Ki67 mRNA Research Articles

Dynamic phenotypic shifts and M2 receptor downregulation in bladder smooth muscle cells induced by mirabegron.

Mirabegron is available for treatment of overactive bladder (OAB). However, mechanisms underlying symptom improvements and long-term effects on bladder smooth muscle cells are uncertain. Contractility and growth of bladder smooth muscle contribute to OAB, and depend on smooth muscle phenotypes, and on muscarinic receptor expression. Here, we examined prolonged exposure to mirabegron (20-48h) on phenotype markers, muscarinic receptor expression, and phenotype-dependent functions in human bladder smooth muscle cells (hBSMC). Expression of markers for contractile (calponin, MYH11) and proliferative (MYH10, vimentin) phenotypes, proliferation (Ki-67), and of muscarinic receptors were assessed by RT-PCR. Proliferation, viability, actin organization and contractions in cultured hBSMC were examined by EdU, CCK-8, phalloidin staining and matrix contraction assays. Calponin-1 mRNA decreased with 100nM and 150nM mirabegron applied for 20h (0.56-0.6 fold of controls). Decreases were resistant to the β3-AR antagonist L-748,337 (0.34-0.55 fold, 100-150nM, 20h). After 40h, decreases occured in the presence of L-748,337, but not without L-748,337. MYH11 mRNA increased with 150nM mirabegron (40h, 1.9 fold). This was partly preserved with L-748,337, but not observed after 20h mirabegron exposure. Vimentin mRNA reduced with 150nM mirabegron after 20h, but not after 40h, with and without L-748,337 (0.71-0.63 fold). MYH10 mRNA expression remained unaffected by mirabegron. Exposure to 150nM mirabegron increased Ki-67 mRNA after 20h in the presence of, but not without L-748,337, and after 40h without, but not with L-748,337. Proliferation rates and actin organization were stable with 50-150nM mirabegron (24h, 48h). Viability increased significantly after mirabegron exposure for 20h, and by trend after 40h, which was fully sensitive to L-748,337. M2 mRNA was reduced by 20h mirabegron, which was resistant to L-748,337. Carbachol (3µM) enhanced time-dependent contractions of hBSMC, which was inhibited by mirabegron (150nM) in late phases (24h), but not in early phases of contractions. Conclusion: Mirabegron induces dynamic phenotype alterations and M2 downregulation in hBSMC, which is paralleled by time-shifted anticontractile effects. Phenotype transitions may be involved in improvements of storage symptoms in OAB by mirabegron.

Graphene oxide-functionalized molecular beacon for real-time interference-free detection of Ki-67 mRNA in living cells

Molecular beacons (MBs) based on hairpin-shaped oligonucleotides are captivating owing to their capability to enable effective real-time detection of cytosolic mRNA in living cells. However, DNase in the nucleus and lysosome could induce the degradation of oligonucleotides in MBs, leading to the generation of false-positive signals. Herein, a graphene oxide (GO) nanosheet was applied as a nanocarrier for MBs to greatly enhance the anti-interference of the easily designed nanoprobe. Advantageously, the absorption capacity of GO for MBs increased with the decrease in pH values, providing the MB-GO nanoprobe with the ability to detect the expression of cytosolic Ki-67 mRNA without interference from DNase Ⅱ in lysosomes. Moreover, the size of GO nanosheets was considerably higher than that of the nuclear pore complex (NPC), which prevented nanoprobes from transition through the NPCs, thereby avoiding the generation of false-positive signals in the nucleus. Altogether, the present work affords a convenient approach for the successful detection of Ki-67 mRNA expression in the cytosol without interference from DNase Ⅰ/Ⅱ in the nucleus/lysosome, which may be potentially further applied for the detection of other cytosolic RNAs.

Screening of the Skin-Regenerative Potential of Antimicrobial Peptides: Clavanin A, Clavanin-MO, and Mastoparan-MO.

Skin wound healing is coordinated by a delicate balance between proinflammatory and anti-inflammatory responses, which can be affected by opportunistic pathogens and metabolic or vascular diseases. Several antimicrobial peptides (AMPs) possess immunomodulatory properties, suggesting their potential to support skin wound healing. Here, we evaluated the proregenerative activity of three recently described AMPs (Clavanin A, Clavanin-MO, and Mastoparan-MO). Human primary dermal fibroblasts (hFibs) were used to determine peptide toxicity and their capacity to induce cell proliferation and migration. Furthermore, mRNA analysis was used to investigate the modulation of genes associated with skin regeneration. Subsequently, the regenerative potential of the peptides was further confirmed using an ex vivo organotypic model of human skin (hOSEC)-based lesion. Our results indicate that the three molecules evaluated in this study have regenerative potential at nontoxic doses (i.e., 200 μM for Clavanin-A and Clavanin-MO, and 6.25 μM for Mastoparan-MO). At these concentrations, all peptides promoted the proliferation and migration of hFibs during in vitro assays. Such processes were accompanied by gene expression signatures related to skin regenerative processes, including significantly higher KI67, HAS2 and CXCR4 mRNA levels induced by Clavanin A and Mastoparan-MO. Such findings translated into significantly accelerated wound healing promoted by both Clavanin A and Mastoparan-MO in hOSEC-based lesions. Overall, the data demonstrate the proregenerative properties of these peptides using human experimental skin models, with Mastoparan-MO and Clavanin A showing much greater potential for inducing wound healing compared to Clavanin-MO.

Role of p57KIP2 in Stem and Progenitor Leydig Cells of Mouse Testes.

Precise control of proliferation and differentiation of Leydig cells is important for gonadal androgenesis and spermatogenesis. Though cyclin-dependent kinase inhibitors are crucial for cell proliferation and differentiation, their role in the development of early adult Leydig cells (ALCs) remained unanswered. To understand mechanism for ALC development, functional expression of p57KIP2 (cdkn1c) was investigated in the stem Leydig cells (SLCs) and progenitor Leydig cells (PLCs) in mice. The roles of p57KIP2 in the proliferation, differentiation, apoptosis, and steroidogenesis in SLCs and PLCs were investigated by antibodies and bromodeoxyuridine (BrdU) labeling in the early neonatal testes and p57kip2 siRNA in the isolated SLCs and PLCs. Steroidogenic differentiation of PLCs was examined by progesterone and testosterone production in cell culture. From postnatal day (PND) 1 to 14, p57KIP2(+) spindle-shaped cells in the testis interstitium were α-smooth muscle actin (αSMA)(-), a peritubular myoid cells marker, suggesting that they are SLCs and PLCs. Besides, p57KIP2 was also expressed in HSD3β(+) fetal Leydig cells. From PND1 to 14, BrdU(+)/αSMA(-), Ki67(+)/p57KIP2(+), and BrdU(+)/p57KIP2(+) spindle-shaped cells were gradually decreased. From PND1 to 14, p57KIP in the αSMA(-)/p57KIP2(+) cells was peaked at PND7 and decreased thereafter. In THY1(+) isolated SLCs, p57kip2 siRNA significantly increased ki67 and pcna mRNA and pdgfrα mRNA, a differentiation marker and decreased nestin mRNA, a SLC marker. No significant difference in apoptosis related genes mRNA was found after p57kip2 siRNA treatment. In HSD3β(+) PLCs, p57kip2 siRNA increased proapoptotic genes mRNA, annexin V(+) early-apoptotic cells. Importantly, p57kip2 siRNA significantly decreased hsd3β6 and cyp17a1 mRNA and progesterone production. p57KIP2 may suppress proliferation and support stemness of SLCs. In PLCs, p57KIP2 may suppress apoptosis and potentiate the steroidogenic differentiation.

Isoform-independent promotion of contractility and proliferation, and suppression of survival by with no lysine/K kinases in prostate stromal cells.

With no lysine/K kinases (WNKs) promote vasocontraction and vascular smooth muscle cell proliferation. In the prostate, smooth muscle contraction and growth may be critical for the development and medical treatment of voiding symptoms in benign prostatic hyperplasia. Here, we examined the effects of isoform-specific WNK silencing and of the WNK inhibitor WNK463 on growth-related functions and contraction in prostate stromal cells, and in human prostate tissues. Impacts of WNK silencing by transfection of cultured stromal cells with isoform-specific siRNAs were qualitatively and quantitatively similar for each WNK isoform. Effects of silencing were largest on cell death (3-5 fold increase in annexin V-positive/7-AAD-positive cells), on proliferation rate, Ki-67 mRNA expression and actin organization (reduced around two-thirds). Contraction in matrix contraction assays and viability were reduced to a lower degree (approximately half), but again to a similar extent for each WNK isoform. Effects of silencing were quantitatively and qualitatively reproduced by 10 μM WNK463, while 1 μM still induced cell death and breakdown in actin organization, without affecting proliferation or viability. Using 500 nM and 10 μM, WNK463 partly inhibited neurogenic and U46619-induced contractions of human prostate tissues (around half), while inhibition of α1-adrenergic contractions (around half) was limited to 10 μM. All four WNK isoforms suppress cell death and promote proliferation in prostate stromal cells. WNK-driven contraction of stromal cells appears possible, even though to a limited extent. Outcomes of isoform-specific WNK silencing can be fully reproduced by WNK463, including inhibition of smooth muscle contraction in human prostate tissues, but require high concentrations.

MiRNA and mRNA-Controlled Double-Cascaded Amplifying Circuit Nanosensor for Accurate Discrimination of Breast Cancers in Living Cells, Animals, and Organoids.

Metastasis is the leading cause of death in patients with breast cancer. Detecting high-risk breast cancer, including micrometastasis, at an early stage is vital for customizing the right and efficient therapies. In this study, we propose an enzyme-free isothermal cascade amplification-based DNA logic circuit in situ biomineralization nanosensor, HDNAzyme@ZIF-8, for simultaneous imaging of multidimensional biomarkers in live cells. Taking miR-21 and Ki-67 mRNA as the dual detection targets achieved sensitive logic operations and molecular recognition through the cascade hybridization chain reaction and DNAzyme. The HDNAzyme@ZIF-8 nanosensor has the ability to accurately differentiate breast cancer cells and their subtypes by comparing their relative fluorescence intensities. Of note, our nanosensor can also achieve visualization within breast cancer organoids, faithfully recapitulating the functional characteristics of parental tumor. Overall, the combination of these techniques offers a universal strategy for detecting cancers with high sensitivity and holds vast potential in clinical cancer diagnosis.

Effect of beta-cypermethrin on the reproductive capacity of female mice in advanced age

The aim of the present study was to investigate whether exposure to pesticides beta-cypermethrin (β-CYP) harms the reproductive capacity of advanced-age female mice. The results evidenced that peri-implantation β-CYP exposure significantly reduced the number of fetuses per advanced-age female in the first litter, and the number and weight of implantation sites. The levels of decidualization markers were significantly reduced in β-CYP-administered advanced-age mice. Lower expression of Pcna, Cdk6, Foxo1, Ki67, and p62 protein and mRNA was found in the decidua of β-CYP-treated advanced-age mice. The levels of Bax, cleaved caspase-3, Lc3a/b, Atg, mTOR, and p-mTOR protein, and the ratio of p-mTOR/mTOR protein expression were clearly downregulated by peri-implantation β-CYP exposure. These results indicated that peri-implantation β-CYP exposure may elevate the decline in reproductive capacity of early pregnant mice in advanced age.

Inhibition of growth and contraction in human prostate stromal cells by silencing of NUAK1 and -2, and by the presumed NUAK inhibitors HTH01-015 and WZ4003.

Background: NUAKs promote myosin light chain phosphorlyation, actin organization, proliferation and suppression of cell death in non-muscle cells, which are critical for smooth muscle contraction and growth. In benign prostatic hyperplasia (BPH), contraction and growth in the prostate drive urethral obstruction and voiding symptoms. However, a role of NUAKs in smooth muscle contraction or prostate functions are unknown. Here, we examined effects of NUAK silencing and the presumed NUAK inhibitors, HTH01-015 and WZ4003 on contraction and growth-related functions in prostate stromal cells (WPMY-1) and in human prostate tissues. Methods: Effects of NUAK1 and -2 silencing, HTH01-015 and WZ4003 on matrix plug contraction, proliferation (EdU assay, Ki-67 mRNA), apoptosis and cell death (flowcytometry), viability (CCK-8) and actin organization (phalloidin staining) were examined in cultured WPMY-1 cells. Effects of HTH01-015 and WZ4003 on smooth muscle contraction were assessed in organ bath experirments with human prostate tissues. Results: Effects of silencing were most pronounced on proliferation and cell death, resulting in decreases of proliferation rate by 60% and 70% by silencing of NUAK1 and NUAK2 (compared to scramble siRNA-transfected controls), decreases in Ki-67 by 75% and 77%, while numbers of dead cells after silencing of NUAK1 and NUAK2 amounted to 2.8 and 4.9 fold of scramble-transfected controls. Silencing of each isoform was paralleled by reduced viability, breakdown in actin polymerization, and partial decreases in contractility (maximally 45% by NUAK1 silencing, 58% by NUAK2 silencing). Effects of silencing were mimicked by HTH01-015 and WZ4003, with numbers of dead cells amounting up to 16.1 fold or 7.8 fold with HTH01-015 or WZ4003, compared to solvent-treated controls. Using concentrations of 500nM, neurogenic contractions of prostate tissues were inhibited partly by HTH01-015 and U46619-induced contractions were inhibited partly by HTH01-015 and WZ4003, while α1-adrenergic and endothelin-1-induced contractions remained unaffected. Using 10μM, inhibition of endothelin-1-induced contractions by both inhibitors and inhibition of α1-adrenergic contractions by HTH01-015 added to effects seen by 500nM. Conclusion: NUAK1 and -2 suppress cell death and promote proliferation in prostate stromal cells. A role in stromal hyperplasia appears possible in BPH. Effects of NUAK silencing are mimicked by HTH01-015 and WZ4003.

PD02-01 NUAK1 AND -2 PROMOTE CONTRACTION, PROLIFERATION AND SUPPRESSION OF CELL DEATH IN HUMAN PROSTATE STROMAL CELLS: EVIDENCE FROM ISOFORM-SPECIFIC SILENCING

You have accessJournal of UrologyCME1 Apr 2023PD02-01 NUAK1 AND -2 PROMOTE CONTRACTION, PROLIFERATION AND SUPPRESSION OF CELL DEATH IN HUMAN PROSTATE STROMAL CELLS: EVIDENCE FROM ISOFORM-SPECIFIC SILENCING Yuhan Liu, Ru Huang, Ruixiao Wang, Alexander Tamalunas, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Yuhan LiuYuhan Liu More articles by this author , Ru HuangRu Huang More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In benign prostatic hyperplasia (BPH), contraction and growth in the prostate contribute to urethral obstruction and voiding symptoms. NUAK1 and -2 are serine/threonine kinases and promote myosin light chain phosphorlyation, actin organization, proliferation and suppression of cell death in non-muscle cells, which are critical for smooth muscle contraction and growth. Here, we examined effects of NUAK silencing on contraction and growth-related functions of human prostate stromal cells (WPMY-1). METHODS: Effects of NUAK1 and -2 silencing on matrix plug contraction, proliferation (EdU, Ki-67 mRNA), apoptosis and cell death (flowcytometry), viability (CCK-8) and actin organization (phalloidin) were examined in cultured WPMY-1 cells. Effects of NUAK inhibitors were examined in matrix contraction and in colony formation assays. RESULTS: Silencing of NUAK1 and -2 by transfection of WPMY-1 cells with isoform-specific siRNAs was confirmed by RT-PCR. Effects of silencing were most pronounced on proliferation and cell death. Proliferation rates were decreased by 60% and 70% by silencing of NUAK1 and NUAK2 (compared to scramble siRNA-transfected controls, 48 h after transfection), paralleled by decreases in Ki-67 mRNA by 75% and 77%. Numbers of dead cells amounted to 2.8 fold of scramble-transfected controls after silencing of NUAK1, and 4.9 fold of scramble controls after NUAK2 silencing, while no effect on apoptosis was observed. Viability was reduced by 31% and 49%, respectively, 24 h after transfection with NUAK1 and NUAK2 siRNA, compared to scramble controls. Polymerized actin was decreased by 66% and 70% by silencing of NUAK1 and -2. Time-dependent contractions in matrix contraction assays were reduced up to 45% by silencing of NUAK1, and up to 58% by silencing of NUAK2. The presumed NUAK inhibitors HTH01-015 and WZ4003 (both 10 µM) reduced contractions of WPMY-1 cells by up to 54% and 57%, and reduced the number of colonies in colony formation assays up to 78% and 64%, respectively. CONCLUSIONS: NUAK1 and -2 suppress cell death, and promote proliferation and contraction in prostate stromal cells. A role of NUAKs for BPH-relevant prostate functions driving voiding symptoms appears possible. NUAK inhibitors mimic effects of silencing on contraction, and inhibit the growth of stromal cells. Source of Funding: Deutsche Forschungsgemeinschaft (DFG), grant HE 5825/9-1; China Scholarship Council (CSC), grant 201908440478. Conflicts of Interest: Nothing to disclose © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e69 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuhan Liu More articles by this author Ru Huang More articles by this author Ruixiao Wang More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Temporal changes of genes associated with intestinal homeostasis in broiler chickens following a single infection with Eimeria acervulina

Infection with the protozoan parasite Eimeria can cause the economically devastating disease coccidiosis, which is characterized by gross tissue damage and inflammation resulting in blunted villi and altered intestinal homeostasis. Male broiler chickens at 21 d of age were given a single challenge with Eimeria acervulina. Temporal changes in intestinal morphology and gene expression were investigated at 0, 3, 5, 7, 10, and 14 d postinfection (dpi). There were increased crypt depths for chickens infected with E. acervulina starting at 3 dpi and continuing to 14 dpi. At 5 and 7 dpi, infected chickens had decreased Mucin2 (Muc2), and Avian beta defensin (AvBD) 6 mRNA at 5 and 7 dpi and decreased AvBD10 mRNA at 7 dpi compared to uninfected chickens. Liver-enriched antimicrobial peptide 2 (LEAP2) mRNA was decreased at 3, 5, 7, and 14 dpi compared to uninfected chickens. After 7 dpi, there was increased Collagen 3a1 and Notch 1 mRNA compared to uninfected chickens. Marker of proliferation Ki67 mRNA was increased in infected chickens from 3 to 10 dpi. In addition, the presence of E. acervulina was visualized by in situ hybridization (ISH) with an E. acervulina sporozoite surface antigen (Ea-SAG) probe. In E. acervulina infected chickens, Ea-SAG mRNA was only detectable on 5 and 7 dpi by both ISH and qPCR. To further investigate the site of E. acervulina infection, Ea-SAG and Muc2 probes were examined on serial sections. The Muc2 ISH signal was decreased in regions where the Ea-SAG ISH signal was present, suggesting that the decrease in Muc2 by qPCR may be caused by the loss of Muc2 in the localized regions where the E. acervulina had invaded the tissue. Eimeria acervulina appears to manipulate host cells by decreasing their defensive capabilities and thereby allows the infection to propagate freely. Following infection, the intestinal cells upregulate genes that may support regeneration of damaged intestinal tissue.

Profiling intestinal stem and proliferative cells in the small intestine of broiler chickens via in situ hybridization during the peri-hatch period

Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5, and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.

The antineoplastic potential of crotoxin isolated from Crotalus durissus terrificus snake venom on oral squamous cell carcinoma

This study investigated the antineoplastic effects of crotoxin isolated from snake venom of the South American Crotalus durissus terrificus in oral cancer cell lines and in an animal model of chemically induced oral cancer. We analyzed cell viability and death, clonogenic formation, DNA fragmentation, migration assay, and gene expression of MMP2, MMP9, COL1A1, and CASP3. In the animal model, after induction of oral cancer by 4-nitroquinoline-1-oxide carcinogen, mice were treated with crotoxin to investigate its effects on tumor development in tongue and oral mucosa. Crotoxin inhibited cell proliferation, viability, colony formation, and migration, favoring cell death. Furthermore, crotoxin increased caspase-3 expression, decreased Ki-67 protein and mRNA expression of MMP2, MMP9, and COL1A1. Mice treated with crotoxin at 10 μg/kg did not alter biochemical parameters total cholesterol, very-low-density lipoprotein, high-density lipoprotein, liver transaminases, glycemia, creatinine, and urea. Crotoxin treatment significantly reduced the frequency of oral squamous cell carcinoma lesions by 50%. Thus, this study highlights crotoxin as a promising chemotherapeutic substance, considering its effects on controlling the neoplastic cell population, reducing cell migration, and inhibiting tumor development. Clinical studies are necessary to understand better the impact of crotoxin as a potential adjuvant therapeutic agent for oral cancer patients.

PSUN134 Loss of Glp-1r Signaling in the β-Cell Impairs Adaptive Proliferation and Insulin Secretion

Abstract Type 2 diabetes mellitus is characterized by insulin resistance and loss of pancreatic b-cell mass. b-cells are located in the pancreatic islets and secrete insulin. Though many therapies exist that address insulin resistance and to augment b-cell insulin secretion, there is a critical need to find therapies to preserve b-cell mass in order to decrease the incidence and severity of diabetes. In non-diabetic obesity, an early adaptive response to insulin resistance is increased b-cell proliferation. This compensatory mechanism leads to increased b-cell mass and increased insulin secretion from b-cells. This increase in b-cell mass is seen in as little as 4 days of high fat diet (HFD) feeding in murine models. A promising therapeutic for the preservation of b-cell mass is glucagon-like peptide-1 receptor (Glp-1r) agonists. The ability of GLP-1 to stimulate b-cell proliferation and inhibit apoptosis is largely based on studies using pharmacologic treatment, but the importance of GLP-1 in b-cell mass regulation in normal physiology or pathophysiology has not been well studied. Notably, GLP-1 is secreted from alpha cells in the local islet environment and this paracrine signaling pathway is important for islet function. The goal of this work is to investigate the contribution of b-cell Glp-1r signaling to b-cell mass regulation in the metabolic stress condition of a one-week HFD. We hypothesize that b-cell Glp-1r signaling is necessary for the compensatory mechanisms needed to maintain glucose homeostasis during metabolic stress conditions and that lack of b-cell Glp-1r will lead to decreased proliferation of b-cells. Understanding the role of β-cell Glp-1r signaling in adaptive b-cell mass expansion will allow for development of new strategies to augment β-cell mass in type 2 diabetes. We used a newly generated murine model with a b-cell specific knockout of Glp-1r, where the Ins1-Cre knock-in transgene drives recombination in b-cells. Glp-1r fl/fl mice (WT) and Glp-1r fl/fl – Ins1Cre mice (KO) were fed a HFD for one week. There was a trend toward elevated fasting (171+/-29 mg/dl vs 205+/-36 mg/dl, p=0.0475, n=9) and non-fasting blood glucose (195+/-24 mg/dl vs 216+/-40 mg/dl, p=0.0537, n=18) and impaired glucose tolerance in the KO mice. Notably, we found that insulin secretion in response to intraperitoneal glucose is impaired in KO mice. KO mice have a blunted proliferation response to a 1-week HFD stress, as measured by Ki67 mRNA levels (1.5-fold induction of Ki67 in WT vs. 0.6-fold induction in KO, p=0.0167, n=8-10). b-cell proliferation will also be measured by immunofluorescent image analysis of whole pancreas sections. These data suggest there is a critical role for b-cell Glp-1r signaling in the early proliferative response of b-cells in response to metabolic stress. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

RANKL down-regulates the mast cell proliferation through inducing senescence.

An increase in the number of mast cells could contribute to inflammatory diseases and pathologic conditions. A receptor activator of NF-κB ligand (RANKL)/RANK system is one of the key signaling pathways accelerating mast cell-mediated allergic inflammatory reactions. However, the biological functions of RANKL in mast cell proliferation remains to be clarified. The aim of the present study is to clarify the role of RANKL in mast cell proliferation. Surprisingly, RANKL remarkably reduced the proliferation of human mast cell line, HMC-1 cells through the inhibition of murine double minute 2 (MDM2) and Ki-67 mRNA expressions in a dose-dependent manner. RANKL significantly reduced cell viability, whereas it increased cellular senescence via increasing levels of p53, phosphorylated(p)-p53, p21, and p16 and decreasing levels of retinoblastoma protein (pRb) and p-pRb in HMC-1 cells. Even in rat peritoneal mast cells, RANKL induced cellular senescence by increasing filamentous-actin polymerization. In addition, RANKL remarkably reduced thymic stromal lymphopoietin (TSLP)-induced mast cell proliferation via the downregulation of MDM2 and Ki-67. RANKL decreased levels of p-signal transducer and activator of transcription 6 in TSLP-stimulated HMC-1 cells. The mast cell growth factor, interleukin-13 was remarkably down-regulated by treatment with RANKL in TSLP-stimulated HMC-1 cells. Furthermore, RANKL increased the number of senescence-associated β-galactosidase-stained cells and protein levels of p53, p-p53, and p21 in TSLP-stimulated HMC-1 cells. These data suggest that RANKL down-regulates mast cell proliferation by inducing senescence.

Individual Variabilities in Adipose Stem Cell Proliferation, Gene Expression and Responses to Lipopolysaccharide Stimulation.

Adipose stem cells (ASCs) are reported to play a role in normal physiology as well as in inflammation and disease. The objective of this work was to elucidate inter-individual differences in growth, gene expression and response to inflammatory stimuli in ASCs from different donors. Human ASC1 (male donor) and ASC2 (female donor) were purchased from Lonza (Walkersville, MD). Cell proliferation was determined by the sulforhodamine B assay. After time-dependent treatment of ASCs with or without bacterial lipopolysaccharide (LPS), marker gene mRNAs for proliferation, steroid hormones, and xenobiotic and immune pathways were determined using RT-PCR, and secreted cytokine levels in media were measured using the Bio-Plex cytokine assay kit. ASCs from both donors expressed androgen receptors but not estrogen receptors. ASC2 had a 2-fold higher proliferation rate and a 6-fold higher level of proliferation marker Ki67 mRNA than ASC1. ASC2 exhibited significantly greater fold induction of TNF-α and CCL2 by LPS compared to ASC1. TNF-α and GM-CSF protein levels were also significantly higher in the LPS-induced ASC2 media, but IL-6 secretion was higher in the LPS-induced ASC1 media. Our findings suggest that inter-individual variability and/or possible sex differences exist in ASCs, which may serve as a key determinant to inflammatory responses of ASCs.

Characterization of Migratory Cells From Bioengineered Bovine Cartilage in a 3D Co-culture Model

Background: Chondrocyte migration in native cartilage is limited and has been implicated as one of the reasons for the poor integration of native implants. Through use of an in vitro integration model, it has previously been shown that cells from bioengineered cartilage can migrate into the native host cartilage during integration. Platelet-rich plasma (PRP) treatment further enhanced integration of bioengineered cartilage to native cartilage in vitro. However, it is not known how PRP treatment of the bioengineered construct promotes this. Hypothesis: PRP supports cell migration from bioengineered cartilage and these migratory cells have the ability to accumulate cartilage-like matrix. Study Design: Controlled laboratory study. Methods: Osteochondral-like constructs were generated by culturing primary bovine chondrocytes on the top surface of a porous bone substitute biomaterial composed of calcium polyphosphate. After 1 week in culture, the constructs were submerged in PRP and placed adjacent, but 2 mm distant, to a native bovine osteochondral plug in a co-culture model for 2 weeks. Cell migration was monitored using phase-contrast imaging. Cell phenotype was determined by evaluating the gene expression of matrix metalloprotease 13 (MMP-13), Ki67, and cartilage matrix molecules using quantitative polymerase chain reaction. When tissue formed, it was assessed by histology, immunohistochemistry, and quantification of matrix content. Results: PRP treatment resulted in the formation of a fiber network connecting the bioengineered cartilage and native osteochondral plug. Cells from both the bioengineered cartilage and the native osteochondral tissue migrated onto the PRP fibers and formed a tissue bridge after 2 weeks of culture. Migratory cells on the tissue bridge expressed higher levels of collagen types II and I (COL2, COL1), Ki67 and MMP-13 mRNA compared with nonmigratory cells in the bioengineered cartilage. Ki67 and MMP-13–positive cells were found on the edges of the tissue bridge. The tissue bridge accumulated COL1 and COL2 and aggrecan and contained comparable collagen and glycosaminoglycan content to the bioengineered cartilage matrix. The tissue bridge did not reliably develop in the absence of cells from the native osteochondral plug. Conclusion: Bioengineered cartilage formed by bovine chondrocytes contains cells that can migrate on PRP fibers and form cartilaginous tissue. Clinical Relevance: Migratory cells from bioengineered cartilage may promote cartilage integration. Further studies are required to determine the role of migratory cells in integration in vivo.

Sortilin 1 Promotes Hepatocellular Carcinoma Cell Proliferation and Migration by Regulating Immune Cell Infiltration.

Objectives Recent evidence suggests that Sort1 promotes carcinogenesis and tumor progression in multiple types of cancers. This study investigates the role of Sort1 in hepatocellular carcinoma (HCC). Methods The differentially expressed gene was screened through GEO and TCGA databases. The Sort1 gene was identified and its expression was then verified by TCGA and HCCDB (a database of hepatocellular carcinoma expression atlas) databases. The Human Protein Atlas database was used to assess the gene expression in tissues. The TCGA and KM-plotter databases were used to study the relationship between Sort1 and HCC. The correlation between Sort1 and immune cells was evaluated through the TIMER database. GO and KEGG enrichment analysis was used to investigate the possible mechanism. The role of Sort1 in cell proliferation and invasion of HCC was further explored through in vitro experiments. Result The differentially expressed molecule obtained from database screening was Sort1. Its expression was higher in cancer tissues than in paracancerous ones, and it was mainly located in the cytoplasm. The TCGA, KM-plotter databases, and our study data showed that low expression of Sort1 in HCC patients had better overall survival (OS), progression-free survival (PFI), and disease-specific survival (DSS). Further analysis indicated a significant correlation between Sort1 expression and immune cell infiltration. The gene set enrichment analysis (GSEA) analysis showed that Sort1 affected the biological events of HCC by participating in the WNT, TGF-BETA, JAK, STAT, and CALCIUM signaling pathways. In vitro, cytological experiments demonstrated reduced expression of PCNA, Ki-67, Vimentin, N-cadherin, and MMP-9 mRNA after knocking down Sort1, although E-cadherin expression was promoted. Overall, these processes reduced the ability of proliferation and invasion of HCC cells. Conclusion Downregulation of Sort1 can prolong the OS, PFI, and DSS of HCC patients. Furthermore, due to its link with immune cell infiltration, the Sort1 gene represents a potentially novel predictive biomarker of HCC. The growth of HCC can be significantly inhibited by interfering with Sort1; therefore, these results provide a potential target for developing anticancer strategies for HCC.

Runting Stunting Syndrome in Broiler Chickens Is Associated with Altered Intestinal Stem Cell Morphology and Gene Expression.

Runting stunting syndrome (RSS) in broiler chickens is characterized by altered intestinal morphology and gene expression and stunted growth. The objective of this study was to conduct a retrospective study of gene expression in stem and differentiated cells in the small intestine of RSS chicks. Two different models of RSS were analyzed: broiler chicks that were experimentally infected and broiler chicks that were naturally infected. Experimentally infected chicks were exposed to litter from infected flocks (RSS-litter chicks) or infected with astrovirus (RSS-astrovirus chicks). Intestinal samples from naturally infected chicks showing clinical signs of RSS were acquired from commercial farms in Georgia and were brought into a poultry diagnostic lab (RSS-clinical-GA) and from farms in Brazil that had a history of RSS (RSS-clinical-BR). The RSS-clinical-BR chicks were separated into those that were positive or negative for gallivirus based on DNA sequencing. Intestinal morphology and intestinal cell type were identified in archived formalin-fixed, paraffin-embedded tissues. In situ hybridization for cell-specific mRNA was used to identify intestinal stem cells expressing olfactomedin 4 (Olfm4), proliferating cells expressing Ki67, absorptive cells expressing sodium glucose cotransporter 1 (SGLT1) and peptide transporter 1 (PepT1), and goblet cells expressing mucin 2 (Muc2). RSS-litter and RSS-clinical-GA chicks showed 4% to 7.5% cystic crypts, while gallivirus-positive RSS-clinical-BR chicks showed 11.7% cystic crypts. RSS-astrovirus and gallivirus-negative RSS-clinical-BR chicks showed few cystic crypts. RSS-litter and gallivirus-positive RSS-clinical-BR chicks showed an increase in crypt depth compared to control or gallivirus-negative chicks, respectively. There was no expression of Olfm4 mRNA in the stem cells of RSS-litter and RSS-clinical-GA chicks, in contrast to the normal expression of Olfm4 mRNA in RSS-astrovirus and RSS-clinical-BR chicks. All chicks regardless of infection status showed normal expression of Ki67 mRNA in crypt cells, Muc2 mRNA in goblet cells, and SGLT1 or PepT1 mRNA in enterocytes. These results demonstrate that RSS, which can be induced by different etiologies, can show differences in the expression of the stem cell marker Olfm4.

Therapeutic effect of Thymoquinone on behavioural response to UCMS and neuroinflammation in hippocampus and amygdala in BALB/c mice model.

Major depressive disorder is the leading cause of disability worldwide. The corticolimbic system plays a critical role in the emotional and cognitive aspects of major depressive disorder. Owing to the unsatisfactory efficacy of conventional antidepressants, there is a need to explore novel therapies. The current study aimed to explore the antidepressant potential of thymoquinone, a natural compound with anti-inflammatory activity, and propose its underlying mechanism of action in the unpredictable chronic mild stress (UCMS) mouse model. Coat state, forced swim test, elevated plus maze test, novelty suppressed feeding test and social interaction test were performed to quantify the behavioural shift induced by UCMS and the effect of thymoquinone and fluoxetine treatment. In addition, messenger RNA (mRNA) expression levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and BDNF and NeuN were analysed by a quantitative real-time polymerase chain reaction in the hippocampus and amygdala of experimental and control groups. UCMS significantly deteriorated coat state. Thymoquinone reinstated the resignation behaviour and latency to feed affected by UCMS. UCMS induced an increase in inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the hippocampus and amygdala, which was decreased by thymoquinone. UCMS caused an increase in BDNF and NeuN mRNA levels in the amygdala while a decrease in the hippocampus. This opposite effect on BDNF was also compensated by thymoquinone; however, thymoquinone did not significantly change Ki67 and NeuN mRNA levels in the hippocampus. Thymoquinone restored the behavioural changes induced by UCMS. In addition, the antidepressant effect of thymoquinone is in line with changes in inflammatory parameters and changes in BDNF in the hippocampus and amygdala.

Can we use Ki67 expression to predict prostate cancer aggressiveness?

specialists have an urge for biomarkers that can discriminate indolent prostate cancer from aggressive tumors. Ki67 is a proliferation marker, and its expression is associated with the aggressiveness of several cancers. analyze the expression of Ki67 in prostate cancer samples correlating with the aggressiveness of the disease. Ki67 mRNA levels were determined utilizing data from a TCGA cohort (Tumor(n)=492 and control(n)=52). The protein expression was determined on 94 biopsies from patients by immunohistochemical assay. in mRNA, the Ki67 upregulation is associated with cancer tissue (p<0.0001) and worst disease-free survival (p=0.035). The protein upregulation is associated with increase of the ISUP score (p<0.0001), cancer stage (p=0.05), biochemical recurrence (p=0.0006) and metastasis (p<0.0001). We also show a positive correlation between Ki67 expression and ISUP score (r=0.5112, p<0.0001) and disease risk stratification (r=0.3388, p=0.0009). Ki67 expression is a factor independently associated with biochemical recurrence (p=0.002) and metastasis (p<0.0001). Finally, the patients with high Ki67expression shows better survival regarding biochemical recurrence (p=0.008) and metastasis (p=0.056). Patients with high Ki67 expression are 2.62 times more likely to develop biochemical recurrence (p=0.036). Ki67 upregulation is associated with prostate cancer aggressiveness.