Active Cell Proliferation Research Articles

Highly regenerative species-specific genes improve age-associated features in the adult Drosophila midgut

BackgroundThe remarkable regenerative abilities observed in planarians and cnidarians are closely linked to the active proliferation of adult stem cells and the precise differentiation of their progeny, both of which typically deteriorate during aging in low regenerative animals. While regeneration-specific genes conserved in highly regenerative organisms may confer regenerative abilities and long-term maintenance of tissue homeostasis, it remains unclear whether introducing these regenerative genes into low regenerative animals can improve their regeneration and aging processes.ResultsHere, we ectopically express highly regenerative species-specific JmjC domain-encoding genes (HRJDs) in Drosophila, a widely used low regenerative model organism. Surprisingly, HRJD expression impedes tissue regeneration in the developing wing disc but extends organismal lifespan when expressed in the intestinal stem cell lineages of the adult midgut under non-regenerative conditions. Notably, HRJDs enhance the proliferative activity of intestinal stem cells while maintaining their differentiation fidelity, ameliorating age-related decline in gut barrier functions.ConclusionsThese findings together suggest that the introduction of highly regenerative species-specific genes can improve stem cell functions and promote a healthy lifespan when expressed in aging animals.

Role of Anti-Müllerian Hormone in Male Reproduction and Sperm Motility.

Anti-Müllerian hormone (AMH) is secreted by Sertoli cells and is responsible for the regression of Müllerian ducts in the male fetus as part of the sexual differentiation process. Serum AMH concentrations are at their lowest levels in the first days after birth but increase after the first week, likely reflecting active Sertoli cell proliferation. AMH rises rapidly in concentration in boys during the first month, reaching a peak level at ∼6 months of age, and it remains high during childhood, then they will slowly decline during puberty, falling to low levels in adulthood. Serum AMH measurement is used by pediatric endocrinologist as a specific marker of immature Sertoli cell number and function during childhood. After puberty, AMH is released especially by the apical pole of the Sertoli cells toward the lumen of the seminiferous tubules, resulting in higher levels in the seminal plasma than in the serum. Recently, AMH has received increasing attention in research on male fertility-related disorders. This article reviews and summarizes the potential contribution of serum AMH measurement in different male fertility-related disorders.

GINS1 promotes the initiation and progression of bladder cancer by activating the AKT/mTOR/c-Myc signaling pathway

Bladder cancer(BC) is one of the most prevalent cancers in the urinary tract, with high recurrence and fatality rates. Research indicates that go-ichi-ni-san complex subunit 1 (GINS1) crucially influences cancer progression by regulating DNA replication through cell cycle modulation. Thus, suppressing the active proliferation of cells in tumor tissues may require silencing GINS1. However, the consequences of GINS1 in bladder cancer aren't to be determined. In this paper, we examine the role and mechanism of GINS1 in the development of bladder cancer. GINS1 expression levels and prognostic relevance in bladder cancer were validated using Western blotting, immunohistochemistry, and Kaplan-Meier survival analysis. The influence of GINS1 on bladder cancer was investigated using a variety of approaches, including cell transfection, cell counts, transwell migrations, colony formation, and flow cytometry. Immunohistochemistry studies demonstrate that GINS1 expression is increased in bladder cancer tissues. GINS1 silencing resulted in an arrest of the cell cycle at the phase of G0/G1, which inhibited BC cell growth both in vitro and in vivo. GINS1 knockdown also hindered the AKT/mTOR pathway. Furthermore, increased GINS1 expression affects the cell cycle and stimulates the AKT/mTOR pathway, allowing BC to develop more quickly. Consequently, GINS1 occurs as a latent therapeutic target, particularly for individuals with BC.

Development of reliable in vitro long-term culture systems for hematopoietic cells of crayfish Cherax quadricarinatus and virus susceptibility assay

Long-term in vitro cultured crayfish cells can serve as a valuable tool for investigating various aspects of crayfish biology, encompassing cell physiology and viral mechanisms. Regrettably, they are difficult to maintain a highly active state and survive during long-term and continuous culture in vitro. In order to generate highly viable crayfish cell cultures, it is crucial to optimize dependable culture conditions and utilize a suitable cell medium. In this study, we have successfully developed reliable in vitro long-term culture systems for hematopoietic (HPT) cells of crayfish Cherax quadricarinatus by formulating an optimal crayfish cell growth medium (OCCM), including under two-dimensional (2D) and three-dimensional (3D) conditions. The novel medium was optimized by a mix of exogenous nutrients, crayfish plasma (CP) and shrimp serum-derived amino acids and saccharides within the Leibovitz's L-15 (L-15) medium. Our newly developed crayfish cell growth medium (CCM-28) enabled the culturing of HPT cells for over 142 days under 2D and over 60 days under 3D conditions, with consistent active cell proliferation observed over a 30-day incubation period. This proliferation was comparable to in vivo conditions, with a cell division rate exceeding 20%, suggesting that a high mitotic rate has been successfully achieved during long-term in vitro culture. Flow cytometry analysis further elucidated the mitosis-arrest of HPT cells cultured in vitro was predominantly observed in the G0/G1 phase. Furthermore, the cell subculture techniques have been established using mechanical dissociation method (in 2D) and substrate exchange method (in 3D). Additionally, the susceptibility of these cell cultures to covert mortality nodavirus (CMNV) and white spot syndrome virus (WSSV) was confirmed by capturing cytopathic effects (CPE) and analyzing viral copy numbers. Species identification using the cytochrome oxidase subunit I (COI) gene established the origin of the cell cultures from C. quadricarinatus. In conclusion, the successful development of highly viable crayfish cells in long-term in vitro culture systems, as demonstrated in this study, will significantly advance the research on immortalization in crayfish cells and the study of mechanisms related to viruses.

Precocious cell differentiation occurs in proliferating cells in leaf primordia in Arabidopsis angustifolia3 mutant.

During leaf development, the timing of transition from cell proliferation to expansion is an important factor in determining the final organ size. However, the regulatory system involved in this transition remains less understood. To get an insight into this system, we investigated the compensation phenomenon, in which the cell number decreases while the cell size increases in organs with determinate growth. Compensation is observed in several plant species suggesting coordination between cell proliferation and expansion. In this study, we examined an Arabidopsis mutant of ANGUSTIFOLIA 3 (AN3)/GRF-INTERACTING FACTOR 1, a positive regulator of cell proliferation, which exhibits the compensation. Though the AN3 role has been extensively investigated, the mechanism underlying excess cell expansion in the an3 mutant remains unknown. Focusing on the early stage of leaf development, we performed kinematic, cytological, biochemical, and transcriptome analyses, and found that the cell size had already increased during the proliferation phase, with active cell proliferation in the an3 mutant. Moreover, at this stage, chloroplasts, vacuoles, and xylem cells developed earlier than in the wild-type cells. Transcriptome data showed that photosynthetic activity and secondary cell wall biosynthesis were activated in an3 proliferating cells. These results indicated that precocious cell differentiation occurs in an3 cells. Therefore, we suggest a novel AN3 role in the suppression of cell expansion/differentiation during the cell proliferation phase.

Consumption of Citric Acid by Bees Promotes the Gland Development and Enhances Royal Jelly Quality.

The glands of bees are responsible for generating and secreting various biologically active substances that significantly impact bee physiological health and adaptability. This study aimed to investigate the effects of adding citric acid (CA) to bee feed on gland development and royal jelly quality. By formulating feed with varying proportions of CA, evaluation was undertaken of pollen feeding by honeybees under laboratory conditions, along with the impact of CA on the development of major glands, to determine suitable addition proportions. Further optimization of the CA proportion involved feeding colonies and evaluating royal jelly production and quality. The results indicated that feed containing 0.75% CA significantly extended the lifespan of bees and increased their pollen consumption. Gland development in bees showed a positive correlation with CA addition within the range of 0.25% to 0.75%, especially at 0.50% and 0.75%, which notably accelerated the development of mandibular, hypopharyngeal, and cephalic salivary glands, with active proliferation and differentiation of glandular cells and maintenance of normal gland size and morphology. CA added to feed stimulated vigorous secretion of wax glands in worker bees, resulting in prolific wax construction. Colonies consuming feed containing 0.50% CA produced royal jelly with significantly reduced moisture and total sugar content and increased levels of 10-HDA, total phenolic acids, total proteins, and acidity. These findings demonstrate that CA consumption significantly prolongs bee lifespan, increases consumption, promotes gland development, and enhances royal jelly quality. This research provides theoretical guidance for beekeeping practices and feed development, contributing to the sustainable advancement of apiculture.

Pharmacodynamic Interaction of Areca catechu with Gliclazide in Wistar Rats

Background and Purpose In alloxan-induced diabetic rats, this study sought to evaluate the hypoglycemic potential of the hydroalcoholic seed extract of Areca catechu, also referred to as areca nut, both as a stand-alone treatment and in combination with the diabetic drug gliclazide. Materials and Methods The hot percolation method was used to produce the hydroalcoholic seed extract of A. catechu. Rats were given alloxan monohydrate and developed type II diabetes. The hydroalcoholic seed extract of A. catechu (1000 mg/kg p.o.) alone or in different combinations of gliclazide (25 mg/kg p.o.) and A. catechu extract (250, 500, and 1000 mg/kg p.o.) were administered to the six diabetic rats. Body weights and blood glucose levels were measured on days 7, 14, 21, and 28 of the treatment. On the 28th day, the kidney, liver, and pancreas underwent histological investigations in addition to an oral glucose tolerance test (OGTT). Results Blood glucose levels were significantly lowered upon administration of A. catechu’s hydroalcoholic seed extract, both alone and in conjunction with gliclazide. Comparing the diabetic control group to the A. catechu extract-treated group, histopathological examinations showed that high amounts of the extract, either by itself or in conjunction with gliclazide, caused the active proliferation of pancreatic beta cells, protecting against damage to the liver and kidneys. Conclusion The hydroalcoholic seed extract of A. catechu showed promise as a monotherapy for controlling blood glucose levels, and as a co-administer for gliclazide, it showed signs of hypoglycemia. These results indicate A. catechu’s possible therapeutic benefit as an adjuvant diabetic treatment. To investigate the underlying mechanisms of action and evaluate its safety and effectiveness in clinical settings, more study is necessary.

Callus proliferation-induced hypoxic microenvironment decreases shoot regeneration competence in Arabidopsis

Plants are aerobic organisms that rely on molecular oxygen for respiratory energy production. Hypoxic conditions, with oxygen levels ranging between 1% and 5%, usually limit aerobic respiration and affect plant growth and development. Here, we demonstrate that the hypoxic microenvironment induced by active cell proliferation during the two-step plant regeneration process intrinsically represses the regeneration competence of the callus in Arabidopsis thaliana. We showed that hypoxia-repressed plant regeneration is mediated by the RELATED TO APETALA 2.12 (RAP2.12) protein, a member of the Ethylene Response Factor VII (ERF-VII) family. We found that the hypoxia-activated RAP2.12 protein promotes salicylic acid (SA) biosynthesis and defense responses, thereby inhibiting pluripotency acquisition and de novo shoot regeneration in calli. Molecular and genetic analyses revealed that RAP2.12 could bind directly to the SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) gene promoter and activate SA biosynthesis, repressing plant regeneration possibly via a PLETHORA (PLT)-dependent pathway. Consistently, the rap2.12 mutant calli exhibits enhanced shoot regeneration, which is impaired by SA treatment. Taken together, these findings uncover that the cell proliferation-dependent hypoxic microenvironment reduces cellular pluripotency and plant regeneration through the RAP2.12–SID2 module.

The crucial role of SETDB1 in structural and functional transformation of epithelial cells during regeneration after intestinal ischemia reperfusion injury.

Su (var) 3-9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75min of ischemia followed by 3, 24, and 48h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.

MRI radiomics-based interpretable model and nomogram for preoperative prediction of Ki-67 expression status in primary central nervous system lymphoma.

To investigate the value of interpretable machine learning model and nomogram based on clinical factors, MRI imaging features, and radiomic features to predict Ki-67 expression in primary central nervous system lymphomas (PCNSL). MRI images and clinical information of 92 PCNSL patients were retrospectively collected, which were divided into 53 cases in the training set and 39 cases in the external validation set according to different medical centers. A 3D brain tumor segmentation model was trained based on nnU-NetV2, and two prediction models, interpretable Random Forest (RF) incorporating the SHapley Additive exPlanations (SHAP) method and nomogram based on multivariate logistic regression, were proposed for the task of Ki-67 expression status prediction. The mean dice Similarity Coefficient (DSC) score of the 3D segmentation model on the validation set was 0.85. On the Ki-67 expression prediction task, the AUC of the interpretable RF model on the validation set was 0.84 (95% CI:0.81, 0.86; p < 0.001), which was a 3% improvement compared to the AUC of the nomogram. The Delong test showed that the z statistic for the difference between the two models was 1.901, corresponding to a p value of 0.057. In addition, SHAP analysis showed that the Rad-Score made a significant contribution to the model decision. In this study, we developed a 3D brain tumor segmentation model and used an interpretable machine learning model and nomogram for preoperative prediction of Ki-67 expression status in PCNSL patients, which improved the prediction of this medical task. Ki-67 represents the degree of active cell proliferation and is an important prognostic parameter associated with clinical outcomes. Non-invasive and accurate prediction of Ki-67 expression level preoperatively plays an important role in targeting treatment selection and patient stratification management for PCNSL thereby improving prognosis.

Immunoswitch Nanomodulators Enable Active Targeting and Selective Proliferation of Regulatory T Cells for Multiple Sclerosis Therapy.

Interleukin-2 (IL-2) used in multiple sclerosis (MS) therapy modulates the balance between regulatory T (Treg) cells and effector T (Teff) cells. However, the off-target activation of Teff cells by IL-2 limits its clinical application. Therefore, a rapidly prepared immunoswitch nanomodulator termed aT-IL2C NPs was developed, which specifically recognized Treg cells with high TIGIT expression thanks to the presence of an anti-TIGIT and an IL-2/JES6-1 complex (IL2C) being delivered to Treg cells but not to Teff cells with low TIGIT expression. Then, IL2C released IL-2 due to the specific expression of the high-affinity IL-2 receptor on Treg cells, thus enabling the active targeting and selective proliferation of Treg cells. Moreover, the anti-TIGIT of aT-IL2C NPs selectively inhibited the proliferation of Teff cells while leaving the proliferation of Treg cells unaffected. In addition, since the IL-2 receptor on Teff cells had medium-affinity, the IL2C hardly released IL-2 to Teff cells, thus enabling the inhibition of Teff cell proliferation. The treatment of experimental autoimmune encephalomyelitis (EAE) mice with aT-IL2C NPs ameliorated the severity of the EAE and restored white matter integrity. Collectively, this work described a potential promising agent for effective MS therapy.

Single-cell RNA sequencing reveals distinct tumor microenvironment of ground glass nodules and solid nodules in lung adenocarcinoma.

Introduction: Lung adenocarcinoma (LUAD) is the most prevalent lung cancer. LUAD presents as ground glass nodules (GGN) and solid nodules (SN) in imaging studies. GGN is an early type of LUAD with good prognosis. However, SN exhibits a more malignant behavior than GGN, including worse pathological staging and tumor prognosis. The mechanism leading to the different malignancy levels of GGN and SN remains elusive. Methods: Three patients with GGN and three patients with SN diagnosed with early LUAD were enrolled. The tumor samples were digested to a single-cell suspension and analyzed using 10× Genomic Single-cell ribonucleic acid sequences (scRNA-seq) techniques. Results: A total of 15,902 cells were obtained and classified into nine major types. The tumor microenvironment (TME) was subsequently described in detail. ScRNA-seq revealed that ribosome-related pathways and cell adhesion played similar but distinct roles in the two groups. SN also had more active cell proliferation, enriched cell cycle regulatory pathways, and severe inflammatory responses. Conclusion: We observed changes in the cellular composition and transcriptomic profile of GGN and SN. The study improved the understanding of the underlying mechanisms of lung carcinogenesis and contributed to lung cancer prevention and treatment.

Гипотеза о возможных механизмах терапевтического действия гексанового экстракта Serenoa repens на состояние гипертрофированного мочевого пузыря в соответствии с патогенезом его дисфункции при инфравезикальной обструкции.

Introduction. Traditionally it is believed that the therapeutic effect of the hexane extract of Serenoa repens (HSRE) in patients with benign prostatic hyperplasia is realized due to its effect on prostate cells. However, in the literature there is data about the possible HSRE effect not only on the prostate, but also directly on the bladder. This paper summarizes data and analyzes the possible cellular mechanisms of HSRE action on the bladder under the development of infravesical obstruction (bladder outlet obstruction – BOO). Materials and methods. Literature data from the PubMed and eLibrary.ru databases for a 25-years period related to the study of the pathogenesis of bladder dysfunction in the development of BOO, cellular and molecular mechanisms by which pathological and adaptive processes in bladder cells are realized, as well as the influence of HSRE on these processes are analyzed. Results. The concept of the pleiotropic effect of HSRE on various links in the pathogenesis of bladder dysfunction caused by BOO has been formed. The main of which are: the antioxidant and anti-inflammatory effects of HSRE components, as well as the regulatory effect on cell growth and proliferation factors and transcription factors. All this reduces the cytotoxic effect of oxidative stress on the cells of the bladder developing in IVO, contributing to a more active proliferation of detrusor smooth muscle cells. The consequence of the complex action is to maintain the contractile function of the bladder in a state of adaptation to increased stress, due to BOO. Conclusions. The present hypothesis about the direct effect of HSRE on the hypertrophied bladder and possible mechanisms of its therapeutic action is based on an understanding of the general biological mechanisms and allows us to explain better the results of previous clinical studies and to assess the prospects for expanding the scope of further drug application.

Breaking spore dormancy in budding yeast transforms the cytoplasm and the solubility of the proteome.

The biophysical properties of the cytoplasm are major determinants of key cellular processes and adaptation. Many yeasts produce dormant spores that can withstand extreme conditions. We show that spores of Saccharomyces cerevisiae exhibit extraordinary biophysical properties, including a highly viscous and acidic cytosol. These conditions alter the solubility of more than 100 proteins such as metabolic enzymes that become more soluble as spores transit to active cell proliferation upon nutrient repletion. A key regulator of this transition is the heat shock protein, Hsp42, which shows transient solubilization and phosphorylation, and is essential for the transformation of the cytoplasm during germination. Germinating spores therefore return to growth through the dissolution of protein assemblies, orchestrated in part by Hsp42 activity. The modulation of spores' molecular properties are likely key adaptive features of their exceptional survival capacities.

Long non-coding RNAs: The growth controller of vascular smooth muscle cells in cardiovascular diseases.

The active proliferation and migration of vascular smooth muscle cells supports the healing of vessel damage while their abnormal aggression or destitution contribute to the aberrant intima-medial structure and function in various cardiovascular diseases, so the understanding of the proliferation disorders of vascular smooth muscle cell and the related mechanism is the basis of effective intervention and control for cardiovascular diseases. Recently, long non-coding RNAs have stood out as upstream switchers for multiple proliferative signaling pathways and molecules, and many of them have been shown to conduce to the dysregulated proliferation and apoptosis of vascular smooth muscle cells under various pathogenic stimuli. This article discusses the long non-coding RNAs disclosed and linked to atherosclerosis, pulmonary hypertension, and aneurysms, and focuses upon their modulation of vascular smooth muscle cell population affecting three deadly cardiovascular diseases.

The Activation of PPARγ by (2Z,4E,6E)-2-methoxyocta-2,4,6-trienoic Acid Counteracts the Epithelial-Mesenchymal Transition Process in Skin Carcinogenesis.

Cutaneous squamous cell carcinoma (cSCC) is the most common UV-induced keratinocyte-derived cancer, and its progression is characterized by the epithelial-mesenchymal transition (EMT) process. We previously demonstrated that PPARγ activation by 2,4,6-octatrienoic acid (Octa) prevents cutaneous UV damage. We investigated the possible role of the PPARγ activators Octa and the new compound (2Z,4E,6E)-2-methoxyocta-2,4,6-trienoic acid (A02) in targeting keratinocyte-derived skin cancer. Like Octa, A02 exerted a protective effect against UVB-induced oxidative stress and DNA damage in NHKs. In the squamous cell carcinoma A431 cells, A02 inhibited cell proliferation and increased differentiation markers' expression. Moreover, Octa and even more A02 counteracted the TGF-β1-dependent increase in mesenchymal markers, intracellular ROS, the activation of EMT-related signal transduction pathways, and cells' migratory capacity. Both compounds, especially A02, counterbalanced the TGF-β1-induced cell membrane lipid remodeling and the release of bioactive lipids involved in EMT. In vivo experiments on a murine model useful to study cell proliferation in adult animals showed the reduction of areas characterized by active cell proliferation in response to A02 topical treatment. In conclusion, targeting PPARγ may be useful for the prevention and treatment of keratinocyte-derived skin cancer.

Cancerous inhibitor of protein phosphatase 2A enhances chemoresistance of gastric cancer cells to oxaliplatin.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a newly discovered oncogene. It is an active cell proliferation regulatory factor that inhibits tumor apoptosis in gastric cancer (GC) cells. CIP2A is functionally related to chemoresistance in various types of tumors according to recent studies. The underlying mechanism, however, is unknown. Further, the primary treatment regimen for GC is oxaliplatin-based chemotherapy. Nonetheless, it often fails due to chemoresistance of GC cells to oxaliplatin. The goal of this study was to examine CIP2A expression and its association with oxaliplatin resistance in human GC cells. Immunohistochemistry was used to examine CIP2A expression in GC tissues and adjacent normal tissues. CIP2A expression in GC cell lines was reduced using small interfering RNA. After confirming the silencing efficiency, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium and flow cytometry assays were used to evaluate cell proliferation and apoptosis caused by oxaliplatin treatment. Further, the key genes and protein changes were verified using real-time quantitative reverse transcription PCR and Western blotting, respectively, before and after intervention. For bioinformatics analysis, we used the R software and Bioconductor project. For statistical analysis, we used GraphPad Prism 6.0 and the Statistical Package for the Social Sciences software version 20.0 (IBM, Armonk, United States). A high level of CIP2A expression was associated with tumor size, T stage, lymph node metastasis, Tumor Node Metastasis stage, and a poor prognosis. Further, CIP2A expression was higher in GC cells than in normal human gastric epithelial cells. Using small interfering RNA against CIP2A, we discovered that CIP2A knockdown inhibited cell proliferation and significantly increased GC cell sensitivity to oxaliplatin. Moreover, CIP2A knockdown enhanced oxaliplatin-induced apoptosis in GC cells. Hence, high CIP2A levels in GC may be a factor in chemoresistance to oxaliplatin. In human GC cells, CIP2A regulated protein kinase B phosphorylation, and chemical inhibition of the protein kinase B signaling pathway was significantly associated with increased sensitivity to oxaliplatin. Therefore, the protein kinase B signaling pathway was correlated with CIP2A-enhanced chemoresistance of human GC cells to oxaliplatin. CIP2A expression could be a novel therapeutic strategy for chemoresistance in GC.

Identification and Expression Profile of CLE41/44-PXY-WOX Genes in Adult Trees Pinus sylvestris L. Trunk Tissues during Cambial Activity

WUSCHEL (WUS)-related homeobox (WOX) protein family members play important roles in the maintenance and proliferation of the stem cells in the cambium, the lateral meristem that forms all the wood structural elements. Most studies have examined the function of these genes in angiosperms, and very little was known about coniferous trees. Pine is one of the most critical forest-forming conifers globally, and in this research, we studied the distribution of WOX4, WOX13, and WOXG genes expression in Pinus sylvestris L. trunk tissues. Further, we considered the role of TDIF(CLE41/44)/TDR(PXY) signaling in regulating Scots pine cambial activity. The distribution of CLE41/44-PXY-WOXs gene expression in Scots pine trunk tissues was studied: (1) depending on the stage of ontogenesis (the first group of objects); and (2) depending on the stage of cambial growth (the second group of objects). The first group of objects is lingonberry pine forests of different ages (30-, 80-, and 180-year-old stands) in the middle taiga subzone. At the time of selection, all the trees of the studied groups were at the same seasonal stage of development: the formation of late phloem and early xylem was occurring in the trunk. The second group of objects is 40-year-old pine trees that were selected growing in the forest seed orchard. We took the trunk tissue samples on 27 May 2022, 21 June 2022, and 21 July 2022. We have indicated the spatial separation expressed of PsCLE41/44 and PsPXY in pine trunk tissues. PsCLE41/44 was differentially expressed in Fraction 1, including phloem cells and cambial zone. Maximum expression of the PsPXY gene occurred in Fraction 2, including differentiating xylem cells. The maximum expression of the PsCLE41/44 gene occurred on 27 May, when the number of cells in the cambial zone was the highest, and then it decreased to almost zero. The PsPXY gene transcript level increased from May to the end of July. We found that the highest transcript level of the PsWOX4 gene was during the period of active cell proliferation in the cambial zone, and also in the trees with the cambial age 63 years, which were characterized by the largest number of cell layers in the cambial zone. In this study, we have examined the expression profiles of genes belonging to the ancient clade (PsWOXG and PsWOX13) in stem tissues in Scots pine for the first time. We found that, in contrast to PsWOX4 (high expression that was observed during the period of active formation of early tracheids), the expression of genes of the ancient clade of the WOX genes was observed during the period of decreased cambial activity in the second half of the growing season. We found that PsWOX13 expression was shifted to Fraction 1 in most cases and increased from the phloem side, while PsWOXG expression was not clearly bound to a certain fraction. Based on the data, the role of the CLE41/44-PXY-WOX signaling module in regulating P. sylvestris cambial growth is discussed.

Hybrid biofabrication of neurosecretory structures as a model for neurosecretion.

The present study aimed to combine extrusion-based three-dimensional (3D) bioprinting and polymer nanofiber electrospinning technology to fabricate tissue-like structures with neurosecretory function in vitro. Using neurosecretory cells as cell resources, sodium alginate/gelatin/fibrinogen as matrix, polylactic acid/gelatin electrospun nanofibers as diaphragm, and neurosecretory cells-loaded 3D hydrogel scaffolds were bioprinted and then covered with electrospun nanofibers layer-by-layer. The morphology was observed by scanning electron microscopy and transmission electron microscopy (TEM), and the mechanical characteristics and cytotoxicity of the hybrid biofabricated scaffold structure were evaluated. The 3D-bioprinted tissue activity, including cell death and proliferation, was verified. Western blotting and ELISA experiments were used to confirm the cell phenotype and secretory function, while animal in vivo transplantation experiments confirmed the histocompatibility, inflammatory reaction, and tissue remodeling ability of the heterozygous tissue structures. Neurosecretory structures with 3D structures were successfully prepared by hybrid biofabrication in vitro. The mechanical strength of the composite biofabricated structures was significantly higher than that of the hydrogel system (P < 0.05). The survival rate of PC12 cells in the 3D-bioprinted model was 92.849 ± 2.995%. Hematoxylin and eosin-stained pathological sections showed that the cells grew in clumps, and there was no significant difference in the expression of MAP2 and tubulin-β between 3D organoids and PC12 cells. The results of ELISA showed that the PC12 cells in 3D structures retained the ability to continuously secrete noradrenaline and met-enkephalin, and the secretory vesicles around and within the cells could be observed by TEM. In in vivo transplantation, PC12 cells gathered and grew in clusters, maintained high activity, neovascularization, and tissue remodeling in 3D structures. The neurosecretory structures were biofabricated by 3D bioprinting and nanofiber electrospinning in vitro, which had high activity and neurosecretory function. In vivo transplantation of neurosecretory structures showed active proliferation of cells and potential for tissue remodeling. Our research provides a new method for biological manufacture of neurosecretory structures in vitro, which maintains neurosecretory function and lays the foundation for the clinical application of neuroendocrine tissues.

MicroRNA-149 Regulates Proliferation, Migration, and Invasion of Pituitary Adenoma Cells by Targeting ADAM12 and MMP14

ObjectivePituitary adenomas (PAs) can adapt an aggressive phenotype by invading adjacent brain structures with rapid cellular proliferation. Previous studies demonstrated that excessive expression of metalloproteases ADAM12 and MMP-14 is instrumental for the active proliferation and invasiveness of PA cells in vitro and of tumors in vivo. However, the mechanisms regulating ADAM12 and MMP-14 expression in PAs remain unclear.MethodsTarget gene prediction and transcriptomic profiling of invasive vs. noninvasive human PA samples were performed to identify miRNA species potentially involved in the regulation of ADAM12 and MMP14. For cellular analyses of miRNA functions, two mouse PA cell lines (AtT20 and TtT/GF) were transfected with miR-149-3p and miR-149-5p, respectively. The effects of miR-149 (3p and 5p) on expression levels of ADAM12 and MMP14 were determined by Western blotting followed by an analysis of proliferation and colony formation assays, scratch migration assays, and invasion assays.ResultsA significant downregulation of miRNA-149 was observed in invasive vs. noninvasive PA (0.32 vs. 0.09, P<0.0001). In AtT-20 and TtT/GF mouse PAs cells, transfection of mimic miRNA-149 (3p and 5p) caused a significantly reduced cell proliferation and matrigel invasion, whilst the effect on cell migration was less pronounced. Both strands of miRNA-149 (3p and 5p) markedly reduced protein levels of ADAM12 and MMP-14 by at least 40% in both cell lines.ConclusionThis study proved that the invasiveness of PA cells is, at least partly, regulated by miRNA-149-dependent expression of ADAM12 and MMP-14.