Antifungal and Insecticidal Potential of the Essential Oil from Ocimum sanctum L. against Dangerous Fungal and Insect Species and Its Safety for Non-Target Useful Soil Species Eisenia fetida (Savigny, 1826).

The antifungal and insecticidal effect of the essential oil from Ocimum sanctum L. was evaluated using a model set of harmful organisms hazardous for health and the economy. Toxigenic and plant pathogenic filamentous fungi, including causal agents of human infections, were chosen as exemplary fungal groups-Fusarium verticillioides, Penicillium expansum and Aspergillus flavus. Spodoptera littoralis (African cotton leafworm), Culex quinquefasciatus (Southern house mosquito), the lymphatic filariasis vector and potential Zika virus vector, and the common housefly, Musca domestica were chosen as model insects. Major and minor active substances were detected and quantified using GC/MS analysis. Environmental safety was verified using the non-target useful organism Eisenia fetida. Significant antifungal and insecticidal activity, as well as environmental safety, were confirmed. The essential oil showed the highest efficacy against A. flavus according to MIC50/90, and against S. littoralis larvae according to LD50/90. The monoterpenoid alcohol linalool, t-methyl cinnamate, and estragole as phenylpropanoids were detected as effective major components (85.4%). The essential oil from Ocimum sanctum L. was evaluated as universal and significantly efficient, providing a high potential for use in environmentally safe botanical pesticides.

The micronucleus cytome assay - A fast tool for DNA damage screening in human conjunctival epithelial cells.

PURPOSE: To assess whether the micronucleus cytome assay (MCyt) reliably detects DNA damage occurring in control and pathological superficial epithelial cells from human conjunctiva. METHODS: Impression cytology samples from the bulbar conjunctiva of 33 healthy controls, eight patients with conjunctival intraepithelial neoplasia (CIN) and eight with mucous membrane pemphigoid (MMP) were examined using the MCyt modified for the ocular surface. RESULTS: The mean number of micronuclei (MNi) in control samples was 0.94 MNi/1000 epithelial cells, with no significant difference between conjunctival quadrants and independent of sex and age. The MCyt assay applied to CIN-affected eyes showed a significantly higher frequency of MNi (18.63/1000 cells), apoptotic cells, nuclear enlargement, multinucleated cells, and keratolysis compared with the corresponding unaffected paired eyes and with the control value. Although the mean MNi frequency in MMP eyes was also higher (1.73 MNi/1000 cells), it did not prove to be statistically different from the control samples. On the other hand, the MMP-affected eyes revealed significantly elevated percentages of cells with snake-like chromatin, multinucleated cells, apoptotic cells, and nuclear buds compared with controls. CONCLUSIONS: Micronucleus cytome assay was adapted as a rapid screening test for genomic instability on the ocular surface. We have determined reference levels for MNi and other nuclear alterations on healthy conjunctiva and demonstrated that particularly frequencies of MNi are significantly elevated in conjunctiva affected by CIN. We demonstrate that MNi are more specific than other nuclear abnormalities and thus can be used for screening of ocular surface neoplasia.

The NANOTUMOR consortium - Towards the Tumor Cell Atlas.

Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.

Generation of Remosomes by the SWI/SNF Chromatin Remodeler Family.

Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.

Frequency of Complications During Preparation of Corneal Lamellae Used in Posterior Lamellar Keratoplasty Using the Pneumodissection Technique (Big Bubble).

PURPOSE: To determine the frequency of formation of various types of bubbles and the potential impact of donor and lamella parameters on this frequency, and to identify possible risk factors of unsuccessful "big-bubble" creation in preparation of pre-Descemet endothelial keratoplasty and Descemet membrane endothelial keratoplasty with peripheral stromal support. METHODS: Donor age and sex, death to preservation time (DPT), storage time, presence of corneal scars (mainly a condition after cataract surgery), and endothelial cell density of 256 donor corneas were assessed before Descemet membrane endothelial keratoplasty with peripheral stromal support or pre-Descemet endothelial keratoplasty lamella preparation using the big-bubble technique. RESULTS: Mean donor age was 62.3 ± 8.5 years (28.3% women and 71.7% men). Mean endothelial cell density of the donor graft was 2866 ± 255 cells/mm. Mean DPT was 10.12 ± 4.88 hours, and mean storage time of the transplant before surgery was 6.5 ± 4.8 days. Corneal scars were present in 17 donor grafts (6.6%) after cataract surgery. Eleven corneas were devalued because of Descemet membrane rupture during preparation (4.3%). In 182 corneas, standard bubble type I was created (71.7%); in 27 corneas, bubble type II was created; eventually, both types of bubbles formed simultaneously (10.5%); in 47 corneas, no bubble was created (18.4%). CONCLUSIONS: We identified higher endothelial cell density, shorter DPT, and the presence of corneal scars after cataract surgery as risk factors threatening successful bubble formation. The only risk factor for creating type II bubbles was higher donor age in our study.

The enzymatic de-epithelialization technique determines denuded amniotic membrane integrity and viability of harvested epithelial cells.

The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture.

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion.

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.

The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.

Stable polymersomes based on ionic-zwitterionic block copolymers modified with superparamagnetic iron oxide nanoparticles for biomedical applications.

Stable polymersomes with semipermeable membranes were prepared by simple mixing of two oppositely charged diblock copolymers containing zwitterionic and cationic (PMPC20-b-PMAPTAC190) or anionic (PMPC20-b-PAMPS196) blocks. The formation of vesicular structures in the mixed solution of the block copolymers was confirmed by direct observation using the cryo-TEM technique. Superparamagnetic iron oxide nanoparticles coated with a cationic chitosan derivative (SPION/CCh) and decorated with a fluorescent probe molecule were next incorporated into the polymersome structure. The average diameter of SPION/CCh-polymersomes estimated using cryo-TEM was about 250 nm. Surface topography of the SPION/CCh-loaded vesicles was imaged using AFM and the magnetic properties of these objects were confirmed by MFM and MRI measurements. The ability of SPION/CCh-polymersomes to affect T2 relaxation time in MRI was evaluated based on the measurements of r2 relaxivity. The obtained value of r2 (573 ± 10 mM-1 s-1) was quite high. The cytotoxicity and intracellular uptake of the SPION/CCh-loaded vesicles into EA.hy926 cells were studied. The results indicate that the SPION/CCh-polymersomes seem to be internalized by vascular endothelium and are not cytotoxic to endothelial cells up to 1 µg Fe per mL. Therefore, it can be suggested that SPION/CCh-polymersomes could prove useful as T2 contrast agents in the MRI of endothelium.

Rapid cooling of the amniotic membrane as a model system for the vitrification of posterior corneal lamellae.

To vitrify human amniotic membrane specimens so that the maximum of epithelial cells survives in order to develop a procedure for the eventual vitrification of posterior corneal lamellae without using cryoprotective agents. To assess different methods of tissue sample preparation preceding vitrification. In group 1, the amniotic membrane specimens were stretched on nitrocellulose support. In group 2, mechanical pressure was used to remove the excess culture medium between the support and the membrane. The samples were frozen in liquid ethane (-183 °C) and stored in liquid nitrogen. The specimens in the control group were not vitrified. Re-warming was performed at 40 °C. The epithelial cell survival rate was assessed after 1, 3 and 7 days of storage following re-warming using calcein and ethidium homodimer-1 fluorescence. A wide range of values was observed among the different groups and among individual specimens within the groups. Resulting average survival rate was 41 % for group 1 and 53 % for group 2; in several samples the cell survival rate exceeded 70 %. The storage period did not significantly affect the survival rates. The results of the rapid cooling of amniotic membranes in liquid ethane indicate that significant percentage of epithelial cells remain viable after the re-warming.

Curcumin-containing liposomes stabilized by thin layers of chitosan derivatives.

Stable vesicles for efficient curcumin encapsulation, delivery and controlled release have been obtained by coating of liposomes with thin layer of newly synthesized chitosan derivatives. Three different derivatives of chitosan were obtained and studied: the cationic (by introduction of the stable, quaternary ammonium groups), the hydrophobic (by attachment of N-dodecyl groups) and cationic-hydrophobic one (containing both quaternary ammonium and N-dodecyl groups). Zeta potential measurements confirmed effective coating of liposomes with all these chitosan derivatives. The liposomes coated with cationic-hydrophobic chitosan derivative are the most promising curcumin carriers; they can easily penetrate cell membrane and release curcumin in a controlled manner. Biological studies indicated that such systems are non-toxic for murine fibroblasts (NIH3T3) while toxic toward murine melanoma (B16F10) cell line.

Spontaneous formation of densely stacked multilamellar vesicles in dioctadecyldimethylammonium bromide/oleosiloxane mixtures.

A double-tailed surfactant, for example, dioctadecyldimethylammonium bromide (DODAB), tends to form bilayer fragments rather than vesicles in aqueous solution upon sonication. This Letter reports on the morphological transformations of the membrane fragments leading to multilamellar vesicle formation. The surfactant membranes were swollen with tetra(11-methoxy-11-oxoundecyl)tetramethylcyclotetrasiloxane (3) (a cyclosiloxane with covalently attached fatty-acid esters) up to 50 mol % based on the surfactant content. That compound was synthesized by hydrosilylation of 1,3,5,7-tetramethylcyclotetrasiloxane with four molecules of methyl undec-10-enoate. Using cryo-transmission electron microscopy (cryo-TEM), it has been shown that densely stacked multilamellar vesicles form spontaneously in the sonicated DODAB dispersion containing 50 mol % of the compound 3 after adjusting pH to the value of 9.5. This occurs as a result of the hydrolysis of ester bonds of the compound 3 inside the membrane with formation of tetra(11-carboxyundecyl)tetramethylcyclotetrasiloxane (4). The carboxylic groups in compound 4 are ionized under experimental conditions. That results in the appearance of the negative charge at the surface which neutralizes the positively charged molecules of DODAB, thus reducing repulsive interactions between the membrane fragments. Additionally, as was shown using Langmuir monolayer measurements, the presence of compound 4 expands the DODAB film and decreases its bending rigidity. That makes the bending process considerably easier and promotes formation of multilamellar vesicles.

Vesicles to concentrate iron in low-iron media: an attempt to mimic marine siderophores.

Amphiphilic catechol-type iron chelators were studied with the aim of mimicking the properties of marine bacterial siderophores. The Fe(III) complexation constants and aqueous solution speciation of L(S10), a sulfonated catechol unit that has a C(10) lipophilic carbon chain connected by an amide linkage, were determined by spectrophotometric titration. The calculated value of pFe3+ is 18.1 at pH 7.4. Cryogenic transmission electron microscopy showed that the tris(catecholate) ferric complex formed at physiological pH initially assembles into micelles, in which the catecholate-iron units stay on the exterior of the micelle. The average diameter of these micelles was estimated to be 4.2 nm. The micelles then slowly rearrange into clusters of different sizes, which leads to the formation of unilamellar and bilamellar vesicles. The reorganization processes are comparable to those observed by Butler et al. for the marinobactin siderophores produced by marine bacteria, but in contrast to the marinobactins, vesicles of the Fe3+-L(S10) complex form without an excess of iron relative to ligand concentration. The time-dependent micelle-to-vesicle transition is discussed herein.