Cyst Cells Research Articles

Circadian Clock Disruption and Growth of Kidney Cysts in Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, and often progresses to kidney failure. ADPKD progression is not uniform among patients, suggesting that factors secondary to the PKD1/2 gene mutation could regulate the rate of disease progression. Here we tested the effect of circadian clock disruption on ADPKD progression. Circadian rhythms are regulated by cell-autonomous circadian clocks composed of clock proteins. BMAL1 is a core constituent of the circadian clock. To disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1RC/RC (RC/RC) mouse model of ADPKD (RC/RC;Bmal1f/f;Pkhd1cre, called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells (Pkd1Bmal1KO mIMCD3 cells). Only male mice were used. Human nephrectomy ADPKD kidneys showed altered clock gene expression when compared to normal control human kidneys. When compared to RC/RC kidneys, DKO kidneys showed significantly altered clock gene expression, increased cyst growth, cell proliferation, apoptosis and fibrosis. DKO kidneys also showed increased lipogenesis and cholesterol synthesis-related gene expression, and increased tissue triglyceride levels compared to RC/RC kidneys. Similarly, in vitro, Pkd1Bmal1KO cells showed altered clock genes, increased lipogenesis and cholesterol synthesis-related genes, and reduced fatty-acid oxidation-related gene expression compared to Pkd1KO cells. The Pkd1Bmal1KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. Renal collecting duct specific Bmal1 gene deletion disrupted the circadian clock and triggered accelerated ADPKD progression by altering lipid metabolism-related gene expression.

Tumor suppressor miR-317 and lncRNA Peony are expressed from a polycistronic non-coding RNA locus that regulates germline differentiation and testis morphology.

This research focuses on investigating the impact of non-coding RNAs on stem cell biology and differentiation processes. We found that miR-317 plays a role in germline stem cell progeny differentiation. miR-317 and its neighbor, the lncRNA Peony, originate and are co-expressed from a singular polycistronic non-coding RNA locus. Alternative polyadenylation is implicated in regulation of their differential expression. While the increased expression of the lncRNA Peony results in the disruption of the muscle sheath covering the testis, the absence of miR-317 leads to the emergence of germline tumors in young flies. The deficiency of miR-317 increases Notch signaling activity in the somatic cyst cells, which drives germline tumorigenesis. Germline tumors also arise from upregulation of several predicted targets of miR-317 , among which are regulators of the Notch pathway. This implicates miR-317 as a novel tumor suppressor that modulates Notch signaling strength.

Soma-to-germline BMP signal is essential for Drosophila spermiogenesis

In the Drosophila testis, developing germ cells are encapsulated by somatic support cells throughout development. Soma-germline interactions are essential for successful spermiogenesis. However, it is still not fully understood what signaling events take place between the soma and the germline. In this study, we found that a Bone Morphogenetic Protein (BMP) ligand, Glass bottom boat (Gbb), secreted from somatic cyst cells (CCs), signals to differentiating germ cells to maintain proper spermiogenesis. Knockdown of Gbb in CCs or the type I BMP receptor Saxophone (Sax) in germ cells leads to a defect in sperm head bundling and decreased fertility. Our Transmission Electron Microscopy (TEM) analyses revealed that the mutant germ cells have aberrant morphology of mitochondria throughout the stages of spermiogenesis and exhibit a defect in nebenkern formation. Elongating spermatids show uncoupled nuclei and elongating mitochondrial derivatives, suggesting that improper mitochondrial development may cause sperm bundling defects. Taken together, we propose a new role of soma-derived BMP signaling, which is essential for spermiogenesis.

Flagella, palmella and cyst Haematococcus lacustris microalgae cells decorated on graphene oxide and graphene nanoplatelets-activated carbon as novel adsorbents for the removal of lead from water

Industrialization has led to generation of large quantities of waste which constitutes various toxic heavy metals such as lead (Pb). In this work, novel bio-nanostructured graphene-based microalgae nanohybrid adsorbents, using three different cell types of Haematococcus lacustris (i.e., flagella (flg-C), palmella (Pal-C) and cyst (Cyst-C)) to introduce more functional moieties and enhance the surface properties of the nanohybrids. The nanostructured graphene oxide-activated carbon modified with algae cells (GO-AC@algae) and graphene nanoplatelets-activated carbon modified with algae cells (GNPs-AC@algae) nanohybrids were characterized and used for the removal of Pb ions. The GO-AC@algae nanohybrids demonstrated a high lead removal efficiency of over 98.0%, whereas the GNPs-AC@algae nanohybrids achieved more than 85.0%. Among the GO-AC@algae nanohybrids, the nanohybrid with cyst cell (GO-AC@Cyst-C) shown remarkable efficacy as an adsorbent for the removal of Pb2+ ions from aqueous solutions due to its high specific area, abundance of oxygen-nitrogen-based functional moieties, hydrophilicity, and pore structure. Chemisorption was found to be a beneficial process for both GO-AC@algae and GNPs-AC@algae samples, where Pb2+ was adsorbed in a single layer onto the uniform material surface. Among the various adsorbents, GO-AC@Cyst-C achieved the highest monolayer adsorption capacity of 25.58 mg/g according to the Langmuir model, making it the most effective nanoadsorbents. Kinetic studies revealed that the sorption mechanism of GO-AC@algae were better described by the second-order kinetic model. Meanwhile, the first-order kinetic model was found to be suited for GNPs-AC@algae samples. The nanohybrids could be employed as greener adsorbents at industrial scale for wastewater treatment without incurring significant costs.

Quercetin ameliorates oxidative stress-induced apoptosis of granulosa cells in dairy cow follicular cysts by activating autophagy via the SIRT1/ROS/AMPK signaling pathway

BackgroundFollicular cysts contribute significantly to reproductive loss in high-yield dairy cows. This results from the death of follicular granulosa cells (GCs) caused by oxidative stress. Quercetin is known to have significant antioxidant and anti-apoptotic effects. However, the effect of quercetin on follicular cysts has yet been elucidated. Therefore, this study aimed to explore the anti-oxidant and anti-apoptosis effects and potential molecular mechanisms of quercetin in H2O2-induced primary cow GCs and 3-nitropropionic acid (3-NPA)-induced mouse model of oxidative stress and thus treat ovarian cysts in dairy cows.ResultsIn this study, compared with estrus cows, cows with follicular cysts showed heightened levels of oxidative stress and increased follicular cell apoptosis, while autophagy levels were reduced. A model of oxidative stress was induced in vitro by H2O2 and showed significant increases in apoptosis together with reduced autophagy. These effects were significantly ameliorated by quercetin. Effects similar to those of quercetin were observed after treatment of cells with the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC). Further investigations using chloroquine (autophagy inhibitor), rapamycin (autophagy activator), selisistat (SIRT1 inhibitor), and compound C (AMPK inhibitor) showed that chloroquine counteracted the effects of quercetin on oxidative stress-induced apoptosis, while rapamycin had the same effect as quercetin. In addition, the SIRT1/AMPK pathway inhibitors antagonized quercetin-mediated mitigation of the effects of oxidative stress on increased apoptosis and reduced autophagy. Consistent with the results in vitro, in mouse ovarian oxidative stress model induced by 3-NPA, quercetin activated autophagy through the SIRT1/AMPK signaling pathway, while alleviating oxidative stress damage and inhibiting apoptosis in mouse ovaries.ConclusionsThese findings indicate that quercetin can inhibit apoptosis in GCs and restore ovarian function by activating autophagy through the SIRT1/ROS/AMPK signaling pathway, suggesting a new direction for the treatment of ovarian follicular cysts in high-yield dairy cows.

Circadian clock disruption and growth of kidney cysts in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, and often progresses to kidney failure. ADPKD progression is not uniform among patients, suggesting that factors secondary to the PKD1/2 gene mutation could regulate the rate of disease progression. Here we tested the effect of circadian clock disruption on ADPKD progression. Circadian rhythms are regulated by cell-autonomous circadian clocks composed of clock proteins. BMAL1 is a core constituent of the circadian clock. To disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1 RC/RC (RC/RC) mouse model of ADPKD (RC/RC; Bmal1 f/f ; Pkhd1 cre , called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells ( Pkd1Bmal1 KO mIMCD3 cells). Only male mice were used. Human nephrectomy ADPKD kidneys and Pkd1 KO mIMCD3 cells showed reduced Bmal1 gene expression compared to normal controls. When compared to RC/RC kidneys, DKO kidneys showed significantly altered clock gene expression, increased cyst growth, cell proliferation, apoptosis and fibrosis. DKO kidneys also showed increased lipogenesis and cholesterol synthesis-related gene expression, and increased tissue triglyceride levels compared to RC/RC kidneys. Similarly, in vitro, Pkd1Bmal1 KO cells showed altered clock genes, increased lipogenesis and cholesterol synthesis-related genes, and reduced fatty-acid oxidation-related gene expression compared to Pkd1KO cells. The Pkd1Bmal1 KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. Renal collecting duct specific Bmal1 gene deletion disrupts the circadian clock and triggers accelerated ADPKD progression by altering lipid metabolism-related gene expression. Lack of BMAL1, a circadian clock protein in renal collecting ducts disrupted the clock and increased cyst growth and fibrosis in an ADPKD mouse model.BMAL1 gene deletion increased cell proliferation by increasing lipogenesis in kidney cells.Thus, circadian clock disruption could be a risk factor for accelerated disease progression in patients with ADPKD.

HL156A, an AMP-Activated Protein Kinase Activator, Inhibits Cyst Growth in Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic kidney disorder. While metformin has demonstrated the ability to inhibit cyst growth in animal models of ADPKD via activation of adenosine monophosphate-activated protein kinase (AMPK), its effectiveness in humans is limited due to its low potency. This study explored the impact of HL156A, a new and more potent AMPK activator, in a mouse model of ADPKD. To investigate whether HL156A inhibits the proliferation of renal cyst cells in ADPKD in vitro, exogenous human telomerase reverse transcriptase (hTERT)-immortalized renal cyst cells from ADPKD patients were treated with HL156A, and an MTT (dimethylthiazol-diphenyltetrazolium bromide) assay was performed. To assess the cyst-inhibitory effect of HL156A in vivo, we generated Pkd1 conditional knockout (KO) mice with aquaporin 2 (AQP2)-Cre, which selectively expresses Cre recombinase in the collecting duct. The effectiveness of HL156A in inhibiting cyst growth and improving renal function was confirmed by measuring the number of cysts and blood urea nitrogen (BUN) levels in the collecting duct-specific Pkd1 KO mice. When cyst cells were treated with up to 20 µM of metformin or HL156A, HL156A reduced cell viability by 25% starting at a concentration of 5 µM, whereas metformin showed no effect. When AQP2-Cre male mice were crossed with Pkd1flox/flox female mice, and when AQP2-Cre female mice were crossed with Pkd1flox/flox male mice, the number of litters produced by both groups was comparable. In collecting duct-specific Pkd1 KO mice, HL156A was found to inhibit cyst growth, reducing both the number and size of cysts. Furthermore, it was confirmed that kidney function improved as HL156A treatment led to a reduction in elevated BUN levels. Lastly, it was observed that the increase in AMPK phosphorylation induced by HL156A decreased ERK phosphorylation and α-SMA expression. HL156A has potential as a drug that can restore kidney function in ADPKD patients by inhibiting cyst growth.

Expression of Fascin and SALL4 in odontogenic cysts and tumors: an immunohistochemical appraisal.

Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Expression of Fascin and SALL4 in odontogenic cysts and tumors: an immunohistochemical appraisal.

Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

L(1)10Bb serves as a conservative determinant for soma-germline communications via cellular non-autonomous effects within the testicular stem cell niche

The testicular stem cell niche is the central regulator of spermatogenesis in Drosophila melanogaster. However, the underlying regulatory mechanisms are unclear. This study demonstrated the crucial role of lethal (1) 10Bb [l(1)10Bb] in regulating the testicular stem cell niche. Dysfunction of l(1)10Bb in early-stage cyst cells led to male fertility disorders and compromised cyst stem cell maintenance. Moreover, the dysfunction of l(1)10Bb in early-stage cyst cells exerted non-autonomous effects on germline stem cell differentiation, independently of hub signals. Notably, our study highlights the rescue of testicular defects through ectopic expression of L(1)10Bb and the human homologous protein BUD31 homolog (BUD31). In addition, l(1)10Bb dysfunction in early-stage cyst cells downregulated the expression of spliceosome subunits in the Sm and the precursor RNA processing complexes. Collectively, our findings established l(1)10Bb as a pivotal factor in the modulation of Drosophila soma-germline communications within the testicular stem cell niche.

Identification of Early Transcriptional Markers of Autosomal Dominant Polycystic Kidney Disease Cystic Epithelial Cells

Study objective: Mechanisms driving autosomal dominant polycystic kidney disease (ADPKD) cystogenesis, a hallmark of this lethal monogenic disease, are not well understood and reliable biomarkers are scarce. We sought to explore transcriptional changes in ADPKD models with suppressed and non-suppressed cystic phenotype to explore mechanisms driving ADPKD cystogenesis and identify potential biomarkers in disease progression. Hypothesis: Early-stage kidneys from a model in which cystic disease is suppressed by expression of the polycystin-1 C-terminal tail (CTT), a 200 amino acid portion of the larger polycystin-1 protein that is dysregulated in ADPKD, exhibit a differential transcriptional profile from early-stage kidneys in a non-suppressed model. Methodology: Orthologous doxycycline-inducible Pkd1fl/fl;Pax8rtTA;Teto-Cre ADPKD C57BL/6N mouse models expressing CTT ( Pkd1-KO+CTT) or not ( Pkd1-KO) were induced between 4-6 weeks and aged to 10 weeks. We performed single cell RNA sequencing (scRNA-seq) with single cell kidney suspensions from biological replicates that were pooled and processed for cDNA synthesis and library preparation with 10X Chromium technology. Reads were aligned using the 10X Genomics CellRanger pipeline and further processed using the gene cell-inverse cell frequency (gf-icf) pipeline. Data: Clusters corresponding to tubule segments were identified using established annotations. Several novel clusters defined by expression of multiple markers, including Jarid2, Tmtc2, and Filip1l, were identified in the Pkd1-KO model. These clusters were sparsely populated in Pkd1-KO+CTT samples, suggesting that they may correspond to cyst epithelial cells that are suppressed by CTT expression. Each of these novel clusters included cells whose markers identified them from distinct tubule segments (e.g. Sglt2, Umod, S100g), suggesting that cells of different segments are clustered together by virtue of sharing the putative cyst cell transcriptional signature. Importantly, at 10 weeks there are no significant differences in disease endpoints such as kidney weight to body weight, serum creatinine, blood urea nitrogen, or kidney morphology between the Pkd1-KO+CTT and Pkd1-KO models, suggesting that the cells assigned to the cystic epithelial cell clusters exhibit early identifiable differences in transcriptional programming. Summary of results: scRNA-seq at an early timepoint reveals novel transcriptional makers of putative cystic epithelial cells. The protein expression levels of individual markers are being assessed. Conclusions: These results suggest that there are epithelial cells early in disease progression exhibiting a unique transcriptional signature that may elucidate the mechanisms of cyst formation and that may provide new candidate biomarkers. This work was funded in part by the NIH F31 DK135356 (VR), NIH RC2 DK120534-01 (MJC), PR191158 Department of Defense (MJC), and PKD 905682 Foundation for Research in Polycystic Kidney Disease Research Grant (MJC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis.

Metabolism participates in the control of stem cell function and subsequent maintenance of tissue homeostasis. How this is achieved in the context of adult stem cell niches in coordination with other local and intrinsic signaling cues is not completely understood. The Target of Rapamycin (TOR) pathway is a master regulator of metabolism and plays essential roles in stem cell maintenance and differentiation. In the Drosophila male germline, mTORC1 is active in germline stem cells (GSCs) and early germ cells. Targeted RNAi-mediated downregulation of mTor in early germ cells causes a block and/or a delay in differentiation, resulting in an accumulation of germ cells with GSC-like features. These early germ cells also contain unusually large and dysfunctional autolysosomes. In addition, downregulation of mTor in adult male GSCs and early germ cells causes non-autonomous activation of mTORC1 in neighboring cyst cells, which correlates with a disruption in the coordination of germline and somatic differentiation. Our study identifies a previously uncharacterized role of the TOR pathway in regulating male germline differentiation.

Integrin restriction by miR-34 protects germline progenitors from cell death during aging.

During aging, regenerative tissues must dynamically balance the two opposing processes of proliferation and cell death. While many microRNAs are differentially expressed during aging, their roles as dynamic regulators of tissue regeneration have yet to be described. We show that in the highly regenerative Drosophila testis, miR-34 levels are significantly elevated during aging. miR-34 modulates germ cell death and protects the progenitor germ cells from accelerated aging. However, miR-34 is not expressed in the progenitors themselves but rather in neighboring cyst cells that kill the progenitors. Transcriptomics followed by functional analysis revealed that during aging, miR-34 modifies integrin signaling by limiting the levels of the heterodimeric integrin receptor αPS2 and βPS subunits. In addition, we found that in cyst cells, this heterodimer is essential for inducing phagoptosis and degradation of the progenitor germ cells. Together, these data suggest that the miR-34-integrin signaling axis acts as a sensor of progenitor germ cell death to extend progenitor functionality during aging.

A transcriptomics-based RNAi screen for regulators of meiosis and early stages of oocyte development in Drosophila melanogaster.

A properly regulated series of developmental and meiotic events must occur to ensure the successful production of gametes. In Drosophila melanogaster ovaries, these early developmental and meiotic events include the production of the 16-cell cyst, meiotic entry, synaptonemal complex (SC) formation, recombination, and oocyte specification. In order to identify additional genes involved in early oocyte development and meiosis, we reanalyzed 3 published single-cell RNA-seq datasets from Drosophila ovaries, using vasa (germline) together with c(3)G, cona, and corolla (SC) as markers. Our analysis generated a list of 2,743 co-expressed genes. Many known SC-related and early oocyte development genes fell within the top 500 genes on this list, as ranked by the abundance and specificity of each gene's expression across individual analyses. We tested 526 available RNAi lines containing shRNA constructs in germline-compatible vectors representing 331 of the top 500 genes. We assessed targeted ovaries for SC formation and maintenance, oocyte specification, cyst development, and double-strand break dynamics. Six uncharacterized genes exhibited early developmental defects. SC and developmental defects were observed for additional genes not well characterized in the early ovary. Interestingly, in some lines with developmental delays, meiotic events could still be completed once oocyte specificity occurred indicating plasticity in meiotic timing. These data indicate that a transcriptomics approach can be used to identify genes involved in functions in a specific cell type in the Drosophila ovary.

A synthetic agent ameliorates polycystic kidney disease by promoting apoptosis of cystic cells through increased oxidative stress

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of chronic kidney disease and the fourth leading cause of end-stage kidney disease, accounting for over 50% of prevalent cases requiring renal replacement therapy. There is a pressing need for improved therapy for ADPKD. Recent insights into the pathophysiology of ADPKD revealed that cyst cells undergo metabolic changes that up-regulate aerobic glycolysis in lieu of mitochondrial respiration for energy production, a process that ostensibly fuels their increased proliferation. The present work leverages this metabolic disruption as a way to selectively target cyst cells for apoptosis. This small-molecule therapeutic strategy utilizes 11beta-dichloro, a repurposed DNA-damaging anti-tumor agent that induces apoptosis by exacerbating mitochondrial oxidative stress. Here, we demonstrate that 11beta-dichloro is effective in delaying cyst growth and its associated inflammatory and fibrotic events, thus preserving kidney function in perinatal and adult mouse models of ADPKD. In both models, the cyst cells with homozygous inactivation of Pkd1 show enhanced oxidative stress following treatment with 11beta-dichloro and undergo apoptosis. Co-administration of the antioxidant vitamin E negated the therapeutic benefit of 11beta-dichloro in vivo, supporting the conclusion that oxidative stress is a key component of the mechanism of action. As a preclinical development primer, we also synthesized and tested an 11beta-dichloro derivative that cannot directly alkylate DNA, while retaining pro-oxidant features. This derivative nonetheless maintains excellent anti-cystic properties in vivo and emerges as the lead candidate for development.

Integrated transcriptomic and metabolomic investigation of the genes and metabolites involved in swine follicular cyst formation.

Follicular cysts are a common reproductive disorder in mammals that is usually caused by stress. However, the pathogenesis of follicular cysts in sows remains unclear. To provide new insights into the mechanisms of follicular cyst formation in pigs, we conducted a combined transcriptomic and metabolomic analysis on theca interna and mural granulosa cells of follicular cysts and mature follicles. We identified 2,533 up-regulated and 1,355 down-regulated genes in follicular cysts, compared with mature follicles. These differentially expressed genes were mainly found in signaling pathways related to tumor formation and cortisol synthesis and secretion as shown by Ingenuity Pathway Analysis, which predicted 4,362 upstream regulatory factors. The combined gene expression and pathway analysis identified the following genes as potential biomarkers for porcine follicular cysts: cytochrome P450 family 2 subfamily C polypeptide 18, L-lactate dehydrogenase, carbamoyl-phosphate synthase, fibroblast growth factor 7, integrin binding sialoprotein, interleukin 23 receptor, prolactin receptor, epiregulin, interleukin 1 receptor type II, arginine vasopressin receptor 1A, fibroblast growth factor 10, claudin 7, G Protein Subunit Gamma 3, cholecystokinin B receptor and cytosolic phospholipase A2. Metabolomics analysis found significant differences in 87 metabolites, which were enriched in unsaturated fatty acid biosynthesis, and sphingolipid signaling pathways. These results provide valuable information on the molecular mechanisms of follicular cyst formation, which may facilitate the development of new therapeutics to prevent and treat follicular cysts.

The IL-1β-p65 axis stimulates quiescent odontogenic epithelial cell rests via TGF-β signalling to promote cell proliferation of the lining epithelia in radicular cysts: A laboratory investigation.

Cyst formation of the jaws is frequently accompanied by the proliferation of odontogenic epithelial cells located in the periodontal ligament (PDL), which consists of heterozygous cells and includes the most fibroblasts. The lining epithelium of radicular cyst, an odontogenic cyst of inflammatory origin, is derived from the proliferation of the remnants of the Hertwig epithelial root sheath (odontogenic epithelial cell rests of Malassez; ERMs) in the PDL. ERMs are maintained at a lower proliferative state under physiological conditions, but the regulatory mechanisms underlying the inflammation-dependent enhanced-proliferative capabilities of ERMs are not fully understood. The aim of this study was to evaluate the effects of cytokine pathway association between TGF-β signalling and IL-1β signalling on the regulation of odontogenic epithelial cell proliferation using radicular cyst pathological specimens and odontogenic epithelial cell lines. Immunofluorescence analyses were performed to clarify the expression levels of Smad2/3 and Ki-67 in ERMs of 8-week-old mouse molar specimens. In radicular cyst (n = 52) and dentigerous cysts (n = 6) specimens from human patients, the expression of p65 (a main subunit of NF-κB), Smad2/3 and Ki-67 were investigated using immunohistochemical analyses. Odontogenic epithelial cells and PDL fibroblastic cells were co-cultured with or without an inhibitor or siRNAs. Odontogenic epithelial cells were cultured with or without TGF-β1 and IL-1β. The proliferative capabilities and Smad2 phosphorylation levels of odontogenic epithelial cells were examined. Immunohistochemically, Smad2/3-positivity was increased, and p65-positivity and Ki-67-positivity were decreased both in ERMs and in the epithelial cells in dentigerous cysts, a non-inflammatory developmental cyst. In contrast, p65-positive cells, along with the expression of Ki-67, were increased and Smad2/3-positive cells were decreased in the lining epithelia of radicular cysts. Co-culture experiments with odontogenic epithelial cells and PDL fibroblastic cells revealed that PDL cells-derived TGF-β1/2 and their downstream signalling suppressed odontogenic epithelial cell proliferation. Moreover, TGF-β1 stimulation induced Smad2 phosphorylation and suppressed odontogenic epithelial cell proliferation, while IL-1β stimulation reversed these phenotypes through p65 transactivation. These results suggest that IL-1β-p65 signalling promotes odontogenic epithelial cell proliferation through suppressing TGF-β-Smad2 signalling, which would be involved in the pathogenesis of radicular cysts.

Pathology of Spinal Tumours

Abstract Spinal tumours are uncommon lesions, but they may cause significant morbidity and mortality. Majority of the spinal tumours are metastatic, that is, spread from tumours in other organs, which are mostly seen in older patients. Primary sarcomas involving the spinal column are rare. Benign tumours and tumour-like lesions of the musculoskeletal system are mostly seen in young patients. Spinal tumours can be subdivided into primary tumours, which are rare, and secondary tumours or metastases, which are much common. Primary vertebral tumours may be benign or malignant. Common benign spinal tumours include osteoid osteoma, osteoblastoma, osteochondroma, aneurysmal bone cyst and giant cell tumour. There are many malignant primary spinal tumours such as- osteosarcoma, chondrosarcoma, and chordoma, Ewing’s sarcoma, plasmacytoma and multiple myeloma. However, all of them, except multiple myeloma, are rare. Intraspinal tumours may be extradural or intradural. Common intradural extramedullary tumours include meningioma, schwannoma and intradural neurofibroma, whereas astrocytoma, ependymoma and haemangioblastoma are a few examples of intradural intramedullary tumours.

Expression of Fascin and SALL4 in odontogenic cysts and tumors: an immunohistochemical appraisal.

Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Diagnostic Test of Transforming Growth Factor-Beta 1 (TGF-β1) in Menstrual Blood with Endometriosis.

Endometriosis is a benign disorder that is generally defined as the presence of endometrial glands and stroma outside their normal location. TGF-β1 is found in stromal cells and its expression is increased in epithelial cells of endometriotic cysts. Endometriosis diagnostics take a long time, so new markers are needed to diagnose endometriosis. This study aims to determine the diagnostic value of TGF-β1 in menstrual blood in diagnosing endometriosis. Diagnostic tests to compare eutopic endometrial TGF-β1 levels from menstrual blood of patients with suspected endometriosis were undertaken in the Obstetrics and Gynecology Department of Dr. Mohammad Hoesin General Hospital, Faculty of Medicine, Sriwijaya University, Palembang, from July 2019 to November 2020. 50 patients who were suspected with endometriosis met the inclusion criteria. Comparison of TGF-β1 levels between endometriosis and nonendometriosis patients was analyzed using the Mann-Whitney test. The cutoff point of the TGF-β1 level towards the histopathological outcome was obtained using the ROC curve. Data analysis was performed by using SPSS version 22.0. In this study, endometriosis patients were 31.6 ± 6.55 years of age with a range of 20 to 46 years. In statistical analysis, there was no difference in BMI (p = 0.181) and BMI classification (p = 0.207), the history of contraception (p = 0.097), infertility (p = 1.000), and dysmenorrhoea (p = 1.000) between endometriosis and nonendometriosis patients. In the study, there were differences in TGF-β1 between endometriosis and nonendometriosis patients (p ≤ 0.001). By using the ROC curve, the cutoff point for TGF-β1 levels has the best sensitivity and specificity, which is 515 ng/ml. The TGF-β1 level has a sensitivity of 80%, a specificity of 90%, a positive predictive value (PPV) of 0.969, a negative predictive value (NPV) of 0.529, a positive likelihood ratio of 8, a negative likelihood ratio of 0.222, and an accuracy of 0.820 to the endometriosis outcome. It can be concluded that the TGF-β1 level has a very good diagnostic value in establishing endometriosis diagnostics. This trial is registered with ISRCTN72218532.