Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools.Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to  Real time cellular fluorescence imaging requires a probe that displays high degrees of localisation, low toxicity and good photostability. Here, the authors report a near infrared fluorophore that displays pH-sensitive fluorescence based on phenol/phenolate interconversion, showing real time imaging of cellular processes. Ehrlich's use of synthetic dyes as a means of staining biological samples can be viewed as one of the foundation stones of modern scientific research. A century later, the use of fluorescence imaging as a technique to visualize specific regions of live cellular12357910The major shortcomings of fluorescence imaging using molecular fluorophores are interference from nonspecific background fluorescence outside the region of interest (ROI), insufficient photostability and cytotoxicity. Poor ROI selectivity necessitates a time delay to allow background fluorophore clearance and/or a washing procedure between fluorophore administration and image acquisition. This can limit imaging to fixed cells or static snapshots, without the possibility of continuous data acquisition throughout the experiment. An innovative approach to enhance target-to-background signal ratio is to exploit a mechanism of selective fluorescence quenching in the background areas, while establishing the emitting potential of the fluorophore only in the ROI12in vitro or in vivo settings. Continuous live-cell imaging places very high demands on photostability of the fluorophore, as the same cell(s) are repeatedly imaged over time. Fluorophore dark toxicity must be low, so that cell viability is not compromised and normal cellular processes are unperturbed. To minimize light-induced toxicity, it is preferable to use low-energy wavelengths in the near-infrared (NIR) spectral region (λ=700–900 nm). For in vivo imaging, the use of NIR fluorophores is essential. This spectral region is required for effective light transmission through body tissue, as there are reduced levels of absorption and scattering at these longer wavelengths and less intrinsic autofluorescence. In addition, if on/off NIR fluorescence switching could be accomplished in vivo, then similar imaging advantages could be gained as for in-vitro cell imaging.Developing a responsive fluorophore suitable for real-time live-cell imaging poses a series of challenges. Stringent criteria are required, such as near-perfect response selectivity, exceptional photostability and low dark and light toxicities. Obtaining selective fluorescence responses for intracellular analytes is not trivial, as analyte selectivity observed in a controlled homogeneous environment of a cuvette does not necessarily translate to far more complex 2-chelated azadipyrromethene class 1 (1516171 (R=Ph) has an absorption/emission λmax at 696 and 727 nm in aqueous solutions, high fluorescence quantum yields (0.3–0.4) and excellent photostabilityin vivo translation.Currently, there is a small yet growing selection of NIR fluorophore classes but they often suffer from insufficient photostability and lack emission wavelengths above 700 nm class 1 15161718.vide infra).The goal of our current work was to develop an NIR fluorophore capable of a lysosomal-induced off-to-on fluorescence response, thereby permitting real-time imaging of cellular uptake, trafficking and efflux without perturbing function20232425ortho-nitro phenolic group was chosen to impart the pH-responsive feature of the probe. It would be expected that the electron withdrawing o-nitro group would result in the ionized phenolate dominating at pH 7.2, resulting in fluorescence quenching due to a non-emissive intramolecular charge transfer excited state -3-phenylpropenone (3 was achieved to produce 4 by reaction with t-butyl bromoacetate and CsF in dimethylsulfoxide (DMSO) at 30 °C. After isolation, compound 4 was then subjected to ortho-nitration of the remaining phenol ring with KHSO4/KNO3 to provide 5. Next, hydrolysis of the t-butyl ester of 5 with trifluoroacetic acid (TFA) gave the carboxylic acid 6, which was converted into its activated ester 7 by reaction with N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide in DMSO. Formation of the activated ester was monitored by 1H NMR via the diagnostic CH2 peaks at 5.48 for 7 and 4.83 p.p.m. for carboxylic acid 6, which showed complete conversion within 2 h  was effective, with the final fluorophore 2 obtained in high yield  showed a single peak for 2 with retention time differing from that of both the acid 6 and ester 7  and TFA to cycle between the two (f)5, giving similar results and indicating that this positive feature is general to the fluorophore class  nm for 2 and 540(40) nm for lysotracker red and pH-rhodo red for 2 h, and their fluorescence intensity monitored. Encouragingly, no photobleaching for 2 was observed, whereas both other fluorophores were ∼80% degraded within that time frame  nm for 2 and 563(9) nm for lysotracker red and pH-rhodo red) were used, allowing images to be acquired at various time intervals. Graphing the average cell fluorescence intensity versus time showed that 2 was the most photostable with 50% loss of signal in 94 s and lysotracker red being the least photostable with 50% of signal loss in just 6 s  before its use in imaging studies. Fluorescence output of 2 was negligible at pH 7.4, but became highly fluorescent at acidic pH with its pKa determined as 4.0  and HEK (human embryonic kidney) cell lines was determined. Following a 24-h incubation of cells with 2, an MTT -2,5-diphenyltetrazolium bromide) assay was performed and EC50 values of 0.43 and 0.44 mM, respectively, were obtained  and imaging with confocal laser scanning microscopy (CLSM)  . These ithin 2 h . The flu2) and green channel (GFP) emissions to the lysosomes  for MNIR and 0.95 (05) for Mgreen and a Pearson's coefficient of 0.79 (09)2 showed no significant co-localization  fused to green fluorescent protein (GFP)ysosomes . Examina2 is physically located exclusively in the lysosomes or it is present in other organelles along the endocytic pathway and is only emissive from the lysosomes due to its lower pH, with the remaining 2 being fluorescent silent. To visualize all intracellular 2 in its fluorescent on state, HeLa cells were incubated with 2 in media at pH 7.4 for 2 h, nuclei stained with 4,6-diamidino-2-phenylindole (DAPI) for 15 min, fixed and imaged using CLSM  with identical microscope settings showed a significantly increased fluorescence with saturation of the field of view within the cells  voltage and it became clear that the additional fluorescence of 2 was predominantly from other cellular organelles of higher pH . Images were continuously acquired every 30 s for 90 min and then combined to form a movie  representation of the cell in real time, a z-stack of 25 focal planes through the cell was acquired every minutex,y,z and time dimensions, from which a time-lapse video of the 3D cellular volume can be created. Using HeLa cells, a 4D data set of the uptake of 2 over a 60-min time period was acquired followed by deconvolution of the data set to correct for motion of fluorescence objects between focal planes during the 1-min time period required to complete a z-stack of the cell. This experiment showed punctuated regions of fluorescence over the 60-min time period starting from a non-fluorescent background at time zero with individual lysosome movement clearly observable  using a widefield microscope. Image analysis software was used to tag individual lysosomes as white spheres to facilitate visualization and the movement of these lysosomes was tracked over the 35-min time period , images were acquired at regular intervals over the course of 24 h. A plot of tumour to background NIR fluorescence showed that the fluorescence signal was low in the beginning in both the background and tumour. Emission from the liver peaked at 1 h and subsided over the following 24 h  to the fluorophore, to further broaden the possibilities of in-vivo imaging targets with potential applications for fluorescence-guided surgery.In conclusion, the development of 1H and 13C spectra were recorded on 300, 400, 500 or 600 MHz NMR spectrometers and chemical shifts were reported in p.p.m. using solvent residual peak as standard. For spectra of compounds 4, 5, 6 and 7, see 19F NMR chemical shifts are referenced to CFCl3. All 11B NMR chemical shifts are referenced to BF3.Et2O/CDCl3. High-resolution mass spectrometry and tandem mass spectrometry experiments were carried on an electrospray ionisation and MALDI–TOF instruments. Infrared spectra were recorded as a KBr pellet using a Fourier Transform infrared spectrometer. Absorbance spectra were recorded with a Varian Cary 50 Scan ultraviolet–visible spectrometer. Fluorescence spectra were recorded with a Varian Cary Eclipse Fluorescence Spectrometer. Solvents for absorbance and fluorescence experiments were of HPLC quality. SigmaPlot, MestreNova, ChemDraw, Zeiss LSM and ImageJ software were used for data analysis. Phenol red-free imaging DMEM medium was used for all experiments.All commercially available solvents and reagents were used as supplied, unless otherwise stated. All reactions were performed under nitrogen or argon atmosphere in oven-dried glassware. Gel chromatography was performed with Davisil 60 silica (230–400 mesh). On the basis of NMR and reverse-phase HPLC, all final compounds were >95% pure. tButyl bromoacetate  was then added via syringe in one go and the solution was stirred at 30 °C for 20 min. The mixture was partitioned between AcOEt (100 ml) and PBS buffer at pH 7 (100 ml). The organic phase was washed with water (3 × 100 ml), brine (50 ml), dried over Na2SO4, filtered and evaporated to dryness. The crude product was purified by silica gel chromatography, eluting with CH2Cl2:AcOEt (99:1→90:10) to yield the product 4 as a red metallic solid . mp: 183–187 °C; 1H NMR : δ 10.58 , 8.19–8.10 , 7.65 , 7.57–7.43 , 7.10 , 6.95 , 4.81 , 1.46 ; 13C NMR : δ 167.5, 161.5, 160.0, 158.9, 155.7, 145.0, 143.8, 142.6, 141.0, 132.5, 132.0, 131.7, 131.4, 129.6, 129.3, 129.1, 129.0, 128.7, 128.6, 124.1, 121.3, 120.3, 119.1, 116.0, 114.8, 81.6, 65.0, 27.7; 11B NMR  δ: 1.00 ; 19F NMR : δ −130.44 ; ultraviolet–visible: λmax (CHCl3): 680 nm ; emission: λmax (CHCl3): 708 nm, ΦF (CHCl3)=0.31; high resolution mass spectrometry (HRMS) (m/z): [M-H]− calcd. for C38H31BN3O4F2, 642.2376; found, 642.2357.Compound ref.   in acetonitrile (6 ml) was heated under reflux for 5 min. A solution of KNO3  and KHSO4  in water (1 ml) was added and the mixture was heated under reflux for 5 min. The suspension was cooled to room temperature (rt) and partitioned between AcOEt (200 ml) and water (100 ml). The organic phase was washed with water (100 ml), brine (2 × 100 ml), dried over Na2SO4, filtered and evaporated to dryness. The crude was purified by silica gel chromatography, eluting with CH2Cl2:AcOEt (99: 1) to yield the product 5 as a dark red metallic solid . mp: 192–194 °C; 1H NMR : δ 11.88 , 8.69 , 8.27 , 8.21 , 8.19–8.13 , 7.71 , 7.60 , 7.57–7.43 , 7.27 , 7.12 , 4.84 , 1.45 ; 13C NMR : δ 167.4, 160.9, 159.3, 154.1, 153.9, 145.4, 143.9, 143.3, 141.5, 137.3, 135.6, 132.1, 131.8, 131.5, 129.9, 129.5, 129.2, 129.0, 128.7, 128.7, 126.6, 123.3, 121.8, 120.8, 119.4, 119.1, 115.0, 81.7, 65.1, 27.7; 11B NMR : δ 0.92 ; 19F NMR : δ −130.31 ; ultraviolet–visible: λmax (CHCl3) 675 nm ; emission: λmax (CHCl3) 703 nm, ΦF (CHCl3)=0.15; HRMS (m/z): [M-H]− calcd. for C38H30BN4O6F2, 687.2226; found, 687.2229.A solution of 5  in dichloromethane (DCM) (9 ml) and the solution was stirred at rt for 3 h. The solvent was removed under vacuo and the residual TFA was removed azeotropically with serial additions of DCM and subsequent removal under vacuo. The solid was suspended in DCM, filtered and washed with DCM, to yield the product as a dark purple solid . The product was pure enough to proceed to the next synthetic step. To remove the last trace of starting material, the solid was partitioned between AcOEt (90 ml) and Na2CO3 sat. (180 ml). The organic layer was discarded and the water layer was extracted with AcOEt (90 ml), separated, carefully acidified with 5 M HCl and extracted again with AcOEt (180 ml). The organic layer was separated, dried over anhydrous Na2SO4, filtered and evaporated to dryness. The product 6 was obtained as a dark purple solid . mp: 214–219 °C; 1H NMR : δ 13.15 , 8.68 , 8.28 , 8.24–8.13 , 7.71 , 7.60 , 7.57–7.44 , 7.28 , 7.14 , 4.87 ; 13C NMR : δ 169.7, 161.0, 159.4, 154.1, 153.8, 145.4, 143.9, 143.4, 141.5, 137.3, 135.5, 132.1, 131.9, 131.5, 129.9, 129.5, 129.2, 129.0, 128.7 (2C), 126.6, 123.2, 121.9, 120.8, 119.5, 119.1, 115.1, 64.6; 11B NMR : δ 0.93 ; 19F NMR : δ −130.34 ; HRMS (m/z): [M-H]− calcd. for C34H22BN4O6F2, 631.1600; found, 631.1603.TFA (1 ml) was added dropwise to a solution of 5 , N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride  and N-hydroxysuccinimide  was placed in a sealed dry flask. Anhydrous deuterated DMSO-d6 (1.2 ml) was added to the mixture and the solution was stirred at rt under N2 atmosphere. Samples (50 μl) were withdrawn at 15, 30, 60, 120 min and 19 h, diluted with DMSO-d6 in an NMR tube (650 μl) and 1H spectra were recorded at a 600-MHz spectrometer.A mixture of 6 , N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride  and N-hydroxysuccinimide  was dissolved in anhydrous DMSO (1 ml) and stirred at rt for 3 h under N2 atmosphere. The solution was partitioned between with DCM (50 ml) and 0.5 M HCl (50 ml). The organic phase was washed with 0.5 M HCl (50 ml), acidic brine (50 ml), dried over Na2SO4, filtered and evaporated to dryness, keeping the temperature of the bath below 35°C. The product 7 was obtained as a purple metallic solid . m.p.: 177–183 °C; 1H NMR : δ 8.69 , 8.29 , 8.23 , 8.21–8.14 , 7.72 , 7.64 , 7.59–7.45 , 7.28 , 7.23 , 5.53 , 2.85 ; 13C NMR : δ 169.9, 165.2, 159.8, 158.6, 154.5, 145.2, 144.2, 143.1, 142.0, 137.4, 135.5, 132.0, 131.8, 131.6, 129.8, 129.6, 129.2, 129.1, 128.7, 126.8, 124.1, 121.5, 120.6, 119.6, 119.5, 115.2, 63.0, 25.5; 11B NMR : δ 0.93 ; 19F NMR : δ −130.34 ; HRMS (m/z): [M-H]− calcd. for C38H25BF2N5O8, 728.1764; found, 728.1730.A mixture of 7  and O-(2-aminoethyl)polyethylene glycol 5000 (CAS 32130–27–1)  was dissolved in anhydrous DMSO (0.88 ml) and stirred at rt for 18 h under a N2 atmosphere. The solvent was removed by short-path distillation at rt overnight and the crude was partitioned between DCM (20 ml) and 1 M Na2CO3 (20 ml). The aqueous phase was extracted with DCM (2 × 20 ml). The organic layers were combined, washed with slightly acidic (HCl) water (20 ml), brine (20 ml), dried over anhydrous Na2SO4, filtered and evaporated to dryness. The residue was dissolved in HPLC grade water (8 ml) and the dark solution was passed through a Sep Pak C18 reverse-phase cartridge, then freeze dried. The product 2 was obtained as a dark green solid . mp: 43–45 °C; 1H NMR : δ 8.74–8.72 , 8.28 , 8.24–8.15 , 7.68 , 7.58–7.52 , 7.52–7.45 , 7.25–7.19 , 7.16 , 4.66 , 3.70–3.65 , 3.50 ; Ultraviolet–visible: λmax (CHCl3) 670 nm, ; emission: λmax (CHCl3) 702 nm, ΦF (CHCl3)=0.18; HRMS (m/z): MALDI–TOF distribution maximum centred at 5410.3999 Da.A mixture of 3 (50 ml) to prepare a stock solution (10−4 M). The stock was diluted to concentrations 2, 4, 6, 8 and 10 × 10−7 M with CHCl3 and each solution was analysed with an ultraviolet–visible spectrometer and a fluorescence spectrometer against CHCl3 background. Excitation=640 nm; emission range=660–900 nm; slit width=5/5 nm; scan rates=600 nm min−1. Plots of absmax versus conc and fluorescence area versus abs (640 nm) allowed the calculation of extinction coefficient and fluorescence quantum yield, respectively. Compound 1  was used as standard for fluorescence quantum yields with ΦF=0.36 (refs The compound of interest (0.005 mmol) was dissolved in CHCl.36 refs , 42.2 or 5 was dissolved in toluene, tetrahydrofuran, dimethylformamide and DMSO (25 ml) to a final concentration of 5 μM. A solution of DBU (29.5 mg in 100 ml of CHCl3) was added (64 μl=1 eq) gradually, and absorbance and fluorescence spectra were recorded before and after each addition. The addition was stopped once spectra remained unchanged. At this stage, an excess of DBU was added and the spectra were recorded. Subsequently, a higher excess of TFA was added and the spectra were recorded. The area below the last two curves was plotted for off/on histogram (shown in 2 contained 1% CHCl3 for solubility).Compound shown in . (Note: 2 (2.8 mg) was dissolved in PBS (500 μl). The stock solution (1 mM) was diluted with DMEM supplemented with 10% FBS to the concentration of 5 μM. The pH of the solution was adjusted with diluted HCl or NaOH, to obtain a range from 8 to 2 at regular intervals, each of which was recorded, and the respective solution analysed by ultraviolet–visible absorption and fluorescence emission. Excitation=625 nm; emission range=635–900 nm.Compound −7 M DMEM solutions at pH 4.0 of 2; lysotracker red and pH-rodo red were continuously irradiated with light of wavelength 620(30) nm for 2 and 540(40) nm for lysotracker red and pH-rhodo red at 25 °C for 2 h. Filtered light from a 150-W light source used with complete cuvette irradiation via a fibre optic with attached light diffuser. Fluorophore fluorescence intensities were recorded every 20 min. The average fluorescence intensity from three independent experiments were normalized and plotted with sigmaplot 8.Entire fluorescence cuvettes contain 1 × 102 (20 μM) for 60 min or lysotracker red (150 nM) for 30 min, or pH rhodo red (15 μM) for 30 min. DMEM was replaced with fluorophore-free media and cells constantly irradiated with a Lumencor SPECTRA light engine LED used as the light source set to a maximum power for 400 s. Excitation filter 563/9 nm was used for lysotracker red and pH-rhodo red and excitation filter 640(14) nm was used for 2. Cells were imaged with the shutter open, a time intervals of either 0.1 or 1.0 or 5 s with exposure of 10 ms and individual frames complied into movie format. The average cellular ROI fluorescence intensities from three independent experiments were plotted. An Olympus × 60 PLANAPO/1.42 objective and Andor iXon 888 ultra were used for signal detection. Acquisition and analysis performed with MetaMorph v7.8.Ten thousand HeLa-Kyoyo cells in DMEM were seeded onto chamber slides and incubated with 2 (4.0 mg) was dissolved in sterile PBS (71 μl) to prepare a stock solution 10 mM. This was serially diluted to prepare samples at 5, 1, 0.5, 0.1 and 0.05 mM. Each of the stock solutions was diluted 1:10 with DMEM medium, which was co-incubated with HeLa or HEK293 cells at 5,000 cells per well on a 96-well plate for 24 h. The solution was removed and substituted with MTT solution (5 mg ml−1 in DMEM). The cells were incubated for 3 h. The medium was removed and the wells were treated with DMSO for 10 min. The absorbance of each well was read with a plate reader at 540 nm.Compound An expression plasmid encoding the LAMP1-GFP fusion protein was generated via the complete open-reading frame coded by a I.M.A.G.E. Fully Sequenced cDNA Clone  of the human LAMP1 (GenBank accession number BC021288) was amplified by PCR using primers designed to append an XhoI site upstream of the translation initiation site and to replace the translation termination site by a segment encoding EcoRI site followed by a linker sequence CTCCTC (single-letter nucleotide code). The PCR product was gel purified and cloned in the XhoI–EcoRI sites of a pEGFP-N1 vector (BD Biosciences Clontech). Constructs were verified by DNA sequencing.−1 G418 was added. The medium was changed every day to remove the G418 non-resistant cells and when the cell number looked stabilized the G418 was lowered to 0.5 g l−1. Cells displaying resistance to G418 and expressing LAMP1-GFP (as judged by fluorescence microscopy) were cloned by limiting dilution and, sorted on a BD FACSAria flow cytometer (Becton Dickinson). The clones were validated by immunostaining, by western blotting and by two functional assays (lysotracker uptake and dextran uptake).For stable transfection, HeLa cells were grown at 37 °C in complete DMEM supplemented with 10% FBS and 1% glutamine, to 30–40% confluency, and subsequently transfected with the LAMP1–GFP-encoding plasmid using FuGENE 6 (Roche) following the manufacturer's instruction. One day later, 0.6 g lConfocal images , 6, 7 we2 to be maintained at 37 °C and 5%, respectively. Fluorophore 2 channel was recorded using a 649-nm emission long-pass filter, GFP was imaged using a 520/50 emission bandpass filter.Live-cell images , 9, 10 w2 (10 μM) in media and incubated for the appropriate time at 37 °C. Cells were counterstained with Hoechst 33342 or DAPI for 15 min. Cells were then washed once with PBS and fixed in 3.7% paraformaldehyde in PBS solution for 3 min and washed thoroughly with PBS. Images were collected by using an Olympus Fluoview 1,000 CLSM. The fluorescence arising from 2 was detected by a Cy5.5 filter. DAPI and GFP channels were used in parallel when cells were counterstained and/or transfected.Cells were seeded onto an eight-well chambered glass slide and allowed to attach for 24 h. The media was then replaced with 200 μl of 2 (10 μM) in media incubated for 2 h at 37 °C. Cells were counterstained with DAPI for 15 min. Cells were then washed once with PBS and fixed in 3.7% paraformaldehyde in PBS solution for 3 min, and washed thoroughly with PBS. A collection of cell were Z-stack imaged using CLSM  and while maintaining focus of the microscope on the same cells the medium was exchanged with medium acidified to pH 4.9 (by addition of HCl (aq)). After allowing 15 min for equilibration the same cells were re-imaged (PMT voltage=782v) using the same laser power. Following which the same cells were imaged for the third time following the adjustment of the PMT voltage 512v to obtain a non-saturated image. Mean total cell fluorescence was determined from two independent experiments using ImageJ.Cells were seeded onto an eight-well chambered glass slide and allowed to attach for 24 h. The media was then replaced with 200 μl of 2 (10 μM) in media incubated for 30 min at 4 °C. Cells were counterstained with DAPI for 15 min. Cells were then washed once with PBS and fixed in 3.7% paraformaldehyde in PBS solution for 3 min and washed thoroughly with PBS. A collection of cell were imaged using CLSM, and while maintaining focus of the microscope on the same cells the medium was exchanged with medium acidified to pH 4.9 (by addition of HCl (aq)). After allowing 15 min for equilibration, the same cells were re-imaged using the same exposure times and laser power.Cells seeded onto an 8-well chambered glass slide and allowed to attach for 24 h. The media was then replaced with 200 μl of 2  was added and fluorescence images (Cy5.5 filter) were acquired at regular intervals. Images were deconvolved and combined in a video format.HeLa Kyoto cells in Dulbecco's cell growth media containing 10% FBS were seeded onto an eight-well chambered glass slide and incubated for 24 h. The slides were placed on the microscope platform and the microscope was focused on a collection of cells. Next, 2 (10 μM) in media incubated for 2 h at 37 °C. Media was replaced with fresh media and the loss of fluorescence monitored over time. Lysosome counting was carried out at 1, 15, 30 and 120 min using ImageJ.Cells seeded onto an eight8-well chambered glass slide and allowed to attach for 24 h. The media was then replaced with 200 μl of Deconvolution of widefield data sets was performed using AutQuant X3 deconvolution software with ten iterations of adaptive point spread function calculations. Lysosome detection and tracking were performed using Imaris 7.7.1 software (Bitplane Scientific). Background subtraction was applied to all images before lysosome detection. The Spots module of Imaris was used to detect lysosomes with an estimated diameter of 1.27 μm. Detected spots were filtered using the ‘quality' algorithm. Only spots with values higher than the set threshold value (>91.76) were analysed. Quality is defined as the intensity at the centre of the spot, Gaussian filtered by the spot radius. The success and accuracy of Spot detection was judged by visual inspection. Tracking lysosome movement over the course of the video was performed using an autoregressive motion algorithm. A maximum search distance of 1 μm was defined to disallow connections between a spot and a candidate match if the distance between the predicted future position of the spot and the candidate position exceeded the maximum distance. A gap-closing algorithm was also implemented to link track segment ends to track segment starts, to recover tracks that were interrupted by the temporary disappearance of particles. The maximum permissible gap length was set equal to three frames. Tracking all the lysosomes in the cell were selected by applying filters, which were based on ‘Track Length' (>0.2 μm) and ‘Track Duration' (>60 s).2 emissions were calculated using the Image J plugin ‘Coloc2'. Rolling ball background subtraction (50 pixel diameter) and a Gaussian Filter (1 pixel diameter) were applied to all images before running the ‘Coloc2' plugin. The ROI surrounding the cell was selected manually using the freeform drawing tool. Analysis was performed on six cells from two independent experiments.Manders' and Pearson's coefficients used to show co-compartmentalization of LAMP1–GFP and Z-stack data acquired on the Olympus FLuoview100 was compressed into a single plane using the ‘Sum Slice' function in Image J. Individual cells were selected using the freeform drawing tool to create a ROI (ROI). Selecting the ‘Measure' function provided the area, the mean grey value and integrated density of the ROI. The mean background level was obtained by measuring the intensity in three different regions outside the cells and averaging the values obtained. The CTCF for each cell was calculated using the formula: CTCF=Integrated density of cell ROI−(Area of ROI × Mean fluorescence of background). The FEF was calculated by dividing the CTCF value of a cell at pH 7 into the CTCF value of the same cell at pH 4.9.Corrected total cell fluorescence (CTCF) in Z-stack data acquired on the Olympus Fluoview100, which was compressed into a single plane using the ‘Sum Slice' function in Image J. A Max Entropy Threshold 15,000–40,000 was applied to each slice followed by use of the ‘Despeckle', ‘Erode' and ‘Dilate' functions to remove noise. To count the number of lysosomes in each image the ‘Analyze Particles' function was used to count objects with a circularity of 0.75–1.00 and size from 0 to 200 pixels.The number of lysosomes per field of view  after efL-glutamine, 50 U ml−1 penicillin, 50 μl ml−1 streptomycin, 1% (v/v) sodium pyruvate and 1% (v/v) non-essential amino acids. All cells were maintained in 5% CO2 (v/v) and 21% O2 (v/v) at 37 °C. Balb/C nu/nu mice (Harlan) were housed in the Biomedical Facility (UCD) in individually ventilated cages in temperature and humidity controlled rooms with a 12-h light–dark cycle. Two to five million MDA-MB-231-luc-D3H2LN cells in 100 μl of a DPBS:Matrigel (50:50) solution were injected subcutaneously behind the fore limb of the 5-week-old mice using a 25-g needle. Tumours reached an average diameter of 6 mm before injection. All animal protocols were approved by University College Dublin's local Animal Research Ethics Committee and under the licence from the Department of Health and Children. Animals were split into two groups (n=4) and 2 dissolved in PBS (200 μl) was administered through the lateral tail vein at a concentration of 2 mg kg−1. Optical imaging was performed with an IVIS Spectrum small-animal in-vivo imaging system  with integrated isoflurane anaesthesia. A non-injected control animal was included. Images were acquired at regular intervals post injection of 2 with excitation 675 nm (30 nm band-pass filter) and emission 720 nm (20 nm band-pass filter) narrow band-pass filters and were analysed using Living Image Software v3.0 .MDA-MB-231-luc-D3H1, a luciferase-expressing human breast adenocarcinoma cell line, was obtained from Caliper Life Sciences. Cells were maintained as a monolayer culture in minimum essential medium containing 10% (v/v) FBS and supplemented with 1% (v/v) How to cite this article: Grossi, M. et al. Lysosome triggered near infra red fluorescence imaging of cellular trafficking processes in real time. Nat. Commun. 7:10855 doi: 10.1038/ncomms10855 (2016).Supplementary Figures 1-13 and Supplementary MethodsIn vitro photobleaching movies of 2.In vitro photobleaching movies of lysotracker red.In vitro photobleaching movies of pH-rhodo red.3D View of Fig. 5.Z-stack of Fig. 6.Z-stack movies of Fig. 7a.Z-stack movies of Fig. 7b.Z-stack movies of Fig. 7c.Movie of Fig. 8.Movie of Fig. 9.Movie of Fig. 10.Movie tracking all lysosomes within the cell shown in Fig. 10.


Tribolium castaneum (Herbst) is a major pest of stored grain-based products, and cause severe damage to cereal grains throughout the world. The present investigation was aimed to determine the pesticidal and pest repellent activities of 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against T. castaneum. The compound 2α,3β,21β,23,28-penta hydroxyl 12-oleanene is a triterpenoid which was isolated from the roots of Laportea crenulata Gaud. Surface film technique was used for pesticidal screening, whereas, pest repellency property of the triterpenoid was determined by filter paper disc method.2. No significant change in mortality records was observed when duration of exposure was increased up to 48 hours. The triterpenoid showed significant repellency activity at doses 0.47 and 0.94 mg/cm2.At 24 hours of exposure duration, significant mortality records (80% and 86%) were observed at doses 0.88 and 1.77 mg/cmT. castaneum and can be used in controlling the pest of grain-based products.These data suggest that the triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene possess both pesticidal and pest repellency activities against The online version of this article (doi:10.1186/0717-6287-47-68) contains supplementary material, which is available to authorized users. Tribolium castaneum (Herbst) is a primary pest of flour and other milled products of cereals, and a secondary pest of stored wheat, causing severe damages to these food grains by both quantity and quality [T. castaneum and have been used to control the pest [T. castaneum have become resistant to malathion. In recent years, there has been increasing information on the use of alternative methods [C. sinensis, C. aurantifolia, C. reticulata, C. limon) (Rutaceae) have been also reported as a source of botanical insecticides [The red flour beetle,  quality –4. A numthe pest . Howeverthe pest . Dyte anthe pest ) reporte methods , and pla methods –14. Diff methods , 15. Pla methods , 17. Cer methods –24. Somecticides . A variecticides , 28.Laportea crenulata Gaud. (Synonym Urtica crenulata Roxb.) is an evergreen shrub of Urticaceae family [Urticaceae are monoecious or dioecious herbs or infrequently shrubs or small trees comprising 45 genera and 700 species, often with specialized stinging hairs [L. crenulata locally known as Agnichutra, also distributed in India and Malay island [T. castaneum, the present study was aimed to determine the insecticidal and insect repellency activities of 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against T. castaneum.e family . The Urtng hairs . About 9ng hairs . In Bangy island . The rooy island . The rooy island . Such try island . In sear2, respectively  at dose 0.22 mg/cm2; both were not significant for pesticidal uses. The higher mortality records with higher doses of the compound suggesting its dose dependent pesticidal activity against T. castaneum. When exposure duration was increased 24 to 48 hours, the mortality records were minutely increased for doses 0.22, 0.88 and 1.77 mg/cm2, however, for 0.44 mg/cm2 dose no change in mortality was observed (Table 50) of the compound for 24 and 48 hours exposure duration were 0.38 and 0.34 mg/cm2, respectively , moderate pest repellency activity (53.2 and 66.6% repulsion) was observed for doses 0.47 and 0.94 mg/cmT. castaneum is a major paste of most grain, widely spread worldwide and very destructive [. Moharrammipour et al. [Ferula asafetida L. extract and the essential oil of Artemisia aucheri Boiss. had antifeedant property on T. castaneum adults.Insects/Pests are a problem in stored grain throughout the world, and cause serious losses in weight and quality of the stored products , 34. T. tructive , 16. As tructive . In manytructive . Moreovetructive , 6, 36.r et al.  and Shahr et al.  demonstrL. crenulata, has been shown both insecticidal and insect repellency properties against T. castaneum. The control of pest in stored food products can be achieved by insecticidal and insect repellency activities [2, whereas, sufficient pest repellency activity was observed at doses 0.47 and 0.94 mg/cm2. Identification of pesticidal compounds in plant is not new; Luo et al. [Tripterygium wilfordii Hook and confirmed activity against Mythimna separate. Pungitore et al. [Junellia aspera (Verbenaceae) and confirmed their toxic effect against T. castaneum. However, only a small number of pest control products directly obtained from plants [T. castaneum. However, further investigations are necessary to know toxicity of the compound to mammals as well as to know the effect on nutritional status of grain.Triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene was isolated from roots of tivities , 39. Exco et al.  isolatede et al.  identifim plants , 42. Botm plants . In the T. castaneum at doses 0.88 and 1.77 mg/cm2. In contrast, significant pest repellency against the pest was observed at doses 0.47 and 0.94 mg/cm2. In the light of the findings of present study, it can be stated that good pesticidal and pest repellency activities of 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against T. castaneum suggesting its suitability as botanical pesticide in controlling the pest of stored grains and grain products.The compound 2α,3β,21β,23,28-penta hydroxyl 12-oleanene has induced significant pesticidal activity against L. crenulata was collected from various part of Rangpur district of Bangladesh in the month of October to November. Taxonomically the plant was identified by Professor A. T. M. Naderuzzaman, Department of Botany, University of Rajshahi, Rajshahi, Bangladesh and a voucher specimen (No. 1239) had been deposited in the Department.The roots of The fresh roots was first washed with water to remove adhering dirt, cut into small pieces, sun dried for three days and finally dried at 45°C for 36 h in an electrical oven . The driin vacuo with an rotary evaporator at 40–50°C to afford a blackish green mass (45 g), which was further extracted with petroleum ether (3×50 mL), chloroform (3×50 mL), and methanol (3×50 mL) to afford petroleum ether (3 g), chloroform (7 g), and methanol (9 g) fractions, respectively [Dried powdered roots (900 g) of the plant were extracted (cold) with ethanol (5 L) in flat bottom glass containers, through occasional shaking and stirring for 10 days. The whole extract was filtered and the solvent was evaporated to dryness een mass  g, whichectively .1H-NMR, 13C-NMR, COSY, HSQC, HMBC, JMOD and NOESY the compound 1 was identified as a new triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene  was subjected to column chromatography using n-hexane, chloroform and methanol of increasing polarity. Column chromatography fractions eluting with 100% chloroform to 40% methanol in chloroform was subjected to preparative TLC (Silica gel PF254) with solvent system ethyl acetate: cyclohexane (2 : 1) to afford compound 1. Using mass spectroscopy (HR/ES-MS), IR, T. castaneum (Herbst) was originally collected from the Crop Protection Department of the University of Newcastle, U.K., and were maintained in the Crop Protection and Toxicology Laboratory of Department of Zoology, University of Rajshahi, Bangladesh. T. castaneum was reared in 1 L glass jar containing food medium, and a filter paper was placed inside each jar for easy movement of the pest. The jar was covered with a filter paper at the top, and kept in an incubator at 30 ± 0.5°C.The pest As food medium wheat flour and powdered brewers yeast in the ratio of 19 : 1 was used to culture the pest. Both flour and yeast were previously passed through a 250 micrometer aperture sieve and mixed thoroughly using an electric blender. The food was sterilized in an oven at 120°C for 6 h. Food was not used until at least 15 d after sterilization to allow its moisture content to equilibrate with that of environment.Screening of insecticidal activity was carried out by surface film method , 39, 47.2) were taken for each sample. Four petridishes were marked by 50 mg, 25 mg, 12.5 mg and 6.25 mg. One ml working solution was poured into the 50 mg petridish and agitated clockwise, anticlockwise, left to right and right to left to further confirm the uniform dispersion, then 1 ml solvent (50% chloroform + 50% methanol) was added to that vial from which 1 ml had been used and mixed uniformly. From this vial, 1 ml solution was poured into the 25 mg petridish and agitated similarly for uniform dispersion. Using this serial dilution technique, likewise sample was poured into 12.5 mg and 6.25 mg petridishes and agitated similarly for uniform dispersion. The above processes were continued two times further using two remaining vials of working solution and eight remaining petridishes. Then the layers of dispersed sample into the petridishes were air dried. Further, 1 ml solvent (50%chloroform + 50%methanol) was poured and dispersed into control petridish and air dried.Thirteen clean and dried petridishes  and Nine filter papers (size-90 mm) were taken for each sample. Three petridishes were marked by 30 mg, 15 mg and 7.5 mg. Three filter papers were taken for these three petridishes and each filter paper was cut (by scissors) into equal two parts through centre, where one part can be used as control part and other part can be used as treated part. For 30 mg petridish with its filter paper, treated part of filter paper was taken at outer background of the petridish and 1 ml working solution (prepared previously) was dispersed uniformly thorough out this part of filter paper and air dried. Then this part of filter paper was joined with its control part using transparent adhesive tape and placed into the 30 mg petridish using forceps. For 15 mg petridish with its filter paper, treated part of filter paper was taken at outer background of 15 mg petridish. One ml solvent (50% chloroform + 50% methanol) was added to that vial from which 1 ml had been used and mixed uniformly. From this vial, 1 ml solution was dispersed uniformly throughout the treated part of filter paper and air dried. Then this part of filter paper was joined with its control part using transparent adhesive tape and placed into the 15 mg petridish using forcep. Similar works was done for 7.5 mg petridish with its filter paper. The above processes were continued two times further using two remaining vials of working solution and six remaining petridishes and filter papers.Ten insects were applied at the center of each filter paper that present in the petridish. This process was continued for each petridish. Then the number of insects have repelled were counted per hour interval up to 5 h. The percentages of repellency were determined and results were analyzed through ANOVA after transforming them into arcsin percentage value.50 of the compound against the stored product insect T. castaneum. The repellency data were calculated for percent repellency, which was again transformed using arcsine transformation for the calculation of analysis of variances (ANOVA). Mean values were compared using one way ANOVA (two factors without replication)  to estimate LD to estimAdditional file 1: Table S1: Two factors ANOVA (without replication) for repellency records through Arcsin transformation data. (DOCX 16 KB)


Recovery from alopecia is rare in autoimmune polyglandular syndrome (APS). A 41-year-old male was admitted to our hospital with hyperglycemia. He developed alopecia areata (AA) 5 months before admission and developed thirst, polyuria, and anorexia in 2 weeks. His plasma glucose level upon admission was 912 mg/dl (50.63 mmol/l) and HbA1c was 13.7%. Although urinary and plasma C-peptide levels showed that insulin secretion was not depleted, anti-insulinoma-associated antigen 2 antibody was present. In addition, measurement of thyroid autoantibodies revealed the presence of Hashimoto's thyroiditis. These findings suggested a diagnosis of APS type 3. The patient has showed signs of improvement with the continuation of insulin therapy. During the successful control of diabetes, he had total hair regrowth within 2–3 months. Human leukocyte antigen typing showed that DRB1*1501-DQB1*0602 and DQB1*0301 were present. Similar cases should be accumulated to clarify the association of APS type 3 with recovery from AA.Alopecia in diabetic patients is a suspicious manifestation of autoimmune type 1 diabetes.Patients with autoimmune type 1 diabetes specifically manifesting alopecia should be further examined for diagnosis of APS.Insulin-mediated metabolic improvement may be a factor, but not the sole factor, determining a favorable outcome of alopecia in patients with autoimmune type 1 diabetes. Alopecia areata (AA) is a tissue-specific, T cell-mediated autoimmune disease of the hair follicles Herein, we present a case of APS type 3 with a severe form of AA (76–99% hair loss) that might not be predicted to show an improvement from hair loss A 41-year-old male was admitted to our hospital with hyperglycemia on 3rd February 2009. During a health check in 2007, his fasting plasma glucose was 115 mg/dl (6.38 mmol/l) and HbA1c was 6.3%, indicating impaired glucose tolerance (IGT). In September 2008, he abruptly developed AA and complained of upper abdominal pain. He visited another hospital where he was diagnosed with a duodenal ulcer. At this time, his random plasma glucose level was 163 mg/dl (9.05 mmol/l). On 10th December 2008, his random plasma glucose level was elevated to 303 mg/dl (16.70 mmol/l), but he did not receive any anti-diabetic medication. In January 2009, his alopecia gradually worsened and he rapidly developed thirst, polyuria, and anorexia at 2 weeks before admission. He was referred to our hospital on 3rd February 2009. His plasma glucose level upon admission was 912 mg/dl (50.63 mmol/l) and HbA1c was 14.1%. Physical examination on admission showed that his skin and tongue were dry due to dehydration. He showed ∼80% of scalp hair loss A and B, 4/mm3, hemoglobin of 16.1 g/dl, and hematocrit of 43.7%. The white blood cell count was 13 500/mm3 with 79% neutrophils, and his platelet count was 20.7×104/mm3. Serum electrolytes were as follows: Na 129 mEq/l (mmol/l); K 4.7 mEq/l (mmol/l); Cl 92 mEq/l (mmol/l); and Ca 8.4 mg/dl (2.10 mmol/l). Serum blood urea nitrogen was 12.6 mg/dl (4.75 mmol/l); creatinine, 0.73 mg/dl (64.5 μmol/l); and uric acid, 6.8 mg/dl (404.5 μmol/l). Total serum protein was 6.2 g/dl with 63.6% albumin; AST, 85 IU/l; ALT, 135 IU/l; LDH, 251 IU/l; γ-GTP, 97 IU/l; and total cholesterol, 204 mg/dl (5.28 mmol/l). Arterial blood gas analysis had a pH of 7.368; PaO2, 81.4 mmHg; PaCO2, 40.9 mmHg; HCO3, 23.0 mEq/l (mmol/l); and base excess of −2.1 mEq/l (mmol/l).His height was 175 cm and weight was 84.0 kg. His blood pressure was 126/80 mmHg. Laboratory findings revealed a red blood cell count of 509×10As shown in Intensive insulin therapy was initiated and plasma glucose levels were normalized in a couple of days. Since the initiation of treatment, the patient has improved following the continuation of insulin therapy. The HbA1c levels are controlled at 6.1–6.5% over 6–8 units/day insulin. He has not developed major diabetic complications such as neuropathy, nephropathy, or retinopathy over the course of disease. Anti-IA-2 antibodies are continuously positive but plasma C-peptide levels are maintained within the normal range, suggesting slowly progressive type 1 diabetes. During successful diabetic control, he had total hair regrowth within 2–3 months. Over the last 5 years, the patient has been in a good condition for both diabetes and AA C.APS is characterized by a combination of at least two endocrine organ failures mediated by autoimmune mechanisms. It is classified into four subtypes, namely APS types 1, 2, 3, and 4 et al. Type 1 diabetes is classified as fulminant, acute-onset, or slowly progressive, depending on the way of onset and progression et al. In this patient, total hair regrowth was observed within 2–3 months after the initiation of insulin therapy. Aw & Cheah et al. There are a number of reports showing that alopecia is a manifestation of APS types 2 and 3 His HLA typing showed that DRB1*1501-DQB1*0602, a protective haplotype for Japanese APS type 3 Written informed consent was obtained from the patient for publication of this case report.S Makino was the physician responsible for the patient and he reviewed and edited the manuscript. T Uchihashi, Y Kataoka, and M Fujiwara were the patient's physicians.


Aframomum plant species  against nine sensitive and MDR cancer cell lines. We have also identified the bioactive constituents of A. arundinaceum.The search for natural products as potential cytotoxic agents has yielded promising candidates. However multidrug resistance (MDR) is still a major hurdle for patients receiving chemotherapy. In the present study, we evaluated the cytotoxicity of the methanol extracts of four dietary A. arundinaceum.The cytotoxicity of the methanol extracts of the above plants was determined using a resazurin reduction assay. Chromatographic techniques were used to isolate the constituents of A. kayserianum and A. alboviolaceum as well as the isolated compounds namely compounds aframodial (1), 8(17),12-labdadien-15,16-dial (2), galanolactone (3), 1-p-menthene-3,6-diol (6) and 1,4-dimethoxybenzene (7) were less active, inducing more than 50% growth of this cell line contrary to A. polyanthum and A. arundinaceum extracts, galanals A (4) and B (5), naringenin (8) and kaempferol-3,7,4’-trimethylether (9). The IC50 values below or around 30 μg/mL were recorded with A. arundinaceum extract against eight of the nine tested cancer cell lines. This extract as well as compound 8 displayed IC50 values below 40 μg/mL towards the nine tested cancer cell lines whilst A. polyanthum extract, compounds 4, 5 and 9 showed selective activities. Collateral sensitivity (hypersensitivity) was observed with A. arundinaceum extract towards leukemia CEM/ADR5000 cells and glioblastoma U87MG.ΔEGFR compared to their respective sensitive counterparts CEM/CEM and U87MG.A preliminary experiment on leukemia CCRF-CEM cells at 40 μg/mL showed that the extracts from Aframomum species as well as a baseline information for the potential use of Aframomum arundinaceum in the fight against drug sensitive and otherwise drug-resistant cancers.The results of this study provide evidence of the cytotoxicity selected  Aframomum, belonging to the Zingiberaceae family have 40 species and is most common in tropical and subtropical regions [Aframomum arundinaceum K. Schum are used as laxative and as anti-helmintic. The fresh juice of the rhizomes is used against body odor. The rhizomes are used against toothache and the crushed seeds against fungal infections [Aframomum melegueta K. Schum together with the leaves of Momordica charantia and Sorghum arundinaceum cereal in local dry gin  is recommended to be taken one dose daily against cholera [Aframomum species such as Aframomum angustifolium, A. danielli, A. sanguineum, and A. sulcatum are also traditionally used to treat fevers in Africa [A. sceptrum and A. latifolium was demonstrated [Aframomum kayserianum[Aframomum polyanthum[Afromomum species such as A. citratum and A. melegueta towards leukemia CCRF-CEM and ADR5000 cell lines was also reported [Aframomum species commonly used as spices in Cameroon, Aframomum alboviolaceum (Ridl.) K. Schum, A. arundinaceum (Oliver & Hanbury) K. Schum, Aframomum kayserianum K. Schum and Aframomum polyanthum K. Schum towards sensitive and multi-factorial drug resistant cancer cell lines. The study was extended to the identification of the bioactive constituents of A. arundinaceum.Chemotherapy remains the major treatment of cancers but often fails due to cells multidrug resistance (MDR) , 2. MDR  regions . Twenty  regions . The Seefections . The dec cholera . Severaln Africa , and recnstrated . The antserianum and Afralyanthum were alsAframomum species, A. alboviolaceum, A. kayserianum and A. polyanthum were purchased from Bafoussam local market (West region of Cameroon) in January 2012. Aframomum arundinaceum was collected in Yaoundé (Centre region) in March 2012. The plants were further identified at the National Herbarium  where voucher specimens were deposited under the reference numbers 11704/SFR/CAM (A. arundinaceum), 34888/HNC , 18884/SRFC (A. kayserianum) and 3981/SRFK (A. polyanthum). The air dried fruits of A. kayserianum, A. polyanthum (100 g) and A. arundinaceum (3000 g) as well as the roots of A. alboviolaceum (100 g) were powdered and macerated with methanol for 48 h at room temperature. The methanol extract was concentrated in vacuo to give 18.7 g, 21.2 g, 25.3 and 100 g of the crude extracts of A. kayserianum, A. polyanthum, A. alboviolaceum and A. arundinaceum respectively. The extracts were then conserved at 4°C until further use.The tested A. arundinaceum (100 g) was successively extracted with petroleum ether, chloroform and methanol at room temperature. The petroleum ether fraction (25 g) was column chromatographed on 100 g of silica gel  using hexane and hexane-choloroform mixture with increasing polarity. Fractions of 300 mL were collected, concentrated, and pooled on the basis of their thin layer chromatography (TLC) profile. The obtained fractions (frs) directly afforded a yellow oil  and amorphous powders 2 , 3 , 4 (frs 30 to 33) and 5 . The chloroform extract (20 g) was also column chromatographed on 250 g of silica gel  using hexane (Hex) and mixture of hexane-chloroform (Hex-CHCl3). Fractions of 400 mL were collected, concentrated and pooled after TLC analysis to give five sub-fractions (sub-frs A-E).Crude extract of 3; 10 to 25; 6 g) was subjected to column chromatography (CC) to afford a white crystal . Sub-fraction C (8.0 g) obtained with Hexane-CHCl3 4:6 was subjected to CC  and eluted with Hex-CHCl3 mixtures of increasing polarity to give 6 new sub-fractions (C1-C6). Sub-fraction C4 obtained with Hex-CHCl3 6:4. afforded a yellow oil . Sub-fraction C5 (Hex-CHCl3 4:6) and C6 (Hex-CHCl3 8:2) were repeatedly filtered through Sephadex LH-20 (CHCl3-MeOH 7:3) to give yellow powders, 8  and 9 .Sub-fraction B (Hex-CHClF254 nm (Merck) was used for thin layer chromatography; the spots were visualized using both ultraviolet light (254 and 366 nm) and 50% H2SO4 spray reagent. NMR spectra were recorded on a Bruker Avance 300  at 300 MHz (1H) and 75 MHz (13C), with the residual solvent peaks as internal references. Mass spectra were recorded with API QSTAR pulsar mass . Melting points (m.p) were recorded using a Stuart Scientific  melting point apparatus (SMP1) and are uncorrected. The chemical structures of the compounds were confirmed by comparing with reference data from available literature  and dissolved in PBS  at a concentration of 10 mM. Geneticin >98% .BCRP clone 23) [p53+/+) colon cancer cells and its knockout clones HCT116 (p53-/-), the U87MG glioblastoma cells and its resistant subline U87MG.ΔEGFR, HepG2 hepatocarcinoma cells and AML12 normal hepatocytes [2 atmosphere at 37°C. Sensitive and resistant cells were kindly provided by Dr. Axel Sauerbrey . The generation of the resistant subline was previously described [BCRP (MDA-MB-231-BCRP clone 23), were maintained under standard conditions as described above for CCRF-CEM cells. Human wild-type HCT116 (p53+/+) colon cancer cells as well as knockout clones HCT116 (p53-/-) derived by homologous recombination were a generous gift from Dr. B. Vogelstein and H. Hermeking . Human glioblastoma multiforme U87MG cells (non-transduced) and U87MG cell line transduced with an expression vector harboring an epidermal growth factor receptor (EGFR) gene with a genomic deletion of exons 2 through 7 (U87MG.ΔEGFR) were kindly provided by Dr. W. K. Cavenee . MDA-MB-231-BCRP, U87MG.ΔEGFR and HCT116 (p53-/-) were maintained in DMEM medium containing 10% FBS (Invitrogen) and 1% penicillin (100 U/mL)-streptomycin (100 μg/mL) (Invitrogen) and were continuously treated with 800 ng/mL and 400 μg/mL geneticin, respectively. Human HepG2 hepatocellular carcinoma cells and normal AML12 heptocytes were obtained from the American Type Culture Collection . The above medium without geneticin was used to maintain MDA-MB-231, U87MG, HCT116 (p53+/+), HepG2 and AML12 cell lines. The cells were passaged twice weekly. All experiments were performed with cells in the logarithmic growth phase.The cell lines used the present work, their origins and their treatments were previously reported , 19. Theatocytes . The CCRescribed . Breast 4 cells were allowed to attach overnight and then treated with different studied samples. Samples were preliminary tested at 40 μg/mL (extract and isolated compounds) and doxorubicin (20 μg/mL) against the sensitive leukemia CCRF-CEM cell line and those inducing less than 50% growth proliferation were further tested for IC50 determinations towards all the studied cell lines. For suspension cells, aliquots of 2 × 104 cells per well were seeded in 96-well-plates in a final volume of 200 μL. Extracts and compounds were prior diluted in DMSO and tested in a final concentration below 0.1% . The tested concentrations ranges were 0.16 μg/mL to 40 μg/mL for crude extracts and isolated compounds and 0.08 μg/mL to 20 μg/mL for doxorubicin. After 72 h incubation and a resazurin  staining, fluorescence was measured on an Infinite M2000 Pro™ plate reader  using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each assay was done at least two times, with six replicates each. IC50 values represent the sample’s concentrations required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel [The cytotoxicity of the studied samples was performed by resazurin reduction assay as we previously described , 24–26. Aframomum arundinaceum were established using spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments, COSY, HMQC, HMBC, and direct comparison with published information and with authentic specimens obtained in our research group for some cases. The compounds isolated from the fruits of A. arundinaceum  [20H30O2  [20H30O3  [20H30O3  [20H30O3  [p-menthene-3,6-diol C10H18O2  [6H6O2  [15H12O5  [18H16O6  [1-9 as well as the crude extracts was determined towards drug sensitive and resistant cancer cell lines.The structures of the compounds isolated from m Figure  were idez 318.2) , 817),1,1Aframomz 318.2) , galanalz 318.2) , and galz 318.2) , 1-p-men5-248°C)  and kaem7-158°C) . The cytAframomum species and compounds isolated from A. arundinaceum, we tested a single concentration of 40 μg/mL for each sample and 20 μg/mL for doxorubicin against the sensitive CCRF-CEM leukemia cell line  and A. arundinaceum (24.68%) as well as compounds 4 (47.78%), 5 (49.81%), 8 (38.49%) and 9 (39.58%) displayed less than 50% growth proliferation of CCRF-CEM cells. The IC50 values of the above samples were further determined on nine cancer cell lines, including both sensitive and MDR phenotypes  than cancer cell lines, suggesting their good selectivity. Importantly, collateral sensitivity (hypersensitivity) was also observed with A. arundinaceum extract towards CEM/ADR5000 cells (degree of resistance of 0.76) and U87MG.ΔEGFR (degree of resistance of 0.95) compared to their respective sensitive counterparts CEM/CEM and U87MG. This extract was also more active against hepatocarcinoma HepG2 as compared to AML12 normal hepatocytes, confirming its selectivity to cancer cells  and BCRP-expressing U87MG.ΔEGFR cells were less cross-resistant towards the best samples namely A. arundinaceum and compound 8 than towards the positive drug, doxorubicin, highlighting their possible therapeutic potential in the fight against multidrug resistance. This report also highlights the importance of the plants of the genus Aframomum as potential source of cytotoxic compounds. The results obtained collaborate with previous investigations. In effect, Aframomum melegueta previously inhibited the proliferation of the leukemia ADR5000 cell lines with a reported IC50 value of 7.80 μg/mL [8) has shown cytotoxicity in various human cancer cell lines and induced apoptosis via a transient induction of caspase-3/CPP32 activity, in the human promyeloleukemia cell line HL-60 [4; IC50 of 18 μM or 5.62 μg/mL) and B  towards human T lymphoma Jurkat cells was also reported [In a preliminary investigation of the four studied  extract . We theror 10 μM , 36 afteotherapy –39. P-gpne HL-60 . The modreported .Figure 2Aframomum species and highlights the good activity of Aframomum arundinaceum on sensitive and drug-resistant cancer cell lines. Bioactive constituents of this plant include galanals A and B, naringenin and kaempferol-3,7,4’-trimethylether. Aframomum arundinaceum could be explored in more detail in the future to develop novel anticancer drugs against sensitive and resistant phenotypes.Finally, this work provides further evidence of the cytotoxic potential of


High altitude often comprises hypobaric hypoxia and cold ambient temperatures. However, research examining human performance during these stressors in combination is sparse . PreviouNine physically active males were exposed to four conditions in a balanced order. The conditions were control/normoxic thermoneutrality, hypoxic thermoneutrality, normoxic cold and hypoxic cold. Thermoneutral conditions were 23°C and cold conditions were 5°C. Hypoxic exposures were 13% oxygen (~4000 m). Subjects were dressed in shorts and socks. After a 40 minute rest period, participants carried out dynamic knee extension at a fixed intensity (35[6] W) until failure. After every 110 seconds of exercise, participants performed an isometric maximal voluntary contraction  with twitch interpolation to quantify voluntary and peripheral fatigue. To test data at each time point for significance, a two-way (2 × 2) repeated measures ANOVA was used.-1  (0.9) and 1.6%.min-1  (0.8) respectively). The combined effect of hypoxic-cold on peripheral fatigue rate was additive (7.6%.min-1 (1.1)) with no interaction (p = 0.525). Volitional  fatigue was unaffected by time (p = 0.327) or condition (p > 0.15) in this study.Rectal temperature was unaffected by condition (p > 0.3). Muscle (3 depth mean) and skin (7 point mean) temperature decreased by 3.8°C (0.4) and 5.4°C (0.1) in cold conditions, compared to 0.8°C (0.3) and 0.3°C (0.1) in neutral conditions (effect of temperature = p < 0.001). There was no effect of hypoxia on body temperature (effect of hypoxia = p > 0.2). Peripheral arterial oxygen saturation was significantly reduced to 85% (1) in hypoxia compared to 99% (1) in normoxia (effect of hypoxia = p < 0.001). In response to exercise, independent exposure to hypoxia and cold reduced time to task failure by 505 (105) seconds (p = 0.002) and 190 (73) seconds (p = 0.006) respectively, compared to 915 (122) seconds in control. During combined hypoxic cold, exercise time was reduced further (589 (110) seconds compared to control); however there was no significant interaction between stressors (p=0.198). The absolute reduction in time to task failure was not additive (e.g. 695-seconds); however the relative influence of hypoxia and cold were similar in the presence of the other stressor (-48% (6) and -51% (5) for hypoxia; -21% (7) and -20% (3) for cold), supporting an independent effect. The rate of increase in peripheral fatigue was also faster (p < 0.005) during independent exposure to hypoxia and cold compared to control (4.9%.minThe results indicate that when compared to exercise in thermoneutral or normoxic conditions, both cold and hypoxia induce significant reductions in time to task failure. Additionally, the time to failure is decreased further when these stressors are combined, and their relative influence is not interactive. The data suggest that the rate of peripheral fatigue development is the primary factor behind the additive effects of hypoxic-cold exposure on fatigue and task failure.


Trypanosoma cruzi, affecting millions of people worldwide. Because of severe side effects and variable efficacy, the current treatments for Chagas’ disease are unsatisfactory, making the search for new chemotherapeutic agents essential. Previous studies have reported various biological activities of naphthoquinones, such as the trypanocidal and antitumor activity of vitamin K3. The combination of this vitamin with vitamin C exerted better effects against various cancer cells than when used alone. These effects have been attributed to an increase in reactive oxygen species generation. In the present study, we evaluated the activity of vitamin K3 and vitamin C, alone and in combination, against T. cruzi. The vitamin K3 + vitamin C combination exerted synergistic effects against three forms of T. cruzi, leading to morphological, ultrastructural, and functional changes by producing reactive species, decreasing reduced thiol groups, altering the cell cycle, causing lipid peroxidation, and forming autophagic vacuoles. Our hypothesis is that the vitamin K3 + vitamin C combination induces oxidative imbalance in T. cruzi, probably started by a redox cycling process that leads to parasite cell death.Chagas’ disease is an infection that is caused by the protozoan  Trypanosoma cruzi, millions of people are still infected worldwide ) has anticancer effects [3 and ascorbic acid (vitamin C [VC]) has been shown to have tumor-specific antitumor effects against many cancer cells both in vitro and in vivo [Novel compounds, including natural and synthetic drugs and drug combinations, has been intensively studied in an attempt to find the most effective chemotherapies with better activity and fewer side effects –8. The l effects , 17. The in vivo –25. The  in vivo , 27, wit in vivo , 29.3 [T. cruzi parasites compared with mammals [3 combination has synergistic effects against all three forms of T. cruzi. Additionally, based on the morphological and ultrastructural alterations that were observed and the results of different probes and compounds that were used to evaluate the effects of VC + VK3 on T. cruzi, we hypothesized that this vitamin combination may trigger an initial processes that are related to increases in the generation of reactive species and reduction of reduced thiol levels, followed by irreversible oxidative imbalance that triggers alterations that are incompatible with T. cruzi survival.Considering the redox imbalance that is induced by VC + VK3 , 27 and  mammals , 31, we 3, 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), monodansylcadaverine (MDC), and wortmannin (WTM) were purchased from Sigma-Aldrich . Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Giemsa were obtained from Invitrogen . Propidium iodide/RNase A (PI-RNase A), diphenyl-1-pyrenylphosphine (DPPP), and 4-amino-5-methylamino-2’,7’-difluorofluorescein (DAF-FM) diacetate were obtained from Invitrogen . All of the other reagents were of analytical grade.VC, VKT. cruzi [2 cells  in DMEM supplemented with 2 mM L-glutamine and 10% FBS, buffered with sodium bicarbonate in a 5% CO2 air mixture at 37°C.All of the experiments were performed using the Y strain of T. cruzi . EpimastT. cruzi . Trypoma3 + VC combination on epimastigotes, trypomastigotes, and amastigotes, we applied the Combination Index method as described by Chou and Talalay [et al. [To evaluate the effects of the VK Talalay  and revi [et al. . The exp6 parasites/ml) in the exponential growth phase were resuspended in LIT medium supplemented with 10% FBS. The vitamins were added to the cell suspension, alone or in combination (1.0–9.0 μM VK3 and 0.35–2.84 mM VC), in 24-well plates and incubated at 28°C. The number of epimastigote forms was determined by counting in a Neubauer hemocytometer after 96 h.Epimastigote forms (1 × 102 cells. Trypomastigote forms (1 × 107 parasites/ml) were resuspended in the presence of DMEM supplemented with 10% FBS and different concentrations of both vitamins, alone or in combination (0.14–4.65 μM VK3 and 0.09–1.42 mM VC), in 96-well plates. Parasites were incubated for 24 h at 37°C in a 5% CO2 atmosphere. After incubation, the viability of the parasites was determined by examining mobility under a light microscope (Olympus CX31) using the Pizzi-Brener method [To evaluate activity against trypomastigote forms, parasites were obtained from the supernatant of infected LLCMKr method .2 cells (2.5 × 105 cells/ml) were harvested, resuspended in DMEM supplemented with 10% FBS, and plated in 24-well plates that contained round glass coverslips. When confluent growth was achieved, the cells were infected with trypomastigotes (1 × 107 parasites/ml) that were obtained from preinfected cultures. After 24 h, the medium that contained the parasites was removed. The cells were then washed in phosphate-buffered saline (PBS), and DMEM with different concentrations of both vitamins, alone or in combination (0.29–4.65 μM VK3 and 0.18–2.84 mM VC), was added. The cells were maintained for 96 h at 37°C in a 5% CO2 atmosphere. Afterward, the glass coverslips were subjected to fixation with methanol and Giemsa staining and permanently prepared with Entellan . The number of infected cells and amastigotes was determined by randomly counting 200 cells. The results were calculated as the survival index, which was obtained by multiplying the percentage of infected cells by the number of amastigotes per infected LLCMK2 cell and then determining the percentage of inhibition. The treated groups were compared with the untreated control, the survival index observed in the control without treatment was considered 100%.To evaluate activity against intracellular amastigote forms, LLCMK50 VK3 combined / IC50 VK3 alone) + CI =  + CI =  were treated with 1.90 μM VK3 and 0.61 mM VC, alone or in combination, for 72 h at 28°C. Trypomastigote forms (1 × 107 parasites/ml) were treated with 0.35 μM VK3 and 0.20 mM VC, alone or in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Intracellular amastigotes were treated with 0.30 μM VK3 and 0.18 mM VC, alone or in combination, for 24 h at 37°C in a 5% CO2 atmosphere. After incubation, the parasites were harvested, washed twice in PBS, and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C. The parasites were then placed on a glass support that was covered with poly-L-lysine, dehydrated in an ascending series of ethanol, critical-point dried with CO2, coated with gold, and observed in a Shimadzu SS-550 scanning electron microscope. For intracellular amastigotes, we used the fracture tape method.For scanning electron microscopy (SEM), epimastigote forms (1 × 106 parasites/ml) were treated with 1.90 μM VK3 and 0.61 mM VC, alone or in combination, for 72 h at 28°C. Trypomastigote forms (1 × 107 parasites/ml) were treated with 0.35 μM VK3 and 0.20 mM VC, alone or in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Intracellular amastigotes were treated with 0.30 μM VK3 and 0.18 mM VC, alone or in combination, for 24 h at 37°C in a 5% CO2 atmosphere. After incubation, the parasites were harvested, washed twice in PBS, fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C, and postfixed in a solution of 1% OsO4, 0.8% potassium ferrocyanide, and 10.0 mM CaCl2 in 0.10 M cacodylate buffer. The samples were then dehydrated in an increasing acetone gradient and embedded in Polybed 812 resin. Ultrathin sections were then obtained, stained with uranyl acetate and lead citrate, and observed in a JEOL JM 1400 transmission electron microscope.For transmission electron microscopy (TEM), epimastigote forms (1 × 103 and VC using the probe H2DCFDA. Epimastigote forms (1 × 106 parasites/ml) were evaluated after exposure to 1.90 μM VK3 and 0.61 mM VC, alone and in combination, for 24 h at 28°C. Trypomastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.35 μM VK3 and 0.20 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Amastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.30 μM VK3 and 0.18 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Hydrogen peroxide  was used as a positive control. Afterward, the parasites were centrifuged, washed, and resuspended in PBS. Parasites were loaded with 10.0 μM of the permeant probe H2DCFDA in the dark for 45 min. Total ROS were measured as an increase in fluorescence that is caused by the conversion of nonfluorescent dye to highly fluorescent 2’,7’-dichlorofluorescein (DCF) in a fluorescence microplate reader  at λexcitation = 488 nm and λemission = 530 nm.The production of total reactive oxygen species  was evaluated in parasitic forms after exposure to VK3 and VC using the probe DAF-FM diacetate. Epimastigote forms (1 × 106 parasites/ml) were evaluated after exposure to 1.90 μM VK3 and 0.61 mM VC, alone and in combination, for 24 h at 28°C. Trypomastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.35 μM VK3 and 0.20 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Amastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.30 μM VK3 and 0.18 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Afterward, the parasites were centrifuged, washed, and resuspended in PBS. The parasites were then loaded with 1.0 μM of the probe DAF-FM diacetate in the dark for 30 min at 37°C. Afterward, the parasites were washed and resuspended in PBS and incubated for an additional 15 min. DAF-FM diacetate is cell-permeant that is deacetylated inside cells to become DAF-FM. This compound in the presence of NO is converted to form fluorescent benzotriazole, which was detected in a fluorescence microplate reader  at λexcitation = 495 nm and λemission = 515 nm.The production of nitric oxide (NO) was evaluated in parasitic forms after exposure to VK3 and VC. Epimastigote forms (1 × 106 parasites/ml) were evaluated after exposure to 1.90 μM VK3 and 0.61 mM VC, alone and in combination, for 24 h at 28°C. Trypomastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.35 μM VK3 and 0.20 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Amastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.30 μM VK3 and 0.18 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Afterward, the parasites were centrifuged. Tris-HCl buffer  was then added and the cells were sonicated. Acidic pH was used during sonication to prevent oxidation of the free thiol groups. Cellular debris was removed by centrifugation, and 100 μl of the supernatant and 100 μl of 500.0 mM phosphate buffer (pH 7.5) were taken from each well, followed by the addition of 20 μl of 1.0 mM DTNB. Free thiol levels were determined using DTNB. Absorbance was measured at 412 nm [Trypanothione reductase (TR) activity plays an important role in the antioxidant activity of trypanosomatids. Its depletion decreases reduced thiol level . Reduced3 and VC. The extent of lipid peroxidation was evaluated by DPPP, which is essentially nonfluorescent until it is oxidized to a phosphine oxide (DPPP-oxide) by peroxides. Epimastigote forms (1 × 106 parasites/ml) were evaluated after exposure to 1.90 μM VK3 and 0.61 mM VC, alone and in combination, for 24 h at 28°C. Trypomastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.35 μM VK3 and 0.20 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Amastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.30 μM VK3 and 0.18 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. After incubation, the parasites were centrifuged, washed, and resuspended in PBS. The parasites were loaded with 50.0 μM DPPP in the dark for 15 min. The direct fluorometric detection was measured as an increase in the fluorescence of the DPPP oxide in a fluorescence microplate reader  at λexcitation = 355 nm and λemission = 460 nm.Lipid peroxidation was evaluated in parasitic forms after exposure to VK3 and 0.61 mM VC, alone and in combination, for 24 h at 28°C. Trypomastigote forms (14 mg/ml) were evaluated after exposure to 0.35 μM VK3 and 0.20 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Amastigote forms (14 mg/ml) were evaluated after exposure to 0.30 μM VK3 and 0.18 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. After incubation, the samples (0.50 mg protein) were heated in a solution that contained 0.37% thiobarbituric acid, 15.0% trichloroacetic acid, and 0.25 N HCl for 45 min at 90–95°C. After cooling, absorbance was read at 532 nm, and the TBARS concentration was calculated based on an ε value of 153,000 M-1cm-1 [We also determined the amount of thiobarbituric acid-reactive substances (TBARS) in terms of malondialdehyde (MDA) levels. Epimastigote forms (14 mg/ml) were evaluated after exposure to 1.90 μM VK M-1cm-1 .T. cruzi after exposure to VK3 and VC. Epimastigote forms (1 × 106 parasites/ml) were evaluated after exposure to 1.90 μM VK3 and 0.61 mM VC, alone and in combination, for 24 h at 28°C. After incubation, the cells were fixed in 70% cold methanol at 4°C for 1 h. Afterward, the parasites were washed in PBS, and 10 μl of PI-RNase A was added, followed by incubation at 37°C for 45 min. Data acquisition and analysis were performed using a FACSCalibur flow cytometer equipped with CellQuest software. A total of 10,000 events were acquired in the region that corresponded to the parasites. The percentages of cells in each stage of the cell cycle were determined.The cell cycle was evaluated in epimastigote forms of 3 and VC using MDC labeling, a fluorescent probe that accumulates in autophagic vacuoles [6 parasites/ml) were evaluated after exposure to 1.90 μM VK3 and 0.61 mM VC, alone and in combination, for 24 h at 28°C. Trypomastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.35 μM VK3 and 0.20 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. Amastigote forms (1 × 107 parasites/ml) were evaluated after exposure to 0.30 μM VK3 and 0.18 mM VC, alone and in combination, for 24 h at 37°C in a 5% CO2 atmosphere. The cells were then incubated with 0.05 mM MDC in PBS for 1 h at 37°C. After incubation, the cells were washed twice in PBS. MDC staining was analyzed using an Olympus BX51 fluorescence microscope, images were captured using a UC30 camera, and fluorescence intensity was evaluated by ImageJ 1.44o. In some of the experiments, the parasites were pretreated with 500.0 nM WTM before the induction of autophagy [Autophagic vacuoles were evaluated in parasitic forms after exposure to VKvacuoles . Epimastutophagy . This coutophagy .post hoc tests, respectively. Values of p ≤ 0.05 were considered statistically significant. The statistical analyses were performed using GraphPad software.All of the quantitative experiments were performed at least three times on independent occasions. Data were evaluated using one- or two-way analysis of variance (ANOVA) with significant differences among means identified by Tukey and Bonferroni 3 and VC, alone and in combination, on the growth of epimastigotes and intracellular amastigotes and viability of trypomastigotes using in vitro assays. We found that this vitamin combination had dose-dependent and robust synergistic effects on the three forms of T. cruzi  caused increases in total ROS production of 67.0%, 108.0%, and 65.0%, respectively, compared with the control group (2O2) also increased ROS production in epimastigotes  of cells in the sub-G0/G1 phase  compared with 13.2% in the control group and a significant reduction of the percentage (13.5%) of cells in the G2/M phase (DNA duplication) compared with 36.4% in the control group  was developed as a new therapeutic strategy for cancer treatment [3 + VC combination has low systemic toxicity [12 combination against epimastigote forms of T. cruzi, in which the presence of VC reduced the IC50 of vitamin B12 [3 against bloodstream trypomastigotes [Several studies have demonstrated the potential of VKer cells –25, whicer cells . It is wenerated . Thus, Areatment , 43. Pretoxicity , 44 and toxicity , 46. Oneamin B12 . Other samin B12  and the stigotes .T. cruzi parasites compared with mammals [3 + VC has activity against the epimastigote, trypomastigote, and amastigote forms of T. cruzi. The most important finding of the present study was the synergistic effects of this vitamin combination against T. cruzi. We also showed the likely mechanism by which these vitamins exert their trypanocidal effects.Considering the different antioxidant capacity of  mammals , 31, the3 + VC combination induced several alterations in T. cruzi parasites, but the most characteristic lesion was swelling in the mitochondrion-kinetoplast region. Previous studies reported the potent effects of naphthoquinones and its derivatives on mitochondrial function [3 + VC combination induced oxidative imbalance, demonstrated by two findings: increase in reactive species production and a decrease in reduced thiol levels. This vitamin combination is well known to induce a redox cycling process [T. cruzi, followed by the depletion of key antioxidant enzymes, such as TR, reflected herein by the decrease in reduced thiol levels. Thus, we believe that the increase in ROS formation via redox cycling might be the main effector mechanism of VK3 + VC that interferes with maintaining redox homeostasis and mediates T. cruzi death. T. cruzi also has the ability to synthesize and capture VC [3 is able to cross the membrane of the parasite. Based on this, ROS production could occur not only outside the parasite but also inside the parasite, which could explain the high efficacy of this vitamin combination. Other notable characteristics of T. cruzi are its single mitochondrion [T. cruzi thiol-redox system [By SEM and TEM we showed that the VKfunction , 51. Impfunction , 53. We  process , 27. Addpture VC , and VK3hondrion  and diffhondrion , 31, 56,hondrion . Studiesx system . Interesx system . Howeverx system , 60.3 + VC induced the lipid peroxidation and arrested the cell cycle of T. cruzi. Alterations in the composition of certain lipids may impair the cell cycle of parasites [3 [3 + VC [3 + VC combination also induced the formation of autophagic vacuoles. An increase in reactive species might be a critical event that triggers lipid oxidation, and this alteration may activate cell autophagy machinery [3 + VC combination against T. cruzi described herein appear to be part of a cascade of events that is triggered by redox imbalance, leading to the loss of homeostasis and culminating in parasite death.Additionally, VKarasites . The celsites [3  and VK3  [3 + VC . The VK3achinery . All of 3 + VC on the three forms of T. cruzi. These effects were marked by morphological, ultrastructural, and functional changes in this parasite. The data clearly indicate the potential therapeutic utility of this combination for the treatment of Chagas’ disease and open the way for further studies of Apatone® and other naphthoquinones combined with VC.In summary, the present study demonstrated the synergic trypanocidal effect of VKS1 Table(PDF)Click here for additional data file.


Thalassospira australica” NP3b2T, a potential poly (PET) plastic biodegrader. This genomic information will enhance information on the genetic basis of metabolic pathways for the degradation of PET plastic.Here, we present the draft genome of “ Plastic pollution in marine ecosystems is a growing environmental concern. Accumulation of large plastic “islands” in ocean gyres demonstrates the abundance of plastic waste in marine environments , 2. PlasThalassospira more than 20 years ago  (PET) from seawater collected from the first meter below the water’s surface at St. Kilda Beach, Port Phillip Bay, Tasman Sea, Victoria, Australia . The genome of strain NP3b2T was sequenced using the IlluminaHiSeq 2000 (100-bp paired-end read) sequencing platform . The generated reads were trimmed and assembled de novo using VELVET, version 0.7.63 (http://www.migap.org/index.php/en/) , 5 rRNAs, and 55 tRNAs.On the basis of the data generated from n 0.7.63 . The resphp/en/)  and NCBIphp/en/) , RNAmmerphp/en/) , and tRNT and the four strains already published, Thalassospira profundimaris WP0211   and the s WP0211 , T. xiamnsis M-5 , T. permC 106175 , and T. is QMT2T , were 82is QMT2T , respectJRJE00000000 for “Thalassospira australica” NP3b2T.The genome data have been deposited at NCBI under BioProject number PRJNA257045 and accession number


The South East Regional Fire Service requested an investigation into the effect of different undergarments worn by fire service instructors, to help improve thermoregulation and reduce the strain experienced. Literature suggests that wearing shorts and t-shirt may reduce heat strain , whilst Eight males  were monitored during three 45 mins sessions in a heat chamber (49.5 ± 1.4 °C and 16.9 ± 4.3 % rh) whilst performing intermittent exercise [5 mins walking  and 5 mins rest]. Participants wore fire service kit and a rucksack to replicate a breathing apparatus, weighing 17 kg in total. Physiological and perceptual measures were recorded every 5 min. Venous blood samples were collected before and after heat exposure for analysis of interleukin (IL)-6.re), volume of oxygen uptake (VO2), physiological strain index (PSI) and IL-6, p < 0.05. IL-6 was significantly decreased for COMPRESSION (6.45 ± 0.43 pg.mL¯¹) and SHORTS (6.59 ± 0.30 pg.mL¯¹) compared to BOILER (6.96 ± 0.28 pg.mL¯¹), p < 0.05. Significant differences were also present between garment types at 45min for PSI and ΔTre, with trends suggesting COMPRESSION caused the lowest levels of strain  compared to SHORTS  and BOILER , p < 0.05. Thermal sensation (TSS) trends suggest that COMPRESSION (7.13 ± 0.52) generated less perceptual stress in comparison to SHORTS (7.43 ± 0.45) and BOILER (7.75 ± 0.27), p > 0.05.Two way repeated measures ANOVA's were conducted, and revealed significant interactions for change in heart rate, change in rectal temperature (ΔTPrevious studies have noted no thermoregulatory improvement whilst wearing COMPRESSION in sporting situations,3. HowevIn comparison to standard issue boiler suits or shorts and t-shirt, wearing compression garments underneath protective clothing, during fire-fighting operations, significantly improves thermoregulation, reducing physiological strain and inflammation. Undergarment selection has a less pronounced effect on perceptions of stress; however differences may be meaningful to fire service instructors.


AbstractDiopatramarocensis in the eastern Mediterranean. This species is small-sized and inhabited muddy bottom near the opening of rivers or lagoons [salinity range: 33−39‰] in the Aegean and Levantine Seas, and reached a maximum density of 90 ind.m-2 in Mersin Bay. This species might be an alien species that was introduced from the East Atlantic  to the eastern Mediterranean via ballast water of ships, as it has never been reported from the western Mediterranean Sea.The present study deals with the presence of  PageBreakDiopatra Audouin & Milne-Edwards, 1833 is represented by 59 species world-wide  in the Mediterranean Sea  and has been overexploited by diggers for the fish bait trade  that were first identified as Diopatra sp.  and at 4 stations in the Aegean Sea  between 2005 and 2012, using a Van Veen grab (sampling an area of 0.1 m-2) and a dredge (stations D13 and D14) , width (excluding parapodia), H+10 (length comprising the head and the first 10 chaetigers) and the length of chaetae were measured by using an ocular micrometer. Drawings were made with the aid of a camera lucida. Specimens were deposited in the Museum of Faculty of Fisheries, Ege University (ESFM).Specimens of PageBreakTaxon classificationAnimaliaEunicidaOnuphidaePaxton, Fadlaoui & Lechapt, 1995Diopatramarocensis : 36°43'33"N, 34°52'11"E, 9 m, mud; ESFM−POL/2005−1511, 1 September 2005, Kale, K41, near Beymelek Lagoon [salinity: 39‰], 36°14'29"N, 30°01'33"E, 5 m, gravelly sand, 1 specimen; ESFM−POL/2005−107, 10 September 2005, D13, near the opening of Ceyhan River [salinity: 39‰], 36°33'22"N, 35°34'17"E, 10 m, muddy sand, 8 specimens; ESFM−POL/05−295, 10 September 2005, İskenderun Bay, D14, near the opening of Ceyhan River [salinity: 38‰], 36°32'51"N, 35°34'37"E, 25 m, 3 specimens; ESFM−POL/2005−1396, 17 September 2005, Mersin Bay, G11, near the opening of Seyhan River [salinity: 38‰], 36°45'47"N, 34°51'54"E, 5 m, mud, 14 specimens; ESFM−POL/2005−2086, 8 October 2005, Kuşadası [salinity: 39‰], G40, 37°52'00"N, 27°15'27"E, 10 m, sandy mud, 3 specimens; ESFM−POL/2009−34, 4 February 2009, Mersin Bay, station 34, near the opening of Seyhan River [salinity: 38‰], 36°43'33"N, 34°52'11"E, 9 m, mud, 2 specimens; ESFM−POL/2009−300, PageBreak29 April 2009, Mersin Bay, station 34, near the opening of Seyhan River [salinity: 34‰], 36°43'33"N, 34°52'11"E, 9 m, mud, 4 specimens; ESFM−POL/2009−281, 20 October 2009, Mersin Bay, station 34, near the opening of Seyhan River [salinity: 38‰], 36°43'33"N, 34°52'11"E, 10 m, mud, 2 specimens; ESFM−POL/2011−48, 23 August 2011, Enez, near the opening of Meriç River [salinity: 33‰], station 3, PageBreak40°43'41.5"N, 26°02'03.1"E, 4 m, sandy mud, 1 specimen; ESFM−POL/2011−256, 28 August 2011, near the opening of Bakırçay River [salinity: 39‰], station 15, 38°55'11"N, 26°58'50"E, 4 m, sandy mud, 1 specimen; ESFM−POL/2011−255, 1 September 2011, near the opening of Büyük Menderes River [salinity: 39‰], station 37, 37°32'39"N, 27°10'28"E, 5 m, sandy mud, 2 specimens (juvenile); ESFM−POL/2011−254, 10 September 2011, Mersin Bay, near the opening of Seyhan River, station 71 [salinity: 37‰], 36°47'00"N, 34°38'06"E, 6 m, sandy mud, 1 specimen; ESFM−POL/2012−1, 17 June 2012, Iskenderun Bay, Yumurtalık [salinity: 39‰], station 11, 36°50'27"N, 35°54'32"E, 12 m, mud, 6 specimens.ESFM−POL/2009−196, 29.04.2009, Mersin Bay, near the opening of Seyhan River [salinity: 34‰], station 34, All specimens incomplete, except for juvenile specimens from stations G40 and 37, and a mature specimen from station 71 (ESFM−POL/2011−254); having 27 mm body length, 1.3 mm body width (chaetiger 5) and 102 chaetigers. Large specimen incomplete, 35 mm long (H+10 = 5 mm), 2.1 mm wide, with 83 chaetigers. Body somewhat semicircular, anterio-ventral side flat in most specimens; a ventral groove in some highly contracted specimens. Anterior end including first 2 chaetigers larger than following chaetigers Figures , 5A. AftAntennae and palps covering dorsum of prostomium, emerging near posterior part of prostomium. One pair of pyriform, subulate or digitiform frontal lips on anterior part of prostomium; large specimen having an anomaly; with three frontal lips; on left side of prostomium, two lips attached with each other at base present. Upper lips, massive, medially distinctly separated; with a distinct, elongated papilla between lips ; each lip cushion shaped, with large, bulbous distal papilla Figure , 5B. MouPageBreakPageBreakrio-dorsal side of peristomium; digitiform, longer than peristomium, extending to posterior part of prostomium ; decreasing gradually in number of whorls and in size after chaetiger 12 ; having 2 filaments after chaetiger 35, one filament after chaetiger 37, becoming a short papilla between chaetiger 40 and 46, absent posteriorly.Mandibles partly sclerotinized  with calcareous distal cutting figs, rounded tip with distal indentations. Maxillae distinctly sclerotinized along cutting edges; supporting structures barely sclerotinized. Maxillary formula: Mx I = 1 + 1; Mx II = 8 + 7; Mx III = 7 + 0; Mx IV = 7 + 9; Mx V = 1 + 1.PageBreakPygidium with two pairs of pygidial cirri; ventral pair (0.5 mm) longer than dorsal pair (0.2 mm).Abraalba  and Parvicardiumexiguum , and plant debris drifted from rivers.Tube parchment-like, cylindrical, surface covered by debris comprising mud with shell fragments of -2).This species was found near the opening of rivers or lagoons [salinity range: 33−39‰] in the area between 4 and 25 m depth, and attained its highest density at station 34  in the parental tube .PageBreakDiopatramarocensis collected from stations D14 and 34 were densely infected by a parasite (?Entoprocta). The parasite is attached to the dorsal side, parapodia and branchiae of the worms. One specimen (ESFM−POL/2009−34) with 15 chaetigerous segments (posterior part is missing) had almost 40 parasites. Diopatramarocensis and a peritricous protozoan (the genus Epistylis) from northern Spain.Specimens of PageBreakDiopatraneapolitana. These explanations can be refuted or proved when more data regarding this species in different basins of the Mediterranean are accumulated. As there is a big gap between the Atlantic and Mediterranean populations of this species, this species can be regarded as a new alien species for the Mediterranean Sea, at least for now.This species is only known from the East Atlantic  and the eastern Mediterranean. The presence of this species in the eastern Mediterranean and absence in the western Mediterranean is interesting. There could be two reasonable explanations for its presence in the eastern Mediterranean; 1) this species might have been introduced to the area by ballast water of ships; 2) it might be an Atlanto-Mediterranean species that widely occurs near openings of river mouths in the area, but has been overlooked up to now, or misidentified as a juvenile specimen of the large Mediterranean species Diopatramarocensis can be distinguished from other Diopatra species  reported from the north-eastern coast of the Atlantic Ocean and Mediterranean Sea, in having pectinate chaetae that have oblique combs with 16−18 thin teeth. The original description of Diopatramarocensis differs from the re-description of the species based on the eastern Mediterranean specimens in having different pigmentation in the anterior end  and branchiae with fewer numbers of whorls . However, such characters of the eastern Mediterranean specimens were also noted on the Spanish specimens .