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Bistable biological regulatory systems need to cope with stochastic noise to fine tune their function close to bifurcation points. Here, we study stability properties of this regime in generic systems to demonstrate that cooperative interactions buffer system variability, hampering noise-induced regime shifts. Our analysis also shows that, in the considered cooperativity range, impending regime shifts can be generically detected by statistical early warning signals from distributional data. Our generic framework, based on minimal models, can be used to extract robustness and variability properties of more complex models and empirical data close to criticality. Our generic framework, based on minimal models, can be used to extract robustness and variability properties of more complex models and empirical data close to criticality.

In most human primary cancers, the expression, or telomerase activity, of telomerase reverse transcriptase (TERT) is detectable. However, the mechanism ofTERTactivity within oncogenesis of thyroid cancer remains largely unknown. In this study, we identified miR-195-5p as having involvement in cell proliferation, apoptosis, and invasion in human thyroid cancer. MTT was used to measure cell proliferation, Transwell chamber was used to measure invasion. Western blotting was used to detect the expressions of TERT, PCNA, and Ki67. Target gene prediction software predicted that TERT may be the target gene of miR-195-5p. Luciferase reporting system was used to identify the targeting relationship. A significant increase of in TERT expression was observed by immunohistochemistry compared with normal tissue, however, a decrease in miR-195-5p expression using qRT-PCRand western blot compared with normal cells. Functional analysis demonstrates that miR-195-5p negatively correlated withTERTand inhibitedTERTexpression through its interaction with theTERT3ʹ-untranslatedregion (3ʹ-UTR). Overexpression of miR-195-5p was shown to inhibit proliferation and invasion, and promote apoptosis of CAL-62 thyroid cancer cells. miR-195-5p-mediatedeffects were rescued by the overexpression ofTERT. Altogether, our data demonstrate that miR-195-5p regulates cell proliferation, apoptosis, and invasion in human thyroid cancer viaTERT, providing evidence of a new potential therapeutic target for further investigation.

Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes’ functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells’ culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we established a new ovarian cell line from the young Asian yellow pond turtle (Mauremys mutica), which exhibited a normal diploid karyotype with high alkaline phosphatase activity. The cell line, designated as YTO2, was then characterized through the analysis of gene expression profiles. The transcriptome analysis and the reverse transcription polymerase chain reaction (RT-PCR) showed that the cells expressed germline genes such as tdrd7, nanos1, klf5, igtb1, hsd17b4 and rad51. Moreover, the immunostaining showed that the germ cell markers, Tdrd7 and Rad51 proteins, were detected predominant in cytoplasm of perinuclear region, while proliferation marker, PCNA, was dominantly observed in the nuclei of cultured cells. Intriguingly, the cells could respond to the retinoic acid induction with significantly increasing the expression level of some meiosis genes, including vasa, dazl, figla, and dmc1. Furthermore, YTO2 cells could be efficiently transfected with the pHBAd-BHG-EGFP adenovirus and properly expressed the exogenous genes. To sum up, an ovarian cell line of the Asian yellow pond turtle had been established and could be stably propagated under in vitro culture condition, as well as being capable of efficiently expressing the exogenous gene tdrd7. This cell line would provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle, even in reptiles.

MicroRNA-107 (miR-107) plays a regulatory role in obesity and insulin resistance, but the mechanisms of its function in adipocytes have not been elucidated in detail. Here we show that overexpression of miR-107 in pre- and mature human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes attenuates differentiation and lipid accumulation. Our results suggest that miR-107 controls adipocyte differentiation via CDK6 and Notch signaling. CDK6 is a validated target of miR-107 and was downregulated upon miR-107 overexpression. Notch3, a signaling receptor involved in adipocyte differentiation, has been shown to decrease upon CDK6 depletion; Here Notch3 and its target Hes1 were downregulated by miR-107 overexpression. In mature adipocytes, miR-107 induces a triglyceride storage defect by impairing glucose uptake and triglyceride synthesis. To conclude, our data suggests that miR-107 has distinct functional roles in preadipocytes and mature adipocytes; Its elevated expression at these different stages of adipocytes may on one hand dampen adipogenesis, and on the other, promote ectopic fatty acid accumulation and reduced glucose tolerance.

NOTCH1 is a well-established lineage specifier for T cells and among the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology, allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of preleukemic cells to full-blown disease.

Numerous sophisticated diagnostic techniques have been designed to monitor electrode-electrolyte interfaces that mainly govern the lifetime and reliability of batteries. Among them is the electrochemical quartz crystal microbalance (EQCM) that offers valuable insights of the interfaces once the required conditions of the deposited film in terms of viscoelastic and hydrodynamic properties are fulfilled. Herein, we propose a friendly protocol that includes the elaboration of a homogeneous deposit by spray coating followed by QCM measurements at multiharmonic frequencies to ensure the film flatness and rigidity for collecting meaningful data. Moreover, for easiness of the measurements, we report the design of a versatile and airtight EQCM cell setup that can be used either with aqueous or non-aqueous electrolytes. We also present, using a model battery material, LiFePO4, how dual frequency and motional resistance monitoring during electrochemical cycling can be used as a well-suitable indicator for achieving reliable and reproducible electrogravimetric measurements. We demonstrate through this study the essential role of the solvent assisting lithium-ion insertion at the LiFePO4 interface with a major outcome of solvent-dependent interfacial behavior. Namely, in aqueous media, we prove a near-surface desolvation of lithium ions from their water solvation shell as compared with organic molecules. This spatial dissimilarity leads to a smoother Li-ion transport across the LFP-H2O interface, hence accounting for the difference in rate capability of LFP in the respective electrolytes. Overall, we hope our analytical insights on interfacial mechanisms will help in gaining a wider acceptance of EQCM-based methods from the battery community.

As an essential and fundamental process of life, cell development involves large‐scale reorganization of the 3D genome architecture, which forms the basis of gene regulation. Here, a landscape‐switching model is developed to explore the microscopic chromosomal structural origin of embryonic stem cell (ESC) differentiation and somatic cell reprogramming. It is shown that chromosome structure exhibits significant compartment‐switching in the unit of topologically associating domain. It is found that the chromosome during differentiation undergoes monotonic compaction with spatial repositioning of active and inactive chromosomal loci toward the chromosome surface and interior, respectively. In contrast, an overexpanded chromosome, which exhibits universal localization of loci at the chromosomal surface with erasing the structural characteristics formed in the somatic cells, is observed during reprogramming. An early distinct differentiation pathway from the ESC to the terminally differentiated cell, giving rise to early bifurcation on the Waddington landscape for the ESC differentiation is suggested. The theoretical model herein including the non‐equilibrium effects, draws a picture of the highly irreversible cell differentiation and reprogramming processes, in line with the experiments. The predictions provide a physical understanding of cell differentiation and reprogramming from the chromosomal structural and dynamical perspective and can be tested by future experiments., Chromosomes are the structural scaffolds for gene functions, which determine the cell developmental process. A non‐equilibrium landscape model is developed to identify the chromosomal structural and dynamical origins of cell differentiation and reprogramming. The irreversible pathways in differentiation and reprogramming show distinct dynamical rearrangements for the chromosome structure to facilitate the formation of specific gene expression patterns and associated functions.

Proliferating cells must coordinate central metabolism with the cell cycle. How central energy metabolism regulates bacterial cell cycle functions is not well understood. Our forward genetic selection unearthed the Krebs cycle enzyme citrate synthase (CitA) as a checkpoint regulator controlling the G1→S transition in the polarized alpha-proteobacterium Caulobactercrescentus, a model for cell cycle regulation and asymmetric cell division. We find that loss of CitA promotes the accumulation of active CtrA, an essential cell cycle transcriptional regulator that maintains cells in G1-phase, provided that the (p)ppGpp alarmone is present. The enzymatic activity of CitA is dispensable for CtrA control and functional citrate synthase paralogs cannot replace CitA in promoting S-phase entry. Our evidence suggests that CitA was appropriated specifically to function as a moonlighting enzyme to link central energy metabolism with S-phase entry. Control of the G1-phase with a central metabolic enzyme may be a common mechanism of cellular regulation.

Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks post-conception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD191 B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form > 40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% +/- 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid-restricted progenitor in human fetal life. Although fetalBMPreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid-restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.

Recent studies have identified CD49a+Eomes− and CD49a+Eomes+ subsets of tissue-resident NK (trNK) cells in different organs of the mouse. However, the characteristics of CD49a+Eomes−/+ NK cell development and the regulation of Eomes expression in NK cells remain unclear. Here, we established an in vitro cytokine-based feeder-free system in which bone marrow progenitor cells differentiate into CD49a+ NK cells. IL-15 was identified as being the key cytokine in this system that supported the development and maintenance of CD49a+ NK cells. The CD49a+ NK cells generated were Eomes−CD49b− and shared the same phenotype as hepatic trNK cells. IL-4 induced the expression of Eomes in generated NK cells and converted them into CD49a+Eomes+ cells, which were phenotypically and functionally similar to uterine trNK cells. Moreover, the IL-4/STAT6 axis was identified as being important in the generation of CD49a+Eomes+ induced NK cells. Collectively, these studies describe an approach to generate CD49a+Eomes−/+ subsets of NK cells and demonstrate important roles for IL-15 and IL-4 in the differentiation of these cells. These findings have potential for developmental research underlying the generation of different subsets of NK cells and the application of adoptive NK cell transfer therapies.

A 7-bp deletion in the Cd4 gene, present in the strain MOLF/Ei of Mus musculus molossinus and absent in laboratory mouse strains (Mus musculus musculus), provided the means to distinguish the parental origin of the Cd4 alleles expressed in single cells of F-1 (AKR x MOLF/Ei) and F-1 (Balb/C x MOLF/Ei) hybrids. Single-cell RT-PCR showed that the individual CD4(+) lymphocyte expresses either the maternal or the paternal Cd4 allele, never both. In situ hybridization proved that Cd4 alleles replicate asynchronously, as expected in the case of genes expressed monoallelically.

Epithelial-mesenchymal transition of tubular epithelial cells in human renal biopsies. Background In recent studies performed on cultured cells and experimental nephropathies, it has been hypothesized that tubular epithelial cells (TEC), via epithelial-mesenchymal transformation (EMT), can become collagen-producing cells. According to this theory, they should proceed through several activating steps, such as proliferation and phenotype changes, to eventually synthesize extracellular matrix (ECM). Methods To evaluate whether EMT operates in human TECs, 133 renal biopsies of different renal diseases were studied, analyzing by immunohistochemistry and in situ hybridization the possible expression of markers of proliferation (PCNA, Mib-1), cellular phenotype (vimentin, α-SMA, cytokeratin, ZO-1) and ECM production (prolyl 4-hydroxylase, HSP47, interstitial collagens). Results Independently of histological diagnosis, variable degrees of TEC positivity for PCNA (2.7 ± 2.4 cells/field) and Mib-1 (1.9 ± 2.3) were present. TECs expressing vimentin (1.4 ± 4.7) and α-smooth muscle actin (α-SMA; 0.04 ± 0.4) also were detected. It was possible to observe loss of epithelial antigens from 8 to 10% of the tubular cross sections. Moreover, TECs were stained by prolyl 4-hydroxylase (3.6 ± 4.3), heat shock protein-47 (HSP47; 2.9 ± 5.4), collagen type I (0.2 ± 2.7) and type III (0.3 ± 2.0). Collagen types I and III mRNAs were found in 0.8 to 1.4 cells/field. The number of TEC with EMT features were associated with serum creatinine and the degree of interstitial damage (P≤ 0.03), and even considering the 45 cases with mild interstitial lesions, the tubular expression of all markers remained strictly associated with renal function (P≤ 0.01). Conclusions Our results suggest that, via transition to a mesenchymal phenotype, TEC can produce ECM proteins in human disease and directly intervene in the fibrotic processes. Moreover, the association of EMT features with serum creatinine supports the value of these markers in the assessment of disease severity.

Although autophagic pathways are essential to developmental processes, many questions still remain regarding the initiation signals that regulate autophagy in the context of differentiation. To address these questions we studied the ocular lens, as the programmed elimination of nuclei and organelles occurs in a precisely regulated spatiotemporal manner to form the organelle-free zone (OFZ), a characteristic essential for vision acuity. Here, we report our discovery that inactivation of MAPK/JNK induces autophagy for formation of the OFZ through its regulation of MTORC1, where MAPK/JNK signaling is required for both MTOR activation and RPTOR/RAPTOR phosphorylation. Autophagy pathway proteins including ULK1, BECN1/Beclin 1, and MAP1LC3B2/LC3B-II were upregulated in the presence of inhibitors to either MAPK/JNK or MTOR, inducing autophagic loss of organelles to form the OFZ. These results reveal that MAPK/JNK is a positive regulator of MTORC1 signaling and its developmentally regulated inactivation provides an inducing signal for the coordinated autophagic removal of nuclei and organelles required for lens function.

Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains. Background The glomerular basement membrane (GBM) originates in development from fusion of subendothelial and subepithelial matrices. Subsequently, newly synthesized subepithelial matrix is added as glomerular capillary loops expand. During GBM assembly, the laminin-1 heterotrimer (α1, β;1, and γ1 chains), initially expressed in vascular clefts of comma- and S-shaped bodies, is eventually replaced by laminin-11 (α5, β;2, and γ1 chains), which persists into maturation. The cellular source(s) of these laminins is not known and prompted this study. Methods To determine which cells synthesize the various laminin chains, postfixation immunoelectron microscopy of developing mouse kidney was performed using monoclonal and polyclonal antibodies that specifically recognized laminin α1, β;1, α5, or β;2 chains. Results Intracellular labeling for laminin α1, β;1 (laminin-1), and α5 and β;2 (laminin-11) chains was observed in developing glomerular endothelial cells and podocytes of comma- and S-shaped nephric figures. Laminin-1 was also seen in unfused GBMs at this stage, whereas laminin-11 was only found intracellularly. In capillary loop stage GBMs, laminin α1 chain was completely absent, whereas labeling for laminin α5 was intense, indicating rapid substitution between α chains. In contrast, laminin β;1 chain labeling remained strong both intracellularly and in GBMs of capillary loop stage glomeruli, and β;2 was up-regulated as well. In maturing stage glomeruli, β;1 labeling declined, and α5 and β;2 remained at high levels intracellularly in both endothelial cells and podocytes and in GBMs. Conclusions Our results show that both endothelial cells and podocytes synthesize laminin-1 and -11 chains throughout glomerular development. The sustained and comparatively high level of laminin synthesis by endothelial cells was unexpected, suggesting that the endothelium may be an important source of GBM proteins in glomerular disease.

Distinct structural forms of type I collagen modulate cell cycle regulatory proteins in mesangial cells. Background Extracellular matrix molecules profoundly regulate cell behavior, including proliferation. In glomerulonephritis, type I collagen accumulates in the mesangium and is constantly structurally modified and degraded during the course of the disease. Methods We studied how two structurally distinct forms of type I collagen, monomer versus polymerized fibrils, affect cell proliferation, mitogen-activated protein kinase (MAPK) activation, and expression of G 1 -phase regulatory proteins in cultured rat mesangial cells (MCs). To analyze the possible involvement of collagen-binding integrins in type I collagen-derived growth signals further, distribution patterns of integrin chains were examined by immunocytochemistry. Results Polymerized type I collagen completely prevented the increase of DNA synthesis and cell replication induced by 5% fetal calf serum (FCS) or 25 ng/mL platelet-derived growth factor (PDGF) in MCs on monomer type I collagen. Protein expression of cyclins D1 and E was markedly down-regulated in MCs plated on polymerized type I collagen for eight hours in 5% FCS, as compared with MCs on monomer type I collagen. Incubation with 5% FCS reduced expression of the cdk-inhibitor protein p27 Kip1 on monomer but not on polymerized type I collagen. Moreover, polymerized type I collagen markedly reduced cyclin E-associated kinase activity in the presence of 5% FCS. Polymerized type I collagen diminished the PDGF-induced phosphorylation and nuclear translocation of p42/p44 MAPK, but did not affect phosphorylation of PDGF β-receptors. In MCs plated on monomer type I collagen, α 1 , α 2 , and β 1 integrin chains were recruited into focal contacts. However, on polymerized type I collagen, α 2 and β 1 , but not α 1 , integrin chains were condensed into focal contacts. Conclusions The growth-inhibitory effect of polymerized type I collagen is characterized by rapid changes of expression and/or activation of MAPK and G 1 -phase regulators and could result from the lack of α 1 β 1 integrin signaling in MCs on polymerized type I collagen. Conceivably, deposition of polymerized type I collagen might reflect a reparative response to control MC replication in glomerular inflammation.

The use of monoclonal antibodies for specific pectic epitopes is an important tool in the study of the cell wall. Throughout the development of mucilage cells of Araucaria angustifolia (Bertol.) Kuntze, a gradient of distribution was observed in relation to the pectic de-esterification, as well as to the increase of galactan and arabinan epitope distribution, and to the reduction of arabinogalactans proteins (AGPs) epitope at maturity. AGP and methyl-esterified homogalacturonan (HGA) were present in the mucilage. Galactans and arabinans were also observed in the mucilage, though with weak labelling. Degradation of AGP in the maturity of mucilage cells, in cell wall, as well as in the secretion, could be involved in the programmed cell death (PCD). Different labellings found among parenchyma and mucilage cells suggested differences in the cell wall properties of the mucilage cells. A utilização de anticorpos monoclonais para epitopos pécticos específicos é uma importante ferramenta no estudo da parede celular. Ao longo do desenvolvimento das células de mucilagem de Araucaria angustifolia (Bertol.) Kuntze foi observado um gradiente de distribuição com relação à de-esterificação de pectinas, bem como um aumento na distribuição dos epitopos de galactanos e arabinanos e uma redução na distribuição de arabinogalactanos proteínas (AGPs) na maturidade. AGP e homogalacturonanos (HGA) metil-esterificados foram detectados na mucilagem. Galactanos e arabinanos foram também observados na mucilagem, embora com fraca marcação. A degradação de AGPs na maturidade das células de mucilagem, tanto na parede celular, como na secreção, poderia estar envolvida no processo de morte celular programada (MCP). Diferentes marcações encontradas entre as células de parênquima e células de mucilagem sugerem diferenças nas propriedades da parede celular das células mucilaginosas.

Ca 2+ signaling induced by sphingosylphosphorylcholine and sphingosine 1-phosphate via distinct mechanisms in rat glomerular mesangial cells. Background To elucidate the molecular mechanism underlying sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) mediated signaling, we compared their effects with those of adenosine triphosphate (ATP) and angiotensin II (Ang II) on the cytosolic free Ca 2+ concentration ([Ca 2+ ] i ), inositol 1,4,5-trisphosphate (IP 3 ) generation and arachidonic acid release in rat glomerular mesangial cells. Methods The fluorescent Ca 2+ indicator, Fura-2, was used to measure the [Ca 2+ ] i changes in cultured rat glomerular mesangial cells either in suspension or attached to the coverslips. Results SPC 5 μm, S1P 5 μm, ATP 100 μm and Ang II 90nm all induced increases in the [Ca 2+ ] i , and the effect showed marked homologous desensitization, while heterologous desensitization was less. After the initial exposure of the cells to SPC, the increase in [Ca 2+ ] i induced by subsequent addition of ATP or Ang II was only reduced by about 14.3% and 4.8%, respectively. After the initial exposure to S1P, a greater reduction was seen (42.1% and 47.7%, respectively). Both arachidonic acid release and IP 3 generation were activated by all four agonists with an identical rank order of effectiveness of SPC > S1P > ATP=Ang II; both were pertussis toxin-sensitive and cholera toxin-resistant. The arachidonic acid release induced by all four agonists showed identical susceptibility to removal of extracellular Ca 2+ , whereas IP 3 generation displayed differential extracellular Ca 2+ dependence. Only SPC-induced IP 3 generation was highly sensitive to extracellular Ca 2+ level, and this Ca 2+ dependence was abolished after pretreatment of cells with arachidonyl trifluoromethyl ketone (AACOCF 3 ), a phospholipase A 2 inhibitor. Furthermore, the Mn 2+ influx was markedly greater in SPC-stimulated cells than in either control or other agonist-stimulated cells, and was decreased by prior exposure of cells to AACOCF 3 . After phospholipase A 2 was inhibited or in the absence of extracellular Ca 2+ , SPC displayed identical effectiveness as S1P on desensitizing the action of ATP or Ang II on the increase in [Ca 2+ ] i . Conclusions Our results indicate that all four agents primarily activate phospholipase C through their receptor occupancies, but that SPC alone also induces further significant Mn 2+ influx and IP 3 generation attributable to its primary stimulatory effect on arachidonic acid release. Thus, the heterologous desensitization to ATP or Ang II induced by SPC was less profound than that induced by S1P, since SPC induced a Ca 2+ influx.

Trabajo presentado en el III International Symposium on Olive Growing, celebrado en Chania, Crete (Grecia) el 22 de septiembre de 1997., Mesocarp cell development was studied in relation to seasonal fruit growth patterns and oil accumulation for 'Picual' olive fruits under irrigated and dry-land conditions. Image analysis was used to determine cell number, cell size and mesocarp area in transverse sections from the central portion of the fruit. At flowering the mesocarp has 12% of its final cell number and less than 5% of its final size. Approximately 80% of mesocarp cells are produced in the first two months of growth, when fruit diameter growth rate is maximum, and 20% between this time and fruit maturity. Cell expansion is high throughout fruit growth, with final cell area at maturity more than double than at 2 months. Water availability affected the seasonal pattern of fruit growth.