Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120(ctn), also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.

The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E-coli strains in human urine

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human urine and show that it can outcompete a representative spectrum of UPEC strains for growth in urine. The unique ability of ABU E. coli 83972 to outcompete UPEC in urine was also demonstrated in a murine model of human UTI, confirming the selective advantage over UPEC in vivo. Comparison of global gene expression profiles of E. coli 83972 grown in lab medium and human urine revealed significant differences in expression levels in the two media; significant down-regulation of genes encoding virulence factors such as hemolysin, lipid A, and capsular pollysaccharides was observed in cells grown in urine. Clearly, divergent abilities of ABU E. coli and UPEC to exploit human urine as a niche for persistence and survival suggest that these key differences may be exploited for preventative and/or therapeutic approaches.

Analysis of Transglutaminase-2 in macrophage function

Programmed cell death, apoptosis, is a highly regulated process that removes damaged or unwanted cells in vivo and has significant immunological implications. Defective clearance of apoptotic cells by macrophages (professional phagocytes) is known to result in chronic inflammatory and autoimmune disease. Transglutaminase-2 (TG2) is a Ca2+-dependent protein crosslinking enzyme known to play an important role in apoptotic cell clearance by macrophages through an ill-defined mechanism. Several studies have implicated TG2 in the apoptosis programme e.g. raised TG2 levels in cells undergoing apoptosis; increased cell death with down-regulation of TG2; up-regulation of TG2 in monocytes upon differentiation into macrophages. Defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be elucidated. Here we aim to characterise the role of TG2 in macrophage function with a focus on apoptotic cell clearance. THP-1 monocytes were induced to differentiate to macrophage-like cells by three different stimulants and were analysed for the presence of TG2. Macrophage-apoptotic cell interaction studies in the presence and absence of irreversible TG2 inhibitors resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. TG2 was expressed at the macrophage cell surface and its association with ß3 integrin expression suggests the possible link between TG2 and ß3 integrins. Our current findings suggest that TG2 had got a crucial but yet to be defined role in apoptotic cell clearance.

Human Synaptic Plasticity Gene Expression Profile and Dendritic Spine Density Changes in HIV-Infected Human CNS Cells: Role in HIV-Associated Neurocognitive Disorders (HAND)

HIV-associated neurocognitive disorders (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occur in approximately 50% of HIV infected individuals. Our current understanding of HAND emanates mainly from HIV-1 subtype B (clade B), which is prevalent in USA and Western countries. However very little information is available on neuropathogenesis of HIV-1 subtype C (clade C) that exists in Sub-Saharan Africa and Asia. Therefore, studies to identify specific neuropathogenic mechanisms associated with HAND are worth pursuing to dissect the mechanisms underlying this modulation and to prevent HAND particularly in clade B infection. In this study, we have investigated 84 key human synaptic plasticity genes differential expression profile in clade B and clade C infected primary human astrocytes by using RT2 Profile PCR Array human Synaptic Plasticity kit. Among these, 31 and 21 synaptic genes were significantly (≥3 fold) down-regulated and 5 genes were significantly (≥3 fold) up-regulated in clade B and clade C infected cells, respectively compared to the uninfected control astrocytes. In flow-cytometry analysis, down-regulation of postsynaptic density and dendrite spine morphology regulatory proteins (ARC, NMDAR1 and GRM1) was confirmed in both clade B and C infected primary human astrocytes and SK-N-MC neuroblastoma cells. Further, spine density and dendrite morphology changes by confocal microscopic analysis indicates significantly decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC neuroblastoma cells compared to uninfected and clade C infected cells. We have also observed that, in clade B infected astrocytes, induction of apoptosis was significantly higher than in the clade C infected astrocytes. In conclusion, this study suggests that down-regulation of synaptic plasticity genes, decreased dendritic spine density and induction of apoptosis in astrocytes may contribute to the severe neuropathogenesis in clade B infection.

Rearing effect of biofloc on antioxidant and antimicrobial transcriptional response in Litopenaeus stylirostris shrimp facing an experimental sub-lethal hydrogen peroxide stress

This study compares the antioxidant and antimicrobial transcriptional expression of blue shrimps reared according to two different systems, BioFloc Technology (BFT) and Clear sea Water (CW) and their differential responses when facing an experimental sublethal hydrogen peroxide stress. After 30 days of rearing, juvenile shrimps were exposed to H2O2 stress at a concentration of 30 ppm during 6 hours. The oxidative stress caused by H2O2 was examined in the digestive glands of the shrimp, in which antioxidant enzyme (AOE) and antimicrobial peptide (AMP) gene expression were analysed by quantitative real-time PCR. Results showed that rearing conditions did not affect the expression of genes encoding AOEs or AMPs. However, H2O2 stress induced a differential response in expression between shrimps from the two rearing treatments (BFT and CW). Comparative analysis of the expression profiles indicates that catalase transcripts were significantly upregulated by H2O2 stress for BFT shrimps while no change was observed for CW shrimps. In contrast, H2O2 caused down-regulation of superoxide dismutase and glutathione transferase transcripts and of the three AMP transcripts studied (penaeidin 2 and 3, and crustin) for CW shrimps, while no effect was observed on BFT shrimp transcript levels. These results suggested that BFT shrimps maintained antioxidant and AMP responses after stress and therefore can effectively protect their cells against oxidative stress, while CW shrimp immune competence seems to decrease after stress.

Synthetic regulation of eukaryotic gene expression by noncoding RNA

Synthetic biological systems promise to combine the spectacular diversity of biological functionality with engineering principles to design new life to address many pressing needs. As these engineered systems advance in sophistication, there is ever-greater need for customizable, situation-specific expression of desired genes. However, existing gene control platforms are generally not modular, or do not display performance requirements required for robust phenotypic responses to input signals. This work expands the capabilities of eukaryotic gene control in two important directions.

For development of greater modularity, we extend the use of synthetic self-cleaving ribozyme switches to detect changes in input protein levels and convey that information into programmed gene expression in eukaryotic cells. We demonstrate both up- and down-regulation of levels of an output transgene by more than 4-fold in response to rising input protein levels, with maximal output gene expression approaching the highest levels observed in yeast. In vitro experiments demonstrate protein-dependent ribozyme activity modulation. We further demonstrate the platform in mammalian cells. Our switch devices do not depend on special input protein activity, and can be tailored to respond to any input protein to which a suitable RNA aptamer can be developed. This platform can potentially be employed to regulate the expression of any transgene or any endogenous gene by 3’ UTR replacement, allowing for more complex cell state-specific reprogramming.

We also address an important concern with ribozyme switches, and riboswitch performance in general, their dynamic range. While riboswitches have generally allowed for versatile and modular regulation, so far their dynamic ranges of output gene modulation have been modest, generally at most 10-fold. We address this shortcoming by developing a modular genetic amplifier for near-digital control of eukaryotic gene expression. We combine ribozyme switch-mediated regulation of a synthetic TF with TF-mediated regulation of an output gene. The amplifier platform allows for as much as 20-fold regulation of output gene expression in response to input signal, with maximal expression approaching the highest levels observed in yeast, yet being tunable to intermediate and lower expression levels. EC50 values are more than 4 times lower than in previously best-performing non-amplifier ribozyme switches. The system design retains the modular-input architecture of the ribozyme switch platform, and the near-digital dynamic ranges of TF-based gene control.

Together, these developments suggest great potential for the wide applicability of these platforms for better-performing eukaryotic gene regulation, and more sophisticated, customizable reprogramming of cellular activity.

Altered transcriptional and epigenetic regulation affects brain cells proliferation and differentiation in the ultra-rare genetic demyelinating disease AGC1 deficiency: a study on in vitro models

AGC1 deficiency is a rare demyelinating disease caused by mutations in the SLC25A12 gene, which encodes for the mitochondrial glutamate-aspartate carrier 1 (AGC1/Alarar), highly expressed in the central nervous system. In neurons, impairment in AGC1 activity leads to reduction in N-acetyl-aspartate, the main lipid precursor for myelin synthesis (Profilo et al., 2017); in oligodendrocytes progenitors cells, AGC1 down regulation has been related to early arrest proliferation and premature differentiation (Petralla et al., 2019). Additionally, in vivo AGC1 deficiency models i.e., heterozygous mice for AGC1 knock-out and neurospheres from their subventricular zone, respectively, showed a global decrease in cells proliferation and a switch in neural stem cells (NSCs) commitment, with specific reduction in OPCs number and increase in neural and astrocytic pools (Petralla et al., 2019). Therefore, the present study aims to investigate the transcriptional and epigenetic regulation underlying the alterations observed in OPCs and NSCs biological mechanisms, in either AGC1 deficiency models of Oli-neu cells (murine immortalized oligodendrocytes precursors cells), partially silenced by a shRNA for SLC25A12 gene, and SVZ-derived neurospheres from AGC1+/- mice. Western blot and immunofluorescence analysis revealed significant variations in the expression of transcription factors involved in brain cells’ proliferation and differentiation, in association with altered histone post-translational modifications, as well as histone acetylases (HATs) and deacetylases (HDACs) activity/expression, suggesting an improper transcriptional and epigenetic regulation affecting both AGC1 deficiency in vitro models. Furthermore, given the large role of acetylation in controlling in specific time-windows OPC maturation (Hernandez and Casaccia; 2015), pharmacological HATs/HDACs inhibitions were performed, confirming the involvement of chromatin remodelling enzymes in the altered proliferation and early differentiation observed in the AGC1 deficiency models of siAGC1 Oli-neu cells and AGC1+/- mice-derived neurospheres.

Impaired Incretin Secretion And Pancreatic Dysfunction With Older Age And Diabetes.

To estimate the impact of aging and diabetes on insulin sensitivity, beta-cell function, adipocytokines, and incretin production. Hyperglycemic clamps, arginine tests and meal tolerance tests were performed in 50 non-obese subjects to measure insulin sensitivity (IS) and insulin secretion as well as plasma levels of glucagon, GLP-1 and GIP. Patients with diabetes and healthy control subjects were divided into the following groups: middle-aged type 2 diabetes (MA-DM), aged Type 2 diabetes (A-DM) and middle-aged or aged subjects with normal glucose tolerance (MA-NGT or A-NGT). IS, as determined by the homeostasis model assessment, glucose infusion rate, and oral glucose insulin sensitivity, was reduced in the aged and DM groups compared with MA-NGT, but it was similar in the MA-DM and A-DM groups. Insulinogenic index, first and second phase insulin secretion and the disposition indices, but not insulin response to arginine, were reduced in the aged and DM groups. Postprandial glucagon production was higher in MA-DM compared to MA-NGT. Whereas the GLP-1 production was reduced in A-DM, no differences between groups were observed in GIP production. In non-obese subjects, diabetes and aging impair insulin sensitivity. Insulin production is reduced by aging, and diabetes exacerbates this condition. Aging associated defects superimposed diabetic physiopathology, particularly regarding GLP-1 production. On the other hand, the glucose-independent secretion of insulin was preserved. Knowledge of the complex relationship between aging and diabetes could support the development of physiopathological and pharmacological based therapies.

Reduced Insulin Clearance And Lower Insulin-degrading Enzyme Expression In The Liver Might Contribute To The Thrifty Phenotype Of Protein-restricted Mice.

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

Morphological Aspects And Cox-2 Expression After Exposure To 780-nm Laser Therapy In Injured Skeletal Muscle: An In Vivo Study.

The effectiveness of low-level laser therapy in muscle regeneration is still not well known. To investigate the effects of laser irradiation during muscle healing. For this purpose, 63 rats were distributed to 3 groups: non-irradiated control group (CG); group irradiated at 10 J/cm(2) (G10); and group irradiated at 50 J/cm(2) (G50). Each group was divided into 3 different subgroups (n=7), and on days 7, 14 and 21 post-injury the rats were sacrificed. Seven days post-surgery, the CG showed destroyed zones and extensive myofibrillar degeneration. For both treated groups, the necrosis area was smaller compared to the CG. On day 14 post-injury, treated groups demonstrated better tissue organization, with newly formed muscle fibers compared to the CG. On the 21(st) day, the irradiated groups showed similar patterns of tissue repair, with improved muscle structure at the site of the injury, resembling uninjured muscle tissue organization. Regarding collagen deposition, the G10 showed an increase in collagen synthesis. In the last period evaluated, both treated groups showed statistically higher values in comparison with the CG. Furthermore, laser irradiation at 10 J/cm(2) produced a down-regulation of cyclooxygenase 2 (Cox-2) immunoexpression on day 7 post-injury. Moreover, Cox-2 immunoexpression was decreased in both treated groups on day 14. Laser therapy at both fluencies stimulated muscle repair through the formation of new muscle fiber, increase in collagen synthesis, and down-regulation of Cox-2 expression.

Clinical Influence Of Different Intracanal Medications On Th1-type And Th2-type Cytokine Responses In Apical Periodontitis.

This clinical study assessed the influence of different intracanal medications on Th1-type and Th2-type cytokine responses in apical periodontitis and monitored the levels of bacteria from primarily infection during endodontic procedures. Thirty primarily infected teeth were randomly divided into 3 groups according to the medication selected: chlorhexidine (CHX), 2% CHX gel; Ca(OH)2/SSL, Ca(OH)2 + SSL; and Ca(OH)2/CHX, Ca(OH)2 + 2% CHX gel (all, n = 10). Bacterial sample was collected from root canals, and the interstitial fluid was sampled from lesions. Culture techniques were used to determine bacterial counts (colony-forming units/mL). Th1 (tumor necrosis factor-α, interferon-γ, and interleukin [IL]-2) and Th2 cytokines (IL-4, IL-5, and IL-13) were measured by enzyme-linked immunosorbent assay. All intracanal medication protocols were effective in reducing the bacterial load from root canals (all P < .05) and lowering the levels of Th1-type cytokines in apical lesions (all P < .05), with no differences between them (P > .05). Both Ca(OH)2 treatment protocols significantly increased the levels of Th2-type cytokines (P < .05), with no differences between them (P > .05). Thus, chlorhexidine medication showed the lowest effectiveness in increasing the levels of Th2-type cytokine. After treatment, regardless of the type of medication, the linear regression analysis indicated the down-regulation of Th2-type cytokines by Th1-type cytokines. All intracanal medication protocols were effective in reducing bacterial load and lowering the levels of Th1-type cytokines. Thus, the use of Ca(OH)2 medications contributed to the increase in the Th2-type cytokine response in apical periodontitis.

Cyclic nitroxides inhibit the toxicity of nitric oxide-derived oxidants: mechanisms and implications

The substantial therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) and related cyclic nitroxides as antioxidants has stimulated innumerous studies of their reactions with reactive oxygen species. In comparison, reactions of nitroxides with nitric oxide-derived oxidants have been less frequently investigated. Nevertheless, this is relevant because tempol has also been shown to protect animals from injuries associated with inflammatory conditions, which are characterized by the increased production of nitric oxide and its derived oxidants. Here, we review recent studies addressing the mechanisms by which cyclic nitroxides attenuate the toxicity of nitric oxidederived oxidants. As an example, we present data showing that tempol protects mice from acetaminophen-induced hepatotoxicity and discuss the possible protection mechanism. In view of the summarized studies, it is proposed that nitroxides attenuate tissue injury under inflammatory conditions mainly because of their ability to react rapidly with nitrogen dioxide and carbonate radical. In the process the nitroxides are oxidized to the corresponding oxammonium cation, which, in turn, can be recycled back to the nitroxides by reacting with upstream species, such as peroxynitrite and hydrogen peroxide, or with cellular reductants. An auxiliary protection mechanism may be down-regulation of inducible nitric oxide synthase expression. The possible therapeutic implications of these mechanisms are addressed.

Genomics and proteomics approaches to the study of cancer-stroma interactions

Background: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression. Methods: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells. Results: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR. Conclusions: A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

High-Density Lipoprotein Inhibits the Uptake of Modified Low-Density Lipoprotein and the Expression of CD36 and Fc gamma RI

Aim: Modified low-density lipoprotein (mLDL), mainly upon oxidative and enzymatic modification, is the major atherogenic lipoprotein. Conversely, high-density lipoprotein (HDL) is considered anti-atherogenic because of its ability to remove cholesterol. The aim of this work was to analyze both the influence of HDL on the uptake of mLDL and the expression of CD36 and Fc gamma I receptors on monocytic cell lines during cell differentiation. Methods: Uptake of fluorescein isothiocyanate (FITC)-conjugated LDL and FITC-conjugated mLDL, i.e., copper-oxidized LDL (oxLDL) or trypsin enzyme modified LDL (enzLDL), was analyzed, as well as the expression of CD36 and Fc gamma RI in THP-1 and U937 cells, using flow cytometry. Results: HDL inhibited the uptake of mLDL, which varied in degree depending on the cell line or type of mLDL. Further, HDL rapidly decreased CD36 and Fc gamma RI involved in the uptake of mLDL. Conclusions: We demonstrate that modified LDL promotes specific LDL receptor-independent uptake by monocytic cell lines, and that the uptake of LDL and enzLDL is less than that of oxLDL. In this process, HDL diminishes the uptake of LDL or mLDL, which may involve the down-regulation of receptors (CD36 and Fc gamma I). This regulatory process represents another way by which HDL can be anti-atherogenic and it depends on the type of modification of LDL and the stage of differentiation of monocytes to macrophages.

Vascular Endothelial Growth Factor Expression in Invasive Papillary Thyroid Carcinoma

Background: The vascular endothelial growth factor (VEGF) is a major promoter of endothelial growth and migration. Some studies have shown a correlation between expression of this growth factor and prognosis in several cancers, including well-differentiated thyroid cancer. Aim: We studied VEGF expression, local invasiveness, and other prognostic factors in papillary thyroid carcinoma (PTC) to test the hypothesis that the expression of VEGF is correlated with the degree of invasion of PTC. Patients and Methods: Clinical and pathological data of 76 patients with PTC were retrospectively reviewed. Group 1 consisted of patients with gross locally invasive tumors, group 2 consisted of patients with only invasion of the thyroid capsule, and group 3 consisted of patients with noninvasive PTC. Results: VEGF expression was noted within the tumor in all groups of PTC patients but was absent in the surrounding normal tissue. Older patients had higher expression of VEGF than younger patients. The age of patients with strong reaction to VEGF was 46 +/- 14 (mean +/- standard deviation), and that in patients with a weaker reaction was 39 +/- 16 (p<0.05). Only 20% of patients with a follicular variant of PTC had a strong reaction to VEGF compared with 68% of patients with classical PTC (p<0.01). Conclusions: VEGF expression appears to be an early event in the development of PTC. Whether VEGF expression promotes the progression of PTC is not known, but the answer to this question may be important in view of its greater expression in older patients, a group whose prognosis in PTC is worse.