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Abstract Objective Haploinsufficiency is widely accepted as the pathogenic mechanism of hereditary spastic paraplegias type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. The aim of this study was to identify the causative variant of SPG4 in a large Chinese family and explore its pathological mechanism. Materials and methods A five-generation family with 49 members including nine affected (4 males and 5 females) and 40 unaffected individuals in Mongolian nationality was recruited. Whole exome sequencing was employed to investigate the genetic etiology. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. Results A novel frameshift variant NM_014946.4: c.483_484delinsC (p.Val162Leufs*2) was identified in SPAST from a pedigree with SPG4. The variant segregated with the disease in the family and thus determined as the disease-causing variant. The c.483_484delinsC variant produced two truncated mutants (mutant M1 and M87 isoforms). They accumulated to a higher level and presented increased stability than their wild-type counterparts and may lost the microtubule severing activity. Conclusion SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The potential toxicity to the corticospinal tract caused by the intracellular accumulation of truncated spastin should be considered as the pathological mechanism of SPG4.

Objective: Haploinsufficiency is widely accepted as the pathogenic mechanism of hereditary spastic paraplegias type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. The aim of this study was to identify the causative variant of SPG4 in a large Chinese family and explore its pathological mechanism. Materials and methods: A five-generation family with 49 members including nine affected (4 males and 5 females) and 40 unaffected individuals in Mongolian nationality was recruited. Whole exome sequencing was employed to investigate the genetic etiology. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. Results: A novel frameshift variant NM_014946.4: c.483_484delinsC (p.Val162Leufs*2) was identified in SPAST from a pedigree with SPG4. The variant segregated with the disease in the family and thus determined as the disease-causing variant. The c.483_484delinsC variant produced two truncated mutants (mutant M1 and M87 isoforms). They accumulated to a higher level and presented increased stability than their wild-type counterparts and may lost the microtubule severing activity. Conclusion: SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The potential toxicity to the corticospinal tract caused by the intracellular accumulation of truncated spastin should be considered as the pathological mechanism of SPG4. [ABSTRACT FROM AUTHOR]

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Drug delivery to the retina is one of the major challenges in ophthalmology due to the biological barriers that protect it from harmful substances in the body. Despite the advancement in ocular therapeutics, there are many unmet needs for the treatment of retinal diseases. Ultrasound combined with microbubbles (USMB) was proposed as a minimally invasive method for improving delivery of drugs in the retina from the blood circulation. This study aimed to investigate the applicability of USMB for the delivery of model drugs (molecular weight varying from 600 Da to 20 kDa) in the retina of ex vivo porcine eyes. A clinical ultrasound system, in combination with microbubbles approved for clinical ultrasound imaging, was used for the treatment. Intracellular accumulation of model drugs was observed in the cells lining blood vessels in the retina and choroid of eyes treated with USMB but not in eyes that received ultrasound only. Specifically, 25.6 ± 2.9% of cells had intracellular uptake at mechanical index (MI) 0.2 and 34.5 ± 6.0% at MI 0.4. Histological examination of retinal and choroid tissues revealed that at these USMB conditions, no irreversible alterations were induced at the USMB conditions used. These results indicate that USMB can be used as a minimally invasive targeted means to induce intracellular accumulation of drugs for the treatment of retinal diseases. [ABSTRACT FROM AUTHOR]

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3′ and 5′ ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2′-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central “window” of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.

A series of novel cobalt bis(dicarbollide)—curcumin conjugates were synthesized. Two conjugates were obtained through the nucleophilic ring-opening reaction of the 1,4-dioxane and tetrahydropyran derivatives of cobalt bis(dicarbollide) with the OH group of curcumin, and using two equiv. of the oxonium derivatives, two other conjugates containing two cobalt bis(dicarbollide) units per molecule were obtained. In contrast to curcumin, the conjugates obtained were found to be non-cytotoxic against both tumor and normal cell lines. The analysis of the intracellular accumulation of the conjugates by flow cytometry showed that all cobalt bis(dicarbollide)—curcumin conjugates entered HCT116 colorectal carcinoma cells in a time-dependent manner. New non-cytotoxic conjugates contain a large amount of boron atoms in the biomolecule and can potentially be used for further biological research into boron neutron capture therapy (BNCT). [ABSTRACT FROM AUTHOR]

Compromised activity is a common impediment for biologics requiring endosome trafficking into target cells. In cancer cells, antibody-drug conjugates (ADCs) are trapped in endosomes or subsequently pumped extracellularly, leading to a reduction in intracellular accumulation. In subsets of dendritic cells (DCs), endosome-engulfed antigens face non-specific proteolysis and collateral damage to epitope immunogenicity before proteasomal processing and subsequent surface presentation. To bypass these shortcomings, we devised Accum™, a conjugable biotechnology harboring cholic acid (ChAc) and a nuclear localization signal (NLS) sequence for endosome escape and prompt nuclear targeting. Combined, these mechanisms culminate in enhanced intracellular accumulation and functionalization of coupled biologics. As proof-of-principle, we have biochemically characterized Accum, demonstrating its adaptability to ADCs or antigens in different cancer settings. Additionally, we have validated that endosome escape and nuclear routing are indispensable for effective intracellular accumulation and guaranteed target cell selectivity. Importantly, we have demonstrated that the unique mechanism of action of Accum translates into enhanced tumor cytotoxicity when coupled to ADCs, and durable therapeutic and prophylactic anti-cancer immunogenicity when coupled to tumor antigens. As more pre-clinical evidence accumulates, the adaptability, unique mechanism of action, and high therapeutic potency of Accum signal a promising transition into clinical investigations in the context of onco-immunotherapy. [ABSTRACT FROM AUTHOR]

Drug delivery to the retina is one of the major challenges in ophthalmology due to the biological barriers that protect it from harmful substances in the body. Despite the advancement in ocular therapeutics, there are many unmet needs for the treatment of retinal diseases. Ultrasound combined with microbubbles (USMB) was proposed as a minimally invasive method for improving delivery of drugs in the retina from the blood circulation. This study aimed to investigate the applicability of USMB for the delivery of model drugs (molecular weight varying from 600 Da to 20 kDa) in the retina of ex vivo porcine eyes. A clinical ultrasound system, in combination with microbubbles approved for clinical ultrasound imaging, was used for the treatment. Intracellular accumulation of model drugs was observed in the cells lining blood vessels in the retina and choroid of eyes treated with USMB but not in eyes that received ultrasound only. Specifically, 25.6 ± 2.9% of cells had intracellular uptake at mechanical index (MI) 0.2 and 34.5 ± 6.0% at MI 0.4. Histological examination of retinal and choroid tissues revealed that at these USMB conditions, no irreversible alterations were induced at the USMB conditions used. These results indicate that USMB can be used as a minimally invasive targeted means to induce intracellular accumulation of drugs for the treatment of retinal diseases.

The combination of ultrasound and microbubbles (USMB) has been applied to enhance drug permeability across tissue barriers. Most studies focused on only one physicochemical aspect (i.e., molecular weight of the delivered molecule). Using an in vitro epithelial (MDCK II) cell barrier, we examined the effects of USMB on the permeability of five molecules varying in molecular weight (182 Da to 20 kDa) and hydrophilicity (LogD at pH 7.4 from 1.5 to highly hydrophilic). Treatment of cells with USMB at increasing ultrasound pressures did not have a significant effect on the permeability of small molecules (molecular weight 259 to 376 Da), despite their differences in hydrophilicity (LogD at pH 7.4 from −3.2 to 1.5). The largest molecules (molecular weight 4 and 20 kDa) showed the highest increase in the epithelial permeability (3-7-fold). Simultaneously, USMB enhanced intracellular accumulation of the same molecules. In the case of the clinically relevant anti- C-X-C Chemokine Receptor Type 4 (CXCR4) nanobody (molecular weight 15 kDa), USMB enhanced paracellular permeability by two-fold and increased binding to retinoblastoma cells by five-fold. Consequently, USMB is a potential tool to improve the efficacy and safety of the delivery of drugs to organs protected by tissue barriers, such as the eye and the brain. [ABSTRACT FROM AUTHOR]

The synthesis of various unique secondary metabolites by lichens is the result of mutualistic symbiotic association between the mycobiont and autotrophic photobiont. The function of these compounds and causal factors for their production are not fully understood. This paper examines the effect of heavy-metal bioaccumulation and physiological parameters related to photosynthesis and carbon metabolism on the production of lichen substances in hyperaccumulator Diploschistes muscorum. The obtained model of secondary metabolite concentrations in the thalli demonstrates that the carbon source provided by the photobiont and associated polyols produced by the mycobiont have positive impact on the production; on the contrary, the increased intracellular load of heavy metals and excessive loss of cell membrane integrity adversely affected secondary metabolite contents. Additionally, the production of secondary metabolites appears to be more dependent on intracellular metal concentrations than on soil pollution level. To compensate for metal stress, both efficient functioning of algal component and sufficient production of secondary metabolites are required. The balanced physiological functioning of mycobiont and photobiont constitutes the complex protective mechanism to alleviate the harmful effects of heavy metal stress on primary and secondary metabolism of lichens. • Diploschistes muscorum has a complex adaptation strategy to heavy-metal stress. • Sugar alcohols have positive impact on lichen secondary metabolite production. • Intracellular load of heavy metals adversely affected secondary metabolite contents. • Efficient photosynthesis and secondary metabolism compensate for metal stress. [ABSTRACT FROM AUTHOR]

ABSTRACT The therapeutic repertoire for tuberculosis (TB) remains limited despite the existence of many TB drugs that are highly active in in vitro models and possess clinical utility. Underlying the lack of efficacy in vivo is the inability of TB drugs to penetrate microenvironments inhabited by the causative agent, Mycobacterium tuberculosis, including host alveolar macrophages. Here, we determined the ability of the phenoxazine PhX1 previously shown to be active against M. tuberculosis in vitro to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. We also investigated the extent of permeation into uninfected and M. tuberculosis-infected human macrophage-like Tamm-Horsfall protein 1 (THP-1) cells directly and by comparing to results obtained in vitro in synergy assays. Our data indicate that PhX1 (4,750 ± 127.2 ng/ml) penetrates more effectively into THP-1 cells than do the clinically used anti-TB agents, rifampin (3,050 ± 62.9 ng/ml), moxifloxacin (3,374 ± 48.7 ng/ml), bedaquiline (4,410 ± 190.9 ng/ml), and linezolid (770 ± 14.1 ng/ml). Compound efficacy in infected cells correlated with intracellular accumulation, reinforcing the perceived importance of intracellular penetration as a key drug property. Moreover, we detected synergies deriving from redox-stimulatory combinations of PhX1 or clofazimine with the novel prenylated amino-artemisinin WHN296. Finally, we used compound synergies to elucidate the relationship between compound intracellular accumulation and efficacy, with PhX1/WHN296 synergy levels shown to predict drug efficacy. Collectively, our data support the utility of the applied assays in identifying in vitro active compounds with the potential for clinical development. IMPORTANCE This study addresses the development of novel therapeutic compounds for the eventual treatment of drug-resistant tuberculosis. Tuberculosis continues to progress, with cases of Mycobacterium tuberculosis (M. tuberculosis) resistance to first-line medications increasing. We assess new combinations of drugs with both oxidant and redox properties coupled with a third partner drug, with the focus here being on the potentiation of M. tuberculosis-active combinations of compounds in the intracellular macrophage environment. Thus, we determined the ability of the phenoxazine PhX1, previously shown to be active against M. tuberculosis in vitro, to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. In addition, the extent of permeation into human macrophage-like THP-1 cells and H37Rv-infected THP-1 cells was measured via mass spectrometry and compared to in vitro two-dimensional synergy and subsequent intracellular efficacy. Collectively, our data indicate that development of new drugs will be facilitated using the methods described herein.

G-quadruplex (G4) ligands attract considerable attention as potential anticancer therapeutics. In this study we proposed an original scheme for synthesis of azole-fused anthraquinones and prepared a series of G4 ligands carrying amino- or guanidinoalkylamino side chains. The heterocyclic core and structure of the terminal groups strongly affect on binding to G4-forming oligonucleotides, cellular accumulation and antitumor potency of compounds. In particular, thiadiazole- and selenadiazole- but not triazole-based ligands inhibit the proliferation of tumor cells (e.g. K562 leukemia) and stabilize primarily telomeric and c-MYC G4s. Anthraselenadiazole derivative 11a showed a good affinity to c-MYC G4 in vitro and down-regulated expression of c-MYC oncogene in cellular conditions. Further studies revealed that anthraselenadiazole 11a provoked cell cycle arrest and apoptosis in a dose- and time-dependent manner inhibiting K562 cells growth. Taken together, this work gives a valuable example that the closely related heterocycles may cause a significant difference in biological properties of G4 ligands. [Display omitted] • A method for synthesis of azole-fused anthraquinone derivatives has been developed. • Selenadiazole- and thiadiazole-based ligands effectively stabilize G4s. • Guanidination of the aminoalkyl side chains increases the thermal shifts of G4s melting. • In vitro anticancer activity of the ligands correlates to G4s binding and intracellular uptake. [ABSTRACT FROM AUTHOR]

A series of novel cobalt bis(dicarbollide)—curcumin conjugates were synthesized. Two conjugates were obtained through the nucleophilic ring-opening reaction of the 1,4-dioxane and tetrahydropyran derivatives of cobalt bis(dicarbollide) with the OH group of curcumin, and using two equiv. of the oxonium derivatives, two other conjugates containing two cobalt bis(dicarbollide) units per molecule were obtained. In contrast to curcumin, the conjugates obtained were found to be non-cytotoxic against both tumor and normal cell lines. The analysis of the intracellular accumulation of the conjugates by flow cytometry showed that all cobalt bis(dicarbollide)—curcumin conjugates entered HCT116 colorectal carcinoma cells in a time-dependent manner. New non-cytotoxic conjugates contain a large amount of boron atoms in the biomolecule and can potentially be used for further biological research into boron neutron capture therapy (BNCT).

The combination of ultrasound and microbubbles (USMB) has been applied to enhance drug permeability across tissue barriers. Most studies focused on only one physicochemical aspect (i.e., molecular weight of the delivered molecule). Using an in vitro epithelial (MDCK II) cell barrier, we examined the effects of USMB on the permeability of five molecules varying in molecular weight (182 Da to 20 kDa) and hydrophilicity (LogD at pH 7.4 from 1.5 to highly hydrophilic). Treatment of cells with USMB at increasing ultrasound pressures did not have a significant effect on the permeability of small molecules (molecular weight 259 to 376 Da), despite their differences in hydrophilicity (LogD at pH 7.4 from −3.2 to 1.5). The largest molecules (molecular weight 4 and 20 kDa) showed the highest increase in the epithelial permeability (3-7-fold). Simultaneously, USMB enhanced intracellular accumulation of the same molecules. In the case of the clinically relevant anti- C-X-C Chemokine Receptor Type 4 (CXCR4) nanobody (molecular weight 15 kDa), USMB enhanced paracellular permeability by two-fold and increased binding to retinoblastoma cells by five-fold. Consequently, USMB is a potential tool to improve the efficacy and safety of the delivery of drugs to organs protected by tissue barriers, such as the eye and the brain.

Heavy metal resistant bacteria are of great interest because of their potential use in bioremediation. Understanding the survival and adaptive strategies of these bacteria under heavy metal stress is important for better utilization of these bacteria in remediation. The objective of this study was to investigate the role of bacterial extracellular polymeric substance (EPS) in detoxifying against different heavy metals in Bacillus sp. S3, a new hyper antimony-oxidizing bacterium previously isolated from contaminated mine soils. The results showed that Bacillus sp. S3 is a multi-metal resistant bacterial strain, especially to Sb(III), Cu(II) and Cr(VI). Toxic Cd(II), Cr(VI) and Cu(II) could stimulate the secretion of EPS in Bacillus sp. S3, significantly enhancing the adsorption and detoxification capacity of heavy metals. Both Fourier transform infrared spectroscopy (FTIR) and three-dimensional excitation–emission matrix (3D-EEM) analysis further confirmed that proteins were the main compounds of EPS for metal binding. In contrast, the EPS production was not induced under Sb(III) stress. Furthermore, the TEM–EDX micrograph showed that Bacillus sp. S3 strain preferentially transported the Sb(III) to the inside of the cell rather than adsorbed it on the extracellular surface, indicating intracellular detoxification rather than extracellular EPS precipitation played an important role in microbial resistance towards Sb(III). Together, our study suggests that the toxicity response of EPS to heavy metals is associated with difference in EPS properties, metal types and corresponding environmental conditions, which is likely to contribute to microbial-mediated remediation. [ABSTRACT FROM AUTHOR]

Exposure is a critically important aspect to consider in the study and management of drug-induced kidney injury. Although blood concentrations of kidney toxicants often may provide a valid surrogate measure of kidney exposure, the kidney has several unique physiological and biochemical properties that lend themselves to accumulation or exclusion of some drugs at sites of toxicity. In such cases, an understanding of these pharmacokinetic mechanisms can be as important as understanding the underlying mechanisms of toxicity. Physiologically based pharmacokinetic models, which mathematically codify such mechanisms in a biologically plausible form, increasingly are being used for developing an understanding of pharmacokinetics across patient populations, drug-drug interactions, and pharmacokinetic-pharmacodynamic relationships. This perspective provides a review of the physiological and biochemical mechanisms as well as the physiochemical properties that theoretically could drive drug accumulation or exclusion within the kidney, along with examples in which these mechanisms have proven important in driving the manifestation of toxicity in vivo. In addition, an overview of the structure, applications, and limitations of existing kidney physiologically based pharmacokinetic models is provided. Finally, a perspective on gaps and associated challenges to such models in the field of toxicology is discussed briefly. [ABSTRACT FROM AUTHOR]

The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1–42 (oAβ1–42) using our donor–acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1–42, 3(pE)-40, 1–40 and 11–42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1–42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1–42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.

A novel strategy was developed to identify hepatotoxic compounds in traditional Chinese medicines (TCMs). It is based on the exposure of HL-7702 cells to a TCM extract, followed by the identification and further determination of potential hepatotoxic compounds accumulated in the cells by liquid chromatography−tandem mass spectrometry (LC−MS/MS). As a case study, potential hepatotoxic components in Chelidonium majus L. were screened out. Five alkaloids (sanguinarine, coptisine, chelerythrine, protopine, and chelidonine) were identified by LC−MS/MS within 10 min, and their intracellular concentrations were first simultaneously measured by LC−MS/MS with a run time of 4 min. A cell viability assay was performed to assess the cytotoxicity of each alkaloid. With their higher intracellular concentrations, sanguinarine, coptisine, and chelerythrine were identified as the main hepatotoxic constituents in Ch. majus. The study provides a powerful tool for the fast prediction of cytotoxic components in complex natural mixtures on a high-throughput basis.

ABSTRACT Aim Nitidine (NTD) has been reported to specifically inhibit the growth of human A549 non-small-cell lung cancer cells via selective intracellular accumulation. Our recent study showed that NTD preferentially accumulates in camptothecin-resistant A549 cells (CRC) compared with normal A549 cells (nA549) in vitro. Here, we examined the in vivo anti-tumor effects of NTD on CRC-derived tumors and oral treatment on nA549 using a xenograft mouse model. Methods Cancer cells were intradermally inoculated into the back of mice. Then, 100 µg NTD was injected i.p. into the nA549- or CRC-derived tumor-bearing mice. To confirm the anti-tumor effects of oral NTD, the nA549-transplanted mice were fed a diet containing NTD concentrate prepared from Toddalia asiatica Lam. Results The anti-tumor effect of NTD (i.p.) manifested earlier in CRC-derived tumors than in the A549-derived tumors. Oral NTD concentrate also significantly inhibited the growth of the nA549-derived tumors. NTD was detected in the tumor but not in other tissues in the NTD concentrate-fed mice. Conclusion CRC-derived tumor was more susceptible to NTD treatment than nA549-derived tumor. Furthermore, the anti-tumor effect of oral NTD and its preferential accumulation in tumor tissues have been demonstrated for the first time. This may be useful for the development of safe anti-cancer drugs. [ABSTRACT FROM AUTHOR]

ABSTRACT Aim Drug resistance has been a major issue in cancer chemotherapy. It is therefore important to screen more potent natural anti-cancer agents for use against the drug-resistant cancer phenotype. The present study investigated the cytotoxicity of the traditional and natural medicinal agent nitidine ( NTD) against the drug-resistant cancer phenotype of A549 human lung adenocarcinoma cells. Methods The camptothecin ( CPT) tolerant phenotype was selected after A549 cells were exposed to CPT for 1 week. The cytotoxicity of NTD in these CPT-resistant cells ( CRC) was studied in vitro in relation to its accumulation and expression of the ATP binding cassette ( ABC) transporter gene. Results CRC expressing higher levels of ABCC1, ABCC2, ABCC3, and ABCA1 were more resistant to CPT compared with normal A549 cells. CRC were, however, more susceptible to NTD compared with normal A549 cells with preferential accumulation of NTD. A statistically significant difference in protein level between normal A549 and CRC was noted only for ABCC1 and ABCC2. Furthermore, gene expression of ABCC2 and ABCC3 had a positive correlation with NTD accumulation, and negative correlation with the effective dose that inhibited 50% of cell growth ( D50) of this agent in nine cancer cell panels. Conclusion CRC were found to be more susceptible to NTD compared with the normal phenotype. The cytotoxicity of NTD was associated with its accumulation and the expression of ABCC2, suggesting the involvement of this transporter in the selective incorporation of NTD into cancer cells, resulting in enhanced cytotoxicity. [ABSTRACT FROM AUTHOR]

The relationship between the lipophilicity, cytotoxic effects, and intracellular accumulation for potential antitumor agents, viz., amino complexes of platinum(IV) with the redox-active nitroxyl radicals cis, trans, cis-Pt(RNH)(NH)(OCOR´)Cl (R is 2,2,6,6-tetramethyl-1-oxylpiperidin-4-yl, OCOR´ are n-alkylcarboxylates C-C), was studied in comparison with cisplatin. In a series of four platinum nitroxyl complexes (PNCs), an increase in the lipophilicity was found to lead to a considerable increase in the cytotoxicity and the efficiency of accumulation in HeLa cells. However, PNCs are characterized by the slower development of cytotoxic effects after a short time exposure compared with hydrophilic cisplatin. This can be attributed to the fact that Pt complexes are prodrugs and need to be reduced to chemically more active Pt derivatives in order to exhibit cytotoxic effects. [ABSTRACT FROM AUTHOR]

Highlights: [•] Growing cells altered bioaccumulation mechanism under different Cd(II) concentrations. [•] Cell morphology modifications played an important role under higher concentrations. [•] Carboxyl groups were mainly involved in the Cd(II) binding. [•] The efflux mechanism was responsible for reducing intracellular Cd(II) contents. [•] Growing cells had high potential for the Cd(II) bioremediation. [Copyright &y& Elsevier]

Abstract: The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1–42 (oAβ1–42) using our donor–acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1–42, 3(pE)-40, 1–40 and 11–42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1–42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1–42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD. [Copyright &y& Elsevier]

Effective bio-removal of heavy metals is important for water treatment. Although a number of microorganism species demonstrated the ability of living cells to remove cadmium, most of them were tested at fixed concentration of metals, salinity, and temperature. This paper reported a research on the screening and performance of a newly developed deep-sea bacterium, Pseudoalteromonas sp. SCSE709-6, for Cd(II) removal by growing cells under a range of experimental conditions: 0-50 mg/L of Cd(II), 15-30 °C of incubation temperatures, 6.5-8.0 of initial pH, and 1.5-5.0 % of salinity. Study results revealed that Pseudoalteromonas sp. SCSE709-6 could remove more than 96 % of Cd(II) on growth. The Cd(II) bioremoval was in correlation but not in accordance with biomass. As cadmium concentrations increased, the Cd(II) removal by cell adsorption played an increasingly important role compared with that of intracellular accumulation. For the removal mechanism, Fourier transform infrared spectroscopy revealed that carboxyl, amido and hydroxyl of saccharides, and proteins in the extracellular polymeric substances are the most active groups for Cd(II) absorption. The bacterium reported in this study offers a new microbe strain for Cd(II) bioremediation. [ABSTRACT FROM AUTHOR]

The molecular basis of Alzheimer's disease has not been clearly established, but disruption of brain metal ion homeostasis, particularly copper and zinc, might be closely involved in the pathogenesis of this disease and its characteristic β-amyloid neuropathological features. The use of complexes of copper with bis(thiosemicarbazones) ([Cu(btsc)]) has been proposed for the treatment of Alzheimer's disease. Their mode of action could involve modulation of the concentration of copper or zinc, and it has been suggested that the compounds can modulate the production of β-amyloid peptide at the neuron level. Furthermore, it has been reported that [Cu(btsc)] complexes can be reduced inside the cells. However, to our knowledge the intracellular reduction of these compounds has never been demonstrated. Thus, the goal of our study was to increase understanding of the mechanism of intracellular accumulation of [Cu(btsc)] complexes. Our results reveal that the intracellular concentration of copper inside the cells is very high and that these compounds are not P-glycoprotein substrates. This protein is a key element of the low permeability properties of the blood-brain barrier. Furthermore, no intracellular reduction of cupric ions was detected. Finally, once inside the cells, the complexes undergo aggregation, strongly suggesting that aggregation of complexes is the driving force responsible for their intracellular accumulation. [ABSTRACT FROM AUTHOR]

Abstract: Heavy metal pollution has become a major environmental concern nowadays and the bioremediation of polluted habitats is an increasingly popular strategy due to both its efficiency and safety. A screening and selection protocol based on different composting processes was designed in order to isolate heavy metal-resistant microorganisms. A collection of 51 microorganisms was obtained and most of them showed the capability to tolerate heavy metals in multi-polluted aqueous systems (Cd(II), Cr(VI), Ni, Pb, Zn(II)), as well as to remove them. The highest detoxification ratios were observed for Pb. Some of the isolates detoxifying more than a 90% of this metal, while the other metals were removed in a range between 20% and 60%. The best isolates (Graphium putredinis, Fusarium solani, Fusarium sp. and Penicillium chrysogenum) were further assayed in order to determine the predominant removal mechanism and the potential use of their dead biomass as a biosorbent. Intracellular accumulation was the prevalent mechanism for most isolates and metals, with the exception of Ni. In this case, the proportion removed by extracellular adsorption was similar or even higher than that removed by intracellular accumulation. Thus, the efficiency of living cells was higher than that of dead biomass except in the case of Ni. [Copyright &y& Elsevier]

Abstract: Lithium is licensed for the treatment of bipolar disorders and also discussed in relation to neuroprotective properties. Although the drug has a small therapeutic window, its uptake and passage into the brain are poorly understood. We administered lithium to rats, following an acute (3mmol/kg, i.p.) or chronic (3mmol/kg/day, p.o.) regime. Lithium levels were assessed in serum, brain homogenate, cerebrospinal fluid (CSF) and, by means of microdialysis, in the extracellular space (ECS) of the brain 2, 6 and 24h post injection or after 3 weeks of chronic administration. Lithium is detected in brain ECS within minutes of administration and reaches maximum levels in brain extracellular fluid after 30min. In the early phase after lithium administration (2 and 6h), serum levels of lithium do not differ significantly from those assessed in CSF and brain homogenate. Afterwards, however, accumulation in brain tissue occurs. As a consequence, after 24h and 3 weeks, lithium levels in brain homogenate (i.e., intracellular levels) are significantly higher than in CSF or dialysates (i.e., extracellular levels). In conclusion, lithium rapidly reaches the brain, but after prolonged treatment, brain intracellular levels are high and poorly represented by plasma or CSF measurements. [Copyright &y& Elsevier]

Arsenic (As) is a metalloid and considered harmful due to its toxic and carcinogenic effects. Removal of arsenic is of great importance for human welfare. The main objective of this study was to isolate arsenic-resistant bacteria that are capable of removing arsenic from the environment. Soil samples were collected from an arsenic-affected area of West Bengal, India and 10 different bacterial strains were isolated. The minimum inhibitory concentration (MIC) values of the isolates varied widely in the range 50–125 mM (As) as arsenate and 10–100 mM (As) as arsenite. TEM and EDAX analysis were done to confirm intracellular accumulation of arsenic. The 16s RNA and phylogenetic analysis showed that seven isolates belonged to γ-proteobacterium, two isolates belonged to Firmicutes and one was identified as Kocuria genera. Some of these bacteria could oxidize arsenite to arsenate and all others could reduce arsenate to arsenite. The growth pattern of the bacterial strains in presence and absence of arsenic was also observed. All the 10 isolates exhibited multiple heavy metal (like Ni, Zn, Cu, Pb, Co, etc.) tolerances. Thus, these new bacterial strains could conveniently be used for bioremediation of soil and effluents and the enzymes produced by them may be used for commercial exploitation. [ABSTRACT FROM PUBLISHER]

Abstract: The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug’s therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived γ-tocotrienol, the extra-virgin olive oil-derived secoiridoid oleocanthal, and the triterpene acid asiatic acid derived from Melaleuca ericifolia and abundant in several other common plant dietary supplements. Treatment with 25μM of cembratriene, oleocanthal, γ-tocotrienol, or asiatic acid showed 2.3–3.0-fold increase in P-gp expression as demonstrated by Western blotting. These results were consistent with those obtained by quantitative analysis of fluorescent micrographs for P-gp. Accumulation studies demonstrated 31–38% decrease in rhodamine 123 intracellular levels when LS-180 cells were treated with the investigated compounds as a result of P-gp induction. Bioactive natural products can up-regulate the P-gp expression and functionality, which may induce herb/food-drug interactions when concomitantly used with P-gp substrate drugs. [Copyright &y& Elsevier]

Apolipoprotein E (apoE) plays a crucial role in lipid transport in circulation and the brain. The apoE4 isoform is a major risk factor for Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than other apoE isoforms and apoE4 fragments have been found in brains of AD patients. These apoE4 fragments have been hypothesized to be involved in the pathogenesis of AD, although the mechanism is not clear. In this study we examined the effect of lipid-free apoE4 on amyloid precursor protein processing and 40-amino-acid Aβ variant and 42-amino-acid Aβ variant levels in human neuroblastoma SK-N-SH cells. We discovered that a specific apoE4 fragment, apoE4[Δ(166-299)], can promote the cellular uptake of extracellular 40-amino-acid Ab variant and 42-amino-acid Aβ variant either generated after amyloid precursor protein transfection or added exogenously. A longer length fragment, apoE4[Δ(186-299)], or full-length apoE4 failed to elicit this effect. ApoE4[Δ(166-299)] effected a 20% reduction of cellular sphingomyelin levels, as well as changes in cellular membrane micro-fluidity. Following uptake, approximately 50% of 42-amino-acid Aβ variant remained within the cell for at least 24 h, and led to increased formation of reactive oxygen species. Overall, our findings suggest a direct link between two early events in the pathogenesis of AD, apoE4 proteolysis and intraneuronal presence of amyloid beta peptide. [ABSTRACT FROM AUTHOR]

Abstract: Human Down syndrome (DS) represents the most frequent cause of mental retardation associated to a genetic condition. DS also exhibits a characteristic early onset of neuropathology indistinguishable from that observed in Alzheimer''s disease (AD), namely the deposition of the β-amyloid peptide. Early endosomal dysfunction has been described in individuals with DS and AD, suggesting an important role of this subcellular compartment in the onset and progression of the pathology. On the other hand, cholesterol activates the amyloidogenic processing pathway for the amyloid precursor protein, and the lipoprotein receptor-related peptide interacts with the β-amyloid peptide. In the present work, using cell lines derived from the cortex of both normal and trisomy 16 mice (Ts16), an animal model of DS, we showed that the application of exogenous β-amyloid has cytotoxic effects, expressed in decreased viability and increased apoptosis. Supplementation of the culture media with cholesterol associated to lipoprotein increased cell viability in both cell lines, but apoptosis decreased only in the normal cell line. Further, intracellular β-amyloid content was elevated in trisomic cells following cholesterol treatment, with higher values in the trisomic cell line. Immunocytochemical detection showed intracellular accumulation of exogenous β-amyloid in Rab4-positive compartments, which are known to be associated to endosomal recycling. The results suggest that the intracellular β-amyloid pool plays a central role in apoptosis-mediated cell death in the trisomic condition. [Copyright &y& Elsevier]

Biosorption of Cr+6 by Termitomyces clypeatus has been investigated involving kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopic (FTIR) studies. Kinetics experiments reveal that the uptake of chromium by live cell involves initial rapid surface binding followed by relatively slow intracellular accumulation. Of the different chromate analogues tested, only sulfate ion reduces the uptake of chromium to the extent of ¿30% indicating chromate ions accumulation into the cytoplasm using sulfate transport system. Metabolic inhibitors, e.g. N,N¿-dicyclohexylcarbodiimide, 2,4-ditrophenol and sodium azide inhibit chromate accumulation by ¿30% in live cell. This indicates that accumulation of chromium into the cytoplasm occurs through the active transport system. TEM-EDXA analysis reveals that the chromium localizes in the cell wall and also in the cytoplasm. Reduction of chromate ions takes place by chromate reductase activity of cell-free extracts of T. clypeatus. FTIR study indicates that chromate ions accumulate into the cytoplasm and then reduced to less toxic Cr+3 compounds. [Copyright &y& Elsevier]

Abstract: The intracellular hepatitis B surface antigen (HBsAg) content per cell was significantly increased by 7.2-fold in the culture of recombinant CHO cells with 1.5% dimethyl sulfoxide (DMSO), while the HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. The significant accumulation of HBsAg within rCHO cells by DMSO stimulation was testified with flow cytometry measurements. Electron microscopy was applied to show that the dilated areas scattered over whole cytoplasm within rCHO cells in response to DMSO, and further revealed that intracellular HBsAg was localized to these areas with immunogold labeling. The failure of intracellular HBsAg virus-like particle assembly was revealed to be closely associated with the HBsAg accumulation within DMSO-stimulated rCHO cells on the basis of sucrose gradient analysis of cell extract. This work provided the details to further understand the HBsAg accumulation within rCHO cells in response to DMSO. [Copyright &y& Elsevier]

The effects of environmental factors and nutrients on the various possible removal mechanisms (surface adsorption, intracellular accumulation and precipitation to sediments) and partitioning of lead among various compartments (plant biomass, water column and sediments) in Salvinia minima batch-operated lagoons, were evaluated. Surface adsorption was found to be the predominant mechanism for Pb(II) removal under all environmental conditions tested in the absence of nutrients (an average of 54.3%) and in a nutrient medium (modified Hutner 1/10 medium) free of EDTA and phosphates (54.41%) at “high” initial Pb(II) concentrations (in the range of 10.3±0.13 to 15.2±0.05 mg/L). Under these conditions, the bioconcentration factors (BCFs) were 2,431±276 and 2,065±35, respectively. Lead removal was very rapid during the first 4 h and reached 70% in the absence of nutrients at the “medium” light intensity and temperature (LIT) tested, 88% in nutrient medium free of EDTA and supplemented with synthetic wastewater (at the “lowest” LIT tested), and 85% in medium free of EDTA and phosphates. It was concluded that the mechanisms of lead removal by S. minima, and the compartmentalization of this metal in the microcosm of batch-operated lagoons, are primarily a function of the presence of certain nutrients and chelants, with secondary dependence on environmental conditions. In addition, the results indicate that the percentage of lead removed is only a gross parameter and that the complementary use of BCF and compartmentalization analysis is required to gain a full insight into the metal removal process. [ABSTRACT FROM AUTHOR]

Abstract: Several mechanisms have been proposed to explain how algae can tolerate heavy metals. In order to better understand the mechanisms determining metal tolerance, we examined the interaction of copper with two strains of the copper-tolerant green algae Oocystis nephrocytioides, isolated from algal communities differing only in copper exposure. The strains were cultured in chemically-defined media containing 0.04μM Cutotal (pCu 12.4) or 2μM Cutotal (pCu 10.6). Growth, photosynthesis rate, content of chlorophyll a and b, copper accumulation, its cellular distribution and ultrastructural localization, as well as the influence of algal growth on extracellular copper complexation were determined. Both strains had comparable growth and photosynthesis rates. The cellular content of both chlorophyll a and b was reduced, by roughly the same extent, at pCu 10.6 compared to pCu 12.4. Copper titration of the media indicated the production of copper-complexing ligands by O. nephrocytioides cultured at pCu 12.4 that increased with increased algal density during cell growth. No additional ligands were detected at pCu 10.6. Copper-complexing ligands had a conditional stability constant of K =1013 at pH 7.3. The intracellular concentration of copper in O. nephrocytioides was 80μM at pCu 12.4 and increased to 7.5mM at pCu 10.6. The proportion of intracellular Cu accumulated increased from 8% of total Cu content at pCu 12.4 to 60% at pCu 10.6. By electron spectroscopic imaging, intracellular Cu was detected in the thylakoids and the pyrenoid of O. nephrocytioides cells. The results indicate that the tolerance of O. nephrocytioides to Cu is constitutive and does not need to be induced by previous exposure to Cu. We propose that accumulation and sequestration of Cu in thylakoids and, to a lesser extent, adsorption of copper to the algal cell surface represent the most important tolerance mechanism, for O. nephrocytioides. [Copyright &y& Elsevier]

Mutations in the MYOC/TIGR gene are responsible for autosomal dominant primary open angle glaucoma (POAG). Almost all mutations responsible for POAG have been detected in the coding region (in particular at exon 3). By using the techniques of PCR, SSCP, automated sequencing and restriction analysis, we have studied 79 patients suffering from glaucoma. We have found five patients with sequence variants in the consensus region of the promoter. These sequence variants might be involved in the altered association between the consensus region and the corresponding transcription factor. This possibility might be favouring the association of other transcription factors, which would operate as activators or inhibitors of the transcription, altering the MYOC/TIGR expression.European Journal of Human Genetics (2005) 13, 384-387. doi:10.1038/sj.ejhg.5201299 Published online 13 October 2004 [ABSTRACT FROM AUTHOR]

Anthracycline antibiotics are widely used as anticancer agents. Idarubicin (4-demethoxydaunorubicin; Ida), a semisynthetic derivative of daunorubicin (Dnr) is more potent than the parent compound in vitro and in vivo. The equitoxic dose of Ida in patients is about one-fourth of that of Dnr. We compared these drugs regarding cytotoxicity, apoptosis induction, and resistance mechanisms in human leukaemic cell lines. Cytotoxicity was studied by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and drug-induced apoptosis by means of the Annexin V–fluorescein isothiocyanate method at similar intracellular concentrations (extracellular concentrations of 0.35 μM for Ida and 1 μM for Dnr). Ida was at least twice as potent as Dnr in MOLT-4, HL60, CEM, and K562 cell lines. It took 8 h for Ida to induce approximately 20% apoptosis, but at least 22 h for Dnr to reach 20% apoptosis at identical intracellular concentration. Ida induces a faster and higher apoptosis rate compared with Dnr. The human chronic myelogenous leukaemia cell line (K562) was selected for resistance to Dnr and Ida with and without verapamil (Ver). Continuous incubation with Dnr, but not with Ida, led to an increased mdr1 gene expression as assessed by real-time PCR. The development of mdr1 gene expression in Dnr-resistant cells could be reversed by the presence of Ver. Ver also reversed the cytotoxicity to Dnr, but not to Ida, in K562/Dnr cells. The results show that Ida is more effective than Dnr in inducing apoptosis and that there are differences in resistance mechanisms between the drugs. [Copyright &y& Elsevier]

Aims Since α1-acid glycoprotein (AGP) levels may be raised during HIV infection, we have examined in vitro the effect of increasing the concentration of AGP on the intracellular accumulation of the HIV protease inhibitors saquinavir (SQV), ritonavir (RTV) and indinavir (IDV). Methods U937 cells (5 × 106 cells in 5 ml RPMI growth medium) were incubated at 37 °C for 18 h with [14C]-SQV (0.1 µCi), [3H]-RTV and [3H]-IDV (0.135 µCi) to a final concentration of 1 µm in the presence of 0, 0.5 and 2.0 mg ml-1 AGP. Following extraction in 60% methanol the intracellular drug concentration was determined by liquid scintillation counting. Results Binding to AGP (2.0 mg ml-1) reduced the mean intracellular concentration of SQV from 31.5 µm to 7.4 µm (P < 0.0001; 95% CI 19.4–28.8). RTV concentration was also reduced (8.8 µm to 1.6 µm; P < 0.0001; 95% CI 5.4–9.0) as was the concentration of IDV (3.0 µm to 1.5 µm; P < 0.0001; 95% CI 1.1–1.9). Conclusions Reduced intracellular protease inhibitor concentrations in the presence of increasing concentrations of AGP will certainly impact on the antiviral activity in vitro. However, since protease inhibitors are high clearance drugs, free drug concentration will likely remain unaffected in the presence of elevated AGP during chronic oral dosing although there will be an increase in total plasma drug concentration. [ABSTRACT FROM AUTHOR]

Hexavalent chromium (Cr(VI)) is one of the typical heavy metals that pose a great threat to the environment. As a novel biotechnology, algal-bacterial aerobic granular sludge (AGS) possesses the merits of both bacterial AGS and algae. This study firstly evaluated Cr(VI) removal via biosorption by algal-bacterial AGS under different operation conditions and then some environmental factors. Results show that the highest Cr(VI) reduction (99.3%) and total Cr removal (89.1%) were achieved within 6 h at pH 2 and 6, respectively. The coexisting oxyanions exhibited slight effects, while both tested natural organic matters (humic acid and tannic acid) and carbon sources promoted Cr(VI) reduction at some appropriate concentrations. The coexistence of metal cations favored Cr(VI) reduction, achieving the highest enhancement of 8.1% by Cu2+ at 5 mg/L, while the total Cr removal was suppressed to some extent. Salinity > 5 g/L severely inhibited both Cr(VI) reduction and total Cr removal. Moreover, the loaded Cr in algal-bacterial AGS was found to be almost in the form of Cr(III), with 66.8% being contributed by intracellular accumulation. This work suggests that Cr(VI) reduction and intracellular accumulation are the main mechanisms involved in Cr(IV) biosorption onto algal-bacterial AGS. [Display omitted] • Microbial activity and granular stability were maintained after Cr(VI) biosorption. • Optimal pH for Cr(VI) reduction was pH 2 while that for total Cr removal was pH 6. • Metal cations may enhance Cr(VI) reduction while suppress total Cr removal. • Organics may promote Cr(VI) removal, especially for Cr(VI) reduction. • The adsorbed Cr in granules was in form of Cr(III) with 66.8% being intracellular. [ABSTRACT FROM AUTHOR]

The effect of methylammonium on the utilization of other nitrogen sources and the characteristics of methylammonium uptake by Cyclotella cryptica Reimann, Lewin, and Guillard were examined. Methylammonium prevented growth on nitrate, nitrite, and arginine, but did not affect growth on ammonium or glutamine. Methylammonium was taken up rapidly by what appears to be an ammonium transport system. Transport followed saturable kinetics, was specific for ammonium and methylammonium, and appeared to be subject to nitrogen regulation. Transport rates were hyperbolically related to external Na[SUP+] and strongly inhibited by high external concentrations of K[SUP+]. These results support the use of methylammonium as an ammonium analogue, as an inhibitor of the utilization of some forms of nitrogen, and in the analysis of the mechanism and regulation of ammonium transport. [ABSTRACT FROM AUTHOR]

The intraerythrocytic levels of folate and methotrexate were measured in 25 patients on long-term methotrexate therapy for recalcitrant psoriasis. The mean steady state concentration of methotrexate in erythrocytes was 85 nmol/l and the mean erythrocyte folate concentration was 729 nmol/l. A linear correlation was not observed between these parameters, but the greatest methotrexate accumulation was found in cells at the lower end of the erythrocyte folate concentration range. In 5 patients started on methotrexate therapy the erythrocyte concentrations of methotrexate and folate were followed over 6 months. 3-4 days after the first dose, methotrexate had been accumulated against a concentration gradient in the erythrocytes. The methotrexate concentration increased steadily until the steady state level was reached after 4-6 weeks. The steady state level was maintained during the 6 month ovservation period. The erythrocyte folate concentration did not change during this period. The data suggest that methotrexate polyglutamate synthesis whithin the circulating erythrocyte is a major cause of methotrexate accumulation in these cells. [ABSTRACT FROM AUTHOR]

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3' and 5' ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2'-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central "window" of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs., Competing Interests: The authors declare no competing interests., (© 2021 Institute of Chemical Biology and Fundamental Medicine SB RAS.)

Morchella is a genus of edible fungi with strong resistance to cadmium (Cd) and the ability to accumulate it in its mycelia. However, the mechanisms underlying Cd resistance in Morchella remain unknown. In the present study, morphological and physiological responses to Cd were evaluated in the mycelia of Morchella spongiola . Variations in hyphal micromorphology, including twisting, folding, and kinking, in mycelia exposed to different Cd concentrations (0.15, 0.9, 1.5, 2.4, 5.0 mg/L) were observed using scanning electron microscopy. Deposition of Cd precipitates on cell surfaces (at Cd concentrations >2.4 mg/L) was shown by SEM-EDS. Transmission electron microscopy analysis of cells exposed to different concentrations of Cd revealed the loss of intracellular structures and the localization of Cd depositions inside and outside the cell. FTIR analysis showed that functional groups such as C=O, -OH, -NH, and -CH could be responsible for Cd binding on the cell surface of M. spongiola . In addition, intracellular accumulation was observed in cultures at low Cd concentrations (<0.9 mg/L), while extracellular adsorption occurred at higher concentrations. These results provide valuable information on the Cd tolerance mechanism in M. spongiola and provide a robust foundation for further studies on fungal bioremediation strategies.

The therapeutic repertoire for tuberculosis (TB) remains limited despite the existence of many TB drugs that are highly active in in vitro models and possess clinical utility. Underlying the lack of efficacy in vivo is the inability of TB drugs to penetrate microenvironments inhabited by the causative agent, Mycobacterium tuberculosis, including host alveolar macrophages. Here, we determined the ability of the phenoxazine PhX1 previously shown to be active against M. tuberculosis in vitro to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. We also investigated the extent of permeation into uninfected and M. tuberculosis-infected human macrophage-like Tamm-Horsfall protein 1 (THP-1) cells directly and by comparing to results obtained in vitro in synergy assays. Our data indicate that PhX1 (4,750 ± 127.2 ng/ml) penetrates more effectively into THP-1 cells than do the clinically used anti-TB agents, rifampin (3,050 ± 62.9 ng/ml), moxifloxacin (3,374 ± 48.7 ng/ml), bedaquiline (4,410 ± 190.9 ng/ml), and linezolid (770 ± 14.1 ng/ml). Compound efficacy in infected cells correlated with intracellular accumulation, reinforcing the perceived importance of intracellular penetration as a key drug property. Moreover, we detected synergies deriving from redox-stimulatory combinations of PhX1 or clofazimine with the novel prenylated amino-artemisinin WHN296. Finally, we used compound synergies to elucidate the relationship between compound intracellular accumulation and efficacy, with PhX1/WHN296 synergy levels shown to predict drug efficacy. Collectively, our data support the utility of the applied assays in identifying in vitro active compounds with the potential for clinical development. IMPORTANCE This study addresses the development of novel therapeutic compounds for the eventual treatment of drug-resistant tuberculosis. Tuberculosis continues to progress, with cases of Mycobacterium tuberculosis (M. tuberculosis) resistance to first-line medications increasing. We assess new combinations of drugs with both oxidant and redox properties coupled with a third partner drug, with the focus here being on the potentiation of M. tuberculosis-active combinations of compounds in the intracellular macrophage environment. Thus, we determined the ability of the phenoxazine PhX1, previously shown to be active against M. tuberculosis in vitro , to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. In addition, the extent of permeation into human macrophage-like THP-1 cells and H37Rv-infected THP-1 cells was measured via mass spectrometry and compared to in vitro two-dimensional synergy and subsequent intracellular efficacy. Collectively, our data indicate that development of new drugs will be facilitated using the methods described herein.