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Background: Articular cartilage degeneration can result from injury, age, or arthritis, causing significant joint pain and disability without surgical intervention. Currently, the only FDA cell-based therapy for articular cartilage injury is Autologous Chondrocyte Implantation (ACI); however, this procedure is costly, time-intensive, and requires multiple treatments. Mesenchymal stromal cells (MSCs) are an attractive alternative autologous therapy due to their availability and ability to robustly differentiate into chondrocytes for transplantation with good safety profiles. However, treatment outcomes are variable due to donor-to-donor variability as well as intrapopulation heterogeneity and unstandardized MSC manufacturing protocols. Process improvements that reduce cell heterogeneity while increasing donor cell numbers with improved chondrogenic potential during expansion culture are needed to realize the full potential of MSC therapy. Methods: In this study, we investigated the potential of MSC metabolic modulation during expansion to enhance their chondrogenic commitment by varying the nutrient composition, including glucose, pyruvate, glutamine, and ascorbic acid in culture media. We tested the effect of metabolic modulation in short-term (one passage) and long-term (up to seven passages). We measured metabolic state, cell size, population doubling time, and senescence and employed novel tools including micro-magnetic resonance relaxometry (µMRR) relaxation time (T2) to characterize the effects of AA on improved MSC expansion and chondrogenic potential. Results: Our data show that the addition of 1 mM L-ascorbic acid-2-phosphate (AA) to cultures for one passage during MSC expansion prior to initiation of differentiation improves chondrogenic differentiation. We further demonstrate that AA treatment reduced the proportion of senescent cells and cell heterogeneity also allowing for long-term expansion that led to a > 300-fold increase in yield of MSCs with enhanced chondrogenic potential compared to untreated cells. AA-treated MSCs with improved chondrogenic potential showed a robust shift in metabolic profile to OXPHOS and higher µMRR T2 values, identifying critical quality attributes that could be implemented in MSC manufacturing for articular cartilage repair. Conclusions: Our results suggest an improved MSC manufacturing process that can enhance chondrogenic potential by targeting MSC metabolism and integrating process analytic tools during expansion. [ABSTRACT FROM AUTHOR]

Previously, we reported successful cellular expansion of a murine colorectal carcinoma cell line (CT-26) using a three-dimensional (3D) engineered extracellular matrix (EECM) fibrillar scaffold structure. CCL-247 were grown over a limited time period of 8 days on 3D EECM or tissue culture polystyrene (TCPS). Cells were then assayed for growth, electroporation efficiency and Vigil manufacturing release criteria. Using EECM scaffolds, we report an expansion of CCL-247 (HCT116), a colorectal carcinoma cell line, from a starting concentration of 2.45 × 105 cells to 1.9 × 106 cells per scaffold. Following expansion, 3D EECM-derived cells were assessed based on clinical release criteria of the Vigil manufacturing process utilized for Phase IIb trial operation with the FDA. 3D EECM-derived cells passed all Vigil manufacturing release criteria including cytokine expression. Here, we demonstrate successful Vigil product manufacture achieving the specifications necessary for the clinical trial product release of Vigil treatment. Our results confirm that 3D EECM can be utilized for the expansion of human cancer cell CCL-247, justifying further clinical development involving human tissue sample manufacturing including core needle biopsy and minimal ascites samples. [ABSTRACT FROM AUTHOR]

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (T n/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O 2 from day 5 onward, we could generate >60% T n/scm -like cells, with longer time in hypoxia correlating with a higher percentage of T n/scm -like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells. [ABSTRACT FROM AUTHOR]

Abstract Previously, we reported successful cellular expansion of a murine colorectal carcinoma cell line (CT-26) using a three-dimensional (3D) engineered extracellular matrix (EECM) fibrillar scaffold structure. CCL-247 were grown over a limited time period of 8 days on 3D EECM or tissue culture polystyrene (TCPS). Cells were then assayed for growth, electroporation efficiency and Vigil manufacturing release criteria. Using EECM scaffolds, we report an expansion of CCL-247 (HCT116), a colorectal carcinoma cell line, from a starting concentration of 2.45 × 105 cells to 1.9 × 106 cells per scaffold. Following expansion, 3D EECM-derived cells were assessed based on clinical release criteria of the Vigil manufacturing process utilized for Phase IIb trial operation with the FDA. 3D EECM-derived cells passed all Vigil manufacturing release criteria including cytokine expression. Here, we demonstrate successful Vigil product manufacture achieving the specifications necessary for the clinical trial product release of Vigil treatment. Our results confirm that 3D EECM can be utilized for the expansion of human cancer cell CCL-247, justifying further clinical development involving human tissue sample manufacturing including core needle biopsy and minimal ascites samples.

In the quest to combat insulin-dependent diabetes mellitus (IDDM), allogenic pancreatic islet cell therapy sourced from deceased donors represents a significant therapeutic advance. However, the applicability of this approach is hampered by donor scarcity and the demand for sustained immunosuppression. Human induced pluripotent stem cells are a game-changing resource for generating synthetic functional insulin-producing β cells. In addition, novel methodologies allow the direct expansion of pancreatic progenitors and mature β cells, thereby circumventing prolonged differentiation. Nevertheless, achieving practical reproducibility and scalability presents a substantial challenge for this technology. As these innovative approaches become more prominent, it is crucial to thoroughly evaluate existing expansion techniques with an emphasis on their optimization and scalability. This manuscript delineates these cutting-edge advancements, offers a critical analysis of the prevailing strategies, and underscores pivotal challenges, including cost-efficiency and logistical issues. Our insights provide a roadmap, elucidating both the promises and the imperatives in harnessing the potential of these cellular therapies for IDDM.

SUMMARY: As a plant‐specific transcription factor, lateral organ boundaries domain (LBD) protein was reported to regulate plant growth and stress response, but the functional research of subfamily II genes is limited. SlMYC2, a master regulator of Jasmonic acid response, has been found to exhibit high expression levels in fruit and has been implicated in the regulation of fruit ripening and resistance to Botrytis. However, its role in fruit expansion remains unknown. In this study, we present evidence that a subfamily II member of LBD, namely SlLBD40, collaborates with SlMYC2 in the regulation of fruit expansion. Overexpression of SlLBD40 significantly promoted fruit growth by promoting mesocarp cell expansion, while knockout of SlLBD40 showed the opposite result. Similarly, SlMYC2 knockout resulted in a significant decrease in cell expansion within the fruit. Genetic analysis indicated that SlLBD40‐mediated cell expansion depends on the expression of SlMYC2. SlLBD40 bound to the promoter of SlEXPA5, an expansin gene, but did not activate its expression directly. While, the co‐expression of SlMYC2 and SlLBD40 significantly stimulated the activation of SlEXPA5, leading to an increase in fruit size. SlLBD40 interacted with SlMYC2 and enhanced the stability and abundance of SlMYC2. Furthermore, SlMYC2 directly targeted and activated the expression of SlLBD40, which is essential for SlLBD40‐mediated fruit expansion. In summary, our research elucidates the role of the interaction between SlLBD40 and SlMYC2 in promoting cell expansion in tomato fruits, thus providing novel insights into the molecular genetics underlying fruit growth. [ABSTRACT FROM AUTHOR]

The in vitro expansion of stem cells is important for their application in different life science fields such as cellular tissue and organ repair. An objective of this paper was to achieve static cell culture in vitro through peptide hydrogel-supported microspheres (MSs). The peptides, with their gel-forming properties, microstructures, and mechanical strengths characterized, were found to have good support for the MSs and to be injectable. The internal structures of poly(L-lactic acid) microspheres (PLLA-MSs) and polystyrene microspheres (PS-MSs) made in the laboratory were observed and statistically analyzed in terms of particle size and pore size, following which the co-cultured MSs with cells were found to have good cell adhesion. In addition, three-dimensional (3D) culturing of cells was performed on the peptide and microcarrier composite scaffolds to measure cell viability and cell proliferation. The results showed that the peptides could be stimulated by the culture medium to self-assembly form a 3D fiber network structure. Under the peptide-MS composite scaffold-based cell culture system, further enhancement of the cell culture effect was measured. The peptide-MS composite scaffolds have great potential for the application in 3D cell culture and in vitro cell expansion. [ABSTRACT FROM AUTHOR]

Cell expansion in a discrete region called the elongation zone drives root elongation. Analyzing time lapse images can quantify the expansion in kinematic terms as if it were fluid flow. We used horizontal microscopes to collect images from which custom software extracted the length of the elongation zone, the peak relative elemental growth rate (REGR) within it, the axial position of the REGR peak, and the root elongation rate. Automation enabled these kinematic traits to be measured in 1575 Arabidopsis seedlings representing 162 recombinant inbred lines (RILs) derived from a cross of Cvi and Ler ecotypes. We mapped ten quantitative trait loci (QTL), affecting the four kinematic traits. Three QTL affected two or more traits in these vertically oriented seedlings. We compared this genetic architecture with that previously determined for gravitropism using the same RIL population. The major QTL peaks for the kinematic traits did not overlap with the gravitropism QTL. Furthermore, no single kinematic trait correlated with quantitative descriptors of the gravitropism response curve across this population. In addition to mapping QTL for growth zone traits, this study showed that the size and shape of the elongation zone may vary widely without affecting the differential growth induced by gravity. [ABSTRACT FROM AUTHOR]

Bone marrow failure (BMF) has become one of the most studied autoimmune disorders, particularly due to its prevalence both as an inherited disease, but also as a result of chemotherapies. BMF is associated with severe symptoms such as bleeding episodes and susceptibility to infections, and often has underlying characteristics, such as anemia, thrombocytopenia, and neutropenia. The current treatment landscape for BMF requires stem cell transplantation or chemotherapies to induce immune suppression. However, there is limited donor cell availability or dose related toxicity associated with these treatments. Optimizing these treatments has become a necessity. Polymer-based materials have become increasingly popular, as current research efforts are focused on synthesizing novel cell matrices for stem cell expansion to solve limited donor cell availability, as well as applying polymer delivery vehicles to intracellularly deliver cargo that can aid in immunosuppression. Here, we discuss the importance and impact of polymer materials to enhance therapeutics in the context of BMF. [ABSTRACT FROM AUTHOR]

The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull's eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications. [ABSTRACT FROM AUTHOR]

Olive (Olea europaea L.) is one of the major oil fruit tree crops worldwide. However, the mechanisms underlying olive fruit growth remain poorly understood. Here, we examine questions regarding the interaction of endoreduplication, cell division, and cell expansion with olive fruit growth in relation to the final fruit size by measuring fruit diameter, pericarp thickness, cell area, and ploidy level during fruit ontogeny in three olive cultivars with different fruit sizes. The results demonstrate that differences in the fruit size are related to the maximum growth rate between olive cultivars during early fruit growth, about 50 days post-anthesis (DPA). Differences in fruit weight between olive cultivars were found from 35 DPA, while the distinctive fruit shape became detectable from 21 DPA, even though the increase in pericarp thickness became detectable from 7 DPA in the three cultivars. During early fruit growth, intense mitotic activity appeared during the first 21 DPA in the fruit, whereas the highest cell expansion rates occurred from 28 to 42 DPA during this phase, suggesting that olive fruit cell number is determined from 28 DPA in the three cultivars. Moreover, olive fruit of the large-fruited cultivars was enlarged due to relatively higher cell division and expansion rates compared with the small-fruited cultivar. The ploidy level of olive fruit pericarp between early and late growth was different, but similar among olive cultivars, revealing that ploidy levels are not associated with cell size, in terms of different 8C levels during olive fruit growth. In the three olive cultivars, the maximum endoreduplication level (8C) occurred just before strong cell expansion during early fruit growth in fruit pericarp, whereas the cell expansion during late fruit growth occurred without preceding endoreduplication. We conclude that the basis for fruit size differences between olive cultivars is determined mainly by different cell division and expansion rates during the early fruit growth phase. These data provide new findings on the contribution of fruit ploidy and cell size to fruit size in olive and ultimately on the control of olive fruit development. [ABSTRACT FROM AUTHOR]

Selenium (Se) and nitrate have the potential to modify rice root architecture, but it is unclear how Se is linked to changes in the rice seedlings nitrate status. Thus, rice seedlings were grown in nutrient solutions containing either 0- or 10-µM Se that were supplemented with 0.05 (low nitrate condition) or 5.0 mM nitrate (high nitrate condition). Se application to seedlings treated with low nitrate led to sugar accumulation in shoot and root and increased cytokinin concentrations in root, while decreasing cytokinin concentrations in shoot compared with seedlings in 0.05 mM nitrate alone. This, in turn, resulted in decreased shoot growth, while downregulation of OsXTH and OsEXP negatively affected root expansion. On the other hand, Se combined with 5.0 mM nitrate did not affect sugar concentration in tissues compared with seedlings in 5.0 mM nitrate. Moreover, Se negatively regulated the cytokinin biosynthesis in shoot and root of seedlings grown under 5.0 mM nitrate. The reduction in cytokinin concentrations by Se under high nitrate condition decreased shoot growth, but increased root growth through induction of OsXTH and OsEXP. Thus, many of the effects of Se in shoot and root growth are due to a shift in nitrate status of the seedlings.

Background: Olfactory impairment has been reported in patients with depression and in rodent models of depression. Olfactory epithelium (OE) is the only peripheral neural tissue connected to the brain that has the potential for self-renewal. We hypothesized the olfactory deficit during depression may be related to the dysfunction of OE progenitor cells. The aim of the present study was therefore to evaluate the expansion and neuronal differentiation potency of cultured OE progenitor cells obtained from a rat model of depression. Methods: Rats were exposed to chronic unpredictable mild stress procedures to establish a depressive-like state. Depressive-like behavior and olfactory sensing function were then evaluated and compared with control rats. Primary OE progenitor cells were cultured in vitro. The proliferation potency and survival of OE progenitor cells were assessed by 5-Ethynyl-2'-deoxyuridine staining and Cell Counting Kit-8 (CCK8), respectively, while cellular apoptosis was measured by flow cytometry. The neuronal differentiation potency of OE progenitor cells was evaluated by measurement of the protein and mRNA level of β-3 tubulin, a marker of neural cells. mRNA expression associated with neural stemness was examined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Results: Depressive-like rats showed decreased olfactory function. OE progenitor cells from depressive-like rats showed reduced cell proliferation/survival and neuronal differentiation potency. Moreover, OE progenitor cells from depressive-like rats showed decreased expression of mRNA related to neural stemness. Conclusions: These results indicate the impaired function of OE progenitor cells may contribute to the olfactory deficit observed during depression. The OE may therefore provide a window for the study of depression. [ABSTRACT FROM AUTHOR]

Selenium (Se) and nitrate have the potential to modify rice root architecture, but it is unclear how Se is linked to changes in the rice seedlings nitrate status. Thus, rice seedlings were grown in nutrient solutions containing either 0- or 10-µM Se that were supplemented with 0.05 (low nitrate condition) or 5.0 mM nitrate (high nitrate condition). Se application to seedlings treated with low nitrate led to sugar accumulation in shoot and root and increased cytokinin concentrations in root, while decreasing cytokinin concentrations in shoot compared with seedlings in 0.05 mM nitrate alone. This, in turn, resulted in decreased shoot growth, while downregulation of OsXTH and OsEXP negatively affected root expansion. On the other hand, Se combined with 5.0 mM nitrate did not affect sugar concentration in tissues compared with seedlings in 5.0 mM nitrate. Moreover, Se negatively regulated the cytokinin biosynthesis in shoot and root of seedlings grown under 5.0 mM nitrate. The reduction in cytokinin concentrations by Se under high nitrate condition decreased shoot growth, but increased root growth through induction of OsXTH and OsEXP. Thus, many of the effects of Se in shoot and root growth are due to a shift in nitrate status of the seedlings. [ABSTRACT FROM AUTHOR]

Coordination of secondary cell wall deposition and cell expansion during plant growth is required for cell development, particularly in vascular tissues. Yet the fundamental coordination process has received little attention. We observed that the Arabidopsis endo-1,4-mannanase gene, AtMAN6 , is involved in the formation of cell walls in vascular tissues. In the inflorescence stem, the man6 mutant had smaller vessel cells with thicker secondary cell walls and shorter fiber cells. Elongation growth was reduced in the root, and secondary cell wall deposition in vessel cells occurred early. Overexpression of AtMAN6 resulted in the inverse phenotypes of the man6 mutant. AtMAN6 was discovered on the plasma membrane and was specifically expressed in vessel cells during its early development. The AtMAN6 protein degraded galactoglucomannan to produce oligosaccharides, which caused secondary cell wall deposition in vessel and fiber cells to be suppressed. Transcriptome analysis revealed that the expression of genes involved in the regulation of secondary cell wall synthesis was changed in both man6 mutant and AtMAN6 overexpression plants. AtMAN6's C-terminal cysteine repeat motif (CCRM) was found to facilitate homodimerization and is required for its activity. According to the findings, the oligosaccharides produced by AtMAN6 hydrolysis may act as a signal to mediate this coordination between cell growth and secondary cell wall deposition. [ABSTRACT FROM AUTHOR]

There are limited studies on the cytology of bamboo leaf development from primordium to maturity. This study delves into the leaf morphological characteristics and growth patterns of Sasaella kogasensis 'Aureostriatus' and provides a three-dimensional anatomical analysis of cell division, expansion, and degradation. Leaves on the same branch develop bottom-up, while individual leaves develop the other way around. Like bamboo shoots and culms, the leaves follow a "slow–fast–slow" growth pattern, with longitudinal growth being predominant during their development. The growth zones of individual leaves included division, elongation, and maturation zones based on the distribution of growth space. By measuring 13,303 epidermal long cells and 3293 mesophyll cells in longitudinal sections of rapidly elongating leaves, we observed that in the rapid elongation phase (S4–S5), the division zone was located in the 1–2 cm segment at the bottom of the leaf blade and maintained a constant size, continuously providing new cells for leaf elongation, whereas in the late rapid elongation phase (S6), when the length of the leaf blade was approaching that of a mature leaf, its cells at the bottom of the blade no longer divided and were replaced by the ability to elongate. Furthermore, to gain an insight into the dynamic changes in the growth of the S. kogasensis 'Aureostriatus' leaves in the lateral and periclinal directions, the width and thickness of 1459 epidermal and 2719 mesophyll cells were counted in the mid-cross section of leaves at different developmental stages. The results showed that during the early stages of development (S1–S3), young leaves maintained vigorous division in the lateral direction, while periplasmic division gradually expanded from the bottom to the top of the leaf blade and the number of cell layers stabilized at S4. The meristematic tissues on both sides of the leaf were still able to divide at S4 but the frequency of the division gradually decreased, while cell division and expansion occurred simultaneously between the veins. At S6, the cells at the leaf margins and between the veins were completely differentiated and the width of the leaf blade no longer expanded. These findings revealed changes in cell growth anisotropically during the leaf development of S. kogasensis 'Aureostriatus' and demonstrated that leaf elongation was closely related to the longitudinal expansion of epidermal cells and proliferative growth of mesophyll cells, whereas the cell division of meristematic tissues and expansion of post-divisional cells contributed to the increases in blade width and thickness. The presented framework will facilitate a further exploration of the molecular regulatory mechanisms of leaf development in S. kogasensis 'Aureostriatus' and provide relevant information for developmental and taxonomic studies of bamboo plants. [ABSTRACT FROM AUTHOR]

Summary: Anisotropic cell expansion is crucial for the morphogenesis of land plants, as cell migration is restricted by the rigid cell wall. The anisotropy of cell expansion is regulated by mechanisms acting on the deposition or modification of cell wall polysaccharides. Besides the polysaccharide components in the cell wall, a layer of hydrophobic cuticle covers the outer cell wall and is subjected to tensile stress that mechanically restricts cell expansion. However, the molecular machinery that deposits cuticle materials in the appropriate spatiotemporal manner to accommodate cell and tissue expansion remains elusive.Here, we report that PpABCB14, an ATP‐binding cassette transporter in the moss Physcomitrium patens, regulates the anisotropy of cell expansion. PpABCB14 localized to expanding regions of leaf cells. Deletion of PpABCB14 resulted in impaired anisotropic cell expansion.Unexpectedly, the cuticle proper was reduced in the mutants, and the cuticular lipid components decreased. Moreover, induced PpABCB14 expression resulted in deformed leaf cells with increased cuticle lipid accumulation on the cell surface.Taken together, PpABCB14 regulates the anisotropy of cell expansion via cuticle deposition, revealing a regulatory mechanism for cell expansion in addition to the mechanisms acting on cell wall polysaccharides. [ABSTRACT FROM AUTHOR]

Plant cell wall researchers were asked their view on what the major unanswered questions are in their field. This article summarises the feedback that was received from them in five questions. In this issue you can find equivalent syntheses for researchers working on bacterial, unicellular parasite and fungal systems.

Cancer cell line is commonly used for discovery and development of anti-cancer drugs. It is generally considered that drug response remains constant for a certain cell line due to the identity of genetics thus protein patterns. Here, we demonstrated that cancer cells continued dividing even after reaching confluence, in that the proteomics was changed continuously and dramatically with strong relevance to cell division, cell adhesion and cell metabolism, indicating time-dependent intrinsically reprogramming of cells during expansion. Of note, the inhibition effect of most anti-cancer drugs was strikingly attenuated in culture cells along with cell expansion, with the strongest change at the third day when cells were still expanding. Profiling of an FDA-approved drug library revealed that attenuation of response with cell expansion is common for most drugs, an exception was TAK165 that was a selective inhibitor of mitochondrial respiratory chain complex I. Finally, we screened a panel of natural products and identified four pentacyclic triterpenes as selective inhibitors of cancer cells under prolonged growth. Taken together, our findings underscore that caution should be taken in evaluation of anti-cancer drugs using culture cells, and provide agents selectively targeting overgrowth cancer cells.

Bone marrow failure (BMF) has become one of the most studied autoimmune disorders, particularly due to its prevalence both as an inherited disease, but also as a result of chemotherapies. BMF is associated with severe symptoms such as bleeding episodes and susceptibility to infections, and often has underlying characteristics, such as anemia, thrombocytopenia, and neutropenia. The current treatment landscape for BMF requires stem cell transplantation or chemotherapies to induce immune suppression. However, there is limited donor cell availability or dose related toxicity associated with these treatments. Optimizing these treatments has become a necessity. Polymer-based materials have become increasingly popular, as current research efforts are focused on synthesizing novel cell matrices for stem cell expansion to solve limited donor cell availability, as well as applying polymer delivery vehicles to intracellularly deliver cargo that can aid in immunosuppression. Here, we discuss the importance and impact of polymer materials to enhance therapeutics in the context of BMF.

Determining the influence of vapor pressure deficit (VPD) on fruit growth is a key issue under a changing climate scenario. Using a comparative approach across different fruit tree species may provide solid indications of common or contrasting plant responses to environmental factors. Knowing fruit growth responses to VPD may also be useful to optimize horticultural management practices under specific atmospheric conditions. Climate data to calculate VPD and fruit relative growth rates (RGR) by fruit gauges were monitored in peach at cell division, pit hardening and cell expansion stages; in two mango cultivars at cell division, cell expansion and maturation stages; in two olive cultivars, either full irrigated or rainfed, at early and late cell expansion stages; in ‘Valencia’ orange at early and late cell division stage, before and after mature fruit harvest; in loquat at cell expansion and maturation stages. At the fruit cell division stage, sensitivity of fruit growth to VPD seems to vary with species, time, and probably soil and atmospheric water deficit. ‘Keitt’ mango and ‘Valencia’ orange fruit growth responded to VPD in opposite ways, and this could be due to very different time of the year and VPD levels in the monitoring periods of the two species. At pit hardening stage of peach fruit growth, a relatively weak relationship was observed between VPD and RGR, and this is not surprising as fruit growth in size at this stage slows down significantly. A consistent and marked negative relationship between VPD and RGR was observed at cell expansion stage, when fruit growth is directly depending on water intake driving cell turgor. Another behavior common to all observed species was the gradual loss of relationship between VPD and RGR at the onset of fruit maturation, when fruit growth in size is generally programmed to stop. Finally, regardless of fruit type, VPD may have a significant effect on fruit growth and could be a useful parameter to be monitored for tree water management mainly when the cell expansion process prevails during fruit growth. [ABSTRACT FROM AUTHOR]

With the objective of improving the ex vivo production of therapeutic chimeric antigen receptor (CAR) T cells, we explored the addition of three-dimensional (3D) polystyrene scaffolds to standard suspension cell cultures. We aimed to mimic the structural support given by the lymph nodes during in vivo lymphocyte expansion. We observed an increase in cell proliferation compared with standard suspension systems as well as an enhanced cytotoxicity toward cancer cells. Moreover, we directly obtained the CAR T cells from peripheral blood mononuclear cells, thus minimizing the ex vivo manipulation of the therapeutic cells and opening the way to synergies among different cell populations. We propose the use of commercially available 3D polystyrene systems to improve the current immune cell cultures and resulting cell products for emerging cellular (immuno)therapies. [Display omitted] [ABSTRACT FROM AUTHOR]

SUMMARY: Chickpea is among the top three legumes produced and consumed worldwide. Early plant vigor, characterized by good germination and rapid seedling growth, is an important agronomic trait in many crops including chickpea, and shows a positive correlation with seed size. In this study, we report a gamma‐ray‐induced chickpea mutant with a larger organ and seed size. The mutant (elm) exhibits increased early vigor and contains higher proline that contributes to a better tolerance under salt stress at germination, seedling, and early vegetative phase. The trait is governed as monogenic recessive, with wild‐type allele being incompletely dominant over the mutant. Genetic mapping of this locus (CaEl) identified it as a previously uncharacterized gene (101503252) in chromosome 1 of the chickpea genome. There is a deletion of this gene in the mutant with a complete loss of expression. In silico analysis suggests that the gene is present as a single copy in chickpea and related legumes of the galegoid clade. In the mutant, cell division and expansion are affected. Transcriptome profiling identified differentially regulated transcripts related to cell division, expansion, cell wall organization, and metabolism in the mutant. The mutant can be exploited in chickpea breeding programs for increasing plant vigor and seed size. [ABSTRACT FROM AUTHOR]

Background and Aims Gigantism is a key component of the domestication syndrome, a suite of traits that differentiates crops from their wild relatives. Allometric gigantism is strongly marked in horticultural crops, causing disproportionate increases in the size of edible parts such as stems, leaves or fruits. Tomato (Solanum lycopersicum) has attracted attention as a model for fruit gigantism, and many genes have been described controlling this trait. However, the genetic basis of a corresponding increase in size of vegetative organs contributing to isometric gigantism has remained relatively unexplored. Methods Here, we identified a 0.4-Mb region on chromosome 7 in introgression lines (ILs) from the wild species Solanum pennellii in two different tomato genetic backgrounds (cv. 'M82' and cv. 'Micro-Tom') that controls vegetative and reproductive organ size in tomato. The locus, named ORGAN SIZE (ORG), was fine-mapped using genotype-by-sequencing. A survey of the literature revealed that ORG overlaps with previously mapped quantitative trait loci controlling tomato fruit weight during domestication. Key Results Alleles from the wild species led to lower cell number in different organs, which was partially compensated by greater cell expansion in leaves, but not in fruits. The result was a proportional reduction in leaf, flower and fruit size in the ILs harbouring the alleles from the wild species. Conclusions Our findings suggest that selection for large fruit during domestication also tends to select for increases in leaf size by influencing cell division. Since leaf size is relevant for both source–sink balance and crop adaptation to different environments, the discovery of ORG could allow fine-tuning of these parameters. [ABSTRACT FROM AUTHOR]

Plant roots elongate when cells produced in the apical meristem enter a transient period of rapid expansion. To measure the dynamic process of root cell expansion in the elongation zone, we captured digital images of growing Arabidopsis roots with horizontal microscopes and analyzed them with a custom image analysis program (PatchTrack) designed to track the growth-driven displacement of many closely spaced image patches. Fitting a flexible logistics equation to patch velocities plotted versus position along the root axis produced the length of the elongation zone (mm), peak relative elemental growth rate (% h−1), the axial position of the peak (mm from the tip), and average root elongation rate (mm h−1). For a wild-type root, the average values of these kinematic traits were 0.52 mm, 23.7% h−1, 0.35 mm, and 0.1 mm h−1, respectively. We used the platform to determine the kinematic phenotypes of auxin transport mutants. The results support a model in which the PIN2 auxin transporter creates an area of expansion-suppressing, supraoptimal auxin concentration that ends 0.1 mm from the quiescent center (QC), and that ABCB4 and ABCB19 auxin transporters maintain expansion-limiting suboptimal auxin levels beginning approximately 0.5 mm from the QC. This study shows that PatchTrack can quantify dynamic root phenotypes in kinematic terms. [ABSTRACT FROM AUTHOR]

Summary: Grain notching is a common deformation that decreases rice (Oryza sativa) quality; however, the underlying molecular basis causing grain notching remains unclear.We report mechanisms underlying grain notching in Small and notched grain (Sng) mutants, which contained an arginine to histidine substitution at amino acid position 422 (R422H) of the α‐tubulin protein OsTUBA3. The R422H mutation decreased cell length and increased cell width/height of glumes and caryopses, but led to elongated caryopses compressed within shortened glumes, thus giving rise to notched and small grains. Glume and caryopsis cells had different dimensional orientations relative to the directions of organ elongation. Thus, the abnormal cell expansion induced in glumes and caryopses by the R422H mutation had different effects on elongation of these organs.The R422H mutation in OsTUBA3 compromised β‐tubulin binding and led to formation of defective heterodimers. This in turn affected tubulin incorporation and microtubule (MT) nucleation and regrowth, consequently leading to MT instability and reducing the transverse orientation. The defective MT dynamics affected cell expansion and shape, causing different alterations in glume and caryopsis dimensions and resulting in grain notching.These data indicate that Arg422 in OsTUBA3 is crucial for MT dynamics and that substitution with His causes grain notching, reducing grain quality and yield. These findings offer valuable insights into the molecular regulation underlying grain development in rice. [ABSTRACT FROM AUTHOR]

Endoreduplication is the major source of somatic endopolyploidy in higher plants, and leads to variation in cell ploidy levels due to iterative rounds of DNA synthesis in the absence of mitosis. Despite its ubiquitous occurrence in many plant organs, tissues, and cells, the physiological meaning of endoreduplication is not fully understood, although several roles during plant development have been proposed, mostly related to cell growth, differentiation, and specialization via transcriptional and metabolic reprogramming. Here, we review recent advances in our knowledge of the molecular mechanisms and cellular characteristics of endoreduplicated cells, and provide an overview of the multi-scale effects of endoreduplication on supporting growth in plant development. In addition, the effects of endoreduplication in fruit development are discussed, since it is highly prominent during fruit organogenesis where it acts as a morphogenetic factor supporting rapid fruit growth, as illustrated by case of the model fleshy fruit, tomato (Solanum lycopersicum). [ABSTRACT FROM AUTHOR]

As the primary natural barrier that protects against adverse environmental conditions, the skin plays a crucial role in the innate immune response of fish, particularly in relation to bacterial infections. However, due to the diverse functionality and intricate anatomical and cellular composition of the skin, deciphering the immune response of the host is a challenging task. In this study, single nuclei RNA-sequencing (snRNA-seq) was performed on skin biopsies obtained from Chinese longsnout catfish (Leiocassis longirostris), comparing Aeromonas hydrophila-infected subjects to healthy control subjects. A total of 19,581 single nuclei cells were sequenced using 10x Genomics (10,400 in the control group and 9,181 in the treated group). Based on expressed unique transcriptional profiles, 33 cell clusters were identified and classified into 12 cell types including keratinocyte (KC), fibroblast (FB), endothelial cells (EC), secretory cells (SC), immune cells, smooth muscle cells (SMC), and other cells such as pericyte (PC), brush cell (BC), red blood cell (RBC), neuroendocrine cell (NDC), neuron cells (NC), and melanocyte (MC). Among these, three clusters of KCs, namely, KC1, KC2, and KC5 exhibited significant expansion after A. hydrophila infection. Analysis of pathway enrichment revealed that KC1 was primarily involved in environmental signal transduction, KC2 was primarily involved in endocrine function, and KC5 was primarily involved in metabolism. Finally, our findings suggest that neutrophils may play a crucial role in combating A. hydrophila infections. In summary, this study not only provides the first detailed comprehensive map of all cell types present in the skin of teleost fish but also sheds light on the immune response mechanism of the skin following A. hydrophila infection in Chinese longsnout catfish. [ABSTRACT FROM AUTHOR]

Exogenous gibberellin treatment can promote early growth of grape fruit, but the underlying regulatory mechanisms are not well understood. Here, we show that VvDELLA2 directly regulates the activity of the VvCEB1 transcription factor, a key regulator in the control of cell expansion in grape fruit. Our results show that VvCEB1 binds directly to the promoters of cell expansion-related genes in grape fruit and acts as a transcriptional activator, while VvDELLA2 blocks VvCEB1 function by binding to its activating structural domain. The exogenous gibberellin treatment relieved this inhibition by promoting the degradation of VvDELLA2 protein, thus, allowing VvCEB1 to transcriptionally activate the expression of cell expansion-related genes. In conclusion, we conclude that exogenous GA3 treatment regulates early fruit expansion by affecting the VvDELLA-VvCEB1 interaction in grape fruit development. [ABSTRACT FROM AUTHOR]

Abstract Background Cytokine-induced killer (CIK) cells have shown promising results in adoptive immunotherapy. However, serum may play a determining role in the large-scale expansion of these cells for clinical applications. According to Good Manufacturing Practice (GMP) guidelines to reduce the use of animal products in cell-based therapies; therefore, this study sought to investigate the impact of serum origin and the reduced serum concentration on the pattern of cell expansion and function. Methods Peripheral blood mononuclear cells (PBMCs) isolated from a healthy donor were expanded based on the CIK cell expansion protocol. The cell culture medium was supplemented with three types of sera comprising fetal bovine serum (FBS), human serum (HS), or human-derived platelet lysate (hPL) at different concentrations (10%, 5%, and 2.5%). The proliferation kinetics for each group were investigated for 30 days of cell culture. Results Cell proliferation in 10% concentration of all sera (hPL, FBS, HS) was higher than their lower concentrations. Moreover, hPL was significantly associated with higher expansion rates than FBS and HS in all three concentrations. Furthermore, cells cultured in hPL showed higher viability, cytotoxicity effect, and CIK CD markers expression. Conclusion hPL at a concentration of 10% showed the best effect on CIK cell proliferation and function.

Enriching target cell clones from diverse cell populations is vital for many life science applications. We have developed a novel method to rapidly and efficiently purify specific clonal cell populations from a larger, heterogeneous group using the Enrich TroVo system (Enrich Biosystems Inc., CT, USA). This system takes advantage of microfabrication and optical technologies by utilizing small hydrogel wells to separate desired cell populations and an innovative patching technique to selectively eliminate undesired cells. This method allows the isolation and growth of desired cells with minimal impact on their viability and proliferation. We successfully isolated and expanded clonal cell populations of desired cells using two model cells. Compared with fluorescence-activated cell sorting, Enrich TroVo system offers a promising alternative for isolating of sensitive, adherent cells, that is, patient-derived cells.

Cell expansion in a discrete region called the elongation zone drives root elongation. Analyzing time lapse images can quantify the expansion in kinematic terms as if it were fluid flow. We used horizontal microscopes to collect images from which custom software extracted the length of the elongation zone, the peak relative elemental growth rate (REGR) within it, the axial position of the REGR peak, and the root elongation rate. Automation enabled these kinematic traits to be measured in 1575 Arabidopsis seedlings representing 162 recombinant inbred lines (RILs) derived from a cross of Cvi and Ler ecotypes. We mapped ten quantitative trait loci (QTL), affecting the four kinematic traits. Three QTL affected two or more traits in these vertically oriented seedlings. We compared this genetic architecture with that previously determined for gravitropism using the same RIL population. The major QTL peaks for the kinematic traits did not overlap with the gravitropism QTL. Furthermore, no single kinematic trait correlated with quantitative descriptors of the gravitropism response curve across this population. In addition to mapping QTL for growth zone traits, this study showed that the size and shape of the elongation zone may vary widely without affecting the differential growth induced by gravity.

The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull’s eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications.

Human articular chondrocytes (hACs) are scarce and lose their chondrogenic potential during monolayer passaging, impeding their therapeutic use. This study investigated (a) the translatability of conservative chondrogenic passaging and aggregate rejuvenation on restoring chondrogenic properties of hACs passaged up to P9; and (b) the efficacy of a combined treatment of transforming growth factor-beta 1 (TGF-β1) (T), chondroitinase-ABC (C), and lysyl oxidase-like 2 (L), collectively termed TCL, on engineering functional human neocartilage via the self-assembling process, as a function of passage number up to P11. Here, we show that aggregate rejuvenation enhanced glycosaminoglycan (GAG) content and type II collagen staining at all passages and yielded human neocartilage with chondrogenic phenotype present up to P7. Addition of TCL extended the chondrogenic phenotype to P11 and significantly enhanced GAG content and type II collagen staining at all passages. Human neocartilage derived from high passages, treated with TCL, displayed mechanical properties that were on par with or greater than those derived from low passages. Conservative chondrogenic passaging and aggregate rejuvenation may be a viable new strategy (a) to address the perennial problem of chondrocyte scarcity and (b) to successfully rejuvenate the chondrogenic phenotype of extensively passaged cells (up to P11). Furthermore, tissue engineering human neocartilage via self-assembly in conjunction with TCL treatment advances the clinical use of extensively passaged human chondrocytes for cartilage repair.

Determining the influence of vapor pressure deficit (VPD) on fruit growth is a key issue under a changing climate scenario. Using a comparative approach across different fruit tree species may provide solid indications of common or contrasting plant responses to environmental factors. Knowing fruit growth responses to VPD may also be useful to optimize horticultural management practices under specific atmospheric conditions. Climate data to calculate VPD and fruit relative growth rates (RGR) by fruit gauges were monitored in peach at cell division, pit hardening and cell expansion stages; in two mango cultivars at cell division, cell expansion and maturation stages; in two olive cultivars, either full irrigated or rainfed, at early and late cell expansion stages; in ‘Valencia’ orange at early and late cell division stage, before and after mature fruit harvest; in loquat at cell expansion and maturation stages. At the fruit cell division stage, sensitivity of fruit growth to VPD seems to vary with species, time, and probably soil and atmospheric water deficit. ‘Keitt’ mango and ‘Valencia’ orange fruit growth responded to VPD in opposite ways, and this could be due to very different time of the year and VPD levels in the monitoring periods of the two species. At pit hardening stage of peach fruit growth, a relatively weak relationship was observed between VPD and RGR, and this is not surprising as fruit growth in size at this stage slows down significantly. A consistent and marked negative relationship between VPD and RGR was observed at cell expansion stage, when fruit growth is directly depending on water intake driving cell turgor. Another behavior common to all observed species was the gradual loss of relationship between VPD and RGR at the onset of fruit maturation, when fruit growth in size is generally programmed to stop. Finally, regardless of fruit type, VPD may have a significant effect on fruit growth and could be a useful parameter to be monitored for tree water management mainly when the cell expansion process prevails during fruit growth.

SUMMARY: The steroidal hormone brassinosteroid (BR) has been shown to positively regulate cell expansion in plants. However, the specific mechanism by which BR controls this process has not been fully understood. In this study, RNA‐seq and DAP‐seq analysis of GhBES1.4 (a core transcription factor in BR signaling) were used to identify a cotton cell cycle‐dependent kinase inhibitor called GhKRP6. The study found that GhKRP6 was significantly induced by the BR hormone and that GhBES1.4 directly promoted the expression of GhKRP6 by binding to the CACGTG motif in its promoter region. GhKRP6‐silenced cotton plants had smaller leaves with more cells and reduced cell size. Furthermore, endoreduplication was inhibited, which affected cell expansion and ultimately decreased fiber length and seed size in GhKRP6‐silenced plants compared with the control. The KEGG enrichment results of control and VIGS‐GhKRP6 plants revealed differential expression of genes related to cell wall biosynthesis, MAPK, and plant hormone transduction pathways – all of which are related to cell expansion. Additionally, some cyclin‐dependent kinase (CDK) genes were upregulated in the plants with silenced GhKRP6. Our study also found that GhKRP6 could interact directly with a cell cycle‐dependent kinase called GhCDKG. Taken together, these results suggest that BR signaling influences cell expansion by directly modulating the expression of cell cycle‐dependent kinase inhibitor GhKRP6 via GhBES1.4. Significance Statement: We discovered GhKRP6 is activated by BR hormone and highly expressed in leaves, fibers, and ovules. GhBES1.4 directly binds to the promoter of GhKRP6 and the downregulation of GhKRP6 significantly reduced leaf and seed size and fiber length and decreased cell ploidy. We demonstrated the physical interaction between GhKRP6 and GhCDKG. Our findings suggest that the BR signal directly regulates the cell cycle‐dependent kinase inhibitor GhKRP6 through GhBES1.4, thereby influencing the cell expansion. [ABSTRACT FROM AUTHOR]

Background: Cytokine-induced killer (CIK) cells have shown promising results in adoptive immunotherapy. However, serum may play a determining role in the large-scale expansion of these cells for clinical applications. According to Good Manufacturing Practice (GMP) guidelines to reduce the use of animal products in cell-based therapies; therefore, this study sought to investigate the impact of serum origin and the reduced serum concentration on the pattern of cell expansion and function. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from a healthy donor were expanded based on the CIK cell expansion protocol. The cell culture medium was supplemented with three types of sera comprising fetal bovine serum (FBS), human serum (HS), or human-derived platelet lysate (hPL) at different concentrations (10%, 5%, and 2.5%). The proliferation kinetics for each group were investigated for 30 days of cell culture. Results: Cell proliferation in 10% concentration of all sera (hPL, FBS, HS) was higher than their lower concentrations. Moreover, hPL was significantly associated with higher expansion rates than FBS and HS in all three concentrations. Furthermore, cells cultured in hPL showed higher viability, cytotoxicity effect, and CIK CD markers expression. Conclusion: hPL at a concentration of 10% showed the best effect on CIK cell proliferation and function. [ABSTRACT FROM AUTHOR]

Summary: Grain size is specified by three dimensions of length, width and thickness, and slender grain is a desirable quality trait in rice. Up to now, many grain size regulators have been identified. However, most of these molecules show influence on multi‐dimensions of grain development, and only a few of them function specifically in grain width, a key factor determining grain yield and appearance quality. In this study, we identify the SLG2 (SLENDER GUY2) gene that specifically regulates grain width by affecting cell expansion in the spikelet hulls. SLG2 encodes a WD40 domain containing protein, and our biochemical analyses show that SLG2 acts as a transcription activator of its interacting WOX family protein WOX11. We demonstrate that the SLG2‐associated WOX11 binds directly to the promoter of OsEXPB7, one of the downstream cell expansion genes. We show that knockout of WOX11 results in plants with a slender grain phenotype similar to the slg2 mutant. We also present that finer grains with different widths could be produced by combining SLG2 with the grain width regulator GW8. Collectively, we uncover the crucial role of SLG2 in grain width control, and provide a promising route to design rice plants with better grain shape and quality. [ABSTRACT FROM AUTHOR]

Gibberellins (GA) are a very important signal for fruit set and growth. Exogenous application of these hormones can replace pollination and promote parthenocarpic fruit growth in many species. We have previously shown strong variations between tomato cultivars in the pericarp cell pattern after fruit set, including the number of cell layers, cell size and level of endoreduplication. Here, the effect of GA addition on this pattern was compared to pollination throughout fruit growth in the cherry tomato cultivar WVa106. The effect of a range of GA levels was also investigated in four tomato cultivars, namely WVa106, Micro-Tom, Money Maker and Ailsa Craig, with a different cell pattern in the pericarp. The level of GA was found to be a limiting factor in cell size and ploidy in the tomato pericarp of all cultivars, with dramatic effects in WVa106, Money Maker and Ailsa Craig, and a much more attenuated response in Micro-Tom. GA treatment maintained a robust correlation between cell size and ploidy that was common to all cultivars, and thus at the species level. Radial cell elongation was strongly stimulated by GA treatment in two cultivars only, WVa106 and Ailsa Craig. It was moderately increased in Micro-Tom and Money Maker, indicating a possible uncoupling between cell expansion and elongation. GA also enabled the cell divisions that produce new cell layers in the pericarp of all cultivars, in a way that suggests a different pathway from that regulating cell expansion. Overall, these results clarify our knowledge of the action of GA on the tomato pericarp cell pattern, and beyond on fruit growth in angiosperms. [ABSTRACT FROM AUTHOR]

SUMMARY: Mesocotyl length (ML) is a crucial factor in determining the establishment and yield of rice planted through dry direct seeding, a practice that is increasingly popular in rice production worldwide. ML is determined by the endogenous and external environments, and inherits as a complex trait. To date, only a few genes have been cloned, and the mechanisms underlying mesocotyl elongation remain largely unknown. Here, through a genome‐wide association study using sequenced germplasm, we reveal that natural allelic variations in a mitochondrial transcription termination factor, OsML1, predominantly determined the natural variation of ML in rice. Natural variants in the coding regions of OsML1 resulted in five major haplotypes with a clear differentiation between subspecies and subpopulations in cultivated rice. The much‐reduced genetic diversity of cultivated rice compared to the common wild rice suggested that OsML1 underwent selection during domestication. Transgenic experiments and molecular analysis demonstrated that OsML1 contributes to ML by influencing cell elongation primarily determined by H2O2 homeostasis. Overexpression of OsML1 promoted mesocotyl elongation and thus improved the emergence rate under deep direct seeding. Taken together, our results suggested that OsML1 is a key positive regulator of ML, and is useful in developing varieties for deep direct seeding by conventional and transgenic approaches. Significance Statement: Mesocotyl elongation (ML) is important for seedling emergence under dry direct seeding in rice. Here, we report the identification and functional validation of a gene, OsML1 (LOC_Os07g24090.1), which contributes greatly to the natural variation of ML, has been under selection during domestication and breeding, and functions mainly by affecting cell elongation via H2O2 homeostasis and the expression of expansin genes. [ABSTRACT FROM AUTHOR]

SUMMARY: Grain size is a key factor in determining rice (Oryza sativa) yield, and exploring new pathways to regulate grain size has immense potential to improve yield. In this study, we report that OsCBL5 encodes a calcineurin B subunit protein that significantly promotes grain size and weight. oscbl5 plants produced obviously smaller and lighter seeds. We further revealed that OsCBL5 promotes grain size by affecting cell expansion in the spikelet hull. Biochemical analyses demonstrated that CBL5 interacts with CIPK1 and PP23. Furthermore, double and triple mutations were induced using CRISPR/Cas9 (cr) to analyze the genetic relationship. It was found that the cr‐cbl5/cipk1 phenotype was similar to that of cr‐cipk1 and that the cr‐cbl5/pp23, cr‐cipk1/pp23, and cr‐cbl5/cipk1/pp23 phenotype was similar to that of cr‐pp23, indicating that OsCBL5, CIPK1, and PP23 act as a molecular module influencing seed size. In addition, the results show that both CBL5 and CIPK1 are involved in the gibberellic acid (GA) pathway and significantly affect the accumulation of endogenous active GA4. PP23 participates in GA signal transduction. In brief, this study identified a new module that affects rice grain size, OsCBL5–CIPK1–PP23, which could potentially be targeted to improve rice yield. Significance Statement: The discovery of the OsCBL5–CIPK1–PP23 module and its role in rice (Oryza sativa) yield represents a significant breakthrough not only in reproductive biology but also in the agricultural field for rice breeding. [ABSTRACT FROM AUTHOR]

Petal size is a critical factor in plant reproduction and horticulture, and is largely determined by cell expansion. Gerbera hybrida is an important horticultural plant and serves as a model for studying petal organogenesis. We have previously characterized GhWIP2, a Trp-Ile-Pro (WIP)-type zinc protein, that constrains petal size by suppressing cell expansion. However, the underlying molecular mechanism remains largely unclear. Using yeast two-hybrid screening, bimolecular fluorescence complementation, and co-immunoprecipitation, we identified a TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) family transcription factor, GhTCP7, that interacts with GhWIP2 both in vitro and in vivo. Using reverse genetic approaches, we elucidated the function of the GhTCP7-GhWIP2 complex in controlling petal expansion. GhTCP7 overexpression severely reduced cell expansion and petal size, whereas GhTCP7 silencing resulted in increased cell expansion and petal size. GhTCP7 showed similar expression patterns to GhWIP2 in various types of G. hybrida petals. We further identified GhIAA26 , which encodes an auxin signalling regulator, that is activated by the GhTCP7-GhWIP2 complex, leading to the suppression of petal expansion. Our findings reveal a previously unknown transcriptional regulatory mechanism that involves protein-protein interactions between two different transcription factor families to activate a negative regulator of petal organogenesis. [ABSTRACT FROM AUTHOR]

Main conclusion: NOR-like1 regulates tomato fruit size by targeting SlARF9, SlGRAS2, SlFW3.2, and SlFW11.3 genes involved in cell division and cell expansion. Fruit size is an important agricultural character that determines the yield of crops. Here, we found that NAC transcription factor NOR-like1 regulated fruit size by regulating cell layer number and cell area in tomato. Over-expressing NOR-like1 gene in tomato reduced fruit weight and size, whereas the knock-out of NOR-like1 increased fruit weight and size. At the molecular level, NOR-like1 binds to the promoter of SlGRAS2, SlFW3.2, and SlFW11.3 to repress their transcription, while it also binds to the promoter of ARF9 to activate its transcription. Overall, these results expand the biological function of NOR-like1 and deepen our understanding of the transcriptional network that regulates tomato fruit size. [ABSTRACT FROM AUTHOR]

Olive (Olea europaea L.) is one of the major oil fruit tree crops worldwide. However, the mechanisms underlying olive fruit growth remain poorly understood. Here, we examine questions regarding the interaction of endoreduplication, cell division, and cell expansion with olive fruit growth in relation to the final fruit size by measuring fruit diameter, pericarp thickness, cell area, and ploidy level during fruit ontogeny in three olive cultivars with different fruit sizes. The results demonstrate that differences in the fruit size are related to the maximum growth rate between olive cultivars during early fruit growth, about 50 days post-anthesis (DPA). Differences in fruit weight between olive cultivars were found from 35 DPA, while the distinctive fruit shape became detectable from 21 DPA, even though the increase in pericarp thickness became detectable from 7 DPA in the three cultivars. During early fruit growth, intense mitotic activity appeared during the first 21 DPA in the fruit, whereas the highest cell expansion rates occurred from 28 to 42 DPA during this phase, suggesting that olive fruit cell number is determined from 28 DPA in the three cultivars. Moreover, olive fruit of the large-fruited cultivars was enlarged due to relatively higher cell division and expansion rates compared with the small-fruited cultivar. The ploidy level of olive fruit pericarp between early and late growth was different, but similar among olive cultivars, revealing that ploidy levels are not associated with cell size, in terms of different 8C levels during olive fruit growth. In the three olive cultivars, the maximum endoreduplication level (8C) occurred just before strong cell expansion during early fruit growth in fruit pericarp, whereas the cell expansion during late fruit growth occurred without preceding endoreduplication. We conclude that the basis for fruit size differences between olive cultivars is determined mainly by different cell division and expansion rates during the early fruit growth phase. These data provide new findings on the contribution of fruit ploidy and cell size to fruit size in olive and ultimately on the control of olive fruit development.

There are limited studies on the cytology of bamboo leaf development from primordium to maturity. This study delves into the leaf morphological characteristics and growth patterns of Sasaella kogasensis ‘Aureostriatus’ and provides a three-dimensional anatomical analysis of cell division, expansion, and degradation. Leaves on the same branch develop bottom-up, while individual leaves develop the other way around. Like bamboo shoots and culms, the leaves follow a “slow–fast–slow” growth pattern, with longitudinal growth being predominant during their development. The growth zones of individual leaves included division, elongation, and maturation zones based on the distribution of growth space. By measuring 13,303 epidermal long cells and 3293 mesophyll cells in longitudinal sections of rapidly elongating leaves, we observed that in the rapid elongation phase (S4–S5), the division zone was located in the 1–2 cm segment at the bottom of the leaf blade and maintained a constant size, continuously providing new cells for leaf elongation, whereas in the late rapid elongation phase (S6), when the length of the leaf blade was approaching that of a mature leaf, its cells at the bottom of the blade no longer divided and were replaced by the ability to elongate. Furthermore, to gain an insight into the dynamic changes in the growth of the S. kogasensis ‘Aureostriatus’ leaves in the lateral and periclinal directions, the width and thickness of 1459 epidermal and 2719 mesophyll cells were counted in the mid-cross section of leaves at different developmental stages. The results showed that during the early stages of development (S1–S3), young leaves maintained vigorous division in the lateral direction, while periplasmic division gradually expanded from the bottom to the top of the leaf blade and the number of cell layers stabilized at S4. The meristematic tissues on both sides of the leaf were still able to divide at S4 but the frequency of the division gradually decreased, while cell division and expansion occurred simultaneously between the veins. At S6, the cells at the leaf margins and between the veins were completely differentiated and the width of the leaf blade no longer expanded. These findings revealed changes in cell growth anisotropically during the leaf development of S. kogasensis ‘Aureostriatus’ and demonstrated that leaf elongation was closely related to the longitudinal expansion of epidermal cells and proliferative growth of mesophyll cells, whereas the cell division of meristematic tissues and expansion of post-divisional cells contributed to the increases in blade width and thickness. The presented framework will facilitate a further exploration of the molecular regulatory mechanisms of leaf development in S. kogasensis ‘Aureostriatus’ and provide relevant information for developmental and taxonomic studies of bamboo plants.