillus koreensis and Stenotrophomonas maltophilia achieved the highest TDS activity. Upon using the Plackett–Burman design, the TDS productivity achieved by B. koreensis (18.1 µmol/mg/min) and S. maltophilia (14.6 µmol/mg/min) increased by ~2.2-fold over the control. The enzyme was purified by gel-filtration and ion-exchange chromatography with ~15 overall folds and with molecular subunit structure 65 and 80 kDa from B. koreensis and S. maltophilia, respectively. The chemical identity of taxadiene was authenticated from the GC-MS analyses, which provided the same mass fragmentation pattern of authentic taxadiene. The tds gene was screened by PCR with nested primers of the conservative active site domains, and the amplicons were sequenced, displaying a higher similarity with tds from T. baccata and T. brevifolia. The highest TDS activity by both bacterial isolates was recorded at 37–40 °C. The Apo-TDSs retained ~50% of its initial holoenzyme activities, ensuring their metalloproteinic identity. The activity of purified TDS was completely restored upon the addition of Mg2+, confirming the identity of Mg2+ as a cofactor. The TDS activity was dramatically reduced upon the addition of DTNB and MBTH, ensuring the implementation of cysteine-reactive thiols and ammonia groups on their active site domains. This is the first report exploring the autonomous robust expression TDS from B. koreensis and S. maltophilia with a higher affinity to cyclize GGPP into taxadiene, which could be a novel platform for taxadiene production as intermediary metabolites of Taxol biosynthesis.

LC-MS/MS-QTOF Screening and Identification of Phenolic Compounds from Australian Grown Herbs and Their Antioxidant Potential

Culinary spices and herbs have been used to impart a characteristic flavour and aroma in food due to their appealing fragrance. Recently, bioactive compounds from herbs, especially phenolics, have gained much attention due to their potential health outcomes. The aim of this study was to characterize and quantify the phenolic compounds from 10 widely used Australian-grown herbs (oregano, rosemary, bay, basil, sage, fenugreek, dill, parsley, mint and thyme). For this purpose, liquid chromatography mass spectrometry (LC-MS) was used for the complete profiling of polyphenolic compounds and quantification of abundant phenolic compounds was completed with high-performance liquid chromatography—photodiode array detection (HPLC-PDA). Polyphenols from Australian-grown herbs were estimated through total phenolic content (TP), total flavonoids (TF) and total tannins (TT) along with their in-vitro antioxidant activities. Oregano and mint were estimated with the highest value of TP (140.59 ± 9.52 and 103.28 ± 8.08 mg GAE/g, milligram gallic acid equivalent/gram) while rosemary and mint had the highest TF (8.19 ± 0.74 and 7.05 ± 0.43 mg QE (quercetin equivalent)/g). In this study, eighty-four (84) phenolic compounds were screened and confirmed through LC-MS/MS by comparing their masses and fragmentation pattern with published libraries. The results of this study validate the use of these herbs as bioactives and their positive impact on human health.

Circulating Cell Free DNA and Citrullinated Histone H3 As Useful Biomarkers of NETosis In Endometrial Cancer.

Background. Cancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC).Methods. The experiments were conducted on 21 healthy subject (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and H2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondance analysis (MCA), between cfDNA, mitcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades. Results. We demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen.Conclusion. Our data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.

Genome-wide cell-free DNA termini in patients with cancer.

The structure, fragmentation pattern, length and terminal sequence of cell-free DNA (cfDNA) is under the influence of nucleases present in the blood. We hypothesized that differences in the diversity of bases at the end of cfDNA fragments can be leveraged on a genome-wide scale to enhance the sensitivity for detecting the presence of tumor signals in plasma. We surveyed the cfDNA termini in 572 plasma samples from 319 patients with 18 different cancer types using low-coverage whole genome sequencing. The fragment-end sequence and diversity were altered in all cancer types in comparison to 76 healthy controls. We converted the fragment end sequences into a quantitative metric and observed that this correlates with circulating tumor DNA tumor fraction (R = 0.58, p < 0.001, Spearman). Using these metrics, we were able to classify cancer samples from control at a low tumor content (AUROC of 91% at 1% tumor fraction) and shallow sequencing coverage (mean AUROC = 0.99 at >1M fragments). Combining fragment-end sequences and diversity using machine learning, we classified cancer from healthy controls (mean AUROC = 0.99, SD = 0.01). Using unsupervised clustering we showed that early-stage lung cancer (n = 13) can be classified from control or later stages based on fragment-end sequences. We observed that fragment-end sequences can be used for prognostication (hazard ratio: 0.49) and residual disease detection in resectable esophageal adenocarcinoma patients, moving fragmentomics toward a greater clinical implementation.

LAMBEDA PHAGE

Background: Recognition of bacteriophages in many aspects plays an important role such as controlling the number and variation of bacteria and participation in horizontal gene transfer, which is an important process in bacterial evolution. Bacteriophages use small proteins to take over the host molecular machinery and thus interfere with central metabolic processes in infected bacteria. In general, phages inhibit or reverse host transcription to transcribe their genome. Mechanical and structural studies of phage host transcription may lead to the development of new antibacterial agents. Result: The result shows that phage vB_Eco4M-7 must be a lytic virus. This was confirmed by monitoring faglitic development by a one-step growth test. In addition, phage of relatively small uniform plaques (1 mm in diameter) occur without lysogenesis. Electron microscopic analysis showed that vB_Eco4M-7 belongs to the family Myoviridae. Based on mass spectrometric analysis, including the fragmentation pattern of unique peptides, 33 vB_Eco4M-7 phage proteins are assigned to the annotated reading frames. The results indicate that the phage studied is a potential candidate for phage treatment and / or food protection against E. coli, which produce Shiga toxin, as most of these strains belong to the O157 serotype. Conclusion: In general, phages inhibit or reverse host transcription to transcribe their genome. Mechanical and structural studies of phage host transcription may lead to the development of new antibacterial agents. The result shows that phage vB_Eco4M-7 must be a lytic virus. This was confirmed by monitoring faglitic development by a one-step growth test.

Distinct stress-dependent signatures of cellular and extracellular tRNA-derived small RNAs (tDRs)

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may yield novel biomarkers for different diseases, and potentially identify key signaling pathways important for disease progression. tRNAs and tRNA-derived small RNAs (tDRs) comprise one of the most abundant RNA species in cells and have been associated with cellular stress responses. The presence of RNA modifications on tDRs has been an obstacle for accurately identifying tDRs with conventional small RNA sequencing. Here, we use AlkB-facilitated methylation sequencing (ARM-seq) to uncover a comprehensive landscape of cellular and extracellular tDR expression in a variety of human and rat cells during common stress responses, including nutritional deprivation, hypoxia, and oxidative stress. We found that extracellular tDRs have a distinct fragmentation signature with a predominant length of 31-33 nts and a highly specific termination position when compared with intracellular tDRs. Importantly, we found these signatures are better discriminators of different cellular stress responses compared to extracellular miRNAs. Distinct extracellular tDR signatures for each profiled stressor are elucidated in four different types of cells. This distinct extracellular tDR fragmentation pattern is also noted in plasma extracellular RNAs from patients on cardiopulmonary bypass. The observed overlap of these patient tDR signatures with the signatures of nutritional deprivation and oxidative stress in our cellular models provides preliminary in vivo corroboration of our findings and demonstrates the potential to establish novel extracellular tDR biomarkers in human disease models.

In Vitro and In Vivo Effects of SerpinA1 on the Modulation of Transthyretin Proteolysis

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.