Differential Expression of Stress Adaptation Genes in a Diatom Ulnaria acus under Different Culture Conditions.

Diatoms are a group of unicellular eukaryotes that are essential primary producers in aquatic ecosystems. The dynamic nature of their habitat necessitates a quick and specific response to various stresses. However, the molecular mechanisms of their physiological adaptations are still underexplored. In this work, we study the response of the cosmopolitan freshwater diatom Ulnaria acus (Bacillariophyceae, Fragilariophycidae, Licmophorales, Ulnariaceae, Ulnaria) in relation to a range of stress factors, namely silica deficiency, prolonged cultivation, and interaction with an algicidal bacterium. Fluorescent staining and light microscopy were used to determine the physiological state of cells under these stresses. To explore molecular reactions, we studied the genes involved in the stress response-type III metacaspase (MC), metacaspase-like proteases (MCP), death-specific protein (DSP), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH12), and glutathione synthetase (GSHS). We have described the structure of these genes, analyzed the predicted amino acid sequences, and measured their expression dynamics in vitro using qRT-PCR. We demonstrated that the expression of UaMC1, UaMC3, and UaDSP increased during the first five days of silicon starvation. On the seventh day, it was replaced with the expression of UaMC2, UaGSHS, and UaALDH. After 45 days of culture, cells stopped growing, and the expression of UaMC1, UaMC2, UaGSHS, and UaDSP increased. Exposure to an algicidal bacterial filtrate induced a higher expression of UaMC1 and UaGSHS. Thus, we can conclude that these proteins are involved in diatoms' adaptions to environmental changes. Further, these data show that the molecular adaptation mechanisms in diatoms depend on the nature and exposure duration of a stress factor.

Effects of microbial agents and corn protein ferment on physiological characteristics in leaves and yield of tomato.

Premature senescence in greenhouse tomato is a significant challenge under long-season cultivation, due to suboptimal nutrient management during growth periods. We investigated the effects of microbial agents (T1), corn protein ferment (T2), and their combined application (T3) on photosynthetic characteristics and antioxidant enzyme activities in 'Saint Laurent 3689' tomato leaves, normal management served as the control (CK). We explored the physiological mechanism of delaying leaf senescence. Results showed that applying microbial agents or corn protein ferment individually led to improvements in leaf photosynthetic characteristics and antioxidant enzyme activities. The combined application yielded superior outcomes. Eighty days post the combined application of microbial agents and corn protein ferment (T3), chlorophyll (a+b) content, net photosynthetic rate, and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in leaves increased by 16.4%, 30.9%, 23.4%, 33.0% and 40.3%, respectively, compared with the CK. Furthermore, plant height and stem diameter increased by 8.2% and 7.0%, while the total yield exhibited a significant increase of 9.9% compared with the CK 210 days post-treatment. In conclusion, the combined application of microbial agents and corn protein ferment has promising potential in enhancing chlorophyll content, net photosynthetic rate, and the activities of SOD, POD and CAT in tomato leaves. This approach effectively delayed leaf senescence, thereby promoting tomato growth and remarkably increasing the yield.

Establishment and optimization of a new model organism to study early land plant evolution: Germination, cultivation and oospore variation of Chara braunii Gmelin, 1826.

For studying land plant evolution, the establishment and optimization of model organisms representing streptophytic algae, sister to land plants, is essential. Long-term cultivation experiments with Chara braunii S276 were performed over 8 years, since 4 years (Nov. 2018) under constant conditions. Additionally, short-term experiments for optimization of culture conditions were performed with three strains of C. braunii (S276, NIES-1604 and Lausiger Teiche, LaT-2708). Germination success after application of sterilization agents, addition of gibberellic acid and under different incubation conditions with respect to pre-treatment, irradiance regime and substrate was investigated in order to develop protocols for generative cultivation of at least unialgal cultures. The resulting cultivation protocols for C. braunii S276, allowing maintenance of vegetative as well as generative cultures are presented in detail, including protocols for germination induction and growth of sterilized and unsterilized oospores.

The Effect of Chronic Exposure of Graphene Nanoplates on the Viability and Motility of A549 Cells.

Graphene and its derivatives are popular nanomaterials used worldwide in many technical fields and biomedical applications. Due to such massive use, their anticipated accumulation in the environment is inevitable, with a largely unknown chronic influence on living organisms. Although repeatedly tested in chronic in vivo studies, long-term cell culture experiments that explain the biological response to these nanomaterials are still scarce. In this study, we sought to evaluate the biological responses of established model A549 tumor cells exposed to a non-toxic dose of pristine graphene for eight weeks. Our results demonstrate that the viability of the A549 cells exposed to the tested graphene did not change as well as the rate of their growth and proliferation despite nanoplatelet accumulation inside the cells. In addition, while the enzymatic activity of mitochondrial dehydrogenases moderately increased in exposed cells, their overall mitochondrial damage along with energy production changes was also not detected. Conversely, chronic accumulation of graphene nanoplates in exposed cells was detected, as evidenced by electron microscopy associated with impaired cellular motility.

Evolutionary Adaptation by Repetitive Long-Term Cultivation with Gradual Increase in Temperature for Acquiring Multi-Stress Tolerance and High Ethanol Productivity in Kluyveromyces marxianus DMKU 3-1042.

During ethanol fermentation, yeast cells are exposed to various stresses that have negative effects on cell growth, cell survival, and fermentation ability. This study, therefore, aims to develop Kluyveromyces marxianus-adapted strains that are multi-stress tolerant and to increase ethanol production at high temperatures through a novel evolutionary adaptation procedure. K. marxianus DMKU 3-1042 was subjected to repetitive long-term cultivation with gradual increases in temperature (RLCGT), which exposed cells to various stresses, including high temperatures. In each cultivation step, 1% of the previous culture was inoculated into a medium containing 1% yeast extract, 2% peptone, and 2% glucose, and cultivation was performed under a shaking condition. Four adapted strains showed increased tolerance to ethanol, furfural, hydroxymethylfurfural, and vanillin, and they also showed higher production of ethanol in a medium containing 16% glucose at high temperatures. One showed stronger ethanol tolerance. Others had similar phenotypes, including acetic acid tolerance, though genome analysis revealed that they had different mutations. Based on genome and transcriptome analyses, we discuss possible mechanisms of stress tolerance in adapted strains. All adapted strains gained a useful capacity for ethanol fermentation at high temperatures and improved tolerance to multi-stress. This suggests that RLCGT is a simple and efficient procedure for the development of robust strains.

Revealing dual roles of g-C3N4 in Chlorella vulgaris cultivation.

Booming graphitic carbon nitride (g-C3N4) photocatalyzed water splitting increases crisis of aquatic contamination. However, a controversial understanding regarding effect of g-C3N4 on growth of microalgae still exists. Accordingly, Chlorella vulgaris were cultured in 0-250 mg/L of g-C3N4 with biomass named as C-0, C-50, C-100, C-150, C-200, and C-250, respectively. g-C3N4 below 200 mg/L was beneficial to short-term cultivation of microalgae, while it was harmful to long-time cultivation. Protein factions of C-0, C-100, and C-250 were 41.4, 42.3, and 36.4 wt%, while their lipid factions varied from 21.5, 16.9, to 17.8 wt%, respectively. In short-term cultivation, superoxide dismutase's activity of C-0, C-150, and C-250 increased dramatically, while accumulated H2O2 led to increased activity of catalase. However, it started to decrease once antioxidant enzymes were per-oxidized, leading to increase of malondialdehyde content. In long-term cultivation, activities of superoxide dismutase, catalase and malondialdehyde content decreased dramatically owning to peroxidation of algae. Scavenger tests with tertiary butanol and triethanolamine implied that·OH was dominate parameter affecting growth of microalgae. This work indicates that g-C3N4 below 200 mg/L is propitious to short-term cultivation of microalgae, while it is bad to long-time cultivation of microalgae, revealing dual rules of g-C3N4 in Chlorella vulgaris cultivation.

Primary Extracellular Matrix Enables Long-Term Cultivation of Human Tumor Oral Mucosa Models.

3D tumor models clearly outperform 2D cell cultures in recapitulating tissue architecture and drug response. However, their potential in understanding treatment efficacy and resistance development should be better exploited if also long-term effects of treatment could be assessed in vitro. The main disadvantages of the matrices commonly used for in vitro culture are their limited cultivation time and the low comparability with patient-specific matrix properties. Extended cultivation periods are feasible when primary human cells produce the extracellular matrix in situ. Herein, we adapted the hyalograft-3D approach from reconstructed human skin to normal and tumor oral mucosa models and compared the results to bovine collagen-based models. The hyalograft models showed similar morphology and cell proliferation after 7 weeks compared to collagen-based models after 2 weeks of cultivation. Tumor thickness and VEGF expression increased in hyalograft-based tumor models, whereas expression of laminin-332, tenascin C, and hypoxia-inducible factor 1α was lower than in collagen-based models. Taken together, the in situ produced extracellular matrix better confined tumor invasion in the first part of the cultivation period, with continuous tumor proliferation and increasing invasion later on. This proof-of-concept study showed the successful transfer of the hyalograft approach to tumor oral mucosa models and lays the foundation for the assessment of long-term drug treatment effects. Moreover, the use of an animal-derived extracellular matrix is avoided.

Long-term semi-continuous cultivation of a halo-tolerant Tetraselmis sp. using recycled growth media.

In this study, a halotolerant Tetraselmis sp. was selected for 11-month outdoor semi-continuous cultivation in one sq. m raceway tank in the Qatari desert. A fraction of the culture was harvested using ferric chloride, and the growth media was returned to the tank. The recycling of culture media continued till the culture salinity reached 8% NaCl; 90% culture was then harvested, and the remaining culture fraction was used as inoculum for a new cultivation cycle. The growth of Tetraselmis sp. was not affected by incremental salinity although the intracellular metabolites varied; the average biomass productivity was 17.8 g/m2/d. Harvesting efficiency was slightly affected by the increase in salinity. Iron content in the harvested biomass was in the range of 1.5-3.3%, and acidic solution (pH = 1.48) was able to recover 91.3% iron from the harvested biomass. Nonetheless, Tetraselmis sp. could be grown continuously throughout the year in Qatar's climate condition.

Assessment of long-term cultivated human precision-cut lung slices as an ex vivo system for evaluation of chronic cytotoxicity and functionality.

BACKGROUND: Investigation of basic chronic inflammatory mechanisms and development of new therapeutics targeting the respiratory tract requires appropriate testing systems, including those to monitor long- persistence. Human precision-cut lung slices (PCLS) have been demonstrated to mimic the human respiratory tract and have potential of an alternative, ex-vivo system to replace or augment in-vitro testing and animal models. So far, most research on PCLS has been conducted for short cultivation periods (≤72 h), while analyses of slowly metabolized therapeutics require long-term survival of PCLS in culture. In the present study, we evaluated viability, physiology and structural integrity of PCLS cultured for up to 15 days. METHODS: PCLS were cultured for 15 days and various parameters were assessed at different time points. RESULTS: Structural integrity and viability of cultured PCLS remained constant for 15 days. Moreover, bronchoconstriction was inducible over the whole period of cultivation, though with decreased sensitivity (EC501d = 4 × 10-8 M vs. EC5015d = 4 × 10-6 M) and reduced maximum of initial airway area (1d = 0.5% vs. 15d = 18.7%). In contrast, even though still clearly inducible compared to medium control, LPS-induced TNF-α secretion decreased significantly from day 1 to day 15 of culture. CONCLUSIONS: Overall, though long-term cultivation of PCLS need further investigation for cytokine secretion, possibly on a cellular level, PCLS are feasible for bronchoconstriction studies and toxicity assays.

Urochloa decumbens growth and P uptake as affected by long-term phosphate fertilization, mycorrhizal inoculation and historical land use in contrasting Oxisols of the Brazilian Cerrado / Crescimento e absorção de P por Urochloa decumbens afetado por longos períodos de adubação fosfatada, inoculação micorrízica e histórico de uso em Latossolos contrastantes do Cerrado brasileiro

ABSTRACT In the fertility management of highly weathered-leached Brazilian Oxisols, P is the most limiting macronutrient. A greenhouse experiment was conducted with the objective to evaluate the influence of the interaction between P doses, mycorrhizal inoculation and historical land use on Urochloa decumbens growth and P uptake in four Oxisols with contrasting chemical, physical and mineralogical properties. The plants were cultivated in plastic pots containing 4 kg of soil in a completely randomized design, four replications and 2x2x2 factorial scheme: with two P doses; with and without mycorrhizal inoculation; soils cultivated for long periods and non-cultivated (under native vegetation). There were two plantings of ten weeks each. Shoot dry mater, concentration and accumulation of P in the shoot were evaluated. In the first planting, the Urochloa response was greater in non-cultivated soils associated with inoculation and P addition. However, in the second planting, the inoculation had a greater effect in all soils compared to the first planting associated with the lowest P dose. As the P concentration in the soil increased, P in the shoot dry matter increases. The inoculation did not affect the P concentration and accumulation in the shoot of Urochloa. The growth of Urochloa decumbens was strongly influenced by the interaction among soil class x history of land use x dose of P x inoculation.

RESUMO No manejo da fertilidade de Latossolos brasileiros altamente intemperizados e lixiviados, o P é o macronutriente mais limitante. Assim, conduziu-se um experimento em casa de vegetação, objetivando-se avaliar a influência da interação entre doses de P, inoculação micorrízica e histórico de uso do solo no crescimento de Urochloa decumbens e absorção de P, em quatro Latossolos com atributos químicos, físicos e mineralógicos contrastantes. As plantas foram cultivadas em vasos contendo 4 kg de solo, dispostos em um delineamento inteiramente casualizado, com quatro repetições e esquema fatorial 2x2x2: duas doses de P; com e sem inoculação micorrízica; solos cultivados por longos períodos e não cultivados (sob vegetação nativa). Realizaram-se dois cultivos com duração de 10 semanas cada. Após cada cultivo avaliaram-se a matéria seca da parte aérea, teor e acúmulo de P na parte aérea. No primeiro cultivo, maior resposta ao P aconteceu nos solos sob vegetação nativa em associação à inoculação e adição de P. No segundo cultivo, a inoculação teve maior efeito em todos os solos, associada à menor dose de P. O aumento do P no solo aumenta a concentração desse nutriente na parte aérea. A inoculação não teve efeito na concentração e no acúmulo de P na parte aérea de Urochloa. O crescimento de Urochloa decumbens foi fortemente influenciado pela interação entre classe de solo x histórico de uso da terra x dose de P x inoculação.

Accumulation of spontaneous γH2AX foci in long-term cultured mesenchymal stromal cells.

Expansion of mesenchymal stromal/stem cells (MSCs) used in clinical practices may be associated with accumulation of genetic instability. Understanding temporal and mechanistic aspects of this process is important for improving stem cell therapy protocols. We used γH2AX foci as a marker of a genetic instability event and quantified it in MSCs that undergone various numbers of passage (3-22). We found that γH2AX foci numbers increased in cells of late passages, with a sharp increase at passage 16-18. By measuring in parallel foci of ATM phosphorylated at Ser-1981 and their co-localization with γaH2AX foci, along with differentiating cells into proliferating and resting by using a Ki67 marker, we conclude that the sharp increase in γH2AX foci numbers was ATM-independent and happened predominantly in proliferating cells. At the same time, gradual and moderate increase in γH2AX foci with passage number seen in both resting and proliferating cells may represent a slow, DNA double-strand break related component of the accumulation of genetic instability in MSCs. Our results provide important information on selecting appropriate passage numbers exceeding which would be associated with substantial risks to a patient-recipient, both with respect to therapeutic efficiency and side-effects related to potential neoplastic transformations due to genetic instability acquired by MSCs during expansion.

A new computer-controlled air-liquid interface cultivation system for the generation of differentiated cell cultures of the airway epithelium.

The increased application of in vitro systems in pharmacology and toxicology requires cell culture systems that facilitate the cultivation process and ensure stable, reproducible and controllable cultivation conditions. Up to now, some devices have been developed for the cultivation of cells under submersed conditions. However, systems meeting the requirements of an air-liquid interface (ALI) cultivation for the special needs of bronchial epithelial cells for example are still lacking. In order to obtain in vivo like organization and differentiation of these cells they need to be cultivated under ALI conditions on microporous membranes in direct contact with the environmental atmosphere. For this purpose, a Long-Term-Cultivation system was developed (CULTEX(®) LTC-C system) for the computer-controlled cultivation of such cells. The transwell inserts are placed in an incubator module (24 inserts), which can be adjusted for the medium level (ultrasonic pulse-echosensor), time and volume-dependent medium exchange, and frequency for mixing the medium with a rotating disc for homogeneous distribution of medium and secretion components. Normal primary freshly isolated bronchial epithelial cells were cultivated for up to 38 days to show the efficiency of such a cultivation procedure for generating 3D cultures exhibiting in vivo-like pseudostratified organization of the cells as well as differentiation characteristics like mucus-producing and cilia-forming cells.

Differences in infectivity between endosymbiotic Chlorella variabilis cultivated outside host Paramecium bursaria for 50†years and those immediately isolated from host cells after one†year of reendosymbiosis.

Chlorella variabilis strain NC64A is an intracellular photobiont of the ciliate Paramecium bursaria. NC64A was isolated from P. bursaria nearly 50 years ago and was thereafter cultivated outside the host. This study was undertaken to detect changes in its infectivity to P. bursaria and its auxotrophy for growth outside the host induced during long-term cultivation. NC64A can grow in Modified Bold's Basal Medium but not in C medium, whereas another symbiotic Chlorella variabilis strain, 1N, that was recently isolated from the host grew in C medium but not in Modified Bold's Basal Medium. With regards infectivity, NC64A in the logarithmic phase of growth showed low infectivity to alga-removed P. bursaria cells, whereas those in the early stationary phase showed high infectivity of about 30%. Those in the decay phase of growth showed no infectivity. Results show that NC64A has infectivity, but the infection rate depends on their culture age in the growth curve. Furthermore, NC64A that had been re-infected to P. bursaria for more than one year and isolated from the host showed a nearly 100% infection rate, which indicates that NC64A can recover its infectivity by re-infection to P. bursaria.