Growth Medium Research Articles

Manipulation of Cancer Cell Spheroids by Photovoltaic Tweezers: Determination of Their Charge State

Photovoltaic optoelectronic tweezers (PVOT), based on the bulk photovoltaic effect presented by certain ferroelectric crystals, have emerged as a versatile tool to manipulate and/or trap micro‐ and nano‐objects. Very recently, an ingenious method has enabled to extend PVOT manipulation to aqueous solution droplets opening the door to applications in optofluidics and biotechnology. The photovoltaic crystal is placed on the bottom of a cuvette filled with paraffin oil while the droplet is hanging from the air–oil interphase where it is manipulated. Herein, this method is used to investigate the feasibility of manipulating hybrid droplets as carriers containing a cancer multicellular spheroid inside cell culture medium. Droplets of two cancer cell lines, MCF‐7 and U‐87 MG, have been successfully manipulated by light‐induced electrical fields generated in the photovoltaic platform. From the droplet dynamics, which includes attractive and repulsive behaviors from the illuminated crystal region, the charge state of the cell culture medium and the spheroid are investigated. The results show that they are positively and negatively charged, respectively. Moreover, analyzing the migration kinetics of water, cell culture medium, and hybrid droplets, to the light of a proposed theoretical model, the charge density of both cell line spheroids has been estimated.

Use of the droplet convergence effect of under-medium superlyophilic membranes in designing a system for on-demand separations of oil-in-water and water-in-oil emulsions

Separation using superwetting membranes that have discrepant interactions with water and oil, primarily those that display extreme liquid repellence, is a facile approach for oily wastewater treatment and purification of water-containing oil. However, these kinds of membranes suffer from size-sieving limitation. Owing to greater affinity toward dispersed droplets over liquid media, under-medium superlyophilic membranes have greater flexibility in the design of superwetting interfaces for oil–water separation. Herein, two membranes with completely opposite superwettabilities both in air and under oil/water media were utilized to create an integrated system for effective, on-demand separation of oil-in-water and water-in-oil emulsions. At superwetting interfaces, the under-medium superlyophilic membrane facilitates convergence of emulsion droplets while the other membrane intercepts converged droplets. As a result, the integrated membranes exhibit high purification capabilities of water and oil in surfactant-stabilized emulsions (e.g., > 99.97 % oil purification under gravity). The viscosity-dependent fluxes are > 550 L m−2h−1 when the viscosities of the liquid media are ≤ 1 mPa s. In contrast, an integrated system in which conventional in-air superhydrophilic membranes are utilized in place of the under-medium superlyophilic membranes does not efficiently separate the emulsions. The unique affinity profiles will endow under-medium superlyophilic membranes with a high potential for liquid–liquid separation.

Optimization of Citrus Pulp Waste-Based Medium for Improved Bacterial Nanocellulose Production.

Bacterial nanocellulose (BC) has attracted significant attention across a wide array of applications due to its distinctive characteristics. Recently, there has been increasing interest in leveraging waste biomass to improve sustainability in BC biogenesis processes. This study focuses on optimizing the citrus pulp waste (CPW) medium to enhance BC production using Komagataeibacter sucrofermentans. The screening of initial medium pH, yeast extract, CPW sugar and inoculum concentrations was conducted using the Plackett-Burman design, with BC yield (mgDW/gCPW) as the model response. The significant parameters, i.e., CPW sugars and yeast extract concentrations, were optimized using response surface methodology, employing a five-level, two-factor central composite design. The optimized CPW-based growth medium resulted in a final yield of 66.7 ± 5.1 mgDW/gCPW, representing a 14-fold increase compared to non-optimized conditions (4.3 ± 0.4 mgBC/gCPW). Material characterization analysis indicated that the produced BC showed high thermal stability (30% mass retained at 600 °C) and a crystallinity index value of 71%. Additionally, to enhance process sustainability, spent baker's yeast hydrolysate (BYH) was assessed as a substitute for yeast extract, leading to a final BC titer of 9.3 ± 0.6 g/L.

COLMAR1d: A Web Server for Automated, Quantitative One-Dimensional Nuclear Magnetic Resonance-Based Metabolomics at Arbitrary Magnetic Fields.

The field of metabolomics, which is quintessential in today's omics research, involves the large-scale detection, identification, and quantification of small-molecule metabolites in a wide range of biological samples. Nuclear magnetic resonance spectroscopy (NMR) has emerged as a powerful tool for metabolomics due to its high resolution, reproducibility, and exceptional quantitative nature. One of the key bottlenecks of metabolomics studies, however, remains the accurate and automated analysis of the resulting NMR spectra with good accuracy and minimal human intervention. Here, we present the COLMAR1d platform, consisting of a public web server and an optimized database, for one-dimensional (1D) NMR-based metabolomics analysis to address these challenges. The COLMAR1d database comprises more than 480 metabolites from GISSMO enabling a database query of spectra measured at arbitrary magnetic field strengths, as is demonstrated for spectra acquired between 1H resonance frequencies of 80 MHz and 1.2 GHz of mouse serum, DMEM cell growth medium, and wine. COLMAR1d combines the GISSMO metabolomics database concept with the latest tools for automated processing, spectral deconvolution, database querying, and globally optimized mixture analysis for improved accuracy and efficiency. By leveraging advanced computational algorithms, COLMAR1d offers a user-friendly, automated platform for quantitative 1D NMR-based metabolomics analysis allowing a wide range of applications, including biomarker discovery, metabolic pathway elucidation, and integration with multiomics strategies.

Mimicking and in vitro validating chronic inflammation in human gingival fibroblasts

ObjectiveThe aim of this study was to identify and validate in vitro conditions that may mimic the translational, cytokine and chemokine profiles observed in human inflamed gingiva in vivo. DesignPrimary human gingiva fibroblast cells (HFIB-G) were cultured under serum starvation conditions (0 – 10 %), supplemented with increasing lipopolysaccharide (LPS) concentrations (0.1, 1, or 10 µg/ml) from two bacterial strains E. coli and P. gingivalis and 0.1, 1, or 10 ng/ml recombinant interleukin 1β (IL-1β), alone or in combinations. The levels of cytokines/chemokines were measured in the cell culture medium by Luminex, and gene expression was quantified by Affymetrix microarrays at 24, 48 and 72 h. ResultsInflammation markers were not elevated after stimulation with P. gingivalis LPS, while E. coli LPS and IL-1β individually increased the secretion of interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) to the cell culture medium. IL-1β administration also increased the secretion of several factors, including tumor necrosis factor (TNFα). However, the combination of 1 µg/ml E. coli LPS, 1 ng/ml IL-1β and serum starvation led to increased secretion of IL-6, TNFα, in addition to other factors found in inflamed tissue. Gene expression analyses revealed that this combination not only enhanced the expression interleukins/chemokines genes but also T helper cell signaling and matrix metalloproteinases. ConclusionSerum reduction in cell culture medium together with the administration of E. coli LPS and IL-1β resulted in gene expression and secreted cytokine/chemokine profiles similar to that found in vivo during chronic inflammation.

Accumulation of podophyllotoxin in the root culture of Hyptis suaveolens (L.) Poit: A potential natural lignan for clinically useful anticancer drugs

Podophyllotoxin (PTOX) is a natural antiviral, antirheumatic and anticancer molecule, but it is expensive to chemically synthesize. The present study aimed to evaluate growth and PTOX accumulation in root cultures of Hyptis suaveolens treated with different concentrations of auxins, vitamins and myo-inositol. Root cultures grown in liquid medium supplemented with different concentrations of indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), vitamins and myo-inositol were used to measure biomass production and PTOX content. PTOX quantitation was performed via high-performance liquid chromatography with diode-array detection (HPLC-DAD) using a newly developed analytical method after successful validation. Root culture in MS medium supplemented with 1 mg L−1 IBA +0.5 mg L−1 NAA resulted in the highest root dry weight (248.76 mg) and the highest PTOX concentration in the roots (179.97 μg g−1 root). The roots cultured in liquid MS medium supplemented with 1 mg L−1 IBA +0.5 mg L−1 NAA presented the greatest increase in root biomass and PTOX content. This adequate balance of vitamin and myo-inositol supplementation in liquid MS culture medium increased the root dry weight and PTOX content.

Role of Denitrification in Selenite Reduction by Azospirillum brasilense with the Formation of Selenium Nanoparticles.

Many bacteria are capable of reducing selenium oxyanions, primarily selenite (SeO32-), in most cases forming selenium(0) nanostructures. The mechanisms of these transformations may vary for different bacterial species and have so far not yet been clarified in detail. Bacteria of the genus Azospirillum, including ubiquitous phytostimulating rhizobacteria, are widely studied and have potential for agricultural biotechnology and bioremediation of excessively seleniferous soils, as they are able to reduce selenite ions. Cultures of A.brasilense Sp7 and its derivatives (mutant strains) were grown on the modified liquid malate salt medium in the presence or absence of selenite. The following methods were used: spectrophotometric monitoring of bacterial growth; inhibition of glutathione (GSH) synthesis in bacteria by L-buthionine-sulfoximine (BSO); optical selenite and nitrite reduction assays; transmission electron microscopy of cells grown with and without BSO and/or selenite. In a set of separate comparative studies of nitrite and selenite reduction by the wild-type strain A.brasilense Sp7 and its three specially selected derivatives (mutant strains) with different rates of nitrite reduction, a direct correlation was found between their nitrite and selenite reduction rates for all the strains used in the study. Moreover, for BSO it has been shown that its presence does not block selenite reduction in A.brasilense Sp7. Evidence has been presented for the first time for bacteria of the genus Azospirillum that the denitrification pathway known to be inherent in these bacteria, including nitrite reductase, is likely to be involved in selenite reduction. The results using BSO also imply that detoxification of selenite through the GSH redox system (which is commonly considered as the primary mechanism of selenite reduction in many bacteria) does not play a significant role in A.brasilense. The acquired knowledge on the mechanisms underlying biogenic transformations of inorganic selenium in A.brasilense is a step forward both in understanding the biogeochemical selenium cycle and to a variety of potential nano- and biotechnological applications.

Study of biotechnological potential of hydrocarbon-oxidizing bacteria from oil-contaminated soils of the Uzen oil field

Background: Oil-contaminated soils are one of the significant environmental concerns in Western Kazakhstan. Cleaning soils from oil contamination is becoming extremely important. Biological soil treatment, which uses hydrocarbon-oxiding bacteria for microbiological remediation, is a more environmentally friendly and delicate method of soil treatment than any other currently used technology (physical, chemical, biological). One of the important aspects of microbiological remediation is the use of bacteria endomimic to the treated soil. This guarantees the most effective soil purification, as the bacteria perform in relatively optimal conditions for themselves. The soil of the Mangistau region, especially the coastal regions, is distinctive due to its high mineralisation and low moisture content, which have helped to form a specific microflora that is adapted to these conditions. Aim: The study aims to search and isolate highly effective hydrocarbon-oxidizing bacteria that are native to highly mineralized soils of the Uzen oil field. Materials and methods: The research employs a variety of analytical techniques, including water chemistry analysis and infrared spectrometry, as well as the microbiological culture of aerobic bacteria on liquid and dense media in environments (temperature and salinity) that closely resemble their native ecotopes. Results: Four enrichment cultures of hydrocarbon-oxidizing bacteria were obtained, and three pure cultures of oil-oxidizing bacteria were isolated. Their hydrocarbon-oxidizing efficiency has been studied. On the basis of heavy, extremely viscous paraffinic oil from the Uzen field, their hydrocarbon-oxidizing efficiency was examined. Conclusion: In this study, active enrichment cultures of halophilic hydrocarbon-oxidizing bacteria as well as active accumulative cultures of halophilic and moderately thermophilic aerobic bacteria were obtained. These bacteria are capable of oxidising a wide range of hydrocarbons, including high-molecular polycyclic and sulfur-containing compounds. Their high biotechnological potential will be studied in further studies.

Cold atmospheric plasma activation of human gingival fibroblasts for improved wound healing

Abstract Soft tissue regeneration plays a crucial role after oral surgery, as the successful healing of the soft tissue is a primary indicator of an efficacious intervention. Cold atmospheric plasma (CAP) has recently emerged as a promising therapeutic modality, exhibiting notable effects on cell migration and proliferation. Despite its potential, the dental application of CAP remains underexplored. This in vitro study aims to elucidate the impact of CAP activated medium on human gingival fibroblast responses, for future wound healing strategies. The study was divided into four parts: initial characterization of the plasma Jet, assessment of cell concentration, exploration of treatment distance effects, and treatment time dynamics. Human gingival fibroblasts were exposed to complete DMEM medium (without sodium pyruvate) activated with CAP at treatment distances of 2, 5, 7, and 9 mm, and treatment times of 15, 60, 120, 180, and 300 s for 1, 2 and 3 d of culture. The cell viability was evaluated using resazurin-based method, while wound healing dynamics was assessed via the scratch assay technique using phase-contrast microscopy. The cell morphology was characterised through fluorescence microscopy using propidium iodide and phalloidin staining, complemented by scanning electron microscopy. The results revealed that treatment distances and exposure times can influence the cell behaviour depending on the cell concentration. For the selected concentration of 1 × 104 cells ml−1, a treatment distance of 9 mm appeared to enhance human gingival fibroblast viability compared to a treatment distance of 2 mm and the control group. The images revealed adherent cells with a pattern typical of fibroblasts. However, no differences were observed for exposure times of 15 s and 180 s. The observed results further evidence that the exposure of the medium to the CAP device promoted an increase in cell viability, proliferation, and attachment in human gingival fibroblasts.

The e-Flower: A hydrogel-actuated 3D MEA for brain spheroid electrophysiology.

Traditional microelectrode arrays (MEAs) are limited to measuring electrophysiological activity in two dimensions, failing to capture the complexity of three-dimensional (3D) tissues such as neural organoids and spheroids. Here, we introduce a flower-shaped MEA (e-Flower) that can envelop submillimeter brain spheroids following actuation by the sole addition of the cell culture medium. Inspired by soft microgrippers, its actuation mechanism leverages the swelling properties of a polyacrylic acid hydrogel grafted to a polyimide substrate hosting the electrical interconnects. Compatible with standard electrophysiology recording systems, the e-Flower does not require additional equipment or solvents and is ready to use with preformed 3D tissues. We designed an e-Flower achieving a curvature as low as 300 micrometers within minutes, a value tunable by the choice of reswelling media and hydrogel cross-linker concentration. Furthermore, we demonstrate the ability of the e-Flower to detect spontaneous neural activity across the spheroid surface, demonstrating its potential for comprehensive neural signal recording.

Low toxicity of dissolved silver from silver-coated titanium dental implants to human primary osteoblast cells

Objectivesthe controlled release of silver as a biocide from Ag-coated medical implants is desirable. However, the biocompatibility of Ag leachates is poorly understood. This study investigated the toxicity of silver released from the silver plated titanium implants to human primary osteoblast cells; and the effect of cell culture medium on the silver speciation and bioavailability. Methods: Ti6Al4V discs were coated with Ag nanoparticles (NPs), silver plus hydroxyapatite (HA) nanoparticles (Ag+nHA), or Ag NPs plus microparticles (Ag+mHA). Primary human osteoblast cells were exposed to the leachates from the various discs for up to 7 days. Results: the total Ag concentrations released as leachate from the silver-plated titanium discs were 0.7–1.6 mg L−1, regardless of treatment. Cumulative silver release over 7 days was approximately 3 mg L−1 in all treatments. The concentration of total Ag in the cell homogenates from all the Ag-containing treatments was modest, ∼ 0.1 µg mg protein−1 or less at day 7. Cells showed normal healthy morphology with no appreciable leak of LDH or ALP activity into the external media compared to the reference control. Similarly, there was no significant differences (Kruskal Wallis, p > 0.05) in the LDH or ALP activity in the cell homogenate between treatments. Conclusions: overall, there was a controlled release of Ag into the external media, but this remained biocompatible with no deleterious effects on the osteoblast cells, which means that the released silver to the peri-implant environment is not toxic making the coating potential for clinical use

Biofabricated osteoblast-derived nanovesicles as extracellular vesicle mimics for bone repair

Osteoblast-derived extracellular vesicles (EVs) have demonstrated therapeutic utility for bone repair as transporters of key biomolecules capable of accelerating biomineralisation and tissue repair. The clinical translation of these biologically derived nanoparticles, however remains limited due to scalability, heterogeneity and standardisation issues. Herein we investigate the generation of nanovesicles (NVs) from mineralising osteoblasts by extrusion directly compared against natural EV counterparts from the same parental cells.Mineralising osteoblast-derived EVs (MO-EVs) were isolated via ultracentrifugation from cell culture media. The parental osteoblasts were then processed via serial extrusion to <200 nm. EVs and NVs were characterised by comparing their size, concentration and morphology. The presence of tetraspanin markers was detected by Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). Osteoblasts viability and metabolic activity was assessed after both EV and NV-treatment before comparing their mineralising potency via alizarin red staining.EVs and NVs exhibited similar diameters of approximately 100 nm with a vesicular morphology. EVs were found to be richer in proteins and exhibited a significantly more negative ζ-potential compared to NVs. SP-IRIS analysis confirmed the presence of CD9, CD63 and CD81 in EVs. At both 1 and 10 μg/mL, EVs and NVs reduced the metabolic activity of osteoblasts, however, this was not associated with any cytotoxic effects. The biomineralisation study performed using osteoblasts showed that only EVs significantly increased mineral deposition (p < 0.05) compared to untreated control.In this study, we have established for the first time the biofabrication of cell-derived nanovesicles as a promising alternative to extracellular vesicles derived from mineralising osteoblasts.

The ability of Arabidopsis to recover from Basta and its application in isolating Cas9-free mutants.

After successfully performing Agrobacterium-mediated CRISPR-Cas9-based gene editing in plants, isolation of the Cas9 T-DNA is essential for the stable inheritance of induced mutations. Here, we report a simple technique that allows the isolation of Cas9-free mutants, eliminating the need for outcrossing or other intricate methods. This method is based on the ability of Basta-sensitive Arabidopsis thaliana seedlings, which generally perish, to recover and grow once transplanted to Basta-free growth media. By growing gene-edited heterozygous populations of single-locus insertion Basta-resistant plants on Basta selection media, plants lacking the Cas9 T-DNA can be identified. These pale-looking plants lacking Cas9 are then rescued on media lacking the Basta to recover Cas9-free plants. The ability of seedlings to recover from Basta selection was also studied in camelina, canola, and wheat. All three crops showed different recovery rates, with wheat demonstrating the highest recovery once transplanted from Basta to normal growth media. In summary, our findings demonstrate that by harnessing the recovery capability of Basta-sensitive seedlings, we can effectively identify and rescue plants lacking the Cas9 T-DNA, enabling the isolation of Cas9-free mutants in Arabidopsis and potentially extending to other crops.

Growth, physiological and N, P, K accumulation responses of Erythropalum scandens Bl. Seedlings under different substrates

Erythropalum scandens Bl. is a medicinal woody vegetable found in southern China and parts of Southeast Asia. Studies have shown improper substrate hindered E. scandens seedling growth, causing water accumulation and nutrient deficiency. In pursuit of an ideal growth medium for E. scandens seedlings during the early stages, this study conducted a pot experiment to identify a mixed substrate with optimal water permeability and fertility. In this study, pure Alfisols soil treatment as the control (CK), and two soilless substrates (peat soil and perlite) were combined with Alfisols soil into different volume ratios, in order to better use soil resources from understory space and balance the texture of mixed substrates. The growth, physiological characteristics and nutrient status of 24-month-old E. scandens seedlings were determined after planting in different mixed ratios. The results showed that as the proportion of peat soil increased in the mix, most indexes exhibited an initial increase followed by a decline, while soluble protein content decreased consistently. Conversely, an increasing perlite ratio resulted in a general decline in most growth and physiological indexes. Root growth, biomass accumulation and chlorophyll content, peaked in the 66.67% Alfisols soil + 33.33% perlite (T4) treatment. Notably, T3 (66.67% Alfisols soil + 33.33% peat soil) showcased the best above-ground growth, while T1 (50.00% Alfisols soil + 50.00% peat soil) excelled in element content accumulation. In conclusion, the cultivation substrate should primarily consist of Alfisols soil, constituting at least 50%. The addition of peat soil enhances above-ground growth and nutrients accumulation, while perlite contributes to robust root development. One third of peat soil and a small amount of perlite can be added to the substrate during E. scandens seedling cultivation, and proper fertilization should also be used in order to increase nutrient accumulation in aboveground and underground parts. This research provides valuable insights into maximizing the potential of E. scandens seedlings through precise cultivation methods.

Investigating the Impact of Microgravity on Stem Cells for Cardiovascular Disease Treatment

Cardiovascular disease is a widespread cause of physical disability, and while treatment options result in reduced mortality, they are still limited in their capabilities for cardiac repair. Since their discovery, the regenerative capacities of stem cells have been hypothesized to one day reverse countless diseases. Current stem cell research in cardiovascular disease has delineated the regenerative capabilities of bone marrow–derived mesenchymal stem cells, induced pluripotent stem cells, and cardiac stem cells, but has its limitations. Additionally, current therapies reduce mortality rates and the risk of recurrent myocardial infarctions and damage, but they do not address the apparently irreversible remodeling of myocardial tissue, which is linked to frequent hospitalizations and a diminished quality of life. As researchers continue to investigate the most efficacious growth mediums for stem cells, microgravity appears to be a supportive environment for stem cell growth. Thus, this review aims to catalogue existing research on microgravity’s differing effects on various stem cell lineages and what implications it may have on the treatment of cardiovascular diseases. Cells grown under these conditions have increased expression of cardioprotective proteins and improved proliferative capacities but may have elevated tumorigenic potential and hypertrophic pathways. Additionally, the results of microgravity’s effects on stem cells are mixed. Nonetheless, microgravity’s impact on the cytoskeleton, overexpression of electrical contraction receptors, and increased proliferative and pluripotent effects make these cells promising avenues for potential improved clinical treatment options.

Expression, regulation and physiological roles of the five Rsm proteins in Pseudomonas syringae pv. tomato DC3000

Proteins belonging to the RsmA (regulator of secondary metabolism)/CsrA (carbon storage regulator) family are small RNA-binding proteins that play crucial roles post-transcriptionally regulating gene expression in many Gram-negative and some Gram-positive bacteria. Although most of the bacteria studied have a single RsmA/CsrA gene, Pseudomonas syringae pv. tomato (Pto) DC3000 encodes five Rsm proteins: RsmA/CsrA2, RsmC/CsrA1, RsmD/CsrA4, RsmE/CsrA3, and RsmH/CsrA5. This work aims to provide a comprehensive analysis of the expression of these five rsm protein-encoding genes, elucidate the regulatory mechanisms governing their expression, as well as the physiological relevance of each variant. To achieve this, we examined the expression of rsmA, rsmE, rsmC, rsmD, and rsmH within their genetic contexts, identified their promoter regions, and assessed the impact of both their deletion and overexpression on various Pto DC3000 phenotypes. A novel finding is that rsmA and rsmC are part of an operon with the upstream genes, whereas rsmH seems to be co-transcribed with two downstream genes. We also observed significant variability in expression levels and RpoS dependence among the five rsm paralogs. Thus, despite the extensive repertoire of rsm genes in Pto DC3000, only rsmA, rsmE and rsmH were significantly expressed under all tested conditions (swarming, minimal and T3SS-inducing liquid media). Among these, RsmE and RsmA were corroborated as the most important paralogs at the functional level, whereas RsmH played a minor role in regulating free life and plant-associated phenotypes. Conversely, RsmC and RsmD did not seem to be functional under the conditions tested.

Growth media change Eisenia fetida epithelium thickness: implications for improving earthworm welfare in vermicomposting systems.

Because the earthworm tegument represents their first barrier against environmental aggressions, we hypothesised that substrate-induced changes in earthworm growth would also alter the morphology of this protective layer. Therefore, a histological and morphometric study was performed on Eisenia fetida specimens that have been grown in five different organic residues: cow manure, horse manure, grape marc, coffee grounds, and a mixture of coffee grounds and cooking oil. The results showed that, across all treatments, both cuticle and epidermis were significantly thicker in the pre-clitellar region than after the clitellum, attributed to the fact that the former region is responsible for breaking up the soil. Notably, the growing media significantly altered the thickness of these two integumentary layers, and the greatest thicknesses were measured in grape marc and in the mixture coffee grounds + cooking oil, which makes them suitable organic residues for vermicomposting. By contrary, a significant epidermal thinning was observed when growing on coffee grounds alone, clearly indicating that it is a hostile environment for earthworms. Overall, these results reveal that tegument thickness can be used not only as a trait indicator of substrate quality but also of earthworm welfare, and it should be integrated in future toxicity assessment and captive breeding guidelines.

Establishment, Multiplication, and Biochemical Analysis of Embryogenic Lines of the Amazonian Palm Euterpe precatoria Mart. under Suspension Culture

The palm Euterpe precatoria holds great social, cultural, and environmental importance. The heart of palm and the fruit are the main products used for industrialization due to their energetic properties. Thus, the aim of this study was to establish a suspension cultivation protocol for the species using different explant sources. For this, eight lineages of E. precatoria embryogenic calluses were tested, with five in liquid medium Murashige and Skoog (MS) with 5 μM Picloram and three for comparison in semisolid medium MS with 20 μM Picloram and 5 μM 2iP. The growth curve was obtained by weighing the calli from 60 to 180 days of cultivation. The Gompertz model was applied, and growth kinetics were evaluated. At 100 days, the contents of total soluble sugars (TSSs) and total soluble proteins (TSPs) were determined. Principal components (PCA) were measured. According to the analysis of the data, the cultivation of E. precatoria lineages in liquid medium was successfully carried out, and the establishment was achieved. The model can be considered adequate since the R2 values found describe more than 90% of the growth kinetics of the lineages. In the liquid system, lineages L1 (from leaf explants and multiplied in semisolid medium—SM), L2 (from leaf explants and multiplied in SM), and L6 (from zygotic embryo explants and multiplied in liquid medium—LM) showed the shortest time to double the biomass accumulation. Multivariate analysis reveals a significant increase in masses in liquid cultures, represented by lineages L6 and L2. There was statistical difference in the amount of TSSs extracted; the highest TSS levels were observed in lineages cultivated in LM. The protein content found was very low, showing statistical differences among the lineages. In this work, the establishment and multiplication of embryogenic calli of E. precatoria are described for the first time, and they emerge as viable alternatives for the vegetative propagation of the species.

Orthosilicic acid inhibits human osteoclast differentiation and bone resorption.

Silicon (Si), which is present in the diet in the bioavailable form of orthosilicic acid (OSA) and is detected as a dissolution product of certain bone-substitute materials, is suggested to promote bone health, and enhance bone healing, respectively. Silicon has been shown to stimulate osteoblastic cell differentiation and function, although the effect of Si on human osteoclasts is unclear. The present study investigated the direct effects of Si on human osteoclast differentiation, gene expression, and bone resorption. Human CD14+ monocytes were isolated from buffy coats and cultured with M-CSF and RANKL in medium without or with Si (50 μg/ml; constituting 75% OSA). The effects of Si on osteoclast differentiation were evaluated by TRAP-staining and the expression levels of CtsK, CalcR, TRAP, and DC-STAMP measured by RT-qPCR. The effect of Si on the expression level of AQP9, which encodes a potential Si transporter, was also analyzed. Bone resorption was determined based on the number of resorption pits formed when the RANKL-stimulated monocytes were cultured on bone slices, and by the levels of type I collagen fragments released into the cell culture medium. Silicon significantly inhibited the number of TRAP+ multinucleated cells and the expression of osteoclast related genes but increased the late expression of AQP9. Furthermore, Si significantly inhibited the number of resorption pits and the amount of collagen fragments in the medium when cells were cultured on bone slices. Our results demonstrate that OSA inhibits RANKL-stimulated human osteoclast differentiation, gene expression of osteoclast phenotypic markers, and bone resorption.

A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.

Peptides are widely used in biomaterials due to their easy of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell-secreted enzymes, which creates the possibility of utilizing cell-secreted enzymes for tuning cell-material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography-mass spectrometry (LC-MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC-MS for the multiplexed quantification of cell-induced peptide degradation, including the need for non-degradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. However, removing these constituents can be expensive, time consuming, and increases sample variability. Here we show that directly injecting samples onto the LC-MS without any purification enables rapid and accurate quantification of peptide concentration, and that hundreds of LC-MS runs can be done on a single column without a significantly diminish the ability to quantify the degradation of peptide libraries. We also show that column failure is evident when hydrophilic peptides fail to be retained on the column, and this can be easily identified using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost-effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide-based therapies.