High-density Cultures Research Articles

Soybean reduced internode 1 determines internode length and improves grain yield at dense planting

Major cereal crops have benefitted from Green Revolution traits such as shorter and more compact plants that permit high-density planting, but soybean has remained relatively overlooked. To balance ideal soybean yield with plant height under dense planting, shortening of internodes without reducing the number of nodes and pods is desired. Here, we characterized a short-internode soybean mutant, reduced internode 1 (rin1). Partial loss of SUPPRESSOR OF PHYA 105 3a (SPA3a) underlies rin1. RIN1 physically interacts with two homologs of ELONGATED HYPOCOTYL 5 (HY5), STF1 and STF2, to promote their degradation. RIN1 regulates gibberellin metabolism to control internode development through a STF1/STF2–GA2ox7 regulatory module. In field trials, rin1 significantly enhances grain yield under high-density planting conditions comparing to its wild type of elite cultivar. rin1 mutants therefore could serve as valuable resources for improving grain yield under high-density cultivation and in soybean–maize intercropping systems.

Optimization of Growth Medium of Bacillus sp. TTMP20 by Response Surface Methodology

High-density culture is able to obtain high concentration and activity of microbes, which is a key technology for fermentation industry. This will greatly shorten fermentation cycle and reduce production cost. Bacillus sp. TTMP20 is a strain with a high yield of 2,3,5,6-tetramethylpyrazine (TTMP). In this study, the growth medium of Bacillus sp. TTMP20 was optimized. Different carbon/nitrogen sources were investigated, and the best carbon source (glucose) and nitrogen source (yeast extract) was determined. With the concentrations of glucose, yeast extract and pH as influence factors, response surface method was used to optimize the above-mentioned influence factors to maximize cell density of the strain. The optimal growth medium is as follows: 4 g/L of yeast extract, 15.754 g/L of glucose, 2 g/L of K2HPO4, 10 g/L of NaCl, and 6.50 of pH. The predicted and actual maxima are 1.388 and 1.3, respectively. This is 61.4% higher than the cell density of the basic medium. This will provide the adequate seed culture for the subsequent fermentation of TTMP, and improve the application level of Bacillus sp. TTMP20.

CRISPR-Cas9 Mediated Stable Expression of Exogenous Proteins in the CHO Cell Line through Site-Specific Integration

Chinese hamster ovary (CHO) cells are a popular choice in biopharmaceuticals because of their beneficial traits, including high-density suspension culture, safety, and exogenously produced proteins that closely resemble natural proteins. Nevertheless, a decline in the expression of exogenous proteins is noted as culture time progresses. This is a consequence of foreign gene recombination into chromosomes by random integration. The current investigation employs CRISPR-Cas9 technology to integrate foreign genes into a particular chromosomal location for sustained expression. Results demonstrate the successful integration of enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) near base 434814407 on chromosome NC_048595.1 of CHO-K1 cells. Over 60 successive passages, monoclonal cell lines were produced that consistently expressed all relevant external proteins without discernible variation in expression levels. In conclusion, the CHO-K1 cell locus, NC_048595.1, proves an advantageous locus for stable exogenous protein expression. This study provides a viable approach to establishing a CHO cell line capable of enduring reliable exogenous protein expression.

Depth filtration for clarification of intensified lentiviral vector suspension cell culture.

Depth filtration significantly impacts efficiency of lentiviral (LV) vector purification process. However, it is often deprioritized in the overall scope of viral vector manufacturing process optimization. The demand for LV vectors has increased with the rise in disease indications, making it crucial to improve current manufacturing processes. Upstream bioreactor process intensification has enabled cell densities of over 107 viable cells/mL, creating challenges for harvest unit operations. The larger size of LV vectors and their physiochemical similarity to host cell-DNA (HC-DNA) and poor clarification performance causes significant challenges for the subsequent chromatography-based purifications. As a result, a robust and scalable harvest of LV process is needed, especially for LV in vivo therapeutic quality needs. In this study, we systematically evaluated the overlooked yet important issue of depth filtration systems to improve enveloped LV functional vector recovery. We found that an established depth filtration system in process A that provided 94% (n = 6) LV functional recovery could not be translated to intensified Process B cell culture. Hence, the depth filtration process became a bottleneck for the purification performance in an intensified process. We demonstrated an improvement in LV functional vector recovery from 34% to 82% via filter train optimization for an intensified suspension cell culture system (>107 cells/mL with higher titer), while still maintaining a loading throughput of ≥82 L/m2 and turbidity ≤20 NTU. It was demonstrated that the two or three-stage depth filtration scheme is scalable and more suitable for high cell density culture for large scale for LV manufacturing process.

Reversible translocation of acyl-CoA:cholesterol acyltransferase (ACAT) between the endoplasmic reticulum and vesicular structures.

The enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is normally localized in the endoplasmic reticulum (ER) where it can esterify cholesterol for storage in lipid droplets and/or the formation of lipoproteins. Here, we report that ACAT can translocate from the ER into vesicular structures in response to different ACAT inhibitors. The translocation was fast (within minutes), reversible and occurred in different cell types. Interestingly, oleic acid was able to fasten the re-translocation from vesicles back into the reticular ER network. The process of ACAT translocation could also be induced by cyclodextrins, cholesterol, lanosterol (but not 4-cholestene-3 one), 25-hydroxycholesterol, and by certain stress stimuli such as hyperosmolarity (sucrose treatment), temperature change, or high-density cultivation. In vitro esterification showed that ACAT remains fully active after it has been translocated to vesicles in response to hyperosmotic sucrose treatment of the cells. The translocation process was not accompanied by changes in the electrophoretic mobility of ACAT, even after chemical crosslinking. Interestingly, the protein synthesis inhibitor cycloheximide showed a stimulating effect on ACAT activity and prevented the translocation of ACAT from the ER into vesicles.

Physical and Biological Characterizations of Injectable Thermosensitive Poly(N-Isopropylacrylamide)-Collagen I Hydrogels Intended for Meniscus Repair

There has been an increase in the incidence of meniscus injuries worldwide, as well as growing needs for injectable hydrogels with the potentials to promote in situ meniscal tissue repair. This work developed poly(N-isopropylacrylamide)-collagen I composite hydrogels, with a series of different collagen I contents. The hydrogels were injectable under room temperature and started to solidify around 32°C due to their thermosensitivities, meeting the requirements of the intended applications. The addition of collagen I to the poly(N-isopropylacrylamide) provided physical reinforcement to the hydrogels. Rheological characterizations showed that, under room temperature, the complex viscosity, the storage modulus, and the loss modulus, respectively, increased from 0.71 ± 0.35 Pa•s to 110.95 ± 23.22 Pa•s, from 3.28 ± 2.02 Pa to 658.06 ± 128.07 Pa, and from 3.30 ± 1.01 Pa to 308.78 ± 56.67 Pa, as the collagen I content in the hydrogel increased from 0% to 1%. Cell proliferation tests and high-density culture also revealed that collagen I promoted bioactivity of the hydrogels and induced fibrochondrocyte phenotype of the chondrocytes. The expressions of collagen I protein and collagen I a1 gene in the PNIPAm-0.5Coll group were 3.50-fold and 3.64-fold of those in the PNIPAm group. In contrast, the expressions of collagen II protein and collagen II a1 gene were less prominent (respectively, 2.36-fold and 3.34-fold in the PNIPAm-0.5Coll group compared with the PNIPAm group). This phenotyping was believed to be conducive to the regeneration of meniscal tissues. The findings of this study have provided an important basis for future in vivo studies and clinical applications of this type of composite hydrogels.

Effects of different aquaculture patterns on growth, survival and yield of diploid and triploid Portuguese oysters (Crassostrea angulata)

Bivalve molluscs are increasingly being promoted as an alternative to land-based meat production due to their high levels of high-quality protein and lipids. The Crassostrea angulata, also known as the Portuguese oyster, has been a globally productive and renowned oyster species. In this study, we evaluated the aquaculture performance of C. angulata over two consecutive culture periods (pre-harvest phase, post-harvest phase) at different stocking densities and water depths, and compared the survival, growth and yield of diploid and triploid oysters. The experiment spanned 13 months, beginning in June 2022 and ending in June 2023. The results indicated that both stocking density and water depth significantly affected the aquaculture performance of C. angulata and exhibited distinct patterns in the two culture phases. Overall, compared to diploid C. angulata, triploid oysters showed significant culture advantages in the pre-harvest period, the growth rates and total yield of which were much higher than those of diploids. In the pre-harvest phase, the depth of 1 m and low-density culture (5 substrates/rope) favored the survival and growth of triploid oysters, while diploid C. angulata presented higher growth rates at high-density cultivation (15 substrates/rope), and the total oyster yield under 5 m culture reached a minimum for both diploids and triploids. In the post-harvest phase, the survival rate was stabilized, water depths of 1–3 m were optimal, and diploid oysters grew better at low (50 ind/cage) and medium (100 ind/cage) densities, whereas triploids had a higher growth rate of shell height at medium and high (150 ind/cage) densities. The findings of this research provide theoretical guidelines for exploring efficient C. angulata cultivation methods in the future, which will be beneficial to its commercial production.

Loss of one copy of vhl in zebrafish facilitates hypoxia tolerance

Insufficient oxygen in the water (hypoxia) is one of the limiting factors for high-density culture in modern aquaculture. Hypoxia tolerance in fish has been reported to be related to the hypoxia-inducible factor (HIF)-α-mediated hypoxia signaling pathway. The well-defined function of the von Hippel-Lindau (VHL) tumor suppressor gene VHL is to mediate proteasomal degradation of hydroxylated HIF-α proteins, resulting in the downregulation of hypoxia signaling pathway. However, whether VHL has an impact on hypoxia tolerance is still elusive. In this study, we find that vhl+/- zebrafish have no gross defects in development, growth, reproduction, and movement. In addition, vhl+/- zebrafish also have normal vasculature. However, under hypoxia, vhl+/- zebrafish are more resistant to hypoxia treatment compared with their wildtype siblings. Further assays indicate that the number of erythrocytes is increased and the expression of hypoxia-responsive genes is enhanced in vhl+/- zebrafish compared with their wildtype siblings. These data indicate that loss of one copy of vhl in zebrafish facilitates hypoxia tolerance, revealing a function of vhl in hypoxia tolerance.

Co-infections of Aeromonas dhakensis and Chryseobacterium indologenes in largemouth bass (Micropterus salmoides)

As one of the most important economic fishes, largemouth bass (Micropterus salmoides) is being widely farmed around the world. With the improper expansion and high-density culture, largemouth bass is susceptible to an increasing number of pathogens. Particularly, co-infection of multiple pathogens is ubiquitous, which has become a main obstacle to the development of largemouth bass industry. In the study, a co-infected case caused by Aeromonas dhakensis (A. dhakensis) and Chryseobacterium indologenes (C. indologenes) in largemouth bass was firstly reported. Diseased fish with clinical signs such as exophthalmia, hemorrhage on gill cover, rot or ulceration on gill, skin and fin were collected for further diagnostics. Tissue lesions of liver, spleen and kidney were observed, such as necrosis, hemorrhage, vacuolization and disintegration. Two bacteria strains were isolated from liver, one appeared as a white or yellowish colony, and the other was characterized by golden yellow color. Based on the conjoint analysis of 16S rRNA and gyrB gene sequences, the isolated strains were identified as A. dhakensis and C. indologenes, respectively. Antibiotic sensitivity testing indicated that both A. dhakensis and C. indologenes were sensitive to flumequine, enrofloxacin, ceftazidime and minocycline, and resistant to penicillin, sulfamethoxine sodium, ampicillin and cefamezin. Largemouth bass showed similar clinical signs and tissue lesions to naturally diseased fish following co-infection with the isolated strains, indicating that the isolated strains fulfill Koch's postulates. Besides, a slightly increased pathogenicity was observed following co-infection compared with single infection. The data firstly reveal a co-infected case caused by A. dhakensis and C. indologenes in largemouth bass, not only enriching the relevant information of largemouth bass diseases, but also promoting the diagnosis, prevention and control of A. dhakensis and C. indologenes co-infection.

Transcriptional responses of liver and spleen in Lota lota to polyriboinosinic polyribocytidylic acid.

The cultured Lota lota can meet the market demand in the context of the decline of wild resources, but the disease in the high-density culture process also deserves attention. Therefore, understanding the immune regulation mechanisms of L. lota will be the basis for obtaining high benefits in artificial culture. To explore the viral response mechanism of L. lota, RNA-seq was applied to identify the transcriptomic changes of the liver and spleen in L. lota by poly (I:C) stress. The DEGs (liver: 2186 to 3123; spleen 1542 to 2622) and up-regulated genes (liver: 1231 to 1776; spleen 769 to 1502) in the liver and spleen increased with the prolongation (12h to 48h) of poly (I:C)-stimulation time. This means L. lota needs to mobilize more functional genes in response to longer periods of poly (I:C)-stimulation. Despite the responses of L. lota to poly (I:C) showed tissue-specificity, we hypothesized that both liver and spleen of L. lota can respond to poly (I:C) challenge may be through promoting apoptosis of DNA-damaged cells, increasing the activity of immune-enhancing enzymes, and increasing energy supply based on DEGs annotation information. Our results demonstrate the transcriptional responses of L. lota to poly (I:C)-stimulation, and these data provide the first resource on the genetic regulation mechanisms of L. lota against viruses. Furthermore, the present study can provide basic information for the prevention of viral diseases in L. lota artificial culture process.

Transcriptomics of Lactobacillus paracasei: metabolism patterns and cellular responses under high-density culture conditions.

Lactobacillus paracasei has significant potential for development and application in the environmental field, particularly in addressing malodor pollution. This study aims to investigate the cellular response of L. paracasei B1 under high-density culture conditions. The selected strain has previously shown effective deodorizing and bacteriostatic abilities. Transcriptomics techniques are employed to dissect the nutrient metabolism pattern of L. paracasei B1 and its response mechanism under environmental stress. The study characterizes the functions of key differentially expressed genes during growth before and after optimizing the culture conditions. The optimization of fermentation culture conditions provides a suitable growth environment for L. paracasei B1, inducing an enhancement of its phosphotransferase system for sugar source uptake and maintaining high levels of glycolysis and pyruvate metabolism. Consequently, the strain is able to grow and multiply rapidly. Under acid stress conditions, glycolysis and pyruvate metabolism are inhibited, and L. paracasei B1 generates additional energy through aerobic respiration to meet the energy demand. The two-component system and quorum sensing play roles in the response and regulation of L. paracasei B1 to adverse environments. The strain mitigates oxygen stress damage through glutathione metabolism, cysteine and methionine metabolism, base excision repair, and purine and pyrimidine metabolism. Additionally, the strain enhances lysine synthesis, the alanine, aspartate, and glutamate metabolic pathways, and relies on the ABC transport system to accumulate amino acid-compatible solutes to counteract acid stress and osmotic stress during pH regulation. These findings establish a theoretical basis for the further development and application of L. paracasei B1 for its productive properties.

Genomic selection for hypoxia tolerance in large yellow croaker

The large yellow croaker (Larimichthys crocea) is one of the most economically important marine fishes on the southeast coast of China. However, high-density cultures, inbreeding depression, and environmental pollution have resulted in L. crocea often experiencing hypoxia, which has caused huge economic losses to the L. crocea farming industry. This study aimed to improve the hypoxia tolerance of farmed L. crocea using genomic selection (GS) for breeding. In this study, 399 and 354 samples from the reference and candidate populations, respectively, were genotyped using a 55 K liquid single-nucleotide polymorphism (SNP) array. Based on two different representations (survival status and survival time), the estimated values of heritability for the hypoxia tolerance trait in L. crocea using 38,472 clean SNPs were 0.65 and 0.62, respectively. A comparison of the genomic prediction accuracy under different GS models and SNP densities showed that GBLUP was the best GS model, and the 2500 SNPs selected using a genome-wide association study (GWAS)-based strategy were essentially close to all 38,472 SNPs. Based on the established optimal GS strategy (prediction accuracy was 0.4901 ± 0.0246), genomic estimated breeding values (GEBVs) were calculated for each candidate population. The tolerance capacity tests showed a significant improvement in hypoxia tolerance in the offspring of the candidate population with the top 10% GEBVs, having an 13.40% decrease and a 34.02% increase in the median lethal concentration (LC50) and median lethal time (LT50), respectively, compared to the control group. These results confirmed that GS is an effective genetic breeding technique when selecting for hypoxia tolerance in L. crocea.

Study a natural co-infection case of Largemouth bass ranavirus, Aeromonas vickert, and Aeromonas hydrophila in Micropterus salmoides

The largemouth bass (LMB) is a significant aquaculture species widely cultivated in China. High-density culture has led to an increased susceptibility of LMB to infection by the opportunistic bacterium Aeromonas. This study investigates a case of natural co-infection with the Largemouth bass ranavirus (LMBV), Aeromonas veronii, and Aeromonas hydrophila in Sichuan Province, China. To identify the causative agents of the infection, two bacterial strains were isolated from diseased LMB specimens and identified through 16S rRNA sequencing as Aeromonas vickert and Aeromonas hydrophila, both of which are gram-negative bacteria. The infection of LMBV was confirmed through polymerase chain reaction (PCR) analysis of tissue samples from the infected fish. After two blind passes of the virus fluid, the typical cytopathological effect (CPE) can be observed. Phylogenetic analysis of the virus sequences showed the highest similarity with LMBV isolates (ON418985 and MW630113) from Guangdong Province, China. This study offers important insights into diagnosing mixed infections in LMB and provides a reference for future research.

Papillary and reticular fibroblasts generate distinct microenvironments that differentially impact angiogenesis.

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization corresponding to their specific functions. These characteristics are associated with gene expression patterns of fibroblasts freshly isolated from their native microenvironment. In order to assess the relevance of these fibroblast subpopulations in a tissue engineering context, we investigated their contribution to matrix production and vascularization using cell sheet culture conditions. We first performed RNA-seq differential expression analysis to determine whether several rounds of cell amplification and high-density culture affected their gene expression profile. Bioinformatics analysis revealed that expression of angiogenesis-related and matrisome gene signatures were maintained, resulting in papillary and reticular ECMs that differ in composition and structure. The impact of secreted or ECM-associated factors was then assessed using two independent 3D angiogenesis assays: -1/ a fibrin hydrogel-based assay allowing investigation of diffusible secreted factors, -2/ a scaffold-free cell-sheet based assay for investigation of fibroblast-produced microenvironment. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and dense and branched microvascular network, whereas reticular fibroblasts produced more structural core components of the ECM associated with less branched and larger vessels. These features mimick the characteristics of both the ECM and the vasculature of dermis subcompartments. In addition to showing that skin fibroblast populations differentially regulate angiogenesis via both secreted and ECM factors, our work emphasizes the importance of papillary and reticular fibroblasts for engineering and modelling dermis microenvironment and vascularization. STATEMENT OF SIGNIFICANCE: Recent advances have brought to the forefront the central role of microenvironment and vascularization in tissue engineering for regenerative medicine and microtissue modelling. We have investigated the role of papillary and reticular fibroblast subpopulations using scaffold-free cell sheet culture. This approach provides differentiated cells conditions allowing the production of their own microenvironment. Analysis of gene expression profiles and characterisation of the matrix produced revealed strong and specific angiogenic properties that we functionally characterized using 3D angiogenesis models targeting the respective role of either secreted or matrix-bound factors. This study demonstrates the importance of cell-generated extracellular matrix and questions the importance of cell source and the relevance of hydrogels for developing physio-pathologically relevant tissue engineered substitutes.

Effects of ribonucleotide supplementation in modulating the growth of probiotic Bacillus subtilis and the synergistic benefits for improving the health performance of Asian seabass (Lates calcarifer).

In aquaculture, due to the requirements for high-density culture, the diseases caused by bacterial pathogens have become a serious issue. To solve this problem, we performed synbiotic application of RNA and Bacillus subtilis as a sustainable and eco-friendly approach to improve the health and immunity of Asian seabass (Lates calcarifer) during cultivation without using any harmful antibiotics or chemicals. Among various forms of nucleic acids, such as mononucleotides and DNA, RNA was found to be most effective in promoting the growth performance of probiotic B. subtilis in all the tested minimal medium conditions. Accordingly, we used the synbiotic combination of B. subtilis and RNA for Asian seabass cultivation. After feed supplementation for fourteen days, the fish that received the combination treatment exhibited a significant increase in innate cellular and humoral immune parameters, including phagocytic activity, phagocytic index, respiratory burst, serum lysozyme and bactericidal activities, as well as upregulated expression of immune-related genes, including HEPC1, A2M, C3, CC, CLEC, LYS, HSP70, and HSP90. Furthermore, significant increases were observed in the ileal villus height and goblet cell numbers in the intestinal villi in all fish treatment groups. The combination treatment did not cause histopathological abnormalities in the intestine and liver, suggesting that the synbiotic treatment is safe for use in fish. The treated Asian seabass also exhibited a significantly increased survival rate after Aeromonas hydrophila challenge. These results indicate that the synbiotic mixture of B. subtilis and RNA can be considered a beneficial feed additive and immunostimulant for Asian seabass cultivation.

A molting chemical cue (N-acetylglucosamine-6-phosphate) contributes to cannibalism of Chinese mitten crab Eriocheir sinensis

Under high-density culture, cannibalism occurs frequently during the molting of the Chinese mitten crabs Eriocheir sinensis, resulting in a large reduction in production. We found that the leakage of molting fluid from sexually immature crabs informs conspecifics that they are in a molting process. This hypothesis was verified through metabolomics analyses combined with behavioral experiments. The GlcNAc-6-P was identified as a molting biomarker from the differential metabolites by non-targeted metabolomics. In addition, we found that the concentration of GlcNAc-6-P in the molting fluid was significantly higher than other molting metabolites at different molting stages, reaching 5.84 μmol L−1, indicating that the molting fluid was the source of GlcNAc-6-P. Moreover, the behavioral experiments showed that crabs were actively approached to high concentrations of GlcNAc-6-P (1 μmol L−1), but had no obvious choice tendency at different concentrations of UTP, 20-HE and low concentrations of GlcNAc-6-P (0.1 μmol L−1, 0.01 μmol L−1) compared with the control groups. In conclusion, that E. sinensis by sensing the concentration change of GlcNAc-6-P can locate the source of GlcNAc-6-P release and actively approach the high concentration GlcNAc-6-P area and attack the molting crab, causing cannibalism.Blocking the reception pathway of molting chemical cues in E. sinensis, thereby preventing the perception of signals originating from conspecifics' molting in the vicinity, could lead to a reduction in cannibalistic behavior and an increase in overall production. Additionally, this method presents a prospective solution for addressing cannibalism in other crustacean species where such behavior is prevalent.

Production of retroviral vectors in continuous high cell density culture

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.Key points• A continuous high cell density process for MLV vector production was established• The tubular cell retention membrane allowed for continuous vector harvesting• The established process had a 18-fold higher space time yield compared to a batch

A two-galectin network establishes mesenchymal condensation phenotype in limb development

Previous work showed that Gal-1A and Gal-8, two proteins belonging to the galactoside-binding galectin family, are the earliest determinants of the patterning of the skeletal elements of embryonic chicken limbs, and further, that their experimentally determined interactions in the embryonic limb bud can be interpreted via a reaction–diffusion–adhesion (2GL: two galectin plus ligands) model. Here, we use an ordinary differential equation-based approach to analyze the intrinsic switching modality of the 2GL network and characterize the network behavior independent of the diffusive and adhesive arms of the patterning mechanism. We identify two states: where the concentrations of both the galectins are respectively, negligible, and very high. This bistable switch-like system arises via a saddle–node bifurcation from a monostable state. For the case of mass-action production terms, we provide an explicit Lyapunov function for the system, which shows that it has no periodic solutions. Our model therefore predicts that the galectin network may exist in low expression and high expression states separated in space or time, without any intermediate states. We test these predictions in experiments performed with high density cultures of chick limb mesenchymal cells and observe that cells inside precartilage protocondensations express Gal-1A at a much higher rate than those outside, for which it was negligible. The Gal-1A and -8-based patterning network is therefore sufficient to partition the mesenchymal cell population into two discrete cell states with different developmental (chondrogenic vs. non-chondrogenic) fates. When incorporated into an adhesion and diffusion-enabled framework this system can generate a spatially patterned limb skeleton.

Automated scoring of nematode nictation on a textured background.

Entomopathogenic nematodes, including Steinernema spp., play an increasingly important role as biological alternatives to chemical pesticides. The infective juveniles of these worms use nictation-a behavior in which animals stand on their tails-as a host-seeking strategy. The developmentally-equivalent dauer larvae of the free-living nematode Caenorhabditis elegans also nictate, but as a means of phoresy or "hitching a ride" to a new food source. Advanced genetic and experimental tools have been developed for C. elegans, but time-consuming manual scoring of nictation slows efforts to understand this behavior, and the textured substrates required for nictation can frustrate traditional machine vision segmentation algorithms. Here we present a Mask R-CNN-based tracker capable of segmenting C. elegans dauers and S. carpocapsae infective juveniles on a textured background suitable for nictation, and a machine learning pipeline that scores nictation behavior. We use our system to show that the nictation propensity of C. elegans from high-density liquid cultures largely mirrors their development into dauers, and to quantify nictation in S. carpocapsae infective juveniles in the presence of a potential host. This system is an improvement upon existing intensity-based tracking algorithms and human scoring which can facilitate large-scale studies of nictation and potentially other nematode behaviors.

Paroxetine Attenuates Chondrocyte Pyroptosis and Inhibits Osteoclast Formation by Inhibiting NF-κB Pathway Activation to Delay Osteoarthritis Progression.

Osteoarthritis (OA), a common chronic joint disease, is characterized by cartilage degeneration and subchondral bone reconstruction. NF-κB signaling pathway-activated inflammation and NLRP3-induced pyroptosis play essential roles in the development of OA. In this study, we examine whether paroxetine can inhibit pyroptosis and reduce osteoclast formation, thereby delaying the destruction of knee joints. We employed high-density cultures, along with quantitative polymerase chain reactions and Western blotting techniques, to investigate the effects of paroxetine on extracellular matrix synthesis and degradation. The expression levels of NF-κB and pyroptosis-related signaling pathway proteins were examined by Western blotting and immunofluorescence. Furthermore, the impact of paroxetine on RANKL-induced osteoclast formation was evaluated through TRAP staining and F-actin ring fluorescence detection. To investigate the role of paroxetine in vivo, we constructed a mouse model with destabilization of the medial meniscus (DMM) surgery. Safranin O-Fast Green staining, Hematoxylin-Eosin staining, and immunohistochemistry were conducted to observe the extent of knee joint cartilage deformation. In addition, TRAP staining was used to observe the formation of osteoclasts in the subchondral bone. In the in vitro experiments with ATDC5, paroxetine treatment attenuated IL-1β-induced activation of the pyroptosis-related pathway and suppressed extracellular matrix catabolism by inhibiting the NF-kB signaling pathway. In addition, paroxetine treatment decreased the expression of RANKL-induced osteoclast marker genes and reduced osteoclast formation. In animal experiments conducted in vivo, mice treated with paroxetine exhibited thicker knee cartilage with a smoother surface compared to the DMM group. Additionally, the formation of osteoclasts in the subchondral bone was reduced in the paroxetine-treated mice. Further analysis revealed that paroxetine treatment played a role in preserving the balance of the extracellular matrix and delaying knee joint degeneration. Paroxetine can inhibit pyroptosis and reduce osteoclast formation via inhibiting the NF-κB signaling pathway, suggesting that it may have therapeutic effects in patients with OA.