Early Stages Of Culture Research Articles

Multispectral Imaging of Collagen, NAD(P)H and Flavin Autofluorescence in Mesenchymal Stem Cells Undergoing Trilineage Differentiation.

Understanding the molecular mechanisms of differentiation is important for regenerative medicine and developmental biology. This study aims to characterise the role of the glycolysis/oxidative phosphorylation balance as a driver of mesenchymal stem cell (MSC) differentiation. Cells were maintained in normal conditions or stimulated towards the MSC trilineage cell types over 21 days. Multispectral imaging of cell autofluorescence was applied as a non-invasive methodology to continuously image cultures in situ. Spectral signals for collagen, NAD(P)H, and flavins were unmixed. MSCs cultured under chondrogenic conditions exhibited increased collagen levels relative to controls. Following osteogenic induction, MSCs showed increased collagen levels relative to controls during the earlier stages of culture; however, control cells increased their collagen levels as they became confluent. MSCs cultured under adipogenic conditions exhibited lower levels of collagen than controls. The redox ratio (RR; NAD(P)H/flavins) immediately decreased during chondrogenesis, with this early effect persisting throughout the culture compared to control cells, which appeared to increase their RR, similar to osteogenesis. Adipogenesis resulted in a small increase in RR on day 2 relative to control cells, followed by a persistent decrease. Chondrogenic and adipogenic differentiation favoured oxidative phosphorylation, whereas osteogenesis and MSC overgrowth resulted in a glycolytic metabolism. Following consideration of these findings, as well as the diverse reports in the literature, it is concluded that neither enhanced oxidative phosphorylation nor glycolysis are fundamental to the canonical modes of differentiation, and researchers should avoid interpreting shifts as indicating differentiation.

Characteristics of Soil Respiration and Organic Carbon Mineralization in Dryland Potato Fields Under Different Ridge-furrow Mulching Patterns

Exploring the response mechanism of soil respiration rate to hydrothermal factors and organic carbon mineralization under different ridge-furrow mulching modes is of high importance for the development of the regional carbon cycle and assessment of its ecological benefits. An experimental study was carried out in 2020 in a dry-crop potato field in the mountainous area of southern Ningxia by setting three furrow-ridge ratios [60 cm∶30 cm （R1）, 60 cm∶45 cm （R2）, and 60 cm∶60 cm （R3）] combined with three mulching modes [ridge covered with ordinary plastic film, furrow covered with straw （DJ）, degradable water-permeable plastic film （DS）, and no mulching （DB） in furrows, respectively]. The soil hydrothermal factors, respiration rate, organic carbon content, and mineralization characteristics of potatoes during the reproductive period under different mulching modes were investigated with plain mulching without mulching （CK） as the control. The results showed that different furrow-ridge ratios combined with the mulching mode could significantly increase the soil water storage capacity in the 0-60 cm layer, and the R3DJ treatment had a better effect, with a significant increase of 24.99% compared with that in CK. The R2 treatment had the best effect of increasing temperature during the whole life cycle of the potato. The DS treatment had the effect of increasing temperature, and the DJ treatment had the effect of decreasing temperature under different mulching materials. Different furrow and ridge cover patterns could significantly increase the average soil respiration rate during the reproductive period, and the R3DS treatment was the most significant among the different furrow and ridge cover patterns, with a significant increase of 24.71% compared with that in the CK treatment. The soil organic carbon content in the 0-20 cm and 20-40 cm layers at harvest time was higher in the R2 and R1 ridges, respectively, and higher in the DB and DS treatments for different mulching materials. The soil organic carbon mineralization rate declined rapidly in the early stage of cultivation and then slowly declined and leveled off in the middle and late stages, which was highest in the R3 treatment for the three types of ridge ratios and highest in the DS treatment for different mulching materials. The fitting equations of soil respiration rate with soil hydrothermal factors during the reproductive period revealed that the synergistic effect of soil hydrothermal dual factors on soil respiration was higher than that of a soil hydrothermal single factor and that the quadratic hydrothermal dual factors could well explain 86.4% to 99.9% of the soil respiration. Correlation analysis showed that the average soil respiration rate during the whole life span of the potato was highly significantly and positively correlated with the average soil temperature in the 0-25 cm layer and the average soil organic carbon mineralization rate in the 0-40 cm layer, and the soil temperature was highly significantly and positively correlated with the organic carbon mineralization rate. The furrow-ridge ratio combined with the mulching mode was shown to improve the soil hydrothermal environment and increase the rate of organic carbon mineralization, thus affecting the soil respiration rate, with a furrow-ridge ratio of 60 cm∶45 cm or 60 cm∶60 cm under the ridge mulching mulch ditch covering biodegradable seepage mulch mode being more effective.

Effects of cell culture time and cytokines on migration of dental pulp stem cell-derived chondrogenic cells in collagen hydrogels.

The transplantation of collagen hydrogels encapsulating human dental pulp stem cell (DPSC)-derived chondrogenic cells is potentially a novel approach for the regeneration of degenerated nucleus pulposus (NP) and cartilage. Grafted cell migration allows cells to disperse in the hydrogels and the treated tissue from the grafted location. We previously reported the cell migration in type I and type II hydrogels. It is important to explore further how cell culture time affect the cell motility. In this study, we observed the decreased motility of DPSC-derived chondrogenic cells after culturing for 2 weeks compared with cells cultured for 2 days in these gels. The Alamarblue assay showed the cell proliferation during the two-week cell culture period. The findings suggest that the transitions of cell motility and proliferation during the longer culture time. The result indicates that the early culture stage is an optimal time for cell transplantation. In a degenerated disc, the expression of IL-1β and TNFα increased significantly compared with healthy tissue and therefore may affect grafted cell migration. The incorporation of IL-1β and TNFα into the collagen hydrogels decreased cell motility. The study indicates that the control of IL-1β and TNFα production may help to maintain cell motility after transplantation.

Role of the mechanotransductor PIEZO1 in megakaryocyte differentiation.

From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an invitro model of Mk differentiation from CD34+ cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 μM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41+CD42+ numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis.

The Selection of Lettuce Seedlings for Transplanting in a Plant Factory by a Non-Destructive Estimation of Leaf Area and Fresh Weight

Selecting uniform and healthy seedlings is important to ensure that a certain level of production can be reliably achieved in a plant factory. The objectives of this study were to investigate the potential of non-destructive image analysis for predicting the leaf area and shoot fresh weight of lettuce and to determine the feasibility of using a simple image analysis to select robust seedlings that can produce a uniform and dependable yield of lettuce in a plant factory. To vary the range of the leaf area and shoot fresh weight of lettuce seedlings, we applied two- and three-day irrigation intervals during the period of seedling production and calculated the projected canopy size (PCS) from the top-view images of the lettuce seedlings, although there were no significant growth differences between the irrigation regimes. A high correlation was identified between the PCS and shoot fresh weight for the lettuce seedlings during the period of seedling production, with a coefficient of determination exceeding 0.8. Therefore, the lettuce seedlings were classified into four grades (A–D) based on their PCS values calculated at transplanting. In the early stages of cultivation after transplanting, there were differences in the lettuce growth among the four grades; however, at the harvest (28 days after transplanting), there was no significant difference in the lettuce yield between grades A–C, with the exception of grade D. The lettuce seedlings in grades A–C exhibited the anticipated yield (150 g/plant) at the harvest time. In the correlation between the PCS and leaf area or the shoot fresh weight of lettuce during the cultivation period after transplanting and the entire cultivation period, the R2 values were higher than 0.9, confirming that PCS can be used to predict lettuce growth with greater accuracy. In conclusion, we demonstrated that the PCS calculation from the top-view images, a straightforward image analysis technique, can be employed to non-destructively and accurately predict lettuce leaf area and shoot fresh weight, and the seedlings with the potential to yield above a certain level after transplanting can be objectively and accurately selected based on PCS.

First Report of Fusarium fujikuroi Causing Postharvest Rot of Kiwifruit (Actinidia chinensis) in Jiangxi Province, China.

Kiwifruits (Actinidia chinensis) are among the most widely planted fruit in Jiangxi Province, China. Infected kiwifruits of the cultivars 'Hongyang' and 'Jinyan' were obtained from a commercial orchard in Fengxin county, Jiangxi Province (28°67' N; 115°42' E) from September to November 2022. The 1200 kiwifruits were collected from cold storage (cold stored for 3 months at 2°C), and moved to room temperatures (15 to 20°C), approximately 20% had symptoms of postharvest soft rot 7 days later. The infected fruits had brown or dark gray spots on the peel. Most were round or oval, with a diameter of approximately 1~3 cm. The pulp was milky white, and there was a waterlogged ring at the junction of decay. The pathogen was isolated by removing several small pieces (3×3 mm) of infected tissue from the diseased kiwifruits, which were sterilized with 75% ethanol for 30 s, dipped in 1% NaClO for 1 min, and rinsed three times with sterile distilled water. These pieces were transferred onto potato dextrose agar (PDA) and incubated for 5 days at 28°C, 75% relative humidity (RH), separated, and repurified. Eight unidentified isolates with similar morphology were obtained on PDA (D3-1 to D3-8). These isolates had abundant aerial fluffy mycelia. The colonies were white during the early stage of culture and turned light purple in the later stage. The mycelia grew 5.8 mm day-1 (n=5) on average and produced abundant conidia 10 days later. The microconidia were solitary, transparent, ovoid, with 0 to 1 septa, and 3.6 to 11.2 × 1.6 to 3.5 µm (average 6.5 × 2.9 µm, n = 50). The macroconidia were sickle-shaped, slender and slightly curved, with 3 to 5 septa, and 22.3 to 53.9 × 2.6 to 5.4 µm (average 39.5 × 4.3 µm, n = 50). Chlamydospores were absent. The morphological characteristics enabled the identification of the pathogen as Fusarium spp. (Leslie and Summerell, 2006). Isolate D3-2 was further confirmed, and the primers ITS1/ITS4 (White et al. 1990), 5F2/7CR and EF1/EF2 (O'Donnell et al. 2022) were used to amplify the internal transcribed spacer (ITS) region, RNA polymerase II largest subunit (RPB2) gene and translation elongation factor-1 alpha regions (TEF-1α). The ITS (accession no. PP077075), RPB2 (PP566653) and TEF-1α (PP566654) sequences shared 99.62 to 100% identities with ITS (ON564593.1), RPB2 (ON734380.1) and TEF-1α (ON697186.1) of F. fujikuroi from NCBI, respectively. Thus, the pathogen was identified as F. fujikuroi based on morphological and molecular characteristics. Each of the three isolates was inoculated on surface-disinfected (75% ethanol, 5 min) disease-free kiwifruits of cv. 'Jinyan' and 'Hongyang'. The six kiwifruits were pierced by a sterile inoculation needle and inoculated with 20 μl spore suspension (1×106 spores/ml), and six kiwifruits were treated with spore suspension without any wounds, four control fruits were inoculated with sterile distilled water. All the fruits were sealed in a storage box, kept at an RH of 90%-95%, and incubated at a constant temperature of 28°C for 5 days. After 3 days, the fruit rotted at the inoculation site, and after 5 days, the lesions gradually increased, and the symptoms were the same as those of the original sample. The control fruits remained disease-free. The pathogenicity tests were repeated three times. Koch's postulates were completed by reisolating the fungus from infected kiwifruits, which was identified as F. fujikuroi by sequencing. Although F. solani (Yang et al. 2018) and F. acuminatum (Wang et al. 2015) have been previously reported to rot kiwifruits in China, this is the first report of F. fujikuroi causing postharvest rot on kiwifruits in China. This discovery can alert agronomists to prevent and control this pathogen.

The role of alternative autophagy in cell viability and response to paclitaxel treatment in v-Ha-ras-transformed NIH 3T3 cells

In confluent v-Ha-ras-transformed NIH 3T3 fibroblasts (Ras-NIH 3T3), LC3 downregulation may precede a decrease in canonical autophagy, thus contributing to cell survival. Herein, we aimed to investigate the role of alternative autophagy in the viability of long-term cultures of Ras-NIH 3T3 cells and their parental NIH 3T3 cells. As cell confluence increased with the culture period, the level of alternative autophagy, as assessed through Lamp2-Rab9 co-localization, gradually decreased in both cell lines. However, Ras-NIH 3T3 cells maintained higher levels of alternative autophagy than the parental cells did. Rab9 knockdown minimally affected NIH 3T3 cells while drastically reducing the viability of Ras-NIH 3T3 cells, which suggested that alternative autophagy plays a critical role in Ras-NIH 3T3 cells. In contrast, reactive oxygen species (ROS) production in Ras-NIH 3T3 cells was higher than that in NIH 3T3 cells during long-term culture. Moreover, NIH 3T3 cells exhibited a continual decrease in mitochondrial mass, whereas Ras-NIH 3T3 cells maintained high mitochondrial mass. Immunofluorescence analysis of mitochondrial membrane marker proteins and mitochondrial membrane potential (MMP) also demonstrated a temporal pattern of changes similar to those of mitochondrial mass. This finding could be attributed to the relatively higher level of alternative autophagy in Ras-NIH 3T3 cells facilitating the removal of damaged mitochondria. Paclitaxel treatment in Ras-NIH 3T3 cells induced an increase in canonical autophagy rates along with suppression of alternative autophagy. Ras-NIH 3T3 cells showed high sensitivity to paclitaxel at the early stage of culture, but as cell confluence increased, resistance to paclitaxel increased, showing a similar level of cell viability to the vehicle control group. The study findings suggest that alternative autophagy is more important than canonical autophagy for maintaining cell survival in response to an unfavorable environment, such as during high cell confluence and exposure to anticancer agents.

Alterations in Soil Bacterial Community and Its Assembly Process within Paddy Field Induced by Integrated Rice–Giant River Prawn (Macrobrachium rosenbergii) Farming

Integrated rice–aquatic animal farming has become a vital strategy for enhancing food security. To assess the effects of integrated rice–giant river prawn (Macrobrachium rosenbergii) farming (IRPF) on agricultural ecosystems, we used 16S rRNA gene sequencing to analyze soil bacterial communities in comparison with traditional rice monoculture (RM). Our study revealed that the IRPF did not significantly affect the diversity of the soil bacterial community. However, during the initial culture stage, IRPF markedly increased the relative abundance of the phylum candidate division NC10 and the genus Candidatus Methylomirabilis, enhancing nitrogen-cycling-related functions within the bacterial community. Additionally, IRPF reduced the complexity and stability of these communities in the early to middle culture stages. While stochastic processes usually dominate the assembly of these communities, IRPF restricted bacterial migration and reduced the influence of these stochastic processes. Furthermore, IRPF had a significant impact on environmental factors within paddy soils, strongly correlating with shifts in bacterial communities, particularly through variations in soil nitrite concentration. In conclusion, the influence of IRPF on the bacterial community in paddy soils was primarily observed during the early and middle culture stages, and the impacts of IRPF on the soil bacterial community were primarily driven by environmental changes, especially soil nitrite concentration. These findings provide theoretical insights and a reference for understanding the microbiological impacts of different integrated rice–fish farming systems on agricultural ecosystems.

Microbial community dynamics and nitrogen transformation associated with turbid-turned culture stage of Pacific white shrimp, Litopenaeus vannamei in small greenhouse farms

Small greenhouse farms have experienced rapid expansion of Litopenaeus vannamei culture in China in recent years. To investigate microbial community dynamics and nitrogen transformation during the early critical stage, known as the turbid-turned stage in small greenhouse farms, microbial samples from the water column of small greenhouses were analyzed by 16S rRNA and 18S rRNA high-throughput sequencing, along with the detection of ammonia nitrogen in water. The results of water quality analysis revealed that accumulated ammonia nitrogen was converted to nitrite as the water color transitioned from clear green to turbid brown. High-throughput sequencing data demonstrated a shift in dominant microorganisms from zooplankton to algae and bacteria during the turbid-turned stage, and the main functional microbial taxa, such Marivita and Chlorella, were enriched. The results of Linear Discriminant Analysis Effect Size and canonical correlation analyses indicated that the compositions of key microbial communities were correlated with environmental factors. Furthermore, analysis of functional genes indicated that ammonia-oxidizing bacteria were present in the early growth stage, while the abundance of nitrite-oxidizing bacteria was low during the early culture stage. Overall, the findings suggest that microorganisms during the turbid-turned stage in small greenhouses may play a crucial role in regulating water quality and promoting shrimp health within a stable small greenhouse environment.

Application of fed-batch strategy to fully eliminate the negative effect of lignocellulose-derived inhibitors in ABE fermentation

BackgroundInhibitors that are released from lignocellulose biomass during its treatment represent one of the major bottlenecks hindering its massive utilization in the biotechnological production of chemicals. This study demonstrates that negative effect of inhibitors can be mitigated by proper feeding strategy. Both, crude undetoxified lignocellulose hydrolysate and complex medium supplemented with corresponding inhibitors were tested in acetone–butanol–ethanol (ABE) fermentation using Clostridium beijerinckii NRRL B-598 as the producer strain.ResultsFirst, it was found that the sensitivity of C. beijerinckii to inhibitors varied with different growth stages, being the most significant during the early acidogenic phase and less pronounced during late acidogenesis and early solventogenesis. Thus, a fed-batch regime with three feeding schemes was tested for toxic hydrolysate (no growth in batch mode was observed). The best results were obtained when the feeding of an otherwise toxic hydrolysate was initiated close to the metabolic switch, resulting in stable and high ABE production. Complete utilization of glucose, and up to 88% of xylose, were obtained. The most abundant inhibitors present in the alkaline wheat straw hydrolysate were ferulic and coumaric acids; both phenolic acids were efficiently detoxified by the intrinsic metabolic activity of clostridia during the early stages of cultivation as well as during the feeding period, thus preventing their accumulation. Finally, the best feeding strategy was verified using a TYA culture medium supplemented with both inhibitors, resulting in 500% increase in butanol titer over control batch cultivation in which inhibitors were added prior to inoculation.ConclusionProperly timed sequential feeding effectively prevented acid-crash and enabled utilization of otherwise toxic substrate. This study unequivocally demonstrates that an appropriate biotechnological process control strategy can fully eliminate the negative effects of lignocellulose-derived inhibitors.Graphical

Characterization of CD83 homologs differently expressed during monocytes differentiation in ginbuna crucian carp, Carassius auratus langsdorfii.

CD83 is a costimulatory molecule of antigen-presenting cells (APCs) that plays an important role in eliciting adaptive responses. It is also a well-known surface protein on mature dendritic cells (DCs). Furthermore, monocytes have been reported to differentiate into macrophages and monocyte-derived dendritic cells, which play an important role in innate immunity. CD83 expression affects the activation and maturation of DCs and stimulates cell-mediated immune responses. This study aims to reveal the CD83 expression during monocyte differentiation in teleosts, and the CD83 homologs evolutionary relationship. This study found two distinct CD83 homologs (GbCD83 and GbCD83-L) in ginbuna crucian carp (Gb) and investigated the evolutionary relationship among GbCD83 homologs and other vertebrates and the gene and protein expression levels of the homologs during 4 days of monocyte culture. The phylogenetic tree showed that the two GbCD83 homologs are classified into two distinct branches. Interestingly, only ostariophysians (Gb, common carp, rohu, fathead minnow and channel catfish), but not neoteleosts, mammals, and others, have two CD83 homologs. Morphological observation and colony-stimulating factor-1 receptor (CSF-1R), CD83, CD80/86, and CCR7 gene expressions illustrated that there is a differentiation of monocytes isolated from peripheral blood leukocytes after 4 days. Specifically, gene expression and immunocytochemistry revealed that GbCD83 is mainly expressed on monocytes at the early stage of cell culture, whereas GbCD83-L is expressed in the latter stage. These findings provided the first evidence of differential expression of CD83 homologs during monocytes differentiation in teleost.

Костяные коньки из погребения раннего этапа аланской культуры Северного Кавказа

The article publishes a mortuary complex of the late 3rd – early 4th centuries AD of the early stage of the North Caucasian Alan culture from kurgan 23 of the Oktyabrskiy I burial field in the Republic of North Ossetia –Alania, which contained a pair of bone skates. Bone skates have been known from written iconographic and ethnographic sources since the 12th century until the first half of the 20th century; however, the attribution of these items as skates raises doubts among a number of researchers, most likely due to the fact that almost all such artifacts, along with other similar bone items (tools), were found in the material remains of settlement sites. The researchers’ doubts resulted in a discussion, the main aspects of which are specified in the paper. The skates being published are made from the hind metapodia of the right legs of two domestic horses. Each of the artifacts has two holes for strings to attach the skates to the shoes. The skates are part of the grave goods of a young man or a youth, along with other leisure items (astragals) of young people. Finds of bone skates in burials are few in number, and, apart from the North Caucasian finds, there are only four Viking Age complexes in Swedish burial grounds. Bone skates were not a regular part of grave goods for centuries among any of the peoples, as evidenced by the insignificant number of burials with skates. Most researchers are convinced that bone skates were mainly used for entertainment by children, teenagers, and young people, which, in our opinion, is consistent with the material remains from kurgan 23 of the Oktyabrskiy I burial field. The grave goods also included a set of astragals (48 examples) associated with leisure items.

Bacterial Community Characteristics and Roles in Nitrogen Transformation in Industrial Farming Systems of Litopenaeus vannamei

Industrial farming is one of the main farming systems used for L. vannamei, and it is also the main direction of development in the future. To investigate the role of the bacterial community in nitrogen transformation in industrial L. vannamei farming systems, we studied the concentration of nitrogen compounds, the bacterial community using 16S rRNA sequencing, and nitrogen cycling genes’ abundance using absolute quantitative PCR in aquaculture water (using seawater (WD) and groundwater mixed with seawater (CY)) and analyzed the correlations between them using a person analysis. The results showed that the bacterial α-diversity index (ACE, Shannon, and Sobs) significantly changed during culture in WD but not in CY. Bacterial community species composition analysis and linear discriminant analysis effect size (LEfSe analysis) revealed that Cyanobiaceae and Microbacteriaceae were the dominant bacteria and biomarkers in WD. Vibrionaceae, Ateromonadaceae, Microbacteriaceae, Saprospiraceae, and Cryomorphaceae were the dominant bacteria and biomarkers in CY. A functional annotation of procaryotic taxa (FAPROTAX) analysis revealed that the bacterial community in WD exhibited a greater phototrophic activity at early culture stages, while at the same stage, CY exhibited strong nitrate reduction. A correlation analysis of nitrogen cycling genes and environmental factors revealed that napA, narG, ureC, amoA, and nirK were significantly correlated with the concentrations of total ammonia nitrogen (TAN), nitrite (NO2−-N), nitrate (NO3−-N), and total nitrogen (TN) in WD. In CY, amoA was positively correlated and napA and nirK were negatively correlated with TAN and TN concentrations. A correlation analysis between bacterial abundance and environmental factors revealed that Flavobacteriaceae, Saprospiraceae, Cryomorphaceae, Cyanobiaceae, Halieaceae, and Cyclobacteriaceae were significantly correlated with the concentrations of TAN and NO2−-N. The above results indicated that the bacterial community in industrial shrimp farming systems changed under different conditions, with consequent changes in the abundance of genes being involved in the nitrogen cycle and in this biogeochemical process in the water. Our study facilitates further understanding of microbes and their functions in nitrogen cycling in industrial shrimp farming systems.

Insulin-transferrin-selenium promote formation of tissue-engineered vascular grafts in early stage of culture

To create tissue-engineered vascular grafts (TEVGs) in vitro, vascular smooth muscle cells (VSMCs) must function effectively and produce sufficient extracellular matrix (ECM) in a three-dimensional space. In this study, we investigated whether the addition of insulin-transferrin-selenium (ITS), a medium supplement, could enhance TEVG formation. PGA fabric was used as the scaffold, and 1% ITS was added to the medium. After two weeks, the tissues were examined using electron microscopy and staining. The ITS group exhibited a denser structure and increased collagen production. VSMCs were cultured in two dimensions with ITS and assessed for collagen production, cell growth, and glucose metabolism. The results showed that ITS supplementation increased collagen production, cell growth, glucose utilization, lactate production, and ATP levels. Furthermore, reducing the amount of fetal bovine serum (FBS) in the medium did not affect the TEVGs or VSMCs when ITS was present. In conclusion, ITS improves TEVG construction by promoting VSMCs growth and reducing the need for FBS.

Phytosulfokine alpha enhances regeneration of transformed and untransformed protoplasts of Brassica oleracea.

Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of in vivo plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on in vitro cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four Brassica oleracea cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.

Particle aggregation induced by microorganisms is a key mechanism for the biostabilization of coastal sediment

Biofilm surrounding sediment particles can significantly enhance the stability of coastal sediment. This phenomenon is known as biostabilization. Biofilm mainly consists of extracellular polymeric substances (EPS) and microorganisms. While the role of EPS in biostabilization is well-established, microbial contribution requires further exploration. Herein, an experiment was conducted to compare the stability and morphological characteristics of sediment under the effects of biofilm (with microorganisms) and pure EPS extracted from the biofilm (without microorganisms). The microorganisms used for biofilm culture are composed of diatoms and chlorella. The sediment used is a mixture of well-sorted muddy sand (D50 = 77 μm). Erosion tests showed that in the early stage of biofilm culture (within 17 days), biofilm and pure EPS had a similar ability to stabilize sediment, suggesting that in this stage, biofilm stabilized sediment relying on EPS. However, after 17 days of culture, biofilm showed a stronger ability to stabilize sediment than pure EPS, implying that in this stage, biofilm stabilized sediment by both EPS and microorganisms. These results indicate that microorganisms can also contribute to biostabilization, but during the early stage of culture, microorganisms in biofilm cannot make such contributions. Optical observations found that when the sediment-stabilizing ability of biofilm and pure EPS was comparable, the morphological characteristics of sediment particles under the effects of them were similar, both appearing as scattered single particles. However, when biofilm exhibited a stronger sediment-stabilizing ability, sediment particles with biofilm connected into large-size aggregates, whereas sediment particles with pure EPS remained single and scattered. These results suggest that in the later stage of biofilm culture, microorganisms play a role in the aggregation of sediment particles and hence contribute to biostabilization. Based on these findings, a conceptual framework has been proposed for predicting sediment stability under the effects of biofilm.

An Artificial Intelligence Framework for Disease Detection in Potato Plants

Agricultural products are a fundamental necessity for every country. When plants are afflicted with diseases, it influences the country's agricultural productivity, as well as its economic resources. Diseases are an important problem for potato plants, causing potatoes to be rejected and resulting in financial losses. Viruses and diseases in potatoes and field plants can be missed with the naked eye, particularly in the early stages of cultivation. The use of modern instruments and technology at an early stage of disease diagnosis dramatically reduces costs. This study used deep learning techniques to categorize and detect plant leaf diseases in photos taken from the Plant Village dataset. The dataset consists of 20,636 photos of plants and their diseases. This study focused on potato plants because it is the most common type of plant in the world, particularly in Pakistan. Convolutional Neural Network (CNN) methods were used to categorize plant leaf diseases into 15 classes, including three classes for healthy leaves and classes for several plant diseases such as fungal and bacterial infections, among others. The proposed models were trained and tested, achieving 98.29 and 98.029% accuracy, respectively.

Macromolecular Crowding Enhances Matrix Protein Deposition in Tissue-Engineered Vascular Grafts.

Successful in vitro culture of small-diameter tissue-engineered vascular grafts (TEVGs) requires rapid deposition of biomacromolecules secreted by vascular smooth muscle cells in a polyglycolic acid mesh scaffold's three-dimensional (3D) porous environment. However, common media have lower crowding conditions than in vivo tissue fluids. In addition, during the early stages of construction, most of the biomolecules secreted by the cells into the medium are lost, which negatively affects the TEVG culture process. In this study, we propose the use of macromolecular crowding (MMC) to enhance medium crowding to improve the deposition and self-assembly efficiency of major biomolecules in the early stages of TEVG culture. The addition of carrageenan significantly increased the degree of MMC in the culture medium without affecting cell viability, proliferation, and metabolic activity. Protein analysis demonstrated that the deposition of collagen types I and III and fibronectin increased significantly in the cell layers of two-dimensional and 3D smooth muscle cell cultures after the addition of a MMC agent. Collagen type I in the culture medium decreased significantly compared with that in the medium without a MMC agent. Scanning electron microscopy demonstrated that MMC agents considerably enhanced the formation of matrix protein structures during the early stages of 3D culture. Hence, MMC modifies the crowding degree of the culture medium, resulting in the rapid formation of numerous matrix proteins and fiber structures. Impact Statement Small-diameter tissue-engineered vascular grafts (TEVGs) are one of the most promising means of treating cardiovascular diseases; however, the in vitro construction of TEVGs has some limitations, such as slow deposition of extracellular matrix (ECM), long culture period, and poor mechanical properties. We hypothesized that macromolecular crowding can increase the crowding of the culture medium to construct a more bionic microenvironment, which enhances ECM deposition in the medium to the cell layer and reduces collagen loss, accelerating and enhancing TEVG culture and construction in vitro.

Comparative study of the growth, survival and morphology of the whitefish hybrids and parent species in the first year of rearing in cage aquaculture

The purpose of the work: to estimate fish breeding indicators of three hybrid forms of whitefish in comparison with parental species when grown in cage aquaculture.Methods used: the production and cultivation of hybrids and parental species were carried out using standard technology for whitefish from November 2021 to May 2022. We have studied the survival and growth rate of fingerlings, and we also conducted a comparative assessment of the exterior of yearlings using factor and cluster analyses.Novelty: for the first time, three hybrid forms of peled and parent species (peled, muksun, broad whitefish, nelma) were grown in cages, and a comparative analysis of survival, growth and morphology of hybrids and parent species was performed.Results: all hybrid forms occupy an intermediate position in terms of growth rate, surpassing the maternal species (peled), yielding to the paternal ones. The best results in terms of weight and linear growth among hybrids were noted in pelchir. In terms of survival, oneyearlings of all hybrids show the effect of heterosis, although at the stage of larvae and early juveniles, the superiority in survival of hybrid forms over the parental pair was noted only in pelnelm. The survival rate of hybrid eggs, as well as the survival rate of pelchir and pelmuk at the early stages of cultivation, had intermediate values — lower than that of peled, but significantly higher than that of paternal species. The lowest survival rate of eggs among hybrids is shown in pelchir. The peled has the most significant morphological differences from other pure species and hybrid forms used in the experiment, all hybrid forms show more similarity with the paternal species.Practical significance: results provide a potential assessment of whitefish hybrids for the prospect of their use in commercial aquaculture.

The Addition of Zinc to the ICIE16-Bioactive Glass Composition Enhances Osteogenic Differentiation and Matrix Formation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

An ICIE16-bioactive glass (BG) composition (in mol%: 49.5 SiO2, 6.6 Na2O, 36.3 CaO, 1.1 P2O5, and 6.6 K2O) has demonstrated excellent in vitro cytocompatibility when cultured with human bone marrow-derived mesenchymal stromal cells (BMSCs). However, its impact on the development of an osseous extracellular matrix (ECM) is limited. Since zinc (Zn) is known to enhance ECM formation and maturation, two ICIE16-BG-based Zn-supplemented BG compositions, namely 1.5 Zn-BG and 3Zn-BG (in mol%: 49.5 SiO2, 6.6 Na2O, 34.8/33.3 CaO, 1.1 P2O5, 6.6 K2O, and 1.5/3.0 ZnO) were developed, and their influence on BMSC viability, osteogenic differentiation, and ECM formation was assessed. Compared to ICIE16-BG, the Zn-doped BGs showed improved cytocompatibility and significantly enhanced osteogenic differentiation. The expression level of the osteopontin gene was significantly higher in the presence of Zn-doped BGs. A larger increase in collagen production was observed when the BMSCs were exposed to the Zn-doped BGs compared to that of the ICIE16-BG. The calcification of the ECM was increased by all the BG compositions; however, calcification was significantly enhanced by the Zn-doped BGs in the early stages of cultivation. Zn constitutes an attractive addition to ICIE16-BG, since it improves its ability to build and calcify an ECM. Future studies should assess whether these positive properties remain in an in vivo environment.