Analysis Of Mass Spectra Research Articles

CRMP2 derived from cancer associated fibroblasts facilitates progression of ovarian cancer via HIF-1α-glycolysis signaling pathway

As the predominant stroma cells of tumor microenvironment (TME), cancer associated fibroblasts (CAFs) are robust tumor player of different malignancies. However, less is known about the regulatory mechanism of CAFs on promoting progression of ovarian cancer (OvCA). In the present study, the conditioned medium of primary CAFs (CAF-CM) from OvCA was used to culture cell lines of epithelial ovarian cancer (EOC), and showed a potent role in promoting proliferation, migration and invasion of cancer cells. Mass spectrum (MS) analysis identified that Collapsin response mediator protein-2 (CRMP2), a microtubule-associated protein involved in diverse malignancies, derived from CAFs was a key regulator responsible for mediating these cell events of OvCA. In vitro study using recombinant CRMP2 (r-CRMP2) revealed that the protein promoted proliferation, invasion, and migration of OvCA cells through activation of hypoxia-inducible factor (HIF)-1α-glycolysis signaling pathway. The CRMP2 was abundantly expressed in OvCA, with a well correlation with metastasis and poor prognosis, as analyzed from 118 patients’ samples. Inhibition of the CRMP2 derived from CAFs by neutralizing antibodies significantly attenuated the tumor size, weights, and metastatic foci numbers of mice in vivo. Our finding has provided a novel therapeutic clue for OvCA based on TME.

Colorimetric and fluorescent probe based on coumarin/ thiophene derivative for sequential detection of mercury(II) and cyanide ions in an aqueous medium

A novel colorimetric and fluorescent coumarin hydrazone probe L was prepared by condensing 7-(diethylamino)-coumarin–3-carbaldehyde and (thiophen-2-ylmethyl) hydrazine. Absorbance and emission titrations show that probe L detects Hg2+ rather than other interfering cations. The colorimetric change from yellow to red and the decrease in fluorescence intensity after the addition of Hg2+ cations to probe L are perfectly visible to the naked eye. It could be related to intramolecular charge transfer and d-d transitions of the metal cations. The binding stoichiometry of the probe L to the Hg2+ cation was found to be 1:1, which was confirmed by Job plot and mass spectrum analysis. The resulting complex L.Hg2+ is preferentially fluorescent and presents a distinct blueshift in its absorbance spectrum only with CN− anions, which is caused by the release of the probe L and the production of the Hg(CN)2 complex. The LOD of Hg2+ and CN− were found to be 5.15 nM and 0.208 µM respectively. Therefore, probe L can be employed to detect and quantify Hg2+ and CN− in real solutions.

Oxygenated volatile organic compounds (VOCs) as significant but varied contributors to VOC emissions from vehicles

Abstract. Vehicular emissions are an important source for volatile organic compounds (VOCs) in urban and downwind regions. In this study, we conducted a chassis dynamometer study to investigate VOC emissions from vehicles using gasoline, diesel, and liquefied petroleum gas (LPG) as fuel. Time-resolved VOC emissions from vehicles are chemically characterized by a proton-transfer-reaction time-of-flight mass spectrometer (PTR-ToF-MS) with high frequency. Our results show that emission factors of VOCs generally decrease with the improvement of emission standards for gasoline vehicles, whereas variations in emission factors for diesel vehicles with emission standards are more diverse. Mass spectrum analysis of the PTR-ToF-MS suggests that cold starts significantly influence VOC emissions of gasoline vehicles, while the influences are less important for diesel vehicles. Large differences in VOC emissions between gasoline and diesel vehicles are observed with emission factors of most VOC species from diesel vehicles being higher than gasoline vehicles, especially for most oxygenated volatile organic compounds (OVOCs) and heavier aromatics. These results indicate quantification of heavier species by the PTR-ToF-MS may be important in the characterization of vehicular exhausts. Our results suggest that VOC pairs (e.g., C14 aromatics / toluene ratio) could potentially provide good indicators for distinguishing emissions from gasoline and diesel vehicles. The fractions of OVOCs in total VOC emissions are determined by combining measurements of hydrocarbons from canisters and online observations of the PTR-ToF-MS. We show that OVOCs contribute 9.4 % ± 5.6 % of total VOC emissions for gasoline vehicles, while the fractions are significantly higher for diesel vehicles (52 %–71 %), highlighting the importance of detecting these OVOC species in diesel emissions. Our study demonstrated that the large number of OVOC species measured by the PTR-ToF-MS are important in the characterization of VOC emissions from vehicles.

SYNTHESIS, CHARACTERIZATION, ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF SUBSTITUTED PYRAZOLINES FROM 2-ACETYLFLUORENE BASED CHALCONE

Objective: The present work is based on the above pyrazoline derivative form heterocyclic substituted chalcone. Methods: The elemental analysis, IR, 1H and 13C NMR and mass spectrum analyses were used to characterise the structures of the obtained compounds. Results: The scheme-1 and scheme-2 describe the synthesis of chalcone (thiophene-fluorenyl derivative) and synthesis of substituted pyrazolines respectively. Chalcone has been synthesized from 2-Acetylfluorene, which had been reacted with phenylhydrazine, 4-substituted phenylhydrazine (4-Chloro and 4-Nitro) and 2,4-Dinitrophenylhydrazine to form the substituted pyrazolines (CP01-CP04). Conclusion: The antimicrobial and antioxidant properties were evaluated on those newly synthesised compounds.

A Novel Antimicrobial Metabolite Produced by Paenibacillus apiarius Isolated from Brackish Water of Lake Balkhash in Kazakhstan.

Four aerobic bacteria with bacteriolytic capabilities were isolated from the brackish water site Strait Uzynaral of Lake Balkhash in Kazakhstan. The morphology and physiology of the bacterial isolates have subsequently been analyzed. Using matrix assisted laser desorption ionization-time of flight mass spectrum and partial 16S rRNA gene sequence analyses, three of the isolates have been identified as Pseudomonas veronii and one as Paenibacillus apiarius. We determined the capability of both species to lyse pre-grown cells of the Gram-negative strains Pseudomonas putida SBUG 24 and Escherichia coli SBUG 13 as well as the Gram-positive strains Micrococcus luteus SBUG 16 and Arthrobacter citreus SBUG 321 on solid media. The bacteriolysis process was analyzed by creating growth curves and electron micrographs of co-cultures with the bacteriolytic isolates and the lysis sensitive strain Arthrobacter citreus SBUG 321 in nutrient-poor liquid media. One metabolite of Paenibacillus apiarius was isolated and structurally characterized by various chemical structure determination methods. It is a novel antibiotic substance.

Study on the reaction mechanism of nitrogenous compounds in zeolite-catalyzed pyrolysis of blue-green algae

The ex-situ catalytic fast pyrolysis (CFP) of blue-green algae (BGA) and its nitrogenous model compounds over HZSM-5 was conducted in a microfluidized bed reactor (MFBR) combined with a U-shape fixed bed reactor (U-FBR). A single photoionization mass spectrometer (SPI-MS) was used for on-line studying the dynamics of volatiles formation with the biomass-to-catalyst ratio (BCR). In the CFP of BGA, six different groups of products were differentiated by statistical analysis of SPI mass spectra, which were obtained with the cumulative BCRs. The relative concentration of each group has its own evolution with catalyst deactivation, indicating the different formation pathways of six groups. In combination with results from the CFP of six model compounds, the reaction pathways of nitrogenous compounds in the CFP of BGA over HZSM-5 were proposed. The benzyl nitrile could be fully converted to hydrocarbons while this is hard to achieve N-cyclic compounds (particularly the pyridines). Besides, most of nitrogenous compounds could be interconvertible in the CFP process. Transmethylation is the favorite way for catalytic conversion. In addition, the CFP of BGA and its N-cyclic compounds would lead to the fast coking of HZSM-5, especially the hard coke.

Aqueous secondary organic aerosol formation attributed to phenols from biomass burning

Biomass burning emits large quantities of phenols, which readily partition into the atmospheric aqueous phase and subsequently may react to produce aqueous secondary organic aerosol (aqSOA). For the first time, we quantitatively explored the influence of phenols emitted from biomass burning on aqSOA formation in the winter of Beijing. A typical haze episode associated with significant aqSOA formation was captured. During this episode, aqueous-phase processing of biomass burning promoted aqSOA formation was identified. Furthermore, high-resolution mass spectrum analysis provided molecular-level evidence of the phenolic aqSOA tracers. Estimation of aqSOA formation rate (RaqSOA) with compiled laboratory kinetic data indicated that biomass-burning phenols can efficiently produce aqSOA at midday, with RaqSOA of 0.42 μg m−3 h−1 accounting for 15 % of total aqSOA formation rate. The results highlight that aqSOA formation of phenols contributes the haze pollution. This implies the importance of regional joint control of biomass burning to mitigate the heavy haze.

Development and Validation of a UHPLC-MS/MS Method for Quantitation of Almonertinib in Rat Plasma: Application to an in vivo Interaction Study Between Paxlovid and Almonertinib.

Almonertinib was approved for the first-line treatment of advanced NSCLC patients with EGFR-TKI-sensitive genetic mutations by National Medical Products Administration (NMPA) in 2021.The purpose of this study was to establish and validate a fast, accurate, stable and facile ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of almonertinib in rat plasma, it was employed to explore the effect of Paxlovid on the pharmacokinetics of almonertinib in rats. Zanubrutinib was used as an internal standard (IS), and the plasma samples were prepared by the protein precipitation method using acetonitrile. Chromatographic separation was carried out on a Shimadzu LC-20AT ultra-performance liquid chromatography system using a Shim-pack velox C18 (2.1× 50 mm, 2.7 μM) column. The mobile phase consisted of methanol and 0.1% formic acid-water. Mass spectrum analysis was executed using Shimadzu 8040 Triple quadrupole mass spectrometry. The precursor and product ions of the analyte and internal standard were detected in multiple reaction monitoring (MRM) mode. The typical fragment ions were m/z 526.20 → 72.10 for almonertinib and m/z 472.15 → 290.00 for zanubrutinib (IS). The method was validated to have good linearity for quantifying almonertinib in rat plasma from 0.1–1000 ng/ml (R2 = 0.999), and the LLOQ was 0.1 ng/ml. The validity of this method was sufficiently verified for selectivity, specificity, extraction recovery, matrix effect, accuracy, precision and stability. The validated UHPLC–MS/MS method was successfully applied to the drug interaction study of almonertinib with Paxlovid in rats. Paxlovid significantly inhibits the metabolism of almonertinib and increased the exposure of almonertinib. This study can help us to understand the metabolic profile of almonertinib better, and further human trials should be conducted to validate the results.

Importance of LCMS in Forced Degradation Studies for new Chemical Entities

Forced degradation studies shows the chemical behaviour of the molecule which in turn helps in the development of formulation and package. In addition, the regulatory guidance is very general and does not explain about the performance of forced degradation studies. In pharmaceutical industry, mandatory degradation tests are important to determine the mechanisms and measure the potential degradants during the analysis of drug material and to help to elucidate the composition of degradation materials. The Liquid Chromatography/ Mass Spectrum analysis (L.C.M.S) is a short term widely established liquid chromatography and a methodology utilizing a natural fluid process. L.C.M.S. is commonly employed in the laboratories for qualitative testing of drug substances, drugs and biological samples. Degradation studies, metabolic screening, metabolite identity and in vivo drug screening, impurity identification, amide mapping, glycoprotein mapping were consistently used in pharmaceutical development by LCMS. The FDA and ICH guidelines sets out the requirement for stability testing data to understand how the quality of the drug substance and drug product changes over time under the influence of various environmental factors. Knowledge of molecular stability helps to select the correct formulation and package as well as to provide proper storage conditions and shelf life, which is essential for regulatory documentation. Active pharmaceutical ingredients (APIs) may form impurities when exposed to excipients or environmental variables such as light, high temperatures, acidic or basic conditions, humidity; and the oxidative atmosphere. Considering that these impurities may have an impact on safety and efficacy, it is necessary to know how these impurities are produced from the drug product and to establish the path of their formation. In this context, forced drug degradation studies have been used to characterize the physicochemical stability of APIs. These studies are also essential in the validation of analytical methodologies, in order to demonstrate the selectivity of methodologies. The API and its impurities and to develop strategies to avoid the formation of degradation products. The objective of this review is to demonstrate how forced degradation studies have been conducted and application of LCMS tools in related studies.

Abstract 4176: Histone deacetylase inhibitor Panobinostat enhances efficacy of immune checkpoint inhibitor via upregulating antigen presentation in breast cancer brain metastasis

Abstract Brain metastasis (BrM) affects millions of cancer patients and major cancer types, e.g., lung cancer, melanoma, and breast cancer, have high incidences of BrM. Symptomatic BrM patients have a less than 20% 1-year survival rate. The poor clinical outcome is due to lack of effective treatment for BrM as even immune checkpoint inhibition (ICI) only showed effects in 55% asymptomatic BrM in melanoma patients. Novel effective combination therapies are urgently needed to address metastasis in the unique brain immune microenvironment (BrIME). Interestingly, our RNA-seq data of patients treated at MD Anderson Cancer Center and immunohistochemistry (IHC) analysis of tumors of various mouse models showed that BrM express significantly lower major histocompatibility class I (MHC-I) and higher histone deacetylase (HDAC) 1 compares to extracranial tumors or metastasis. HDAC inhibitors (HDACi) have been reported to upregulate MHC-I in extracranial tumors. In this study, we tested whether HDACi, Panobinostat, can upregulate MHC-I expression in BrM and whether Panobinostat combined with ICI (e.g., anti-PD1) treatment in mouse models can impede BrM development. Clearly, our data showed that HDACi Panobinostat significantly increased MHC-I expression in brain-seeking human breast cancer cells (MDA-231, MDA-435) and mouse mammary tumor cells (4T1, EO771) with or without IFN-γ treatment. We then tested Panobinostat penetration of the blood-brain barrier (BBB) for treating BrM in vivo. C57BL/6 mice were treated with either 10 or 20mpk (i.p.) Panobinostat for three consecutive days. Mass spectrum analysis shows that Panobinostat can be detected in the brain at level of 10-8M after the treatment. We found that Panobinostat treatment increased both CD4+ and CD8+ T cell in the brain by FACS analysis. To test therapeutic efficacy of HDACi and ICI combination therapy in BrM, we induced BrM in C57BL/6 mice by intracarotid artery (ICA) injection of E0771-Luc.GFP tumor cells, then mice were divided to four groups and were treated with i) vehicle, ii) Panobinostat, iii) anti-PD1 and iv) Panobinostat +anti-PD1. We found that combination of Panobinostat with anti-PD1 more effectively inhibited BrM tumor outgrowth compares to either monotherapy and the vehicle control. Furthermore, Immunohistochemistry staining revealed upregulated MHC-I in BrM tumor cells and increased CD8+ T cell infiltration by Panobinostat treatment. In summary, our data indicated that HDACi Panobinostat can increase ICI efficacy in BrM by upregulating MHC-I for enhanced antigen presentation. Citation Format: Shao-Ping Yang, Yimin Duan, Xiangliang Yuan, Lin Zhang, Patrick Zhang, Jason T. Huse, Dihua Yu. Histone deacetylase inhibitor Panobinostat enhances efficacy of immune checkpoint inhibitor via upregulating antigen presentation in breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4176.

Enhanced heat resistance of Listeria innocua as a surrogate of Listeria monocytogenes after sublethal heat treatment

Abstract Its ability to survive under different environmental conditions makes Listeria monocytogenes a critical concern for food safety. When the microorganisms are exposed to sublethal heat treatment above their optimum growth temperature, they increase stress adaptation for further heat treatments. In order to investigate heat stress resistance of L. monocytogenes, L. innocua as a surrogate was exposed to sublethal heat at 46 °C for 30 and 60 min, prior to heat treatment at 60 °C. There was no significant difference in D60°C values between samples exposed to sublethal heat for 30 min and non-pre-heat-treated samples (control) (P > 0.05). In comparison, sublethal heat treatment for 60 min caused a significant increase in D60°C values compared to control samples (P < 0.05). Additionally, cluster analysis of mass spectra obtained from MALDI-TOF was analysed by discriminant analysis of principal components (DAPC) for sublethal heat treatment at 46 °C for 30 min and control group to check stress response at the proteomic level. However, differentiation of stress responses by distinct clusters was not revealing.

Head and Tail Oxidized Terpenoid Esters from Androconia of Heliconius erato Butterflies.

Heliconius erato is a neotropical butterfly species that is part of a complex mimicry ring, with colorful wing patterns. For intraspecific communication, males use pheromones that are released from two different scent-emitting structures. Scent glands located near the abdominal claspers of males, containing antiaphrodisiac pheromones, release a highly complex mixture of compounds that is transferred to females during mating, rendering them unattractive to other males. On the other hand, androconia, scent-emitting scale areas on the wings of male butterflies, release a structurally more restricted set of compounds that likely serves an aphrodisiac role. We report here on two structurally related compounds that are the major androconial constituents, produced in high amounts and are not volatile due to their high molecular mass. Their structures were established by extensive analysis of mass, infrared, and NMR spectra, as well as microderivatization reactions of the natural extract. After establishing synthetic access, the compounds were unequivocally identified as two unusual head and tail oxidized terpenoids, (4E,8E,12E)-4,8,12-trimethyl-16-oxoheptadeca-4,8,12-trien-1-yl oleate (1) and stearate (2). Although behavioral assays are necessary to fully comprehend their role in the chemical communication of the species, hypotheses for their use by the butterflies are also discussed.

TRPC5OS induces tumorigenesis by increasing ENO1-mediated glucose uptake in breast cancer

Breast cancer is the most common malignant tumor worldwide and the leading cause of cancer-related deaths in female. Metabolic reprogramming plays critical roles in breast tumorigenesis and induces enhanced glucose uptake and glycolysis. TRPC5OS is encoded by short transient receptor potential channel 5 opposite strand, and predicted to correlate with tumor metabolic reprogramming. Here we aim to elucidate the function of TRPC5OS in aberrant metabolism mediated tumorigenesis. We detected TRPC5OS expression levels in cell lines and tissues by quantitative real-time polymerase chain reaction and immunohistochemistry. Then we assessed the effects of TRPC5OS on proliferation and cell cycle progression in breast cancer cells by cell counting kit-8, colony-formation, EdU-incorporation assays and flow cytometry. Tumor growth in vivo was observed in a mouse xenograft model. Mass spectrum analyses were performed to identify potential interactors of TRPC5OS in tumor cells, and the interaction between TRPC5OS and interactors was validated by co-immunoprecipitation (CO-IP), western blots, and immunofluorescent staining. Glucose uptake was measured by liquid scintillation spectrometry. TRPC5OS highly expresses both in breast tumors and cell lines, and might be an independent prognostic marker for breast cancer patients. Overexpressed TRPC5OS promotes breast cancer cell proliferation, cell cycle progression, and enhances tumor xenograft growth. Mass spectral and CO-IP data showed that TRPC5OS interacts with ENO1. We also demonstrate that TRPC5OS could enhance ENO1/PI3K/Akt-mediated glucose uptake in breast cancer cells. Our study demonstrated that TRPC5OS promotes breast tumorigenesis by ENO1/PI3K/Akt-mediated glucose uptake. TRPC5OS might be an independent prognostic marker and potential therapeutic target for breast cancer patients.

Macrophage migration inhibitory factor (MIF) acetylation protects neurons from ischemic injury

Ischemia-induced neuronal death leads to serious lifelong neurological deficits in ischemic stroke patients. Histone deacetylase 6 (HDAC6) is a promising target for neuroprotection in many neurological disorders, including ischemic stroke. However, the mechanism by which HDAC6 inhibition protects neurons after ischemic stroke remains unclear. Here, we discovered that genetic ablation or pharmacological inhibition of HDAC6 reduced brain injury after ischemic stroke by increasing macrophage migration inhibitory factor (MIF) acetylation. Mass spectrum analysis and biochemical results revealed that HDAC6 inhibitor or aspirin treatment promoted MIF acetylation on the K78 residue. MIF K78 acetylation suppressed the interaction between MIF and AIF, which impaired MIF translocation to the nucleus in ischemic cortical neurons. Moreover, neuronal DNA fragmentation and neuronal death were impaired in the cortex after ischemia in MIF K78Q mutant mice. Our results indicate that the neuroprotective effect of HDAC6 inhibition and aspirin treatment results from MIF K78 acetylation; thus, MIF K78 acetylation may be a therapeutic target for ischemic stroke and other neurological diseases.

Acinetobacter silvestris sp. nov. discovered in forest ecosystems in Czechia.

We investigated a taxonomically novel group of the genus Acinetobacter , which included five strains isolated from soil and water samples collected in preserved forest areas in Czechia between 2013 and 2021. The whole-genome sequences of the strains were 3.1–3.2 Mb in size, with G+C contents of 38.0–38.2 mol%. Core genome-based phylogenetic analysis showed that they formed a compact and deeply branched clade within the genus. The genomic average nucleotide identity based on blast/digital DNA–DNA hybridization values for the novel strains were 99.2–99.6 %/95.2–98.4 %, whereas those between the novel strains and the type strains of the known Acinetobacter species reached <78 %/<24 %. The results of the genus-wide analysis of whole-cell MALDI-TOF mass spectra supported the sharp distinctness of the group. The five strains were non-glucose acidifying, nonhaemolytic, nonproteolytic and growing at 28 °C, but not at 32 °C; they assimilated acetate, benzoate, ethanol, l-histidine, 4-hydroxybenzoate, dl-lactate and malonate but not 4-aminobutyrate, l-aspartate or 2,3-butanediol; this phenotype is unique among the known Acinetobacter species. We conclude that the five strains represent a novel environmental species, for which the name Acinetobacter silvestris sp. nov. is proposed, with the type strain ANC 4999T (=CCM 9207T=CCUG 75877T=CNCTC 8124T).

Lignans and Other Compounds From the Roots of Pandanus tonkinensis and Their Lipid Peroxidation Inhibitory Activity

A new phenylpropane (1) and 9 known (2-10) compounds were isolated from the methanol extract of Pandanus tonkinensis roots. Their chemical structures were determined as (7 S)-2,6-dimethoxyphenyl-7,9-propanediol-1 -O-β-D-glucopyranoside (1), isorhapontigenin (2), pinoresinol-4,4′-di -O-β-D-glucoside (3), isoeucommin A (4), pinoresinol-4′ -O-β-D-glucoside (5), acanthoside B (6), eucommin A (7), urolignoside (8), benzyl O-α-L-arabinopyranosyl-(1→6) -β-D-glucopyranoside (9), and (6 S,9 S)-roseoside (10) by comprehensive analysis of high-resolution electron spray ionization mass spectrum and nuclear magnetic resonance spectral data, as well as by comparison of their spectral data with those reported in the literature. In addition, the stereochemistry of 1 was successfully determined by both theoretical and calculated CD spectra. All the isolates were tested for their lipid peroxidation inhibitory effects by in vitro assay. Compounds 2-7 exhibited significantly lipid peroxidation inhibitory effects with IC50 values of 21.3 ± 1.7, 61.9 ± 3.9, 57.5 ± 5.5, 10.4 ± 0.7, 28.9 ± 0.3, 54.2 ± 3.5 µM, respectively, compared to that of the positive control, trolox (31.4 ± 2.2 µM).

UCHL1 protects against ischemic heart injury via activating HIF-1α signal pathway

Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1−/− hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1−/− cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1−/− cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling.

Bioactive secondary metabolites from Trichoderma viride MM21: structure elucidation, molecular docking and biological activity.

Four bioactive metabolites; ergosterol (1), peroxy ergosterol (2), α-cyclopiazonic acid (3) and kojic acid (4), were isolated from the fungal sp. Trichoderma viride MM21. Their structures were assigned by cumulative analysis of NMR and mass spectra, and comparison with literature. The antimicrobial activity of the fungus supernatant, mycelial cake, cumulative crude extract and compounds 1-4 was broadly studied against 11 diverse pathogens, revealing auspicious activity results. Based on the molecular docking, ergosterol (1) and peroxy ergosterol (2) were picked up to be computationally tested against topoisomerase IV of Staphylococcus aureus. The nominated enzyme is a possible target for the antibacterial activity of triterpenoidal/steroidal compounds. Compounds 1, 2 showed a deep inserting inside the enzyme groove recording a good binding affinity of -8.1 and -8.4kcal/mol, respectively. Noteworthy that the antibacterial activity of ergosterol was higher (14-17mm) than peroxy ergosterol (11-14mm), although ergosterol formed only one hydrogen bond with the target, while peroxy ergosterol formed three hydrogen bonds. Such higher antibacterial activity of ergosterol may be attributed to its interference with other proteins included in this inhibition. The cytotoxic activity was tested against brine shrimp, revealing 100% mortality for the supernatant, crude extract and whole isolated compounds. Such strong cytotoxicity is attributed most likely to the abundant productivity/concentration of α-cyclopiazonic acid and kojic acid.

Radical fragment ions in collision-induced dissociation-based tandem mass spectrometry

Collision-induced dissociation (CID) is a common fragmentation strategy in tandem mass spectrometry (MS2) analysis. A conventional understanding is that fragment ions generated in low-energy CID should follow the even-electron rule. As such, (de)protonated ([M+H]+/[M−H]−) or even-electron precursor ions should follow heterolytic cleavages and predominately generate even-electron fragment ions with very few radical fragment ions (RFIs). However, the extent to which RFIs present in MS2 spectra has not been comprehensively investigated. This work uses the annotated high-resolution MS2 spectra from the latest NIST 20 tandem mass spectral library to investigate the occurrence of RFIs in CID MS2 experiments. In particular, RFIs were recognized using integer double bond equivalent (DBE) values calculated from their annotated molecular formulas. Our study shows that 65.4% and 68.8% of MS2 spectra of even-electron precursors contain at least 10% RFIs by ion-count (total number of ions) in positive and negative electrospray ionization modes, respectively. Furthermore, we classified chemicals based on their compound classes and chemical substructures, and calculated the percentages of RFIs in each class. As expected, compounds that can stabilize the radical site via resonance, such as aromatic and conjugated double bond-containing chemicals, are more likely to form RFIs. We also found four possible patterns of change in RFI percentages as a function of CID collision energy. Finally, we demonstrate that the inadequate consideration of RFIs in most conventional bioinformatic tools might be problematic during in silico fragmentation and de novo annotation of MS2 spectra. This work provides a further understanding of CID MS2 mechanisms, and the unexpectedly large percentage of RFIs suggests that the even-electron rule seems to be challenged in numerous cases where it is disobeyed.

Synthesis, Characterization and Biological Activity of New Amides Containing Azo Moieties

Three new amide compounds 6, 8 and 9 were derived from diazo dye 4. 4-Hydroxy-3-methoxy-5-(2,6-dinitrophenyl) benzaldehyde 4 was reacted separately with compounds 5 and 7 to obtain 6 and 8, respectively. The amide compound 9 was synthesized by two different methods. The reaction between 6 and a diacyl chloride compound 7 gave compound 9. Also, compound 9 was produced via the reaction of compound 8 and an N-terminus compound 5. All compounds were characterized by their melting points, UV-Vis, and FTIR spectra. Moreover, the mass spectrum and elemental analysis of compound 9 were determined. All three synthesized compounds were tested against P. mirabilis, E. coli, and S. aureus at different concentrations and showed significant results.