Gradual Increase In Expression Research Articles

Dynamic analyses of a soft tissue-implant interface: Biological responses to immediate versus delayed dental implants.

To qualitatively and quantitatively evaluate the formation and maturation of peri-implant soft tissues around 'immediate' and 'delayed' implants. Miniaturized titanium implants were placed in either maxillary first molar (mxM1) fresh extraction sockets or healed mxM1 sites in mice. Peri-implant soft tissues were evaluated at multiple timepoints to assess the molecular mechanisms of attachment and the efficacy of the soft tissue as a barrier. A healthy junctional epithelium (JE) served as positive control. No differences were observed in the rate of soft-tissue integration of immediate versus delayed implants; however, overall, mucosal integration took at least twice as long as osseointegration in this model. Qualitative assessment of Vimentin expression over the time course of soft-tissue integration indicated an initially disorganized peri-implant connective tissue envelope that gradually matured with time. Quantitative analyses showed significantly less total collagen in peri-implant connective tissues compared to connective tissue around teeth around implants. Quantitative analyses also showed a gradual increase in expression of hemidesmosomal attachment proteins in the peri-implant epithelium (PIE), which was accompanied by a significant inflammatory marker reduction. Within the timeframe examined, quantitative analyses showed that connective tissue maturation never reached that observed around teeth. Hemidesmosomal attachment protein expression levels were also significantly reduced compared to those in an intact JE, although quantitative analyses indicated that macrophage density in the peri-implant environment was reduced over time, suggesting an improvement in PIE barrier functions. Perhaps most unexpectedly, maturation of the peri-implant soft tissues was a significantly slower process than osseointegration.

Establishment and identification of a skin cell line from Chinese tongue sole (Cynoglossus semilaevis) and analysis of the changes in its transcriptome upon LPS stimulation

The Chinese tongue sole (Cynoglossus semilaevis) holds significant economic importance within the fishing industry along the eastern coasts of China. In recent years, the frequent outbreaks of bacterial diseases have become a common concern as the aquaculture scale expands. The majority of the diseased fish exhibit symptoms such as skin congestion, damage and skin ulceration. As the skin serves as the first line of defense against bacterial infections, establishing a skin cell line for immunological research on Chinese tongue sole's response to bacterial infection is of utmost importance. In this study, a cell line named CSS (derived from the skin of the Chinese tongue sole) was successfully established. The cells have demonstrated stability during passages and exhibit a multipolar fibroblast-like morphology. They were cultured in L-15 medium with 20% serum and have been successfully passed through 60 passages over a period of 20 months. The identification of the mitochondrial CO1 gene confirmed that the cell originated from Chinese tongue sole. The karyotype detection revealed that the cell had a chromosome number of 2n = 42. After being stored in liquid nitrogen for 15 months, the cells can maintain more than 75% viability upon recovery. After transfecting with cy3-labeled scramble siRNA and pEGFP-N3 plasmid, clear fluorescence was observed in the transfected cells. We observed that lipopolysaccharide (LPS) from Escherichia coli significantly upregulate the gene expression of various immune-related pathways at 2 h in the CSS cell line. Additionally, the differentially expressed genes showed a higher enrichment in immune-related pathways at 2 and 6 h after stimulation compared to the 24 h point. Moreover, we identified 347 genes that exhibited a gradual increase in expression during the 0–24 h stimulation period. These genes were primarily enriched in pathways related to Autophagy, GABAergic synapse, Apelin signaling and Ferroptosis. In general, the CSS cell line established in this study exhibits stable growth and can serve as a valuable tool for in vitro studies of immunology and other basic biologies of Chinese tongue sole.

Genome-wide identification and transcriptome-based expression profiling of E2 gene family: Implication for potential roles in gonad development of Crassostrea gigas

In this study, we investigated the role of E2 ubiquitin conjugating enzymes (E2) in the Pacific oyster Crassostrea gigas, with a focus on their involvement in gonad development. We identified 34 E2 genes clustered into nine subgroups and 24 subfamilies. The gene structure and intron-exon location were conserved within the same subfamily, but motif variation suggested functional diversity. Tissue transcriptome analyses revealed that most E2 genes were broadly expressed, with UBE2CL showing specific expression in the female gonad. Expression profiling of E2 genes during early embryo-larvae development stages suggested that five E2 genes were highly expressed in early embryo development, indicating their involvement in cell division processes. Furthermore, by profiling the expression of E2 genes in different gonadal developmental stages, we observed a gradual increase in expression for four genes during gametogenesis, with significantly higher expression in the female gonad at the maturation stage. Similarly, five E2 genes displayed elevated expression levels in the male gonad at the maturation stage, indicating their crucial roles in gonadal development and gametogenesis. Our study provides valuable insights into the potential functions of the E2 gene family in C. gigas, shedding light on the molecular mechanisms underlying gonad development in oysters.

Mathematical Modeling Reveals That Sucrose Regulates Leaf Senescence via Dynamic Sugar Signaling Pathways.

Sucrose (Suc) accumulation is one of the key indicators of leaf senescence onset, but little is known about its regulatory role. Here, we found that application of high (120–150 mM) and low levels (60 mM) of Suc to young leaf (YL) and fully expanded leaf (FEL) discs, respectively, decreased chlorophyll content and maximum photosynthetic efficiency. Electrolyte leakage and malondialdehyde levels increased at high Suc concentrations (90–120 mM in YL and 60 and 150 mM in FEL discs). In FEL discs, the senescence-associated gene NtSAG12 showed a gradual increase in expression with increased Suc application; in contrast, in YL discs, NtSAG12 was upregulated with low Suc treatment (60 mM) but downregulated at higher levels of Suc. In YL discs, trehalose-6-phosphate (T6P) accumulated at a low half-maximal effective concentration (EC50) of Suc (1.765 mM). However, T6P levels declined as trehalose 6 phosphate synthase (TPS) content decreased, resulting in the maximum velocity of sucrose non-fermenting-1-related protein kinase (SnRK) and hexokinase (HXK) occurring at higher level of Suc. We therefore speculated that senescence was induced by hexose accumulation. In FEL discs, the EC50 of T6P occurred at a low concentration of Suc (0.9488 mM); T6P levels progressively increased with higher TPS content, which inhibited SnRK activity with a dissociation constant (Kd) of 0.001475 U/g. This confirmed that the T6P–SnRK complex induced senescence in detached FEL discs.

Novel regulation mechanism of adrenal cortisol and DHEA biosynthesis via the endogen ERAD inhibitor small VCP-interacting protein

Endoplasmic reticulum-associated degradation (ERAD) is a well-characterized mechanism of protein quality control by removal of misfolded or unfolded proteins. The tight regulation of ERAD is critical for protein homeostasis as well as lipid metabolism. Although the mechanism is complex, all ERAD branches converge on p97/VCP, a key protein in the retrotranslocation step. The multifunctionality of p97/VCP relies on its multiple binding partners, one of which is the endogenous ERAD inhibitor, SVIP (small VCP-interacting protein). As SVIP is a promising target for the regulation of ERAD, we aimed to assess its novel physiological roles. We revealed that SVIP is highly expressed in the rat adrenal gland, especially in the cortex region, at a consistently high level during postnatal development, unlike the gradual increase in expression seen in developing nerves. Steroidogenic stimulators caused a decrease in SVIP mRNA expression and increase in SVIP protein degradation in human adrenocortical H295R cells. Interestingly, silencing of SVIP diminished cortisol secretion along with downregulation of steroidogenic enzymes and proteins involved in cholesterol uptake and cholesterol biosynthesis. A certain degree of SVIP overexpression mainly increased the biosynthesis of cortisol as well as DHEA by enhancing the expression of key steroidogenic proteins, whereas exaggerated overexpression led to apoptosis, phosphorylation of eIF2α, and diminished adrenal steroid hormone biosynthesis. In conclusion, SVIP is a novel regulator of adrenal cortisol and DHEA biosynthesis, suggesting that alterations in SVIP expression levels may be involved in the deregulation of steroidogenic stimulator signaling and abnormal adrenal hormone secretion.

Centromere Protein F (CENPF): A novel marker for salivary gland pathology.

Salivary gland tumours are relatively uncommon, and there exists considerable diagnostic difficulty. This is due to individual lesions having diverse histopathological features, presence of number of types and variants, and overlapping histological features in different tumour entities. The current study aimed at assessing the expression of centromere protein F (CENPF) in benign and malignant salivary gland tumours and to evaluate the efficacy of CENPF as a proliferative marker to aid in the diagnosis of malignancy so that it will help in surgical pathology practice. The study group involved 20 cases of benign salivary gland tumours, 20 cases of malignant salivary gland tumours, and 10 normal salivary gland tissues. All the cases were subjected to immunohistochemical analysis for CENPF expression and were assessed by two independent observers and further taken up for evaluation. The results were analysed statistically among different groups using analysis of variance (ANOVA) or Kruskal-Wallis test with Chi-squared test using IBM's Statistical Package for the Social Sciences (SPSS) version 17.0. CENPF expression in normal salivary gland was negative with gradual increase in expression from benign salivary gland tumours to malignant salivary gland tumours. CENPF expression was high in malignant salivary gland tumours. Findings of the study suggest that CENPF can be regarded as a new cell proliferation marker for malignant salivary gland tumours.

Apoptosis as a mechanism of the albumin-induced kidney damage in childhood nephrotic syndrome

It was shown previously on in vivo studies that proteinuria-induced effects play a crucial role in renal damage in chronic kidney disease (CKD). However, an initial mechanism of irreversible kidney damage in pediatric diseases characterized by chronic proteinuria, i.e. nephrotic syndrome, remains to be unclear. 
 The aim of our work was to study the initial mechanism of kidney cells apoptosis development in nephrotic children. 
 Methods.An examination of renal biopsies of 53 patients (aged 10 to 15 years) with nephrotic syndrome hospitalized in Pediatric Nephrology unit of the Children Clinical Hospital №7 (Kyiv, Ukraine) done. In vitro studies of albumin toxicity performed on rat proximal tubular cells in primary culture (RPTC). 
 Results. Our study showed that albumin overload in nephrotic children leads to high levels of apoptosis. Its distribution and level varies regarding the level of focal segmental glomerulosclerosis (FSGS). The progression of sclerosis as a sign of irreversible kidney damage is accompanied by gradual increase in expression of proapoptotic factor Bax. 
 In vitro studies on rat proximal tubular cells in primary culture (RPTC) showed that excessive albumin uptake into rat primary renal cells causes an almost immediate mitochondrial accumulation of the apoptotic factor Bax. We hypothesize that this might be initial pathway leading to kidney cells apoptosis in childhood nephrotic syndrome. 
 Conclusions. We show thatoverexpression of apoptotic factor Bax has a place in children with nephrotic syndrome. Thus, chronic influence of albumin is a factor predisposing disturbances in system controlling apoptosis in this cohort of patients. Our data demonstrate that there is a dependence between the Bax overexpression level and the stage of CKD. We show the topologic difference between the Bax levels and FSGS degree. This is an indication thatdevelopment of glomerular and tubule-interstitial disorders under the influence of proteinuria occurs in specific range. In vitro data demonstrate that albumin overload causes mitochondrial Bax translocation that could be an initial factor in apoptotic pathway activation.

Differential expression of miRNA-146a and miRNA-155 in gastritis induced by Helicobacter pylori infection in paediatric patients, adults, and an animal model

BackgroundHelicobacter pylori is a major aetiologic agent associated with gastritis. H. pylori infections increase the expression of the Toll-like receptor (TLR), which in turn modulates the expression of microRNA (miRNA)-146a and miRNA-155. The objective of this study was to compare the expression of miRNA-146a and miRNA-155 in gastric lesions of paediatric and adult patients with different pathologies and in Mongolian gerbils (Meriones unguiculatus) infected with H. pylori 26,695.MethodsQuantification of miRNA expression was performed by quantitative real-time polymerase chain reaction (qRT-PCR) of paraffin-embedded gastric lesions of children with or without an infection (n = 25), adults with follicular gastritis and metaplasia (n = 32) and eight-week-old M. unguiculatus males (Hsd:MON) infected with H. pylori 26,695 for 0, 3, 6, 12 and 18 months (n = 25). The genes RNU48 and RNU6 were used as endogenous controls for data normalization. Statistical analyses were performed using Kruskal-Wallis, Mann-Whitney, ANOVA and Student’s t-test.ResultsThe expression of miRNA-146a and miRNA-155 in infected children increased by 247.6- and 79.4-fold (on average), respectively, compared to that observed in the control group. However, these results were not significant (p = 0.12 and p = 0.07 respectively). In some children a gradual increase in expression was observed, while in others, expression was very high. Additionally, the expression levels of miRNA-146a and miRNA-155 increased by an average of 21.7- and 62-fold, respectively, in adult patients with follicular gastritis when compared to those of the controls. In M. unguiculatus infected with H. pylori 26,695, the expression of both miRNAs increased as the infection progressed.ConclusionThis is the first report to show differences in the expression of miRNA-146a and miRNA-155 in paediatric and adult patients with gastritis who were infected with H. pylori. In addition, in M. unguiculatus infected with H. pylori, miRNA expression was associated with the progression of infection and the ability of the bacteria to adapt to the host.

Expression profile of phenylpropanoid pathway genes in Decalepis hamiltonii tuberous roots during flavour development.

To explore the transcriptional control of phenylpropanoid pathway (PPP) involved in vanillin flavour metabolites production in tuberous roots of Decalepis hamiltonii, four PPP key genes expressed during the tuber development were identified and their mRNA expression profiles were evaluated using quantitative real-time PCR. Flavour metabolite quantification by HPLC analysis confirmed 10, 170 and 500µg/g 2-hydroxy-4-methoxy benzaldehyde and 4, 20 and 40µg/g vanillin in first- (3-month-old plant), second- (18-month-old plant) and third-stage tubers (60-month-old matured plant), respectively. The expression of all four genes phenylalanine ammonia lyase (DhPAL), cinnamate-4-hydroxylase (DhC4H), caffeic acid-O-methyltransferase (DhCOMT) and vanillin synthase (DhVAN) increased with flavour development from first stage to second stage. A decrease in expression from 1.9-folds to 0.1-folds and 19.2-folds to 5.2-folds for DhCOMT and DhVAN was recorded for second stage to third stage, respectively. However, a gradual increase in expression of DhPAL (up to 26.4-folds) and a constant expression pattern for DhC4H (up to 7.1-folds) was evident from second stage to third stage of flavour development. The decrease in the expression levels of DhCOMT and DhVAN in third stage shows thatthe second-stage tubers are more transcriptionally active towards flavour biosynthesis.

Chimeric Adeno-Associated Virus-Mediated Cardiovascular Reprogramming for Ischemic Heart Disease.

Here, we demonstrated chimeric adeno-associated virus (chimeric AAV), AAV-DJ-mediated cardiovascular reprogramming strategy to generate new cardiomyocytes and limit collagen deposition in cardiac fibroblasts by inducing synergism of chimeric AAV-expressing Gata4, Mef2c, Tbx5 (AAV-GMT)-mediated heart reprogramming and chimeric AAV-expressing thymosin β4 (AAV-Tβ4)-mediated heart regeneration. AAV-GMT promoted a gradual increase in expression of cardiac-specific genes, including Actc1, Gja1, Myh6, Ryr2, and cTnT, with a gradual decrease in expression of a fibrosis-specific gene, procollagen type I and here AAV-Tβ4 help to induce GMT expression, providing a chimeric AAV-mediated therapeutic cell reprogramming strategy for ischemic heart diseases.

Hyperoxia provokes a time- and dose-dependent inflammatory response in mechanically ventilated mice, irrespective of tidal volumes

BackgroundMechanical ventilation and hyperoxia have the potential to independently promote lung injury and inflammation. Our purpose was to study both time- and dose-dependent effects of supplemental oxygen in an experimental model of mechanically ventilated mice.MethodsHealthy male C57Bl/6J mice, aged 9–10 weeks, were intraperitoneally anesthetized and randomly assigned to the mechanically ventilated group or the control group. In total, 100 mice were tracheotomized and mechanically ventilated for either 8 or 12 h after allocation to different settings for the applied fractions of inspired oxygen (FiO2, 30, 50, or 90%) and tidal volumes (7.5 or 15 ml/kg). After euthanisation arterial blood, bronchoalveolar lavage fluid (BALf) and tissues were collected for analyses.ResultsMechanical ventilation significantly increased the lung injury score (P < 0.05), mean protein content (P < 0.001), and the mean number of cells (P < 0.01), including neutrophils in BALf (P < 0.001). In mice ventilated for 12 h, a significant increase in TNF-α, IFN-γ, IL-1β, IL-10, and MCP-1 (P < 0.01) was observed with 90% FiO2, whereas IL-6 showed a decreasing trend (P for trend = 0.03) across FiO2 groups. KC, MIP-2, and sRAGE were similar between FiO2 groups. HMGB-1 was significantly higher in BALf of mechanically ventilated mice compared to controls and showed a gradual increase in expression with increasing FiO2. Cytokine and chemokine levels in BALf did not markedly differ between FiO2 groups after 8 h of ventilation. Differences between the tidal volume groups were small and did not appear to significantly interact with the oxygen levels.ConclusionsWe demonstrated a severe vascular leakage and a pro-inflammatory pulmonary response in mechanically ventilated mice, which was enhanced by severe hyperoxia and longer duration of mechanical ventilation. Prolonged ventilation with high oxygen concentrations induced a time-dependent immune response characterized by elevated levels of neutrophils, cytokines, and chemokines in the pulmonary compartment.

The Life and Death of Differentiated and Dedifferentiated Intestinal Absorptive and Secretory Cells

Intestinal stem cells (ISC) differentiate into many types of secretory and absorptive progenies which migrate up the villi and are exfoliated after 3–5 d. Because epithelial homeostasis requires balanced rates of proliferation, migration, differentiation and apoptosis, studies increasing our understanding of cell turnover are critical. We used murine primary intestinal organoids to determine lifespan of different intestinal cell types. We tested the hypotheses that organoids directed to differentiate into distinct cell types would exhibit lifespans similar to those previously estimated in vivo, and that forced dedifferentiation of organoids containing mature cells would result in reacquisition of ISC characteristics and extension of lifespan. We directed, via Wnt and Notch signaling, strict ISC differentiation into specialized organoids comprised primarily of stem cells (ISC), enterocytes (ENT), goblet cells (GOB), or Paneth cells (PAN). Cell type enrichment was confirmed via biomarker mRNA expression and immunofluorescence, as follows: stem cells (~95%), enterocytes (~90%), goblet (~85%) or Paneth cells (~65%) which contain relative proportions much higher than in situ.We found that continuously proliferating ISC organoids can be passaged several times; in contrast, ENT and GOB organoids do not live past 5 or 4 d, respectively, and PAN organoids live ~11 d. These findings are similar to known in vivo lifespans. During differentiation, ENT and GOB organoids exhibited a gradual increase in expression of enterocyte biomarker sucrase isomaltase and goblet cell biomarker mucin‐2, respectively, while levels of stem cell markers Leucine Rich Repeat Containing G Protein‐Coupled Receptor 5 (Lgr5) and SRY‐Box 9 (Sox9) decreased dramatically. Forced dedifferentiation of ENT and GOB organoids was characterized by a dramatic decrease in expression of differentiated cell markers paralleled by pronounced increases in stem cell characteristics.Differentiation was accompanied by sharp decreases over time in anti‐apoptotic B‐cell lymphoma 2 (Bcl2) in both ENT and GOB organoids (&gt;90%). Changes in expression of pro‐apoptotic caspase‐3 (Casp3) and Fas Cell Surface Death Receptor (Fas) were insignificant. Dedifferentiated ENT organoids (dENT) expressed altered apoptotic markers compared to their normal ENT and ISC counterparts. Casp3 and Fas levels decreased 50 and 70% respectively in dENT organoids compared to ENT controls while Bcl2 and Tumor protein p53 (P53) increased significantly (1.6 and 5‐fold respectively). In dedifferentiated goblet organoids (dGOB), Bcl2 and P53 also increased 2–3‐fold. Phenotypically, life‐span increased markedly in both dENT and dGOB organoids by more than 2‐fold due to dedifferentiation, but cell morphology was not altered.In conclusion, we were able to differentiate organoids enriched in distinct cell lineages, then to induce these specialized organoids to undergo in vitro dedifferentiation which markedly extended their life span. In vitro lifespan of specific cell type organoids was similar to that in vivo, and was strongly correlated with mRNA levels of Bcl2. Additionally, we were able to induce specialized organoids to undergo in vitro dedifferentiation and markedly extend their lifespan. This study furthers our understanding of organoid physiology, intestinal cell turnover, and plasticity of cell fate.Support or Funding InformationSupported by NSF Grants No. IOS‐1121049 and 1456673 (RPF). RPF also received support from NIH R01‐DK‐102934 (NG).

Age-related Differences in CYP3A Abundance and Activity in the Liver of the Göttingen Minipig.

In view of paediatric drug development, regulatory authorities often request safety studies in juvenile animals, including minipigs. Unfortunately, knowledge on the ontogeny of the biotransformation processes in animal models remains scarce and impedes a correct interpretation of the toxicity findings. CYP3A4 is one of the most important drug-metabolizing enzymes in human beings and shows important similarities with CYP3A in the minipig. Therefore, the aim of this study was to assess the abundance and activity of CYP3A in liver microsomes from foetal, juvenile (days 1, 3, 7 and 28) and adult male and female Göttingen minipigs. CYP3A abundance was studied by an indirect enzyme-linked immunosorbent assay (ELISA), whereas CYP3A activity was assessed by a biotransformation assay with Luciferin-IPA. CYP3A abundance could not be detected until day 3. From day 7 onwards, a gradual increase in expression was noted, leading to the highest abundance in adult animals. CYP3A activity was not detectable in foetuses and 1-day-old animals. The CYP3A activity was detectable, but below the LLOQ in day 3 animals and increased gradually with age to reach the highest level in adults. The CYP3cide and ketoconazole inhibition, and testosterone and midazolam reduction of Luciferin-IPA metabolism in minipig liver microsomes substantiate that Luciferin-IPA is metabolized by CYP3A in minipigs. A positive correlation was found between CYP3A abundance and biotransformation of Luciferin-IPA (Pearson r = 0.863; p < 0.0001). In conclusion, both abundance and activity of CYP3A increased gradually in juvenile minipigs, but remained below the levels observed in adult animals.

Stress and inflammatory gene networks in bovine liver are altered by plane of dietary energy during late pregnancy.

The prepartal dietary energy level is tightly correlated with the degree of tissue mobilization that the animal experiences around parturition (giving birth). To better understand the link between the dry period dietary energy management and the inflammatory status around parturition, 12 multiparous Holstein cows were fed for the entire dry period either a high-wheat straw/lower-energy diet to supply at least 100% of the calculated net energy for lactation (NEL) (control, CON) or a higher-energy diet to supply >140% of NEL (overfed, OVE). The blood was sampled throughout the transition period for biomarker analyses. Liver tissue samples were taken on days -14, 7, 14, and 30 relative to parturition for triacylglycerol (TAG) composition and gene expression analysis. Fifty genes involved in inflammation, endoplasmic reticulum (ER), and oxidative stress, and cell cycle and growth were evaluated. Although blood biomarkers did not reveal signs of a greater inflammatory status compared with OVE, CON cows had a greater activation of the intrahepatic unfolded protein response prepartum. However, postpartum mRNA profiling indicated that the OVE group experienced a mild but sustained level of ER stress, with higher oxidative stress and impairment of antioxidant mechanisms. After parturition, inflammation-related genes were upregulated in OVE cows compared with CON. However, CON cows experienced a gradual increase in expression of key inflammatory transcription regulators up to 30 days postpartum which agreed with the lower plasma albumin and cholesterol, suggesting an inflammatory state. Data underscored that ER stress is not necessarily linked with inflammation during the peripartal period. Gene expression data also suggest that prepartum overnutrition could have negative effects on normal cell cycle activity. Overall, allowing cows to overconsume energy prepartum increased the hepatic pro-inflammatory response prepartum and up to the point of parturition. Subsequently, cows fed the lower-energy diet experienced a gradual increase in the inflammatory response. The lack of differences between groups in voluntary feed intake and lactation capacity suggests that nutritional management prepartum triggers different mechanisms that affect ER and oxidative stress along with inflammation. Although no clinical disorders were detected, these alterations expose animals to the development of immuno-metabolic disorders.

Management of Hypoxia-induced Disorders in Nephrotic Children

Objectives: The objective of this paper was to study the levels of cellular hypoxia, apoptosis controlling factors in relation to the value of kidney function impairment and to evaluate possible protective effects of the treatment with antioxidant agent tocopherol in pediatric patients with nephrotic syndrome. Background: Hypoxia-related disorders play an important role in irreversible tissue damage via activation of certain secondary processes. Methods: An examination of renal biopsies of 53 patients (aged 10 to 15 years) with nephrotic syndrome was done. Conventional clinical investigations, immunohistochemistry, immunoblotting were used in this study. Results: We show that nephrotic patients reveal a high level of marker of cellular hypoxia HIF-1α and its dependence on the level of kidney function impairment and proteinuria. The progression of the sclerosis as a sign of irreversible kidney damage is accompanied by gradual increase in expression of proapoptotic factor Bax. Applied treatment composes conventional scheme and tocopherol showed an improvement in hypoxia level, proapoptotic factor Bax expression. Conclusion: Hypoxia-induces disorders in nephrotic children presented by disturbances in apoptosis controlling system can be attenuated by the antioxidants application

Temporal Colonic Gene Expression Profiling in the Recurrent Colitis Model Identifies Early and Chronic Inflammatory Processes

The recurrent TNBS-colitis model in BALB/c mice has been proposed as a model of Inflammatory Bowel Disease with a shifting pattern of local cytokines with the expression of Th1 cytokines during the early phase, Th17 cytokines during the intermediate phase and Th2 cytokines during late fibrotic stages. In this study, we evaluated the development of pathology in time–in conjunction with genome-wide gene expression in the colons–in response to three weekly intrarectal instillations of TNBS. During this time-frame mice develop colitis with extensive cellular infiltration of (sub)mucosa and mildly to moderately affected crypt architecture. These pathological processes were sensitive to local treatment with budesonide. Gene expression profiling confirmed an acute phase response after each intrarectal TNBS-challenge. In addition, a chronic inflammatory process developed over time particularly evident from a gradual increase in expression of mast cell related genes. The changes in pathological hallmarks were consistent with a temporal expression of mRNA encoding a selection of chemokines. In conclusion, the early stages of the recurrent TNBS-colitis model reflect several aspects of inflammatory bowel disease which are sensitive to immunomodulation.

A continuum of transcriptional identities visualized by combinatorial fluorescent in situ hybridization

Oligonucleotide-based fluorescent in situ hybridization (FISH) coupled with high-resolution high-sensitivity microscopy allows the visualization of single RNA molecules within fixed cells and tissues as distinct foci. We show here that combinatorial labeling of RNA molecules with several fluorescent dyes extends the number of genes that can be targeted simultaneously beyond the number of fluorophores used. This approach also inherently validates the identification of transcripts reducing false positive counts. We have used combinatorial FISH and image analysis to measure the transcript densities of six genes using three fluorophores. This has allowed us to visualize the endothelial maturation of lateral mesoderm in an in vitro ES differentiation assay from a single snapshot of molecular identities. Our observations show that, under these specific conditions, endothelial maturation follows a homogeneous course with a gradual increase in expression of Cdh5 and a concomitant loss of early transcription factors, arguing that maturation is governed in a generally deterministic manner. This methodology is limited by the number of fluorophores that can be used and by the available microscopic resolution, but currently available equipment should allow the visualization of transcripts from 10 or more genes simultaneously.

Dendritic Cells Are Central Coordinators of the Host Immune Response to Staphylococcus aureus Bloodstream Infection

Dendritic cells (DCs) play an important role in integration of the immune responses induced by pathogens. The purpose of this study was to determine the importance of DCs in host defense against Staphylococcus aureus bacteremia. Using a murine infection model, we demonstrated that DCs are rapidly recruited into infected tissue after intravenous inoculation with S. aureus. The recruited DCs were fully functional and in a more advanced stage of maturation than those isolated from uninfected mice. Depletion of DCs in CD11c-DTR transgenic mice resulted in substantial worsening of infection, as indicated by increased bacterial loads in kidneys and lungs, accelerated mortality, and more severe pathology. Furthermore, DC depletion completely abolished IL-12 production in response to infection. The beneficial effect afforded by DCs during S. aureus infection was not mediated by their contribution to direct bacterial killing, nor by increased neutrophil recruitment. Instead, neutrophil influx (along with expression of CXC chemokines) was significantly enhanced in infected tissue after depletion of DCs. We also found that the bactericidal capacity of the recruited neutrophils was significantly impaired in DC-depleted mice. More importantly, the detrimental effect of DC depletion was practically reversed by treatment with exogenous recombinant mouse IL-12. Our results demonstrated that DCs, probably through their production of IL-12, play an important role in coordinating the inflammatory response during S. aureus infection.

Expression of Wnt 3a, β-Catenin, Cyclin D1 and PCNA in Mouse Dentate Gyrus Subgranular Zone (SGZ): a Possible Role of Wnt Pathway in SGZ Neural Stem Cell Proliferation

In mammalian dentate gyrus subgranular zone, the addition of new neurons throughout adult-hood is a remarkable form of structural plasticity. Yet, the molecular controls over subgranular zone neural stem cell proliferation, survival, and differentiation are poorly understood. In this study we analysed the expression of Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen in mouse subgranular zone to elucidate the involvement of Wnt pathway in subgranular zone neural stem cell proliferation. We performed immunohistochemistry and RT-PCR for the above molecules on adult and postnatal developing hippocampal tissues of mice, respectively. RT-PCR analysis showed a gradual increase in expression of mRNA of Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen as the postnatal hippocampus developed, and immunohistochemical analysis showed a highly positive immunoreactive expression for Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen in the subgranular zone cells. Together, our data suggested that the Wnt pathway is activated in subgranular zone and could play an important role in regulating subgranular zone neural stem cell proliferation in mouse hippocampus.

Expression Profile of PIP Kinases During In Vitro Human Erythropoiesis

AbstractAbstract 2079Phosphatidylinositol phosphate kinases (PIPKs) are a family of lipid kinase enzymes that produce the second messenger PI4,5P2 (phosphatidylinositol 4,5-biphosphate), which plays an important role in the regulation of a variety of cellular activities, including gene expression. PIPKs are classified into 3 subfamilies — PIPK I (a, b, g), PIPK II (a, b, g) and PIPK III — which are functionally distinct and are located in different subcellular compartments. In a recent study in our laboratory, the PIPKIIa gene was differentially expressed in reticulocytes from 2 siblings with hemoglobin (Hb) H disease who had the same genotype (-a3.7/–SEA). Expression of both the PIPKIIa and b-globin genes were higher in the patient with the higher Hb H level, suggesting a possible relationship between PIPKIIa and the production of globins, particularly b-globin. In light of these findings, the aim of this study was to determine the gene expression profiles of PIPKs (I and II - with their isoforms a, b and g - and III) during erythropoiesis in peripheral blood hematopoietic CD34+ cell culture from 11 healthy volunteers and 6 patients with hemoglobinopathies [2 with a-thalassemia (Hb H disease), 2 with b-thalassemia (homozygous for the IVS-I-6-T-C mutation) and 2 with sickle cell anemia] using quantitative real time PCR (qRT-PCR) and to compare these profiles with the gene expression profiles of a-, b- and g-globins on the 7th, 10th and 13th days of the erythroid culture. In the cell culture from the normal group, expression of the PIPKIIa and other PIPK genes increased during erythroid differentiation, coinciding with the expression profiles of globin genes and showing in particular that a-globin has a significant effect on PIPKIIa (p<0.0001), as the PIPKIIa on a-globin gene (p=0.0002). In the patients, the expression profile of the PIPKIIa gene also increased during differentiation, whereas the results for the other PIPK genes varied. However, mRNA levels differed between patients, indicating greater complexity in individuals with hemoglobinopathies. PIPKIIa expression level was elevated in the culture from one of the a-thalassemia patients (approximately 12 times higher than in the corresponding control) but was lower than the control in one of the b-thalassemia patients. Expression levels of this gene also varied among sickle cell patients. This is the first study of the gene expression profiles of these kinases during in vitro human erythropoiesis. We identified a standard pattern of gene expression for PIPKs, and PIPKIIa in particular, a gradual increase in expression during erythroid differentiation, similar to the pattern for globin genes. This suggests that PI4,5P2, as an important secondary messenger involved in the regulation of gene expression, may play an important role in the regulation of globin gene expression and the normal process of Hb synthesis in red blood cells. Although our results varied between patients, highlighting the complexity of the regulatory systems involved in Hb production, they reinforce the hypothesis of a relationship between PIPKIIa and globin expression. This work was supported by FAPESP, CNPq and CAPES. Disclosures:No relevant conflicts of interest to declare.