Evaluation of Novel Inhibitors of Tryptophan Dioxygenases for Enzyme and Species Selectivity Using Engineered Tumour Cell Lines Expressing Either Murine or Human IDO1 or TDO2.

Indoleamine 2, 3-dioxygenase 1 (IDO1) is commonly expressed by cancers as a mechanism for evading the immune system. Preclinical and clinical studies have indicated the potential of combining IDO1 inhibitors with immune therapies for the treatment of cancer, strengthening an interest in the discovery of novel dioxygenase inhibitors for reversing tumour-mediated immune suppression. To facilitate the discovery, development and investigation of novel small molecule inhibitors of IDO1 and its hepatic isozyme tryptophan dioxygenase (TDO2), murine tumour cells were engineered to selectively express either murine or human IDO1 and TDO2 for use as tools to dissect both the species specificity and isoenzyme selectivity of newly discovered inhibitors. Lewis lung carcinoma (LLTC) lines were engineered to express either murine or human IDO1 for use to test species selectivity of the novel inhibitors; in addition, GL261 glioma lines were engineered to express either human IDO1 or human TDO2 and used to test the isoenzyme selectivity of individual inhibitors in cell-based assays. The 20 most potent inhibitors against recombinant human IDO1 enzyme, discovered from a commissioned screening of 40,000 compounds in the Australian WEHI compound library, returned comparable IC50 values against murine or human IDO1 in cell-based assays using the LLTC-mIDO1 and LLTC-hIDO1 line, respectively. To test the in vivo activity of the hits, transfected lines were inoculated into syngeneic C57Bl/6 mice. Individual LLTC-hIDO1 tumours showed variable expression of human IDO1 in contrast to GL261-hIDO1 tumours which were homogenous in their IDO1 expression and were subsequently used for in vivo studies. W-0019482, the most potent IDO1 inhibitor identified from cell-based assays, reduced plasma and intratumoural ratios of kynurenine to tryptophan (K:T) and delayed the growth of subcutaneous GL261-hIDO1 tumours in mice. Synthetic modification of W-0019482 generated analogues with dual IDO1/TDO2 inhibitory activity, as well as inhibitors that were selective for either TDO2 or IDO1. These results demonstrate the versatility of W-0019482 as a lead in generating all three subclasses of tryptophan dioxygenase inhibitors which can be applied for investigating the individual roles and interactions between IDO1 and TDO2 in driving cancer-mediated immune suppression.

Junk food: Polymer composition of macroplastic marine debris ingested by green and loggerhead sea turtles from the Gulf of Oman.

Pollution of the marine environment by plastic marine debris has become one of the most pervasive threats impacting marine environments. In this study, for the first time, we evaluate the polymer types of the plastic marine debris ingested by 49 green and 14 loggerhead sea turtle strandings in the Gulf of Oman. Plastic marine debris was ingested by 73.5% of green and 42.9% of loggerhead sea turtles in this study. Overall, evidence suggested that green sea turtles from the Gulf of Oman coast of the United Arab Emirates ingested high levels of plastic marine debris, predominantly Polypropylene (PP) & Polyethylene (PE), followed by Nylon, PP-PE mixture, Polystyrene (PS), Poly vinyl chloride (PVC) and Ethylene vinyl acetate (EVA), respectively. Loggerhead sea turtles also ingested high levels of plastic marine debris, which also predominantly consisted of PP & PE, followed by PP-PE mixture, Nylon and PS. While recent studies were directed into polymer characterization of micro-plastics in aquatic life, our study focuses on macro-plastics which impose significantly greater risks.

Lysozyme and Human Serum Albumin Proteins as Potential Nitric Oxide Cardiovascular Drug Carriers: Theoretical and Experimental Investigation.

Nitric oxide-containing drugs present a critical remedy for cardiovascular diseases. Nitroglycerin (NG, O-NO) and S-nitrosoglutathione (SNG, S-NO) are the most common nitric oxide drugs for cardiovascular diseases. Insights regarding the binding affinity of NO drugs with lysozyme and human serum albumin (HSA) proteins and their dissociation mechanism will provide inquisitive information regarding the potential of the proteins as drug carriers. For the first time, the binding interactions and affinities are investigated using molecular docking, conventional molecular dynamics, steered molecular dynamics, and umbrella sampling to explore the ability of both proteins to act as nitric oxide drug carriers. The molecular dynamics simulation results showed higher stability of lysozyme-drug complexes compared to HSA. For lysozyme, cardiovascular drugs were bound in the protein cavity mainly by the electrostatic and hydrogen bond interactions with residues ASP53, GLN58, ILE59, ARG62, TRP64, ASP102, and TRP109. For HSA, key binding residues were ARG410, TYR411, LYS414, ARG485, GLU450, ARG486, and SER489. The free energy profiles produced from umbrella sampling also suggest that lysozyme-drug complexes had better binding affinity than HSA-drug. Binding characteristics of nitric oxide-containing drugs NG and SNG to lysozyme and HSA proteins were studied using fluorescence and UV-vis absorption spectroscopy. The relative change in the fluorescence intensity as a function of drug concentrations was analyzed using Stern-Volmer calculations. This was also confirmed by the change in the UV-vis spectra. Fluorescence quenching results of both proteins with the drugs, based on the binding constant values, demonstrated significantly weak binding affinity to NG and strong binding affinity to SNG. Both computational and experimental studies provided important data for understanding protein-drug interactions and will aid in developing potential drug carrier systems in cardiovascular diseases.

Parallel discovery of selective and dual inhibitors of tryptophan dioxygenases IDO1 and TDO2 with a newly-modified enzymatic assay.

The expression of tryptophan catabolising enzyme indoleamine 2,3-dioxygenase 1 (IDO1) or tryptophan 2,3-dioxygenase 2 (TDO2) in cancers is associated with suppressed immunity and poor patient prognosis. Results from human clinical trials of IDO1 inhibitors have been disappointing. There is now a strong interest in the development of TDO2-selective or dual IDO1/TDO2 inhibitors that may surpass IDO1 inhibitors by providing broader efficacy and blocking constitutively-expressed hepatic TDO2. To expedite the discovery of novel TDO2-specific and dual inhibitors, an assay that enabled the efficient and accurate measurement of the inhibitory activity of compounds against both IDO1 and TDO2 enzymes, concurrently in the same experiment was established to screen 5,682 compounds that included the National Cancer Institute Diversity set 5, for inhibition of IDO1 and TDO2 activity. This screen identified 82 compounds that inhibited either IDO1, TDO2 or both enzymes > 50% at 20 µM. Thirty Pan Assay Interference compounds were removed from the list and the IC50 of the remaining 52 compounds against IDO1 and TDO2 was subsequently determined using the newly-developed concurrent assay. Ten compounds were confirmed as dual IDO1/TDO2 inhibitors having IC50 values under 50 µM against both enzymes and within 2-fold of each other. Six compounds with IC50 values between 1.39 and 8.41 µM were identified as potential TDO2-selective leads. The use of this concurrent protocol is anticipated to expedite the discovery of novel leads for dual and selective inhibitors against IDO1 and or TDO2 and speed the evaluation of novel analogues that will ensue.

FRET-based fluorescent probe for drug assay from amino acid@gold-carbon nanoparticles.

Biocompatible and luminescent nanostructures synthesized by capping gold-carbon nanoparticles (HOOC-4-C6H4-AuNPs) with amino acids tyrosine, tryptophan, and cysteine were used for the quantitative estimation of ranitidine (RNH), a peptic ulcer and gastroesophageal reflux drug. We applied a fluorescence quenching mechanism to investigate the viability of the energy transfer based on gold-carbon nanosensors. Förster resonance energy transfer (FRET) calculations showed a donor-acceptor distance of 1.69 nm (Tyr@AuNPs), 2.27 nm (Trp@AuNPs), and 2.32 nm (Cys@AuNPs). The constant time-resolved fluorescence lifetime measurements supported the static quenching nature. This method was successfully utilized in the detection and quantification of RNH, with a limit of detection (LOD) of 0.174, 0.56, and 0.332 µM for Tyr@AuNP, Trp@AuNP, and Cys@AuNP bioconjugates, respectively. This approach was also successful in the quantification of RNH in spiked serum samples.

First fatal cerebral phaeohyphomycosis due to Rhinocladiella mackenziei in Iran, based on ITS rDNA.

Black yeast-like fungi and relatives as agents of cerebral phaeohyphomycosis are often encountered in human fatal brain abscesses and lead to almost 100% mortality despite the application of antifungal and surgical therapy. We report to our knowledge the first case of brain infection due to Rhinocladiella mackenziei in a 54-year-old immunocompetent male in Iran where R. mackenziei has not been reported previously. The initial diagnosis was brain fungal infection because of pigmented, irregular, branched, septated hyphae based on histopathological staining. The patient was treated with intravenous amphotericin B deoxycholate (0.5mg/kg/day) combined with oral itraconazole (200mg twice daily), nevertheless, his neurological function deteriorated rapidly and ultimately the patient died due to respiratory failure later two weeks. R. mackenziei was identified based on the sequencing of internal transcribed spacer (ITS rDNA region) (KJ140287). Therefore, considerable attention for this life-threatening infection is highly recommended.

Tropical legume crop rotation and nitrogen fertilizer effects on agronomic and nitrogen efficiency of rice.

Bush bean, long bean, mung bean, and winged bean plants were grown with N fertilizer at rates of 0, 2, 4, and 6 g N m(-2) preceding rice planting. Concurrently, rice was grown with N fertilizer at rates of 0, 4, 8, and 12 g N m(-2). No chemical fertilizer was used in the 2nd year of crop to estimate the nitrogen agronomic efficiency (NAE), nitrogen recovery efficiency (NRE), N uptake, and rice yield when legume crops were grown in rotation with rice. Rice after winged bean grown with N at the rate of 4 g N m(-2) achieved significantly higher NRE, NAE, and N uptake in both years. Rice after winged bean grown without N fertilizer produced 13-23% higher grain yield than rice after fallow rotation with 8 g N m(-2). The results revealed that rice after winged bean without fertilizer and rice after long bean with N fertilizer at the rate of 4 g N m(-2) can produce rice yield equivalent to that of rice after fallow with N fertilizer at rates of 8 g N m(-2). The NAE, NRE, and harvest index values for rice after winged bean or other legume crop rotation indicated a positive response for rice production without deteriorating soil fertility.

Recent advances in gold and silver nanoparticles: synthesis and applications.

In the last decade, gold and silver nanomaterials have received considerable attention due to their attractive electronic and chemical properties and their potential applications in the development of new technologies. Recent advances in the study of various gold and silver nanomaterials have led to their utilization in a number of very important applications including biosensing, diagnostic imaging, and cancer diagnosis and therapy. This review surveys the various synthetic methods of gold and silver nanomaterials. Recent experimental studies focusing on the use of gold and silver nanomaterials in catalysis, food industry, and environmental conservation are also reviewed. This review also highlights the advantages of gold and silver nanomaterials in the development of fluorescence biosensors, glucose biosensors, nucleic acids-based biosensors, and protein-based biosensors. Moreover, the potent in vitro and in vivo anti-microbial and cyto-genotoxic effects of various gold and silver nanomaterials are underlined. Finally, recent advances in the employment of gold and silver nanomaterials as effective drug delivery vehicles and promising cancer therapeutic agents are summarized. Despite their use in remediating numerous medical and health-related conditions, the efficacy and safety of many gold and silver nanomaterials is still under some scrutiny. Needless to say, researchers are facing many challenges and obstacles in their ample attempts to synthesize nanomaterials that are relatively easy to design, inexpensive to fabricate, and effective in treating various diseases, but at the same time display a very low, if any, toxicity to the body. Future investigations should aim at overcoming such challenges in an attempt to design nanomaterials that will prove to be useful in diagnosing and treating life-threatening diseases while ensuring a high degree of efficacy and safety.

Discovery and characterisation of hydrazines as inhibitors of the immune suppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO1).

Screening of a fragment library identified 2-hydrazinobenzothiazole as a potent inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme expressed by tumours that suppresses the immune system. Spectroscopic studies indicated that 2-hydrazinobenzothiazole interacted with the IDO1 haem and in silico docking predicted that the interaction was through hydrazine. Subsequent studies of hydrazine derivatives identified phenylhydrazine (IC50=0.25 ± 0.07 µM) to be 32-fold more potent than 2-hydrazinobenzothiazole (IC50=8.0 ± 2.3 µM) in inhibiting rhIDO1 and that it inhibited cellular IDO1 at concentrations that were noncytotoxic to cells. Here, phenylhydrazine is shown to inhibit IDO1 through binding to haem.

Enhanced accumulation of copper and lead in amaranth (Amaranthus paniculatus), Indian mustard (Brassica juncea) and sunflower (Helianthus annuus).

BACKGROUND: Soil contamination by copper (Cu) and lead (Pb) is a widespread environmental problem. For phytoextraction to be successful and viable in environmental remediation, strategies that can improve plant uptake must be identified. In the present study we investigated the use of nitrogen (N) fertilizer as an efficient way to enhance accumulation of Cu and Pb from contaminated industrial soils into amaranth, Indian mustard and sunflower. METHODS/PRINCIPAL FINDINGS: Plants were grown in a greenhouse and fertilized with N fertilizer at rates of 0, 190 and 380 mg kg⁻¹ soil. Shoots, roots and total accumulation of Cu and Pb, transfer factor (TF), translocation index were assessed to evaluate the transport and translocation ability of tested plants. Addition of N fertilizer acidified the industrial soil and caused the pH to decrease to 5.5 from an initial pH of 6.9. Industrial soil amended with N fertilizer resulted in the highest accumulation of Pb and Cu (for Pb 10.1-15.5 mg kg⁻¹, for Cu 11.6-16.8 mg kg⁻¹) in the shoots, which was two to four folds higher relative to the concentration in roots in all the three plants used. Sunflower removed significantly higher Pb (50-54%) and Cu (34-38%) followed by amaranth and Indian mustard from industrial soils with the application of N fertilizer. The TF was <1 while the shoot and root concentration (SC/RC) ratios of Pb and Cu were between 1.3-4.3 and 1.8-3.8, respectively, regardless of plant species. CONCLUSIONS: Sunflower is the best plant species to carry out phytoextraction of Pb and Cu. In contrast, Pb and Cu removal by Indian mustard and amaranth shows great potential as quick and short duration vegetable crops. The results suggest that the application of N fertilizer in contaminated industrial soil is an effective amendment for the phytoextraction of Pb and Cu from contaminated industrial soils.

Labeling of oxidizable proteins with a photoactivatable analog of the antitumor agent DMXAA: evidence for redox signaling in its mode of action.

The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-alpha production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA.

Neutrophil influx and chemokine production during the early phases of the antitumor response to the vascular disrupting agent DMXAA (ASA404).

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) acts through tumor vascular disruption and cytokine production and is the first of its class to enter phase 3 trials. We characterized leukocytes and cytokines in murine Colon 38 tumors before and after DMXAA treatment. Tumor mass declined 50% 24 hours after DMXAA administration, but the leukocyte count per gram of tumor increased threefold owing to a large influx of Ly6G(+)CD11b(+)F4/80(-) cells with the morphology of neutrophils. However, B and T lymphocytes, natural killer cells, and macrophages in the tumor all decreased in numbers. Seven chemokines were substantially induced in the tumor, spleen, and serum 4 hours after DMXAA administration. Using cultured spleen cell subpopulations, CD11b(+) cells (largely monocytes and macrophages) were shown to be the primary producers of tumor necrosis factor alpha, interleukin 6 (IL-6), and macrophage inflammatory 1alpha (MIP-1alpha). CD49b(+) natural killer cells produced IP-10, whereas CD45R(+) B lymphocytes produced regulated upon activation normal T cell express sequence. T lymphocytes were not major producers of cytokines in the response to DMXAA. Murine peripheral blood leukocytes (PBLs) produced a similar panel of cytokines in culture to that detected in mouse serum after DMXAA treatment. Cytokines in human PBL cultures were subsequently measured with the aim of identifying potential serum markers of the human response to DMXAA. IP-10 (P < .001), monocyte chemoattractant protein 1 (P < .001), and sCD40L (P < .01) were decreased, whereas IL-8 (P < .001) and MIP-1alpha (P = .03) were increased in DMXAA-treated compared with untreated PBL cultures from a group of 12 donors.

Carbon nanotube-ionic liquid composite sensors and biosensors.

A new composite electrode has been fabricated using multiwall carbon nanotubes (MWCNT) and the ionic liquid n-octylpyridinum hexafluorophosphate (OPFP). This electrode shows very attractive electrochemical performances compared to other conventional electrodes using graphite and mineral oil, notably improved sensitivity and stability. One major advantage of this electrode compared to other electrodes using carbon nanotubes and other ionic liquids is its extremely low capacitance and background currents. A 10% (w/w) loading of MWCNT was selected as the optimal composition based on voltammetric results, as well as the stability of the background response in solution. The new composite electrode showed good activity toward hydrogen peroxide and NADH, with the possibility of fabricating a sensitive biosensor for glucose and alcohol using glucose oxidase and alcohol dehydrogenase, respectively, by simply incorporating the specific enzyme within the composite matrix. The marked electrode stability and antifouling features toward NADH oxidation was much higher for this composite compared to a bare glassy carbon electrode. While a loading of 2% MWCNT showed very poor electrochemical behavior, a large enhancement was observed upon gentle heating to 70 degrees C, which gave a response similar to the optimum composition of 10%. The ease of preparation, low background current, high sensitivity, stability, and small loading of nanotubes using this composite can create new novel avenues and applications for fabricating robust sensors and biosensors for many important species.

Semiconducting metal oxide based sensors for selective gas pollutant detection.

A review of some papers published in the last fifty years that focus on the semiconducting metal oxide (SMO) based sensors for the selective and sensitive detection of various environmental pollutants is presented.

Dual WO(3) based sensors to selectively detect DMMP in the presence of alcohols.

A size selective approach to improving selectivity in semiconducting metal oxides (SMO) sensors was obtained by tailoring the architecture of WO(3) powders. The key for achieving high selectivity is based on using a dual sensor configuration where the response on a porous WO(3) powder sensor was compared to the response on a nonporous WO(3) powder sensor. Detection selectivity between methanol and dimethyl methylphosphonate (DMMP) is obtained because the access of a gas molecule in the interior pore structure of WO(3) is size dependent leading to a size dependant magnitude change in the conductivity of SMO sensor.

Determination of the flight range and dispersal of the house fly, Musca domestica (L.) using mark release recapture technique.

In order to control any pest it is essential to study the life cycle, biology and bionomics of the target pest under control. With this respect, we have studied the flight range of the house fly Musca domestica (L.). The flight range of the house fly from two sites i.e, the poultry farm and a stable farm has been studied. The flight range study was conducted using a mark release technique. The approach we used in this study was that the flies collected from the respective farms were marked and released at different distances from the farms. The flies were then re-captured from the poultry farm and the stable farm. Studies conducted elsewhere use the technique of releasing the insect species at one spot and recapturing the insect species with the help of baited traps placed at various locations from the release point. The advantage of the approach used in this study was that the flight range as well as the homing effect was determined. From this study, the flight range of house flies released at the poultry farm was 7 km whereas flight range for flies release from stable farm was 5 km. The recovery rate of house flies at the poultry and stable farm was 0.05% and 0.016%, In this study, marked specimens has been detected up to 8 days in field conditions indicating that under field condition the life expectancy could be in the range of 1-2 weeks.

Bacteria fauna from the house fly, Musca domestica (L.).

The house fly, Musca domestica has long been considered a potential agent for disease transmission ever since its existence. The general truth of this assertion remains undisputed till the present day in spite of increasing awareness toward an improved sanitation and better hygiene. The habitual movement of house fly from filthy substrata such as human faeces, animal excreta, carcasses, garbage, etc. makes them ideal candidates for disease transmission such as cholera, shigellosis, salmonellosis and others when settling on food. Fly as a potential mechanical vector of pathogenic bacteria was elucidated in this study by examining flies from various breeding sites such as food courts, dumping ground, food processing areas and poultry farm in Peninsular Malaysia. The flies were baited with 10% sugar solution on a glass slide in the field. All materials used for collection of samples were sterile. Bacteria from fly sample were isolated using the normal isolation technique. Bacillus sp., Coccobacillus sp., Staphylococcus sp., Microccus sp., Streptococcus sp., Acinetobacter sp., Enterobacter sp., Proteus sp., Escherichia sp., Klebsiella sp. and yeast cells were isolated from feaces, vomitus, external surfaces and internal organs of house fly. Newly emerged house fly did not harbour any bacteria.

Photodecomposition of carbaryl in the presence of silver-doped zeolite Y and Suwannee River natural organic matter.

The synthesis and characterization of a novel catalyst for the photodecomposition of carbaryl (1-naphthyl, N-methylcarbamate) is reported. In the absence of a catalyst, but in the presence of UV light a 30 ppm solution of carbaryl decomposes with a first-order rate constant of (5.6 +/- 0.3) x 10(-5) s(-1) (298 K) and a quantum efficiency of 4.8 x 10(-3) molecules/photon. In the presence of the Ag-zeolite Y catalyst with 2.42% Ag by weight, the photodecomposition rate becomes 80 times faster. The addition of Suwannee River natural organic matter (NOM), which can inactivate photocatalysts, has a minimal effect on this system. In the presence of three different concentrations of NOM and 30 ppm carbaryl, our results indicate that the NOM increases or decreases the catalytic photodecomposition rate by only a factor of 3 at most.