Investigating the antitumor effects of a novel ruthenium (II) complex on malignant melanoma cells: An NMR-based metabolomic approach.

Metals have been used for many years in medicine, particularly for the treatment of cancer. Cisplatin is one of the most used drugs in the treatment of cancer. Although platinum-containing therapeutics have unparalleled efficacy in cancer treatment, they are coupled with adverse effects and the development of tumour resistance. This has led to the exploration of other metal-based modalities including ruthenium-based compounds. Thus, in our previous study, we synthesized and characterized a novel ruthenium (II) complex (referred to herein as GA113) containing a bis-amino-phosphine ligand. The complex was subsequently screened for its anti-cancerous potential against a human malignant melanoma A375 cell line and findings revealed favourable cytotoxicity. In the current study, a nuclear magnetic resonance (NMR)-based cellular metabolomics approach was applied to probe the possible mechanism of GA113 in A375 cells. In addition, other biological assays including light microscopy, Hoechst-33258 and MitoTracker Orange CM-H2TMRos stain were used to assess cellular viability and apoptosis in GA113-treated cells. Consequently, multivariate statistical data analysis was applied to the metabolomic data to identify potential biomarkers. Six signatory metabolites were altered after treatment. Changes in these metabolites were linked to two metabolic pathways, which include the alanine, aspartate, and glutamate metabolic pathway as well as the glycine, serine, and threonine pathway. By means of an NMR-based metabolomic approach, we identified the potential mechanism of action of complex GA113 in A375 cancer cells thus providing new insights into the metabolic pathways affected by complex GA113 and establishing a foundation for further development, research, and eventual application in cancer.

Metabolic profiling of HIV infected individuals on an AZT-based antiretroviral treatment regimen reveals persistent oxidative stress.

Human immunodeficiency viral (HIV) infection and long-term use of combination antiretroviral therapy (cART) have both been associated with the development of metabolic and immunological complications. Despite having many markers for HIV disease progression, the reliability of these markers remains debatable and most of these cannot be used as valid markers for treatment response. As such, it remains important to discover and develop biological markers, which will aid in monitoring disease progression, treatment response and the diagnoses of HIV-related metabolic disorders. Previous HIV-metabonomics studies unravelled the ability to detect and measure potential biological markers of HIV disease progression and treatment response. Several significantly differing metabolites were identified, however, only a small number of studies have investigated the link of specific metabolic disorders to an exact antiretroviral regimen. Here, an NMR-based untargeted metabonomic approach was used to profile metabolic changes in the sera of 24 HIV+ cART+ individuals receiving Zidovudine-based combination antiretroviral therapy compared to their 15 HIV+ ART- and 38 HIV- counterparts. Chemometric analysis identified significant differences in metabolic features related to glutamine, glutamate, glutathione, glucose and arginine. Pathway analysis also revealed the glutamine and glutamate metabolism pathway as the most significantly altered pathway between the HIV+ cART+ and HIV+ cART- group. Findings from this study further confirm the reliability of NMR-based metabonomics in HIV biomarker discovery. In addition, this study contributes to our understandings of the metabolic effect of antiretroviral therapy.

Genomic and Physiological Investigation of Heavy Metal Resistance from Plant Endophytic Methylobacterium radiotolerans MAMP 4754, Isolated from Combretum erythrophyllum.

Combretum erythrophyllum is an indigenous southern African tree species, a metal hyperaccumulator that has been used as a phytoextraction option for tailing dams in Johannesburg, South Africa. In hyperaccumulators, metal detoxification has also been linked or attributed to the activities of endophytes, and, in this regard, metal detoxification can be considered a form of endophytic behavior. Therefore, we report herein on the identification of proteins that confer heavy metal resistance, the in vitro characterization of heavy metal resistance, and the production of plant growth-promoting (PGP) volatiles by Methylobacterium radiotolerans MAMP 4754. Multigenome comparative analyses of M. radiotolerans MAMP 4754 against eight other endophytic strains led to the identification of zinc, copper, and nickel resistance proteins in the genome of this endophyte. The maximum tolerance concentration (MTC) of this strain towards these metals was also investigated. The metal-exposed cells were analyzed by transmission electron microscopy (TEM). The ethyl acetate and chloroform extracts (1:1 v/v) of heavy metal untreated M. radiotolerans MAMP 4754 were also screened for the production of PGP compounds by Gas Chromatography-Mass Spectroscopy (GC/MS). The MTC was recorded at 15 mM, 4 mM, and 12 mM for zinc, copper, and nickel, respectively. The TEM analysis showed the accumulation of metals in the intracellular environment of M. radiotolerans MAMP 4754, while the GC/MS analysis revealed several plant growth-promoting compounds, including alcohols, phthalate esters, alkenes, ketones, sulfide derivatives, phenols, and thiazoles. Our findings suggest that the genetic makeup of M. radiotolerans MAMP 4754 encodes heavy metal resistant proteins that indicate hyperaccumulator-specific endophytic behavior and the potential for application in bioremediation. The production of plant growth-promoting volatiles in pure culture by M. raditotolerans MAMP 4754 is a characteristic feature for plant growth-promoting bacteria.

Raman-based metabonomics unravels metabolic changes related to a first-line tenofovir-based treatment in a small cohort of South African HIV-infected patients.

In addition to immunological disorders, human immunodeficiency virus (HIV) also causes metabolic abnormalities. Though successful in viral suppression and immune restoration, continued use of antiretroviral therapy (ART) has also been linked to the development of several metabolic ailments. Currently, the only clinical markers used to manage and monitor the development of HIV-induced metabolic disorders, disease progression as well as observing individual's response to antiviral treatment are CD4 count, viral loads and several other single variable colometric assays. Despite the common use of these clinical markers, these markers remain unreliable and limited in the ability to monitor the development of metabolic disorders as well as monitor treatment response. Given these limitations, it is imperative to discover and develop reliable biological markers for overall HIV disease management. Here, Raman spectroscopy was used to profile metabolic changes in the plasma of 22 HIV+ receiving a first-line tenofovir-based combination antiretroviral therapy compared to their 8 HIV+ ART- and 10 HIV- counterparts. Multivariate statistical analysis was performed in order to classify the samples into their respective groups and to identify significantly altered metabolites between the control and experimental groups. Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) discriminant analysis identified significant differences (p < 0.05) in 9 different metabolites. Alterations were identified in spectral regions associated with glucose (1124 cm-1), lipids/phospholipids (1116 cm-1, 1098 cm-1, 1077 cm-1), proteins (1120 cm-1), nucleic acids (1081 cm-1) and phenylalanine (1103 cm-1). Pathway analysis also revealed 3 significantly altered pathways. This study presented the reproducible nature of Raman spectroscopy in distinguishing between HIV-infected (treated and untreated) and uninfected blood plasma and allowed for the detection and identification of treatment induced metabolite changes. The results obtained in the study may, therefore, give insights into understanding the metabolic effect of HIV therapy.

Synthesis and anti-cancer activity of bis-amino-phosphine ligand and its ruthenium(II) complexes.

The development of both chemotherapeutic drug resistance as well as adverse side effects suggest that the current chemotherapeutic drugs remain ineffective in treating the various types of cancers. The development of new metallodrugs presenting anti-cancer activity is therefore needed. Ruthenium complexes have gained a great deal of interest due to their promising anti-tumour properties and reduced toxicity in vivo. This study highlighted the effective induction of cell death in a malignant melanoma cell by two novel bis-amino-phosphine ruthenium(II) complexes referred to as GA105 and GA113. The IC50 concentrations were determined for both the complexes, the ligand and cisplatin, for comparison. Both complexes GA105 and GA113 displayed a high anti-cancer selectivity profile as they exhibited low IC50 values of 6.72 µM and 8.76 µM respectively, with low toxicity towards a non-malignant human cell line. The IC50 values obtained for both complexes were lower than that of cisplatin. The new complexes were more effective compared to the free ligand, GA103 (IC50 = >20 µM). Morphological studies on treated cells induced apoptotic features, which with further studies could indicate an intrinsic cell death pathway. Additionally, flow cytometric analysis revealed that the mode of cell death of complex GA113 was apoptosis. The outcomes herein give further insight into the potential use of selected Ru(II) complexes as alternative chemotherapeutic drugs in the future.

Antimicrobial and Antioxidant Properties of a Bacterial Endophyte, Methylobacterium radiotolerans MAMP 4754, Isolated from Combretum erythrophyllum Seeds.

This study reports on the isolation and identification of Methylobacterium radiotolerans MAMP 4754 from the seeds of the medicinal plant, Combretum erythrophyllum, for the purposes of investigating antimicrobial and antioxidant activities from this endophyte. The strain identity was confirmed by 16S rRNA-based phylogeny and Scanning Electron Microscopy (SEM). Ethyl acetate and chloroform (1 : 1 v/v) extracts from the endophyte were tested for antimicrobial and antioxidant activity on a total of 7 bacterial species (3 Gram-positive and 4 Gram-negative) using the standard Minimum Inhibitory Concentration (MIC) protocol and Quantitative Radical Scavenging activity using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay, respectively. The MICs were recorded at 250 µg/mL for B. subtilis ATCC 19659, B. cereus ATCC 1076, E. coli ATCC1053, and 62.5 µg/mL for K. oxytoca ATCC 13182 and M. smegmatis ATCC 21293, while an IC50 of 5.65 µg/mL was recorded with the DPPH assay. Qualitative phytochemical analysis was positive for alkaloids, flavonoids, and steroids. Gas chromatography/mass spectrometry (GC/MS) analysis revealed the presence of 9-octadecene, 2,4-dinitrophenyl acetate, and 2(5H)-furanone, which have been previously reported for the targeted activities. M. radiotolerans MAMP 4754 tested positive for antimicrobial and antioxidant activity and this is linked to the production of plant-derived secondary metabolites by this strain.

Multi-platform metabonomics unravel amino acids as markers of HIV/combination antiretroviral therapy-induced oxidative stress.

Infection by the human immunodeficiency virus (HIV) elicits an immune response wherein neutrophils produce reactive oxygen species (ROS) to defend against pathogen invasion. Consequently, disproportionate levels of ROS in relation to antioxidants lead to oxidative stress (OS), which plays a key role in HIV disease progression and pathogenesis. There is a close relationship between oxidative stress status and HIV infection/progression, both separately and in the presence of combination antiretroviral therapy (cART). Biomarkers of oxidative stress present an additional means of monitoring HIV disease progression and/or management. Thus, the objective of this study was to apply untargeted nuclear magnetic resonance (NMR)-based metabonomics followed by targeted quantitative gas chromatography-mass spectrometry (GC/MS) analyses to identify predictors of oxidative stress in HIV infected individuals, with or without cART. Untargeted NMR-based metabonomics allowed a global profiling of metabolic perturbations in HIV-infected sera. The cohort consisted of 21 HIV-negative control subjects (HIV-) and 113 HIV-infected individuals, of which 100 were on cART. Significant differences in metabolic features corresponding to changes in glucose, lipids, phenylalanine, glutamic acid, aspartic acid and branched amino acids were observed, which point to oxidative stress and insulin resistance. To further confirm oxidative stress, targeted GC/MS-based metabonomics, performed in succession, allowed for a quantitative description of a total of 9 oxidative stress-related metabolites. Significant up-regulation of aspartic acid, phenylalanine and glutamic acid were observed in the HIV-infected cohorts as compared to controls. Tryptophan and tyrosine were down-regulated whereas cystine levels were increased in HIV-infected and untreated individuals as compared to both HIV treated and negative control subjects. Pathway analysis also revealed 11 metabolic pathways to be significantly altered by infection and/or treatment. These pathways included aminoacyl-tRNA biosynthesis, nitrogen metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. This pilot study demonstrated the use of multiplatform metabonomic strategies to elucidate metabolic markers that would be essential in predicting HIV/cART-induced oxidative stress. This could aid and contribute in HIV treatment and management programmes.

HIV/HAART-associated oxidative stress is detectable by metabonomics.

Chronic human immunodeficiency virus (HIV) infection, separately and in combination with highly active antiretroviral therapy (HAART) is closely associated with oxidative stress (OS). Most studies demonstrating redox imbalances in HIV-infected individuals have done so using conventional biochemical methodologies. The limited simultaneous detection of multiple OS markers within one sample is a major drawback of these methodologies and can be addressed through the use of metabonomics. HIV-metabonomic studies utilizing biofluids from HAART cohorts as the investigative source, are on the increase. Data from many of these studies identified metabolic markers indicative of HIV-induced OS, usually as an outcome of an untargeted metabonomics study. Untargeted studies cast a wide net for any and all detectable metabolites in complex mixtures. Given the prevalence of OS during HIV infection and antiviral treatment, it is perhaps not surprising that indicators of this malady would become evident during metabolite identification. At times, targeted studies for specific (non-OS) metabolites would also yield OS markers as an outcome. This review examines the findings of these studies by first providing the necessary background information on OS and the main ways in which free radicals/reactive oxygen species (ROS) produced during OS, cause biomolecular damage. This is followed by information on the biomarkers which come about as a result of free radical damage and the techniques used for assaying these stress indicators. The established links between elevated ROS and lowered antioxidants during HIV infection and the subsequent use of HAART is then presented followed by a review of the OS markers detected in HIV metabonomic studies to date. We identify gaps in HIV/HAART-associated OS research and finally suggest how these research gaps can be addressed through metabonomic analysis, specifically targeting the multiple markers of HIV-induced OS.

Synthesis and Investigation of Novel Spiro-isoxazolines as Anti-Cancer Agents.

A series of structurally diverse 4-bromo spiro-isoxazolines possessing a variety of aromatic and aliphatic substituents at the 3 position, were synthesized through a 1,3-dipolar cycloaddition followed by intramolecular cyclization of a pendant hydroxyl or carboxylic acid group. The biochemical antiproliferative activity was evaluated in vitro by using two breast cancer cell lines (MCF-7 and MDA-MB-231) and two prostate cancer cell lines (PC-3 and DU-145) using the MTT viability assay, and the IC50 values were obtained. Spiro-isoxazoline derivatives bearing a p-chloro or an o-dichloro aromatic substituent at the 3-position of the isoxazoline showed considerable antitumor activities in all four cell lines with IC50 value ranging from 43µM to 56µM.

A combined chemometric and quantitative NMR analysis of HIV/AIDS serum discloses metabolic alterations associated with disease status.

Individuals infected with the human immunodeficiency virus (HIV) often suffer from concomitant metabolic complications. Treatment with antiretroviral therapy has also been shown to alter the metabolism of patients. Although chemometric analysis of nuclear magnetic resonance (NMR) spectra of human sera can distinguish normal sera (HIVneg) from HIV-infected sera (HIVpos) and sera from HIV-infected patients on antiretroviral therapy (ART), quantitative analysis of the discriminating metabolites and their relationship to disease status has yet to be determined. The objectives of the study were to analyze NMR spectra of HIVneg, HIVpos, and ART serum samples with a combination of chemometric and quantitative methods and to compare the NMR data with disease status as measured by viral load and CD4 count. High-resolution magic angle spinning (HRMAS) NMR spectroscopy was performed on HIVneg (N = 10), HIVpos (N = 10), and ART (N = 10) serum samples. Chemometric linear discriminant analysis classified the three groups of spectra with 100% accuracy. Concentrations of 12 metabolites were determined with a semi-parametric metabolite quantification method named high-resolution quantum estimation (HR-QUEST). CD4 count was directly associated with alanine (p = 0.008), and inversely correlated with both glutamine (p = 0.017) and glucose (p = 0.022) concentrations. A multivariate linear model using alanine, glutamine and glucose as covariates demonstrated an association with CD4 count (p = 0.038). The combined chemometric and quantitative analysis of the data disclosed previously unknown associations between specific metabolites and disease status. The observed associations with CD4 count are consistent with metabolic disorders that are commonly seen in HIV-infected patients.

Mid-ATR-FTIR spectroscopic profiling of HIV/AIDS sera for novel systems diagnostics in global health.

Global health, whether in developed or developing countries, is in need of robust systems diagnostics for major diseases, such as HIV/AIDS, impacting the world populations. Fourier transform Infrared (FTIR) spectroscopy of serum is a quick and reagent-free methodology with which to analyze metabolic alterations such as those caused by disease or treatment. In this study, Attenuated Total Reflectance Fourier-Transform (ATR-FTIR) Spectroscopy was investigated as a means of distinguishing HIV-infected treatment-experienced (HIV(pos) ART(pos), n=39) and HIV-infected-treatment-naïve (HIV(pos) ART(neg), n=16) subjects from uninfected control subjects (n=30). Multivariate pattern recognition techniques, including partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA), successfully distinguished sample classes, while univariate approaches identified significant differences (p<0.05) after Benjamini-Hochberg corrections. OPLS-DA discriminated between all groups with sensitivity, specificity, and accuracy of >90%. Compared to uninfected controls, HIV(pos) ART(pos) and HIV(pos) ART(neg) subjects displayed significant differences in spectral regions linked to lipids/fatty acids (3010 cm(-1)), carbohydrates (1299 cm(-1); 1498 cm(-1)), glucose (1035 cm(-1)), and proteins (1600 cm(-1); 1652 cm(-1)). These are all molecules shown by conventional biochemical analysis to be affected by HIV/ART interference. The biofluid metabolomics approach applied here successfully differentiated global metabolic profiles of HIV-infected patients and uninfected controls and detected potential biomarkers for development into indicators of host response to treatment and/or disease progression. Our findings therefore contribute to ongoing efforts for capacity-building in global health for robust omics science and systems diagnostics towards major diseases impacting population health.

Metabonomic analysis of HIV-infected biofluids.

Monitoring the progression of HIV infection to full-blown acquired immune deficiency syndrome (AIDS) and assessing responses to treatment will benefit greatly from the identification of novel biological markers especially since existing clinical indicators of disease are not infallible. Nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) are powerful methodologies used in metabonomic analyses for an approximation of HIV-induced changes to the phenotype of an infected individual. Although early in its application to HIV/AIDS, (biofluid) metabonomics has already identified metabolic pathways influenced by both HIV and/or its treatment. To date, biofluid NMR and MS data show that the virus and highly active antiretroviral treatment (HAART) mainly influence carbohydrate and lipid metabolism, suggesting that infected individuals are susceptible to very specific metabolic complications. A number of well-defined biofluid metabonomic studies clearly distinguished HIV negative, positive and treatment experienced patient profiles from one another. While many of the virus or treatment affected metabolites have been identified, the metabonomics measurements were mostly qualitative. The identities of the molecules were not always validated neither were the statistical models used to distinguish between groups. Assigning particular metabolic changes to specific drug regimens using metabonomics also remains to be done. Studies exist where identified metabolites have been linked to various disease states suggesting great potential for the use of metabonomics in disease prognostics. This review therefore examines the field of metabonomics in the context of HIV/AIDS, comments on metabolites routinely detected as being affected by the pathogen or treatment, explains what existing data suggest and makes recommendations on future research.

Chlorido(1-cyclo-pentyl-idene-4-ethyl-thio-semicarbazidato-κN,S)diphenyl-tin(IV).

The Sn atom in the title compound, [Sn(C(6)H(5))(2)(C(8)H(14)N(3)S)Cl], is penta-coordinated with a trigonal-bipyramidal coordination geometry. The 1-cyclo-pentyl-idene-4-ethyl-thio-semicarbazidate (cpetsc) ligand coordinates through the S atom and the N atom bonds to the cyclo-pentyl group, forming a five-membered ring with the Sn center. The chloride ligand and the coordinated N atom are in axial positions. In the crystal structure, inter-molecular N-H⋯Cl hydrogen bonds form chains along [101].

Rhodamine 6G hexa-chlorido-stannate(IV) acetonitrile disolvate.

In the title compound, bis({6-ethylamino-10-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthen-3-ylidene}ethanaminium) hexachloridotin(IV) acetonitrile disolvate, (C(27)H(29)N(2)O(3))(2)[SnCl(6)]·2C(2)H(3)N, the octa-hedral SnCl(6) (2-) anion lies on an inversion center. The xanthene ring system is essentially planar, with an average deviation of 0.020 Å, and the substituent benzene ring forms a dihedral angle of 85.89 (2)° with it. The Sn-Cl distances are in the range 2.4237 (3)-2.4454 (3) Å. There are N-H⋯Cl hydrogen bonds between SnCl(6) (2-) ions and rhodamine 6G cations as well as π-π stacking inter-actions between rhodamine 6G cations (inter-planar distance of 3.827 Å).