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Background: This study delves into the understudied yet potentially crucial role of paternal zinc deficiency in programming offspring metabolic outcomes. By examining paternal zinc deficiency, we aim to shed light on a previously unexplored avenue with the potential to significantly impact future generations. We investigated the intergenerational effects of paternal zinc deficiency on metabolic parameters in Drosophila melanogaster., Methods: Dietary zinc deficiency was induced by supplementing the diet of Drosophila F0 male flies with TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine) from egg stage. The F0 male flies after eclosion were mated with age-matched virgin female flies from the control group, resulting in the F1 offspring generation. The F1 generation were then cultured on a standard diet for subsequent metabolic analyses, including assessments of body weight, locomotion, and levels of glucose, trehalose, glycogen, and triglycerides as well as the expression of related genes., Results: We observed an increase (p<0.05) in body weight in male parent flies and female offspring. Negative geotaxis performance was also impaired in the female offspring. Paternal zinc deficiency exerted distinct effects on carbohydrate and lipid metabolism, as evidenced by a significant (p<0.05) increase in trehalose and triglyceride levels in both parent and offspring. Additionally, zinc deficiency led to alterations in the expression of key metabolic genes, including significant (p<0.05) increase in DILP2 mRNA levels, highlighting potential links to insulin signaling. Also, there were reduced mRNA levels of SOD1 and CAT in both parental and offspring generations. Parental zinc deficiency also increased the expression of Eiger and UPD2 mRNA in the offspring, suggesting potential perturbations in the immune response system., Conclusion: These findings underscore the link between zinc status and various physiological and molecular processes, revealing both immediate and intergenerational impacts on metabolic, antioxidant, and inflammatory pathways and providing valuable insights on the implications of paternal zinc deficiency in Drosophila melanogaster., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The neurobehavioral assessment of N,N-bis(2-mercapatoethly)-N',N'-diethylenediamine (BMEDA), which can form a chelate with rhenium-188 ( 188 Re) to produce the 188 Re-BMEDA-liposome, was evaluated. The purpose of this study was to evaluate the potential neurobehavioral changes by using the functional observational battery observation procedures when intravenous injection of BMEDA to Sprague-Dawley rats. Rats were administered BMEDA at dose levels of 1, 2, and 5 mg/kg. No mortalities were observed. There are some observations related to BMEDA treatment found in the 5 mg/kg dose group at 10 min post-dose. Tremor was observed in one male rat and seven female rats. The increased respiration, vocalization, not easy to handle and/or loss of tone in the limb were observed in both males and females, and increased body temperature was observed in male animals. Based on the results, a single intravenous dose of BMEDA administered to rats resulted in increased respiration, vocalization, not easy to handle and/or loss of tone in the limb increasing at the dose level of 5 mg/kg. No neurobehavioral effects were noted after BMEDA administration up to the dose level of 2 mg/kg. The information of this study will provides a point of reference to design appropriately therapeutic studies for future human use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The need for a systematic approach in developing new metal-based drugs with dual anticancer-antimicrobial properties is emphasized by the vulnerability of cancer patients to bacterial infections. In this context, a novel organometallic assembly was designed, featuring ruthenium(II) coordination with p-cymene, one chlorido ligand, and a bidentate neutral Schiff base derived from 4-methoxybenzaldehyde and N,N-dimethylethylenediamine. The compound was extensively characterized in both solid-state and solution, employing single crystal X-ray diffraction, nuclear magnetic resonance, infrared, ultraviolet-visible spectroscopy, and density functional theory, alongside Hirshfeld surface analysis. The hydrolysis kinetic was thoroughly investigated, revealing the important role of the chloro-aqua equilibrium in the dynamics of binding with deoxyribonucleic acid and bovine serum albumin. Notably, the aqua species exhibited a pronounced affinity for deoxyribonucleic acid, engaging through electrostatic and hydrogen bonding interactions, while the chloro species demonstrated groove-binding properties. Interaction with albumin revealed distinct binding mechanisms. The aqua species displayed covalent binding, contrasting with the ligand-like van der Waals interactions and hydrogen bonding observed with the chloro specie. Molecular docking studies highlighted site-specific interactions with biomolecular targets. Remarkably, the compound exhibited wide spectrum moderate antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, coupled with low micromolar cytotoxic activity against human colorectal adenocarcinoma cells and significant activity against human leukemic monocyte lymphoma cells. The presented findings encourage further development of this compound, promising avenues for its evolution into a versatile therapeutic agent targeting both infectious diseases and cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present study aims to obtain manganese ferrite nanoparticles functionalized with chitosan (C-MNP) or ethylenediamine (E-MNP) by coprecipitation and polyol one-step methods, characterize their interaction with S. griseus demonstrating cell immobilization, and evaluate the antimicrobial activity of the free cell extracts obtained from immobilized S. griseus fermentation in the presence of different concentrations of MNP. The adsorption isotherms were analyzed mathematically using Langmuir and Freundlich models. The highest coefficient of determination (R 2 ) for the S. griseus cell adsorption isotherm with C-MNP was observed with a linear function of the Langmuir model. The adsorption isotherm of S. griseus cells with E-MNP was better fitted to the Freundlich model. Cell immobilization by adsorption on magnetic nanoparticles was demonstrated in both cases. Different concentrations of C-MNP and E-MNP were used in fermentations to prepare cell-free extracts with antifungal activity. The best results were obtained with E-MNP, with a 91.5% inhibition of radial fungal growth. Magnetic nanoparticles offer potential applications in different fields and easy biomass separation., Competing Interests: Declarations. Ethical Approval: It is an observational study. The Universidad Autonoma de Coahuila Ethics Committee has confirmed that no ethical approval is required to develop the present study. Consent to Participate: Informed consent was not strictly required to develop the present research, as neither humans nor animals were involved. Consent for Publication: Consent to publish was unnecessary for developing the present research work as neither humans nor animals were involved. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Iron(III) complexes based on N,N´-bis(salicylidene)ethylenediamine (salene) scaffolds have demonstrated promising anticancer features like induction of ferroptosis, an iron dependent cell death. Since poor cellular uptake limits their therapeutical potential, this study aimed to enhance the lipophilic character of chlorido[N,N'-bis(salicylidene)-1,2-bis(3-methoxyphenyl)ethylenediamine]iron(III) complexes by introducing lipophilicity improving ligands such as fluorine (X1), chlorine (X2) and bromine (X3) in 5-position in the salicylidene moieties. After detailed characterization the binding to nucleophiles, logP values and cellular uptake were determined. The complexes were further evaluated regarding their biological activity on MDA-MB 231 mammary carcinoma, the non-tumorous SV-80 fibroblast, HS-5 stroma and MCF-10A mammary gland cell lines. Stability of the complexes in aqueous and biological environments was proven by the lack of interactions with amino acids and glutathione. Cellular uptake was positively correlated with the logP values, indicating that higher lipophilicity enhanced cellular uptake. The complexes induced strong antiproliferative and antimetabolic effects on MDA-MB 231 cells, but were inactive on all non-malignant cells tested. Generation of mitochondrial reactive oxygen species, increase of lipid peroxidation and induction of both ferroptosis and necroptosis were identified as mechanisms of action. In conclusion, halogenation of chlorido[N,N'-bis(salicylidene)-1,2-bis(3-methoxyphenyl)ethylenediamine]iron(III) complexes raises their lipophilic character resulting in improved cellular uptake., (© 2024. The Author(s).)

Cell senescence is defined as irreversible cell cycle arrest, which can be triggered by telomere shortening or by various types of genotoxic stress. Induction of senescence is emerging as a new strategy for the treatment of cancer, especially when sequentially combined with a second senolytic drug capable of killing the resulting senescent cells, however severely suffering from the undesired off-target side effects from the senolytic drugs. Here, we prepare a bimetalic platinum-aluminum salen complex (Alumiplatin) for cancer therapy-a combination of pro-senesence chemotherapy with in situ senotherapy to avoid the side effects. The aluminum salen moiety, as a G-quadruplex stabilizer, enhances the salen's ability to induce cancer cell senescence and this phenotype is in turn sensitive to the cytotoxic activity of the monofunctional platinum moiety. It exhibits an excellent capability for inducing senescence, a potent cytotoxic activity against cancer cells both in vitro and in vivo, and an improved safety profile compared to cisplatin. Therefore, Alumiplatin may be a good candidate to be further developed into safe and effective anticancer agents. This novel combination of cell senescence inducers with genotoxic drugs revolutionizes the therapy options of designing multi-targeting anticancer agents to improve the efficacy of anticancer therapies., (© 2024 Wiley-VCH GmbH.)

The insertion of hydrophobic and hydrophilic chains in the chitosan molecule can improve its antibacterial activity, expanding its range of application in several areas of medical-pharmaceutical sciences. Thus, this work aimed to increase the antibacterial activity of chitosan through the modification reaction with phthalic anhydride (QF) and subsequent reaction with ethylenediamine (QFE). The chitosan and derivatives obtained were characterized by elemental analysis, 13 C Nuclear Magnetic Resonance ( 13 C NMR), X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Thermogravimetric Analysis (TG), where it was possible to prove the chemical modification. Both materials showed a greater antibacterial inhibitory effect against Gram-positive bacteria, Staphylococcus aureus, emphasizing antibacterial activity against Gram-negative bacteria, Escherichia coli, with values above 70 % of the inhibitory effect, which is a promising result. Assays with human fibroblast cells by the [3-(4,5-dimethylthiazolyl)-2,5-diphenyl tetrazolium (MTT)] bromide reduction test did not indicate toxicity in the materials. Thus, the derived materials showed promise for biomedical applications since they combined excellent antibacterial activity against gram-positive and gram-negative strains and did not show cytotoxicity., Competing Interests: Declaration of competing interest All authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A series of novel dibutyltin complexes based on salen-like ligands (S01-S03) were synthesized and characterized using ultraviolet-visible spectra，infrared spectra, 1 H, 13 C, and 119 Sn nuclear magnetic resonance, high-resolution mass spectrometry, X-ray crystallography, and thermogravimetric analysis. Complex S03 had excellent anticancer activity in vitro (IC 50  = 1.5 ± 0.2 μM in CAL-27 cell lines), which highly activated ROS expression levels and induced apoptosis and cell cycle arrest at the G2/M phase. Interestingly, complex S03 induced cancer cell death through multiple mechanisms (mitochondrial pathway, ER-stress pathway, and DNA damage pathway). This study reveals new mechanisms of organotin complexes and provides new insights into the development of organotin metal complexes as anticancer drugs in the future, and compounds with multiple anticancer mechanisms may be a new strategy for delaying or overcoming drug resistance to chemotherapy and target therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The impact of methoxy and hydroxyl groups at the salicylidene moiety of chlorido[ N , N '-bis(methoxy/hydroxy)salicylidene-1,2-bis(4-methoxyphenyl)ethylenediamine]iron(III) complexes was evaluated on human MDA-MB 231 breast cancer and HL-60 leukemia cells. Methoxylated complexes ( C1 - C3 ) inhibited proliferation, migration, and metabolic activity in a concentration-dependent manner following the rank order: C2 > C3 > C1 . In particular, C2 was highly cytotoxic with an IC 50 of 4.2 μM which was 6.6-fold lower than that of cisplatin (IC 50 of 27.9 μM). In contrast, hydroxylated complexes C4 - C6 were almost inactive up to the highest concentration tested due to lack of cellular uptake. C2 caused a dual mode of cell death, ferroptosis, and necroptosis, whereby at higher concentrations, ferroptosis was the preferred form. Ferroptotic morphology and the presence of ferrous iron and lipid reactive oxygen species proved the involvement of ferroptosis. C2 was identified as a promising lead compound for the design of drug candidates inducing ferroptosis.

Chemically assisted phytoremediation is suggested as an effective approach to amplify the metal-remediating potential of hyperaccumulators. The current study assessed the efficiency of two biodegradable chelants (S,S-ethylenediamine disuccinic acid, EDDS; nitrilotriacetic acid, NTA) in enhancing the remediation of Cd by Coronopus didymus (Brassicaceae). C. didymus growing in Cd-contaminated soil (35-175 mg kg -1 soil) showed increased growth and biomass due to the hormesis effect, and chelant supplementation further increased growth, biomass, and Cd accumulation. A significant interaction with chelants and different Cd concentrations was observed, except for Cd content in roots and Cd content in leaves, which exhibited a non-significant interaction with chelant addition. The effect of the NTA amendment on the root dry biomass and shoot dry biomass was more pronounced than EDDS at all the Cd treatments. Upon addition of EDDS and NTA, bio-concentration factor values were enhanced by ~184-205 and ~ 199-208, respectively. The tolerance index of root and shoot increased over the control upon the addition of chelants, with NTA being better than EDDS. With chelant supplementation, bio-accumulation coefficient values were in the order Cd 35  + NTA (~163%) > Cd 105  + NTA (~137%) > Cd 35  + EDDS (~89%) > Cd 175  + NTA (~85%) > Cd 105  + EDDS (~62%) > Cd 175  + EDDS (~40%). The translocation factor correlated positively (r ≥ 0.8) with tolerance index and Cd accumulation in different plant parts. The study demonstrated that chelant supplementation enhanced Cd-remediation efficiency in C. didymus as depicted by improved plant growth and metal accumulation, and NTA was more effective than EDDS in reclaiming Cd., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The biological activity of six structurally similar tetradentate Schiff base copper(II) complexes, namely [Cu(ethylenediamine-bis-acetylacetonate)] (CuAA) and five derivatives where two methyl groups are replaced by phenyl, (CuPP), CF3 (CuTT) or by mixed groups CH3/CF3 (CuAT), Ph/CF3 (CuPT), and Ph/CH3 (CuAP) has been investigated. The set of antioxidant assays was performed, and the results were expressed as IC 50 and EC 50 values. The series of complexes showed interesting bioactivity and were investigated for the determination of antioxidant, antifungal, antimicrobial, and cytotoxic activity. A significant antioxidant behavior was exhibited by complex CuAA, greater than Trolox in the Oxygen Radical Absorbance Capacity (ORAC) assay. Antibacterial assay over Gram-positive and Gram-negative pathogenic bacterial strains and some fungal pathogens were studied. Antiproliferative activity of complexes in two human tumor cell lines, breast adenocarcinoma MCF-7, colon adenocarcinoma LS-174, and normal fibroblast cells-MRC-5, examined the effect on cell cycle progression. The significant cytotoxic potential, comparable to cisplatin cytotoxicity, was determined in human breast cancer cell line-MCF-7 with IC 50 values being 17.53-31.40 μM and human colon cancer cell line-LS-174 with IC 50 values being 15.22-23.92 μM. All tested compounds showed nearly twice more selectivity toward cancer cell lines than normal cells. The interactions of complexes with human serum albumin (HSA), the most prominent protein in plasma, were investigated using spectroscopic fluorescence techniques. The complexes bind to human serum albumin at multiple sites (n = 0.2-1.9), displaying a moderate binding constant K a  = 4.1-12.4 × 10 4  M -1 . The molecular docking experiment effectively showed complex binding to HSA and DNA molecular fragments., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Aleksandar Mijatovic reports financial support was provided by University of Belgrade Faculty of Mining And Geology. Aleksandar Lolic reports financial support was provided by University of Belgrade Faculty of Chemistry., (Copyright © 2023 Elsevier Inc. All rights reserved.)

N-geranyl-N΄-(2-adamantyl)ethane-1,2-diamine (SQ109) is a tuberculosis drug that has high potency against Mycobacterium tuberculosis (Mtb) and may function by blocking cell wall biosynthesis. After the crystal structure of MmpL3 from Mycobacterium smegmatis in complex with SQ109 became available, it was suggested that SQ109 inhibits Mmpl3 mycolic acid transporter. Here, we showed using molecular dynamics (MD) simulations that the binding profile of nine SQ109 analogs with inhibitory potency against Mtb and alkyl or aryl adducts at C-2 or C-1 adamantyl carbon to MmpL3 was consistent with the X-ray structure of MmpL3 - SQ109 complex. We showed that rotation of SQ109 around carbon-carbon bond in the monoprotonated ethylenediamine unit favors two gauche conformations as minima in water and lipophilic solvent using DFT calculations as well as inside the transporter's binding area using MD simulations. The binding assays in micelles suggested that the binding affinity of the SQ109 analogs was increased for the larger, more hydrophobic adducts, which was consistent with our results from MD simulations of the SQ109 analogues suggesting that sizeable C-2 adamantyl adducts of SQ109 can fill a lipophilic region between Y257, Y646, F260 and F649 in MmpL3. This was confirmed quantitatively by our calculations of the relative binding free energies using the thermodynamic integration coupled with MD simulations method with a mean assigned error of 0.74 kcal mol -1 compared to the experimental values., (© 2023. The Author(s).)

The linking of ethacrynic acid with ethylenediamine and 1,4-butanediamine gave EDEA and BDEA, respectively, as membrane-permeable divalent pro-inhibitors of glutathione S -transferase (GST). Their divalent glutathione conjugates showed subnanomolar inhibition and divalence-binding to GSTmu (GSTM) (PDB: 5HWL) at ∼0.35 min -1 . In cisplatin-resistant SK-OV-3, COC1, SGC7901 and A549 cells, GSTM activities probed by 15 nM BDEA or EDEA revealed 5-fold and 1.0-fold increases in cisplatin-resistant SK-OV-3 and COC1 cells, respectively, in comparison with the susceptible parental cells. Being tolerable by HEK293 and LO2 cells, BDEA at 0.2 μM sensitised resistant SK-OV-3 and COC1 cells by ∼3- and ∼5-folds, respectively, released cytochrome c and increased apoptosis; EDEA at 1.0 μM sensitised resistant SK-OV-3 and A549 cells by ∼5- and ∼7-fold, respectively. EDEA at 1.7 μg/g sensitised resistant SK-OV-3 cells to cisplatin at 3.3 μg/g in nude mouse xenograft model. BDEA and EDEA are promising leads for probing cellular GSTM and sensitising cisplatin-resistant ovarian cancers.

The proteasome is a promising target for antimalarial chemotherapy. We assessed ex vivo susceptibilities of fresh Plasmodium falciparum isolates from eastern Uganda to seven proteasome inhibitors: two asparagine ethylenediamines, two macrocyclic peptides, and three peptide boronates; five had median IC 50 values <100 nM. TDI8304, a macrocylic peptide lead compound with drug-like properties, had a median IC 50 of 16 nM. Sequencing genes encoding the β2 and β5 catalytic proteasome subunits, the predicted targets of the inhibitors, and five additional proteasome subunits, identified two mutations in β2 (I204T, S214F), three mutations in β5 (V2I, A142S, D150E), and three mutations in other subunits. The β2 S214F mutation was associated with decreased susceptibility to two peptide boronates, with IC 50 s of 181 nM and 2635 nM against mutant versus 62 nM and 477 nM against wild type parasites for MMV1579506 and MMV1794229, respectively, although significance could not be formally assessed due to the small number of mutant parasites with available data. The other β2 and β5 mutations and mutations in other subunits were not associated with susceptibility to tested compounds. Against culture-adapted Ugandan isolates, two asparagine ethylenediamines and the peptide proteasome inhibitors WLW-vinyl sulfone and WLL-vinyl sulfone (which were not studied ex vivo ) demonstrated low nM activity, without decreased activity against β2 S214F mutant parasites. Overall, proteasome inhibitors had potent activity against P. falciparum isolates circulating in Uganda, and genetic variation in proteasome targets was uncommon.

The colocalization of taurine and zinc transporters (TAUT, ZnTs) has not been explored in retina. Our objective is to evaluate the effect of the intracellular zinc chelator N,N,N,N-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) on zinc localization and colocalization TAUT and ZnT-1 (of plasma membrane), 3 (vesicular), and 7 (vesicular and golgi apparatus) in layers of retina by immunohistochemistry. To mark zinc, it was used cell-permeable fluorescent Zinquin ethyl ester. Specific first and secondary antibodies, conjugated with rhodamine or fluorescein-isothiocyanate were used to mark TAUT and ZnTs. The fluorescence results were reported as integrated optical density (IOD). Zinc was detected in all layers of the retina. The treatment with TPEN produced changes in the distribution of zinc in layers of retina less in the outer nuclear layer compared with the control. TAUT was detected in all layers of retina and TPEN chelator produced decrease of IOD in all layers of retina except in the photoreceptor compared with the control. ZnT 1, 3, and 7 were distributed in all retina layers, with more intensity in ganglion cell layer (GCL) and in the layers where there is synaptic connection. For all transporters, the treatment with TPEN produced significant decrease of IOD in layers of retina least in the inner nuclear layer for ZnT1, in the photoreceptor for ZnT3 and in the GCL and outer plexiform layer for ZnT7. The distribution of zinc, TAUT, and ZnTs in the layers of retina is indicative of the interaction of taurine and zinc for the function of the retina and normal operation of said layers. HIGHLIGHTS: Taurine and zinc are two molecules highly concentrated in the retina and with relevant functions in this structure. Maintaining zinc homeostasis in this tissue is necessary for the normal function of the taurine system in the retina. The study of the taurine transporter and the different zinc transporters in the retina (responsible for maintaining adequate levels of taurine and zinc) is relevant and novel, since it is indicative of the interactions between both molecules in this structure., (© 2022 Wiley Periodicals LLC.)

Zinc is a trace metal vital for various functions in nerve cells, although the effect of zinc deficiency on neuronal autophagy remains unclear. This study aimed to elucidate whether zinc deficiency induced by treatment with N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a zinc chelator, affects and alters autophagy activity. In cell viability assays, TPEN showed cytotoxicity in HT-22 cells. TPEN treatment also increased LC3-II levels and the ratio of LC3-II to LC3-I. Western blot analysis showed that phospho-AMP-activated protein kinase levels and the ratio of phospho-AMP-activated protein kinase to total AMP-activated protein kinase increased. Protein levels of the mammalian target of rapamycin and sirtuin 1 decreased following TPEN treatment. When TPEN-treated HT-22 cells were cotreated with autophagy inhibitors, 3-methyladenine (1 mM), or bafilomycin A1 (3 nM), the TPEN-induced decrease in cell viability was exacerbated. Cotreatment with chloroquine (10 μM) partially restored cell viability. The study showed that zinc deficiency induces autophagy and may be cytoprotective in neurons. We expect our results to add a new perspective to our understanding of the neuronal pathology related to zinc deficiency.

Directly Observed Treatment Short-course (DOTs), is an effective and widely recommended treatment for tuberculosis (TB). The antibiotics used in DOTs, are immunotoxic and impair effector T cells, increasing the risk of re-infections and reactivation. Multiple reports suggest that addition of immune-modulators along with antibiotics improves the effectiveness of TB treatment. Therefore, drugs with both antimicrobial and immunomodulatory properties are desirable. N 1 -(Adamantan-2-yl)-N 2 -[(2E)-3,7-dimethylocta-2,6-dien-1-yl]ethane-1,2-diamine (SQ109) is an asymmetric diamine derivative of adamantane, that targets Mycobacterial membrane protein Large 3 (MmpL3). SQ109 dissipates the transmembrane electrochemical proton-gradient necessary for cell-wall biosynthesis and bacterial activity. Here, we examined the effects of SQ109 on host-immune responses using a murine TB model. Our results suggest the pro-inflammatory nature of SQ109, which instigates M1-macrophage polarization and induces protective pro-inflammatory cytokines through the p38-MAPK pathway. SQ109 also promotes Th1 and Th17-immune responses that inhibit the bacillary burden in a murine model of TB. These findings put forth SQ109 as a potential-adjunct to TB antibiotic therapy., (© 2022. The Author(s).)

Natural naphthoquinones and their derivatives exhibit a broad spectrum of pharmacological activities and have thus attracted much attention in modern drug discovery. However, it remains unclear whether naphthoquinones are potential drug candidates for anti-angiogenic agents. The aim of this study was to evaluate the anti-angiogenic properties of a novel naphthoquinone derivative, PPE8, and explore its underlying mechanisms. Determined by various assays including BrdU, migration, invasion, and tube formation analyses, PPE8 treatment resulted in the reduction of VEGF-A-induced proliferation, migration, and invasion, as well as tube formation in human umbilical vein endothelial cells (HUVECs). We also used an aorta ring sprouting assay, Matrigel plug assay, and immunoblotting analysis to examine PPE8's ex vivo and in vivo anti-angiogenic activities and its actions on VEGF-A signaling. It has been revealed that PPE8 inhibited VEGF-A-induced micro vessel sprouting and was capable of suppressing angiogenesis in in vivo models. In addition, PPE8 inhibited VEGF receptor (VEGFR)-2, Src, FAK, ERK1/2, or AKT phosphorylation in HUVECs exposed to VEGF-A, and it also showed significant decline in xenograft tumor growth in vivo. Taken together, these observations indicated that PPE8 may target VEGF-A-VEGFR-2 signaling to reduce angiogenesis. It also supports the role of PPE8 as a potential drug candidate for the development of therapeutic agents in the treatment of angiogenesis-related diseases including cancer.

This paper summarises a study of the application of the synthetic chelate ethylenediaminetetraacetic acid (EDTA), and the natural chelates ethylenediamine-N,N'-disuccinic acid (EDDS) and nitrilotriacetate (NTA) to enhance ryegrass (Lolium multiflorum Lam.) uptake of the heavy metal(oid)s (HMs) (As, Cd, Cu, Pb and Zn) from contaminated soils in mining sites. The study compares the effects of these chelates (EDTA, EDDS and NTA) on the phytoavailability of HMs (As, Cd, Cu, Pb, Zn) using ryegrass (Lolium multiflorum Lam.) through the single addition and sequential addition methods. The results show that application of EDTA, EDDS and NTA significantly increases ryegrass (Lolium multiflorum Lam.)'s shoot uptake of some HMs when compared with no EDTA, EDDS or NTA application, particularly through sequential chelate treatment (EDTA 0.5:1+0.5:1; NTA 0.5:1+0.5:1; EDDS 0.5:1+0.5:1). EDTA 0.5:1+0.5:1 was more effective at increasing the concentration of Pb in shoots than were the other chelates (EDDS and NTA) and controls. Moreover, the concentrations of Zn in the shoots of ryegrass (Lolium multiflorum Lam.) in Hich Village significantly increased with the application of split dose (0.5:1+0.5:1). The plants displayed symptoms of toxicity including yellow and necrotic leaves at the end of the experiment. The selected chelates (EDTA, EDDS and NTA) led to a significant decrease in plant biomass (yield) 28 days after transfer with a maximum decrease in EDTA treatment (0.5:1+0.5:1) soils. This decrease was 3.43-fold in Ha Thuong, 3-fold in Hich Village and 1.59-fold in Trai Cau, respectively, relative to the control. HM concentration and dissolved organic carbon (DOC) in pore water provided an explanation for why fresh weight was significantly reduced with application of chelates in sequential dose (EDTA 0.5:1+0.5:1 and NTA 0.5:1+0.5:1)., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We have synthesized a series of novel substituted sulfonyl ethylenediamine (en) Ru II arene complexes 1-8 of [(η 6 -arene)Ru(R 1 -SO 2 -EnBz)X], where the arene is benzene, HO(CH 2 ) 2 O-phenyl or biphenyl (biph), X = Cl or I, and R 1 is phenyl, 4-Me-phenyl, 4-NO 2 -phenyl or dansyl. The 'piano-stool' structure of complex 3, [(η 6 -biph)Ru(4-Me-phenyl-SO 2 -EnBz)I], was confirmed by X-ray crystallography. The values of their aqua adducts were determined to be high (9.1 to 9.7). Complexes 1-8 have antiproliferative activity against human A2780 ovarian, and A549 lung cancer cells with IC 50 values ranging from 4.1 to >50 μM, although, remarkably, complex 7 [(η 6 -biph)Ru(phenyl-SO 2 -EnBz)Cl] was inactive towards A2780 cells, but as potent as the clinical drug cisplatin towards A549 cells. All these complexes also showed catalytic activity in transfer hydrogenation (TH) of NAD + to NADH with sodium formate as hydride donor, with TOFs in the range of 2.5-9.7 h -1 . The complexes reacted rapidly with the thiols glutathione (GSH) and N -acetyl-L-cysteine (NAC), forming dinuclear bridged complexes [(η 6 -biph) 2 Ru 2 (GS) 3 ] 2- or [(η 6 -biph) 2 Ru 2 (NAC-H) 3 ] 2- , with the liberation of the diamine ligand which was detected by LC-MS. In addition, the switching on of fluorescence for complex 8 in aqueous solution confirmed release of the chelated DsEnBz ligand in reactions with these thiols. Reactions with GSH hampered the catalytic TH of NAD + to NADH due to the decomposition of the complexes. Co-administration to cells of complex 2 [(η 6 -biph)Ru(4-Me-phenyl-SO 2 -EnBz)Cl] with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, partially restored the anticancer activity towards A2780 ovarian cancer cells. Complex 2 caused a concentration-dependent G1 phase cell cycle arrest, and induced a significant level of reactive oxygen species (ROS) in A2780 human ovarian cancer cells. The amount of induced ROS decreased with increase in GSH concentration, perhaps due to the formation of the dinuclear Ru-SG complex.

A nutrition deficiency is one of the various causes of hearing loss. Zinc is an essential element for cell proliferation, antioxidant reactions, and the maintenance of hearing ability. Our previous studies have reported that the auditory brainstem response (ABR) threshold is increased in mice fed with zinc-deficient diets. However, the molecular mechanism of zinc involved in auditory system remains to be elucidated. In the present study, we examined the detrimental effects of zinc deficiency on cell cycle progression in murine auditory cells (HEI-OC1). The treatment of HEI-OC1 cells with 0.5 μM TPEN (N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine) for 24 h inhibited cell proliferation, accumulation of reactive oxygen species (ROS), and induction of apoptosis. The cell proliferation block was caused by a G1/S phase arrest. Supplementation of the cell growth medium with 5 μM ZnCl 2 after exposure to TPEN attenuated ROS accumulation and the arrest caused by the zinc deficiency. The ABR threshold was elevated in mice fed with a zinc-deficient diet. Additionally, we observed an increased expression of p21 and decreased expression of cyclin E and pRb in the spiral ganglion (SG), the organ of Corti (OC), Limbus (L), and stria vascularis (SV) in the zinc-deficient mouse cochlea. These results indicated that zinc is an essential nutrient for proliferation via the cell cycle and that a dysregulation of the cell cycle may cause hearing loss., (Copyright © 2021. Published by Elsevier Inc.)

To understand the role of intracellular zinc ion (Zn 2+ ) dysregulation in mediating age-related neurodegenerative changes, particularly neurotoxicity resulting from the generation of excessive neurotoxic amyloid-β (Aβ) peptides, this study aimed to investigate whether N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn 2+ -specific chelator, could attenuate Aβ 25-35 -induced neurotoxicity and the underlying electrophysiological mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of hippocampal neurons and performed single-cell confocal imaging to detect the concentration of Zn 2+ in these neurons. Furthermore, we used the whole-cell patch-clamp technique to detect the evoked repetitive action potential (APs), the voltage-gated sodium and potassium (K + ) channels of primary hippocampal neurons. The analysis showed that TPEN attenuated Aβ 25-35 -induced neuronal death, reversed the Aβ 25-35 -induced increase in intracellular Zn 2+ concentration and the frequency of APs, inhibited the increase in the maximum current density of voltage-activated sodium channel currents induced by Aβ 25-35 , relieved the Aβ 25-35 -induced decrease in the peak amplitude of transient outward K + currents (I A ) and outward-delayed rectifier K + currents (I DR ) at different membrane potentials, and suppressed the steady-state activation and inactivation curves of I A shifted toward the hyperpolarization direction caused by Aβ 25-35 . These results suggest that Aβ 25-35 -induced neuronal damage correlated with Zn 2+ dysregulation mediated the electrophysiological changes in the voltage-gated sodium and K + channels. Moreover, Zn 2+ -specific chelator-TPEN attenuated Aβ 25-35 -induced neuronal damage by recovering the intracellular Zn 2+ concentration., (© 2021. The Author(s).)

Despite the common use of salens and hydroxyquinolines as therapeutic and bioactive agents, their metal complexes are still under development. Here, we report the synthesis of novel mixed-ligand metal complexes (MSQ) comprising salen (S), derived from (2,2'-{1,2-ethanediylbis[nitrilo(E) methylylidene]}diphenol, and 8-hydroxyquinoline (Q) with Co(II), Ni(II), Cd(II), Al(III), and La(III). The structures and properties of these MSQ metal complexes were investigated using molar conductivity, melting point, FTIR, 1 H NMR, 13 C NMR, UV-VIS, mass spectra, and thermal analysis. Quantum calculation, analytical, and experimental measurements seem to suggest the proposed structure of the compounds and its uncommon monobasic tridentate binding mode of salen via phenolic oxygen, azomethine group, and the NH group. The general molecular formula of MSQ metal complexes is [M(S)(Q)(H 2 O)] for M (II) = Co, Ni, and Cd or [M(S)(Q)(Cl)] and [M(S)(Q)(H 2 O)]Cl for M(III) = La and Al, respectively. Importantly, all prepared metal complexes were evaluated for their antimicrobial and anticancer activities. The metal complexes exhibited high cytotoxic potency against human breast cancer (MDA-MB231) and liver cancer (Hep-G2) cell lines. Among all MSQ metal complexes, CoSQ and LaSQ produced IC 50 values (1.49 and 1.95 µM, respectively) that were comparable to that of cisplatin (1.55 µM) against Hep-G2 cells, whereas CdSQ and LaSQ had best potency against MDA-MB231 with IC 50 values of 1.95 and 1.43 µM, respectively. Furthermore, the metal complexes exhibited significant antimicrobial activities against a wide spectrum of both Gram-positive and -negative bacterial and fungal strains. The antibacterial and antifungal efficacies for the MSQ metal complexes, the free S and Q ligands, and the standard drugs gentamycin and ketoconazole decreased in the order AlSQ > LaSQ > CdSQ > gentamycin > NiSQ > CoSQ > Q > S for antibacterial activity, and for antifungal activity followed the trend of LaSQ > AlSQ > CdSQ > ketoconazole > NiSQ > CoSQ > Q > S. Molecular docking studies were performed to investigate the binding of the synthesized compounds with breast cancer oxidoreductase (PDB ID: 3HB5). According to the data obtained, the most probable coordination geometry is octahedral for all the metal complexes. The molecular and electronic structures of the metal complexes were optimized theoretically, and their quantum chemical parameters were calculated. PXRD results for the Cd(II) and La(III) metal complexes indicated that they were crystalline in nature., Competing Interests: The authors declare no conflict of interest.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic., Competing Interests: Declaration of interests O.A.-W. has served as a consultant for H3B Biomedicine, Foundation Medicine, Merck, Prelude Therapeutics, and Janssen, and is on the scientific advisory board of Envisagenics, Pfizer Boulder, and AIChemy. O.A.-W. has received prior research funding from Loxo Oncology and H3 Biomedicine unrelated to this work. S.X.L. has served as a consultant (uncompensated) for PTC Therapeutics. B.R. has served as a consultant for Bayer and Roche. D.Z. reports clinical research support to his institution from Astra Zeneca, Plexxikon, and Genentech and personal/consultancy fees from Merck, Synlogic Therapeutics, Tesaro, Bristol Myers Squibb (BMS), Genentech, Xencor, Memgen, Calidi Biotherapeutics, and Agenus. L.A.D. is a member of the board of directors of Personal Genome Diagnostics (PGDx) and Jounce Therapeutics. L.A.D. is a compensated consultant to PGDx, 4Paws (PetDx), Innovatus CP, Se’er, Delfi, Kinnate, and Neophore. L.A.D. is an uncompensated consultant for, but has received clinical trial support from, Merck. L.A.D. holds equity in PGDx, Jounce, Se’er, Delfi, Kinnate, and Neophore and divested equity in Thrive Earlier Detection in 2021. His spouse holds equity in Amgen. J.D.W. is a consultant for Amgen, Apricity, Arsenal IO, Ascentage Pharma, AstraZeneca, Astellas, Boehringer Ingelheim, BMS, Chugai, Dragonfly, F Star, Eli Lilly, Georgiamune, IMVAQ, Merck, Polynoma, Psioxus, Recepta, Trieza, Truvax, Sellas, and Werewolf Therapeutics. J.D.W. has grant/research support from BMS and Sephora. J.D.W. reports equity in Tizona Pharmaceuticals, Imvaq, Beigene, Linneaus, Apricity, Arsenal IO, and Georgiamune. T.M. is an inventor on patents involving the use of anti-PD-1 antibodies. T.M. is a consultant for Immunos Therapeutics and Pfizer. T.M. is a cofounder of and equity holder in IMVAQ. T.M. receives research funding from BMS, Surface Oncology, Kyn Therapeutics, Infinity Pharmaceuticals, Peregrine Pharmaceuticals, Adaptive Biotechnologies, Leap Therapeutics, and Aprea Therapeutics. S.X.L., O.A.-W., and R.K.B. are inventors on a patent application submitted by FHCRC related to this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Zinc, an abundant transition metal, serves as a signalling molecule in several biological systems. Zinc transporters are genetically associated with cardiovascular diseases but the function of zinc in vascular tone regulation is unknown. We found that elevating cytoplasmic zinc using ionophores relaxed rat and human isolated blood vessels and caused hyperpolarization of smooth muscle membrane. Furthermore, zinc ionophores lowered blood pressure in anaesthetized rats and increased blood flow without affecting heart rate. Conversely, intracellular zinc chelation induced contraction of selected vessels from rats and humans and depolarized vascular smooth muscle membrane potential. We demonstrate three mechanisms for zinc-induced vasorelaxation: (1) activation of transient receptor potential ankyrin 1 to increase calcitonin gene-related peptide signalling from perivascular sensory nerves; (2) enhancement of cyclooxygenase-sensitive vasodilatory prostanoid signalling in the endothelium; and (3) inhibition of voltage-gated calcium channels in the smooth muscle. These data introduce zinc as a new target for vascular therapeutics.

Sigma-1 receptor (chaperone Sigma1R) is an intracellular protein with chaperone functions, which is expressed in various organs, including the brain. Sigma1R participates in the regulation of physiological mechanisms of anxiety (Su, T. P. et al., 2016) and reactions to emotional stress (Hayashi, T., 2015). In 2006, fabomotizole (ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride) was registered in Russia as an anxiolytic (Seredenin S. and Voronin M., 2009). The molecular targets of fabomotizole are Sigma1R, NRH: quinone reductase 2 (NQO2), and monoamine oxidase A (MAO-A) (Seredenin S. and Voronin M., 2009). The current study aimed to clarify the dependence of fabomotizole anxiolytic action on its interaction with Sigma1R and perform a docking analysis of fabomotizole interaction with Sigma1R. An elevated plus maze (EPM) test revealed that the anxiolytic-like effect of fabomotizole (2.5 mg/kg i.p.) administered to male BALB/c mice 30 min prior EPM exposition was blocked by Sigma1R antagonists BD-1047 (1.0 mg/kg i.p.) and NE-100 (1.0 mg/kg i.p.) pretreatment. Results of initial in silico study showed that fabomotizole locates in the active center of Sigma1R, reproducing the interactions with the site's amino acids common for established Sigma1R ligands, with the ΔG bind value closer to that of agonist (+)-pentazocine in the 6DK1 binding site.

Two new aminodiphosphonic acids derived from salicylic acid and its phosphonic analogue were prepared through a simple and efficient synthesis. 2-[(2-Amino-2,2-diphosphono)ethyloxy]-benzoic acid 8 and 2-[(2-amino-2,2-diphosphono)ethyloxy]-5-ethyl-phenylphosphonic acid 9 were evaluated for their applicability as 68 Ga binding bone-seeking agents. Protonation constants of 8 and 9 and stability constants of the Ga 3+ complexes with 8 and 9 in water were determined. The stability constant of Ga 3+ complex with fully phosphorylated acid 9 (log K GaL = 31.92 ± 0.32) significantly exceeds stability constant of Ga 3+ complex with 8 (log K GaL = 26.63 ± 0.24). Ligands 8 and 9 are as effective for Ga 3+ cation binding as ethylenediamine- N , N' -diacetic- N , N' -bis(methy1enephosphonic) acid and ethylenediamine- N , N , N' , N' -tetrakis(methylenephosphonic) acid, respectively. The labelling process and stability of [ 68 Ga]Ga- 8 and [ 68 Ga]Ga- 9 were studied. Both 8 and 9 readily form 68 Ga-complexes stable to ten-fold dilution with saline. However, in fetal bovine serum, only [ 68 Ga]Ga- 9 was stable enough to be subject to biological evaluation. It was injected into rats with bone pathology and aseptic inflammation of soft tissues. For [ 68 Ga]Ga- 9 in animals with a bone pathology model in 60 and 120 min after injection, a slight accumulation in the pathology site, stable blood percentage level, and moderate accumulation in the liver were observed. For animals with an aseptic inflammation, the accumulation of [ 68 Ga]Ga- 9 in the pathology site was higher than that in animals with bone pathology. Moreover, the accumulation of [ 68 Ga]Ga- 9 in inflammation sites was more stable than that for [ 68 Ga]Ga-citrate. The percentage of [ 68 Ga]Ga- 9 in the blood decreased from 3.1% ID/g (60 min) to 1.5% ID/g (120 min). Accumulation in the liver was comparable to that obtained for [ 68 Ga]Ga-citrate.

We investigated the aluminium-salen complex MBR-8 as a potential anti-cancer agent. To see apoptotic effects induced by MBR-8, alone and in combination with common cytostatic drugs, DNA-fragmentations were studied using the flow cytometric analysis. Western blot analysis and measurement of the mitochondrial membrane potential with a JC-1 dye were employed to identify the pathway of apoptosis. An impressive overcoming of multidrug-resistance in leukemia (Nalm6) cells was observed. Additionally, solid tumor cells including Burkitt-like lymphoma (BJAB) and mamma carcinoma cells (MCF-7) are affected by MBR-8 in the same way. Western blot analysis revealed activation of caspase-3. MBR-8 showed very pronounced selectivity with regard to tumor cells and high synergistic effects in Nalm6 and daunorubicin-resistant Nalm6 cells when administered in combination with vincristine, daunorubicin and doxorubicin. The aluminium-salen complex MBR-8 showed very promising anti-cancer properties which warrant further development towards a cytostatic agent for future chemotherapy. Studies on aluminium compounds for cancer therapy are rare, and our report adds to this important body of knowledge.

Dietary zinc deficiency may lead to olfactory deficits, whose mechanism remains largely elusive. Olfactory ensheathing cells (OECs), a type of glial cells that support the function and neurogenesis in the olfactory bulb (OB), may play a pivotal role in the maintenance of the olfactory system. In the present study, we established a rat model of dietary zinc deficiency and found that severe zinc deficiency, but not marginal zinc deficiency, caused significantly reduced food intake, growth retardation, and apparent olfactory deficit in growing rats. We showed that severe zinc deficiency resulted in the loss of OECs in the olfactory nerve layer (ONL) of the olfactory bulb. In addition, we revealed that the number of TUNEL-positive cells increased markedly in the region, suggesting an involvement of apoptotic cell death in zinc deficiency-induced loss of OECs. Moreover, we found that treatment with zinc chelator N,N,N'N',-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) triggered the apoptosis of in vitro-cultured primary OECs. The apoptosis of OECs was correlated with significantly elevated expression of p53. Importantly, TUNEL and CCK-8 assays both demonstrated that treatment with p53 antagonist pifithrin-α (PFT-α) markedly attenuated TPEN-induced OEC apoptosis. These findings implicated that p53-triggered apoptosis of OECs might play an integral role in zinc deficiency-induced olfactory malfunction.

Plants in the family Lemnaceae are aquatic monocots and the smallest, simplest, and fastest growing angiosperms. Their small size, the smallest family member is 0.5 mm and the largest is 2.0 cm, as well as their diverse morphologies make these plants ideal for laboratory studies. Their rapid growth rate is partially due to the family's neotenous lifestyle, where instead of maturing and producing flowers, the plants remain in a juvenile state and continuously bud asexually. Maturation and flowering in the wild are rare in most family members. To promote further research on these unique plants, we have optimized laboratory flowering protocols for 3 of the 5 genera: Spirodela ; Lemna ; and Wolffia in the Lemnaceae. Duckweeds were widely used in the past for research on flowering, hormone and amino acid biosynthesis, the photosynthetic apparatus, and phytoremediation due to their aqueous lifestyle and ease of aseptic culture. There is a recent renaissance in interest in growing these plants as non-lignified biomass sources for fuel production, and as a resource-efficient complete protein source. The genome sequences of several Lemnaceae family members have become available, providing a foundation for genetic improvement of these plants as crops. The protocols for maximizing flowering described herein are based on screens testing daylength, a variety of media, supplementation with salicylic acid or ethylenediamine- N , N '-bis(2-hydroxyphenylacetic acid) (EDDHA), as well as various culture vessels for effects on flowering of verified Lemnaceae strains available from the Rutgers Duckweed Stock Cooperative.

Despite some advances in the treatment of acute lymphoblastic (ALL) and myeloid leukemia (AML) in recent years, there is still a prominent percentage of pediatric patients with a reduced overall prognosis. Therefore, other therapeutic approaches are needed to treat those patients. In the present study, we report that the metal chelator TPEN affected ΔΨ m and DNA content in isolated CD34 + refractory cells from bone marrow ALL (n = 7; B-cell, n = 4; T-cell, n = 3) and AML (n = 3) pediatric patients. Furthermore, TPEN induced oxidation of hydrogen peroxide (H 2 O 2 ) sensor protein DJ-1, induced up-regulation of BH3-only pro-apoptotic protein PUMA, transcription factor p53 and activated the executor protease CASPASE-3 as apoptosis markers, and reduced the reactivity of the cellular proliferating marker Ki-67 in all acute leukemic groups, and reduced the phosphorylation of c-ABL protein signal in an AML case. Remarkably, bone marrow cells from non-leukemic patients' cells (n = 2) displayed neither loss of ΔΨ m nor loss of DNA content when exposed to TPEN. We conclude that TPEN selectively induces apoptosis in acute leukemic cells via reactive oxygen species (ROS) signaling mechanism. Understanding the pathways of TPEN-induced cell death may provide insight into more effective therapeutic ROS-inducing anticancer agents.

High mobility group box 1 protein (HMGB1) is involved in the pathogenesis of inflammatory bowel disease (IBD). Patients with IBD develop zinc deficiency. However, the detailed roles of HMGB1 and zinc deficiency in the intestinal epithelial barrier and cellular metabolism of IBD remain unknown. In the present study, Caco-2 cells in 2D culture and 2.5D Matrigel culture were pretreated with transforming growth factor-β (TGF-β) type 1 receptor kinase inhibitor EW-7197, epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 and a TNFα antibody before treatment with HMGB1 and inflammatory cytokines (TNFα and IFNγ). EW-7197, AG-1478 and the TNFα antibody prevented hyperpermeability induced by HMGB1 and inflammatory cytokines in 2.5D culture. HMGB1 affected cilia formation in 2.5D culture. EW-7197, AG-1478 and the TNFα antibody prevented the increase in cell metabolism induced by HMGB1 and inflammatory cytokines in 2D culture. Furthermore, ZnSO 4 prevented the hyperpermeability induced by zinc chelator TPEN in 2.5D culture. ZnSO 4 and TPEN induced cellular metabolism in 2D culture. The disruption of the epithelial barrier induced by HMGB1 and inflammatory cytokines contributed to TGF-β/EGF signaling in Caco-2 cells. The TNFα antibody and ZnSO 4 as well as EW-7197 and AG-1478 may have potential for use in therapy for IBD.

A very small number of cobalt complexes is examined in oncology research. In this work, we investigate the cobalt (III) salen complex MBR-60 that turns out to be a promising anticancer drug. It induces apoptosis in Nalm6 leukemia and BJAB lymphoma cells and overcomes multidrug resistances by blocking the drug efflux pump P-glycoprotein. It further develops the apoptotic effects over the intrinsic pathway. An activation of caspase-3, caspase-8 and caspase-9 can be detected by western blot analysis. The independence of CD95 is shown by similar apoptotic inductions in BJAB and BJAB FADDdn cells. MBR-60 displays synergistic effects with daunorubicin and vincristine and has a selectivity to tumor cells. In comparison to the apoptotic effects of MBR-60 in BJAB lymphoma cells, the cobalt-free ligand 5 does not influence these cells. The research highlights that a cobalt complex has a therapeutic potential for cancer treating with a focus on drug-resistant tumors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Spreading of the multidrug-resistant (MDR) strains of the one of the most harmful pathogen Mycobacterium tuberculosis (Mtb) generates the need for new effective drugs. SQ109 showed activity against resistant Mtb and already advanced to Phase II/III clinical trials. Fast SQ109 degradation is attributed to the human liver Cytochrome P450s (CYPs). However, no information is available about interactions of the drug with Mtb CYPs. Here, we show that Mtb CYP124, previously assigned as a methyl-branched lipid monooxygenase, binds and hydroxylates SQ109 in vitro. A 1.25 Å-resolution crystal structure of the CYP124-SQ109 complex unambiguously shows two conformations of the drug, both positioned for hydroxylation of the ω-methyl group in the trans position. The hydroxylated SQ109 presumably forms stabilizing H-bonds with its target, Mycobacterial membrane protein Large 3 (MmpL3). We anticipate that Mtb CYPs could function as analogs of drug-metabolizing human CYPs affecting pharmacokinetics and pharmacodynamics of antitubercular (anti-TB) drugs.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Owing to the limitations in the current therapeutic strategies for treating HCC, development of novel chemotherapeutic drugs is urgently needed. In the present study, we found that QQM, a newly-synthesized quinolinylmethyl substituted ethylenediamine compound, exhibited anti-HCC effects both in vitro and in vivo. QQM inhibited HCC cell growth and induced G0/G1-phase cell cycle arrest and apoptosis in a dose-dependent manner. Our results showed that QQM acted by significantly increasing intracellular reactive oxygen species in HCC cells, which led to cell apoptosis and growth inhibition. Furthermore, QQM treatment resulted in an accumulation of reactive nitric oxide (NO) in HCC cells, and introduction of a NO scavenger, carboxy-PTIO, largely attenuated QQM-induced cytotoxicity. Finally, we found that QQM inhibited growth and induced apoptosis of HCC xenograft tumors in vivo. Taken together, our results indicated that QQM exerted anti-HCC effects by inducing reactive oxygen species and NO accumulation in HCC cells. Thus, QQM exhibits the qualities of a novel, promising anti-tumor candidate for the treatment of HCC., (Copyright © 2020. Published by Elsevier B.V.)

Previously, we demonstrated that the immediate administration of multitarget anxiolytic afobazole slows down the progression of neuronal damage in a 6-hydroxidodamine (6-OHDA) model of Parkinson's disease due to the activation of chaperone Sigma1R. The aim of the present study is to evaluate the therapeutic potential of deferred afobazole administration in this model. Male ICR mice received a unilateral 6-OHDA lesion of the striatum. Fourteen days after the surgery, mice were treated with afobazole, selective Sigma1R agonist PRE-084, selective Sigma1R antagonist BD-1047, and a combination of BD-1047 with afobazole or PRE-084 for another 14 days. The deferred administration of afobazole restored the intrastriatal dopamine content in the 6-OHDA-lesioned striatum and facilitated motor behavior in rotarod tests. The action of afobazole accorded with the effect of Sigma1R selective agonist PRE-084 and was blocked by Sigma1R selective antagonist BD-1047. The present study illustrates the Sigma1R-dependent effects of afobazole in a 6-OHDA model of Parkinson's disease and reveals the therapeutic potential of Sigma1R agonists in treatment of the condition.

This study examined the antibacterial effect of protoporphyrin IX-ethylenediamine derivative (PPIX-ED)-mediated photodynamic antimicrobial chemotherapy (PPIX-ED-PACT) against Pseudomonas aeruginosa in vitro and in vivo. PPIX-ED potently inhibited the growth of Pseudomonas aeruginosa by inducing reactive oxygen species production via photoactivation. Atomic force microscopy revealed that PPIX-ED-PACT induced the leakage of bacterial content by degrading the bacterial membrane and wall. As revealed using acridine orange/ethidium bromide staining, PPIX-ED-PACT altered the permeability of the bacterial membrane. In addition, the antibacterial effect of PPIX-ED-PACT was demonstrated in an in vivo model of P. aeruginosa-infected wounds. PPIX-ED (100 μM) decreased the number of P. aeruginosa colony-forming units by 4.2 log10. Moreover, histological analysis illustrated that the wound healing rate was 98% on day 14 after treatment, which was 10% higher than that in the control group. According to the present findings, PPIX-ED-PACT can effectively inhibit the growth of P. aeruginosa in vitro and in vivo., Competing Interests: The authors have declared that no competing interests exist

Chelants application can increase the bioavailability of metals, subsequently limiting plant growth and reducing the efficiency of phytoremediation. Plant growth-promoting rhizobacteria (PGPRs) and rhizobium have substantial potential to improve plant growth and plant tolerance to metal stress. We evaluated the effects of co-inoculation with a PGPR strain (Paenibacillus mucilaginosus) and a Cu-resistant rhizobium strain (Sinorhizobium meliloti) on the efficiency of biodegradable chelant (S,S-ethylenediaminedisuccinic acid; EDDS) assisted phytoremediation of a Cu contaminated soil using alfalfa. The highest total Cu extraction by alfalfa was observed in the EDDS-treated soil upon co-inoculation with the PGPR and rhizobium strains, which was 1.2 times higher than that without co-inoculation. Partial least squares path modeling identified plant oxidative damage and soil microbial biomass as the key variables influencing Cu uptake by alfalfa roots. Co-inoculation significantly reduced the oxidative damage to alfalfa by mitigating the accumulation of malondialdehyde and reactive oxygen species, and improving the antioxidation capacity of the plant in the presence of EDDS. EDDS application decreased microbial diversity in the rhizosphere, whereas co-inoculation increased microbial biomass carbon and nitrogen, and microbial community diversity. Increased relative abundances of Actinobacteria and Bacillus and the presence of Firmicutes taxa as potential biomarkers demonstrated that co-inoculation increased soil nutrient content, and improved plant growth. Co-inoculation with PGPR and rhizobium can be useful for altering plant-soil biochemical responses during EDDS-enhanced phytoremediation to alleviate phytotoxicity of heavy metals and improve soil biochemical activities. This study provides an effective strategy for improving phytoremediation efficiency and soil quality during chelant assisted phytoremediation of metal-contaminated soils., Competing Interests: Declaration of competing interest This study does not involve human subjects. This manuscript is original and it has not been previously published or submitted in part or whole. No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

Background and Aims: Patients with inflammatory bowel disease [IBD], especially Crohn's disease, often develop zinc deficiency. However, the precise mechanisms by which zinc deficiency affects IBD pathology, particularly intestinal macrophage function, remain unclear. We studied the effects of zinc deficiency on the development and progression of colitis in mice., Methods: To induce colitis, mice were treated with 2,4,6-trinitrobenzene sulphonic acid. Rag1-/- mice were then given injections of naïve CD4+CD62L+ T cells. The respective degrees of mucosal injury of mice that had received a zinc chelator (TPEN; N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) and of control mice were subsequently compared. Colonic lamina propria mononuclear cells were isolated by enzymatic digestion and were examined using flow cytometry. To generate mouse bone marrow-derived macrophages [BMDMs], bone marrow cells were stimulated with mouse macrophage-colony stimulating factor., Results: Zinc deficiency aggravates colonic inflammation through the activation of type 17 helper T [Th17] cells in mice. Flow cytometric analysis revealed that zinc deficiency significantly increases the proportion of pro-inflammatory [M1] macrophages in colonic lamina propria mononuclear cells obtained from inflamed colon. Interferon-γ plus lipopolysaccharide-mediated M1 skewing alters the expression of zinc transporters in BMDMs and thereby decreases the intracellular free zinc. TPEN treatment mimicking the effects of the M1 skewing up-regulates IL-23p19 expression, which is strongly related to Th17 development. Furthermore, the nuclear accumulation of interferon-regulatory factor 5 is closely involved in IL-23p19 induction in zinc-deficient macrophages., Conclusions: Zinc deficiency aggravates colonic inflammation through activation of the IL-23/Th17 axis. This activation is controlled by subcellular distribution of interferon-regulatory factor 5., (Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Nerve regeneration remains a challenge. Patient-derived induced pluripotent stem cell (iPSC)-differentiated neural stem cells (NSCs) provide a promising hope. Zinc is closely involved in central nervous system development and metabolism, but its role on iPSC neural differentiation is elusive and zinc detection methods in live cells are limited. In this study, intracellular zinc was detected in real time by a zinc fluorescent chemosensor and was shown to be increased during the iPSC neural induction process. iPSC neural differentiation was promoted with the addition of zinc chloride (ZnCl 2 ) and inhibited with the addition of zinc chelator N , N , N 0, N 0-tetrakis(2-pyridylmethyl)-ethylenediamine, indicated by western blot and enzyme-linked immunosorbent assay analysis of NSC marker Nestin expression and measurement of neurite-like structures. Mechanistically, the phosphorylation level of ERK1/2 and STAT3 was changed with the zinc level, suggesting that zinc may affect the neural differentiation of iPSCs through ERK-STAT signaling. In conclusion, our study shows the important role of zinc in iPSC neural differentiation and suggests a new idea for iPSC-derived NSC application in nerve regeneration.

Oxidative stress is one of the hallmarks of ischemic stroke. Catalase-based (CAT) biomimetic complexes are emerging as promising therapeutic candidates that are expected to act as neuroprotectants for ischemic stroke by decreasing the damaging effects from H 2 O 2 . Unfortunately, these molecules result in the unwanted production of the harmful hydroxyl radical, HO • . Here, we report a series of salen-based tri-manganese (Mn(III)) metallocryptands ( 1 - 3 ) that function as catalase biomimetics. These cage-like molecules contain a unique "active site" with three Mn centers in close proximity, an arrangement designed to facilitate metal cooperativity for the effective dismutation of H 2 O 2 with minimal HO • production. In fact, significantly greater oxygen production is seen for 1 - 3 as compared to the monomeric Mn(Salen) complex, 1c . The most promising system, 1 , was studied in further detail and found to confer a greater therapeutic benefit both in vitro and in vivo than the monomeric control system, 1c , as evident from inter alia studies involving a rat model of ischemic stroke damage and supporting histological analyses. We thus believe that metallocryptand 1 and its analogues represent a new and seemingly promising strategy for treating oxidative stress related disorders.

Zinc supplementation in cell culture has been shown to inhibit various viruses, like herpes simplex virus, rotavirus, severe acute respiratory syndrome (SARS) coronavirus, rhinovirus, and respiratory syncytial virus (RSV). However, whether zinc plays a direct antiviral role in viral infections and whether viruses have adopted strategies to modulate zinc homeostasis have not been investigated. Results from clinical trials of zinc supplementation in infections indicate that zinc supplementation may be beneficial in a pathogen- or disease-specific manner, further underscoring the importance of understanding the interaction between zinc homeostasis and virus infections at the molecular level. We investigated the effect of RSV infection on zinc homeostasis and show that RSV infection in lung epithelial cells leads to modulation of zinc homeostasis. The intracellular labile zinc pool increases upon RSV infection in a multiplicity of infection (MOI)-dependent fashion. Small interfering RNA (siRNA)-mediated knockdown of the ubiquitous zinc uptake transporter ZIP1 suggests that labile zinc levels are increased due to the increased uptake by RSV-infected cells as an antiviral response. Adding zinc to culture medium after RSV infection led to significant inhibition of RSV titers, whereas depletion of zinc by a zinc chelator, N , N , N ', N '-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) led to an increase in RSV titers. The inhibitory effect of zinc was specific, as other divalent cations had no effect on RSV titers. Both RSV infection and zinc chelation by TPEN led to reactive oxygen species (ROS) induction, whereas addition of zinc blocked ROS induction. These results suggest a molecular link between RSV infection, zinc homeostasis, and oxidative-stress pathways and provide new insights for developing strategies to counter RSV infection. IMPORTANCE Zinc deficiency rates in developing countries range from 20 to 30%, and zinc supplementation trials have been shown to correct clinical manifestations attributed to zinc deficiency, but the outcomes in the case of respiratory infections have been inconsistent. We aimed at understanding the role of zinc homeostasis in respiratory syncytial virus (RSV) infection. Infection of lung epithelial cell lines or primary small-airway epithelial cells led to an increase in labile zinc pools, which was due to increased uptake of zinc. Zinc supplementation inhibited RSV replication, whereas zinc chelation had an opposing effect, leading to increases in RSV titers. Increases in labile zinc in RSV-infected cells coincided with induction of reactive oxygen species (ROS). Both zinc depletion and addition of exogenous ROS led to enhanced RSV infection, whereas addition of the antioxidant inhibited RSV, suggesting that zinc is part of an interplay between RSV-induced oxidative stress and the host response to maintain redox balance., (Copyright © 2020 Khan et al.)

In previous studies we demonstrated that zinc stimulates iron uptake in intestinal Caco-2 cells via Zinc-PI3K-IRP2-DMT1 axis. In the current study we investigated the effect of zinc on basolateral iron release and characterized the associated mechanisms. In Caco-2 cells grown on permeable supports, zinc induced iron transport and expression of DMT1, HEPH mRNA and protein, but not that of FPN1. LY294002, an inhibitor of PI3K, inhibited the zinc-induced iron transport, DMT1, HEPH mRNA and protein expression. In addition, LY294002 also inhibited the basal expression of HEPH and FPN1 resulting in blockade of iron egress from cells. In addition, siRNA-silencing of HEPH led to inhibition of both zinc-induced and basal iron transport. Conversely, TPEN, a chelator of zinc, inhibited iron uptake, DMT1, HEPH and FPN1 mRNA and protein expression. These results suggest that intestinal cell zinc status is a critical determinant of iron absorption and effects are mediated via activation of PI3K. Further, PI3K pathway appears to selectively modulate the expression of iron transporters and iron absorption, therefore this might serve as a therapeutic target in iron overload disorders., Competing Interests: Declaration of competing interest The Authors declare that there are no competing interests associated with the manuscript., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Objective: Trichosporon asahii is the major causative fungus of disseminated or deep-seated trichosporonosis and forms a biofilm on medical devices. Biofilm formation leads to antifungal drug resistance, so biofilm-related infections are relatively difficult to treat and infected devices often require surgical removal. Therefore, prevention of biofilm formation is important in clinical settings. In this study, to identify metal cations that affect biofilm formation, we evaluated the effects of cation chelators on biofilm formation in T. asahii., Results: We evaluated the effect of cation chelators on biofilm formation, since microorganisms must assimilate essential nutrients from their hosts to form and maintain biofilms. The inhibition by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) was greater than those by other cation chelators, such as deferoxamine, triethylenetetramine, and ethylenediaminetetraacetic acid. The inhibitory effect of TPEN was suppressed by the addition of zinc. TPEN also inhibited T. asahii hyphal formation, which is related to biofilm formation, and the inhibition was suppressed by the addition of zinc. These results suggest that zinc is essential for biofilm formation and hyphal formation. Thus, zinc chelators have the potential to be developed into a new treatment for biofilm-related infection caused by T. asahii.

A series of aminocarboxylic acid analogues of aspergillomarasmine A (AMA) and ethylenediamine-N,N'-disuccinic acid (EDDS) were chemoenzymatically synthesized via the addition of various mono- and diamine substrates to fumaric acid catalyzed by the enzyme EDDS lyase. Many of these novel AMA and EDDS analogues demonstrate potent inhibition of the bacterial metallo-β-lactamase NDM-1. Isothermal titration calorimetry assays revealed a strong correlation between the inhibitory potency of the compounds and their ability to bind zinc. Compounds 1a (AMA), 1b (AMB), 5 (EDDS), followed by 1d and 8a, demonstrate the highest synergy with meropenem resensitizing an NDM-1 producing strain of E. coli to this important carbapenem of last resort.

Zinc was discovered to be a novel second messenger in immunoreactive cells. We synthesized a novel free zinc chelator, IPZ-010. Here, we investigated the effects of IPZ-010 in a mouse postoperative ileus model and determined the effects of zinc signal inhibition as a new therapeutic strategy against postoperative ileus. Zinc waves were measured in bone marrow-derived mast cells (BMMCs) loaded with a zinc indicator, Newport green. Degranulation and cytokine expression were measured in BMMCs and bone marrow-derived macrophages (BMDMs). Postoperative ileus model mice were established with intestinal manipulation. Mice were treated with IPZ-010 (30 mg/kg, s.c. or p.o.) 1 h before and 2 h and 4 h after intestinal manipulation. Gastrointestinal transit, inflammatory cell infiltration, and expression of inflammatory mediators were measured. Free zinc waves occurred following antigen stimulation in BMMCs and were blocked by IPZ-010. IPZ-010 inhibited interleukin-6 secretion and degranulation in BMMCs. IPZ-010 inhibited tumor necrosis factor-α mRNA expression in BMMCs stimulated with lipopolysaccharide or adenosine triphosphate, whereas IPZ-010 had no effects on tumor necrosis factor-α mRNA expression in BMDMs stimulated with lipopolysaccharide or adenosine triphosphate. In postoperative ileus model mice, IPZ-010 inhibited leukocyte infiltration and cytokine expression, which ameliorated gastrointestinal transit. Furthermore, ketotifen (1 mg/kg) induced similar effects as IPZ-010. These effects were not amplified by co-administration of IPZ-010 and ketotifen. IPZ-010 inhibited zinc waves, resulting in inhibition of inflammatory responses in activated BMMCs in vitro. Targeting zinc waves in inflammatory cells may be a novel therapeutic strategy for treating postoperative ileus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier Masson SAS.)

Although metallothionein-3 (MT3), a brain-enriched form of metallothioneins, has been linked to Alzheimer's disease, little is known regarding the role of MT3 in glioma. As MT3 plays a role in autophagy in astrocytes, here, we investigated its role in irradiated glioma cells. Irradiation increased autophagy flux in GL261 glioma cells as evidenced by increased levels of LC3-II but decreased levels of p62 (SQSTM1). Indicating that autophagy plays a cytoprotective role in glioma cell survival following irradiation, measures inhibiting autophagy flux at various steps decreased their clonogenic survival of irradiated GL261 as well as SF295 and U251 glioma cells. Knockdown of MT3 with siRNA in irradiated glioma cells induced arrested autophagy, and decreased cell survival. At the same time, the accumulation of labile zinc in lysosomes was markedly attenuated by MT3 knockdown. Indicating that such zinc accumulation was important in autophagy flux, chelation of zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), induced arrested autophagy in and reduced survival of GL261 cells following irradiation. Suggesting a possible mechanism for arrested autophagy, MT3 knockdown and zinc chelation were found to impair lysosomal acidification. Since autophagy flux plays a cytoprotective role in irradiated glioma cells, present results suggest that MT3 and zinc may be regarded as possible therapeutic targets to sensitize glioma cells to ionizing radiation therapy.

Leishmania donovani is the causative agent of visceral leishmaniasis. Annually, 500 million new cases of infection are reported mainly in poor communities, decreasing the interest of the pharmaceutical industries. Therefore, the repositioning of new drugs is an ideal strategy to fight against these parasites. SQ109, a compound in phase IIb/III of clinical trials to treat resistant Mycobacterium tuberculosis, has a potent effect against Trypanosoma cruzi, responsible for Chagas' disease, and on Leishmania mexicana, the causative agent of cutaneous and muco-cutaneous leishmaniasis. In the latter, the toxic dose against intramacrophagic amastigotes is very low (IC 50 ~ 11 nM). The proposed mechanism of action on L. mexicana involves the disruption of the parasite intracellular Ca 2+ homeostasis through the collapse of the mitochondrial electrochemical potential (ΔΨ m ). In the present work, we show a potent effect of SQ109 on L. donovani, the parasite responsible for visceral leishmaniasis, the more severe and uniquely lethal form of these infections, obtaining a toxic effect on amastigotes inside macrophages even lower to that obtained in L. mexicana (IC 50 of 7.17 ± 0.09 nM) and with a selectivity index > 800, even higher than in L. mexicana. We also demonstrated for first time that SQ109, besides collapsing ΔΨ m of the parasite, induced a very rapid damage to the parasite acidocalcisomes, essential organelles involved in the bioenergetics and many other important functions, including Ca 2+ homeostasis. Both effects of the drug on these organelles generated a dramatic increase in the intracellular Ca 2+ concentration, causing parasite death.