Epithelial-specific and stage-specific functions of insulin-like growth factor-I during postnatal mammary development

Postnatal development of the mammary gland requires interactions between the epithelial and stromal compartments, which regulate actions of hormones and growth factors. IGF-I is expressed in both epithelial and stromal compartments during postnatal development of the mammary gland. However, little is known about how local expression of IGF-I in epithelium or stroma regulates mammary growth and differentiation during puberty and pregnancy-induced alveolar development. The goal of this study was to investigate the mechanisms of IGF-I actions in the postnatal mammary gland and test the hypothesis that IGF-I expressed in stromal and epithelial compartments has distinct functions. We established mouse lines with inactivation of the igf1 gene in mammary epithelium by crossing igf1/loxP mice with mouse lines expressing Cre recombinase under the control of either the mouse mammary tumor virus long-terminal repeat or the whey acidic protein gene promoter. Epithelial-specific loss of IGF-I during pubertal growth resulted in deficits in ductal branching. In contrast, heterozygous reduction of IGF-I throughout the gland decreased expression of cyclins A2 and B1 during pubertal growth and resulted in alterations in proliferation of the alveolar epithelium and milk protein levels during pregnancy-induced differentiation. Reduction in epithelial IGF-I at either of these stages had no effect on these indices. Taken together, our results support distinct roles for IGF-I expressed in epithelial and stromal compartments in mediating growth of the postnatal mammary gland.     

Microleakage in conventional and bonded amalgam restorations: influence of cavity volume

This study verified the relationship between the volume and microleakage of conventional and bonded amalgam restorations. Also, the microleakage influence of intermediate materials, substrates and the direction of sectioning was investigated. Fifty-six bovine incisors were selected. Standard Class V cavities were prepared in buccal and lingual surfaces. For each tooth, two cavity sizes were prepared, corresponding to two cavity volumes: one larger (A) and the other smaller (B). The cervical wall was located in cementum/dentin and the incisal wall in enamel. The teeth were distributed in four groups (n=28) according to the intermediate material employed (glass-ionomer cement, resin cement, adhesive system and copal varnish-control). The materials were applied following manufacturers' directions. After restoration, the teeth were submitted to thermal cycling. They were then immersed in a dye solution and sectioned in two directions inciso-cervical (IC) and mesio-distal (MD) sections to evaluate the microleakage. Data were subjected to non-parametric statistical analysis (Wilcoxon's paired test and Kruskal-Wallis test). No significant difference was found between the two cavity sizes. Leakage in enamel was statistically lower than in the cementum/dentin interface (p < 0.05). In some situations, glass-ionomer or resin cement lined amalgam restorations presented less dye leakage than copal varnish lined restorations (p < 0.05). No significant difference was observed in microleakage between IC or MD sectioning. Within the limitations of this study, it was concluded that cavity size and direction of section were not significant factors for microleakage, while substrate and intermediate materials had a significant effect on the sealing ability in amalgam restorations.     

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signaling

Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1–caspase-8 death–inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase–dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1β release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1β neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.

Gasdermins are a family of recently described pore-forming proteins and are emerging as key drivers of cell death and inflammation. Gasdermins comprise a cytotoxic N-terminal domain connected to an inhibitory carboxyl-terminal domain and are activated upon proteolytic cleavage (1, 2). This cleavage event releases the cytotoxic N-terminal fragment, which creates membrane pores and triggers a form of lytic cell death called pyroptosis (3–6). Gasdermin D (GSDMD) is arguably the best characterized family member to date and is activated upon proteolysis by caspase-1, 4, 5, 8, and 11 and serine proteases (7–14). Active GSDMD promotes host defense by eliminating the replicating niche of intracellular pathogens (15) and inducing the extrusion of antimicrobial neutrophil extracellular traps (NETs) (16). In addition, GSDMD pores act as a conduit for bioactive IL-1β release (17–19), a potent proinflammatory cytokine that similarly requires proteolytic cleavage by caspase-1 or -8 to gain biological activity (20). By contrast, gasdermin E (GSMDE [also known as DFNA5]) is activated by apoptotic caspase-3 and 7 and granzyme B, which drives tumor cell pyroptosis and anti-tumor immunity (21–23). The physiological function of GSDME in primary immune cells and its potential role in host defense remain unresolved and have not been reported.Pathogenic Yersinia are a group of Gram-negative extracellular bacteria that causes disease ranging from gastroenteritis (Yersinia pseudotuberculosis) to plague (Y. pestis). A major mechanism by which pathogenic Yersinia establish systemic infection is by injecting the effector protein YopJ, an acetyltransferase that blocks transforming growth factor beta-activated kinase 1 (TAK1), to inhibit host innate immune signaling and proinflammatory cytokine production (24). To counteract this, detection of YopJ activity by myeloid cells induces the assembly of a cytoplasmic death–inducing complex that comprises receptor-interacting serine/threonine protein kinase 1 (RIPK1), fas-associated protein with death domain, and caspase-8 (24–26). During in vivo infection, RIPK1/caspase-8–dependent cell death in myeloid cells restricts bacterial dissemination and replication at distal sites by inducing proinflammatory cytokine production from uninfected bystander cells (24). More recently, GSDMD was identified as a caspase-8 substrate during Yersinia infection that drives antimicrobial defense in vivo (11, 12, 27). However, whether RIPK1 activates additional substrates to restrict Yersinia infection is unclear and is a focus of this study. Here, we identify GSDME as a substrate activated downstream of RIPK1 that confers host resistance against Yersinia. Gsdme-deficient mice failed to control bacterial replication in the spleen and liver and consequently are more susceptible to Yersinia infection than wild-type (WT) animals. Mechanistically, our data reveal that RIPK1 promotes caspase-3–dependent GSDME activation and IL-1β release in neutrophils, but not macrophages. Neutralization of IL-1β impaired bacterial clearance in WT, but not Gsdme^−/−, animals, indicating that IL-1β is mainly secreted through GSDME pores during Yersinia challenge in vivo.     

INFLUENCE OF THERMAL STRESS ON MARGINAL INTEGRITY OF RESTORATIVE MATERIALS

The aim of this study was to evaluate the influence of thermal stress on the marginal integrity of restorative materials with different adhesive and thermal properties. Three hundred and sixty Class V cavities were prepared in buccal and lingual surfaces of 180 bovine incisors. Cervical and incisal walls were located in dentin and enamel, respectively. Specimens were restored with resin composite (RC); glass ionomer (GI) or amalgam (AM), and randomly assigned to 18 groups (n=20) according to the material, number of cycles (500 or 1,000 cycles) and dwell time (30 s or 60 s). Dry and wet specimens served as controls Specimens were immersed in 1% basic fuchsine solution (24 h), sectioned, and microleakage was evaluated under x40 magnification. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests: Thermal cycling regimens increased leakage in all AM restorations (p<0.05) and its effect on RC and GI restorations was only significant when a 60-s dwell time was used (p<0.05). Marginal integrity was more affected in AM restorations under thermal cycling stress, whereas RC and GI ionomer restoration margins were only significantly affected only under longer dwell times.     

Identification of core genes in ovarian cancer by an integrative meta-analysis

Background

Epithelial ovarian cancer is one of the most ?severe public health threats in women. Since it is still challenging to screen in early stages, identification of core genes that play an essential role in epithelial ovarian cancer initiation and progression is of vital importance.

Results

Seven gene expression datasets (GSE6008, GSE18520, GSE26712, GSE27651, GSE29450, GSE36668, and GSE52037) containing 396 ovarian cancer samples and 54 healthy control samples were analyzed to identify the significant differentially expressed genes (DEGs). We identified 563 DEGs, including 245 upregulated and 318 downregulated genes. Enrichment analysis based on the gene ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that the upregulated genes were significantly enriched in cell division, cell cycle, tight junction, and oocyte meiosis, while the downregulated genes were associated with response to endogenous stimuli, complement and coagulation cascades, the cGMP-PKG signaling pathway, and serotonergic synapse. Two significant modules were identified from a protein-protein interaction network by using the Molecular Complex Detection (MCODE) software. Moreover, 12 hub genes with degree centrality more than 29 were selected from the protein-protein interaction network, and module analysis illustrated that these 12 hub genes belong to module 1. Furthermore, Kaplan-Meier analysis for overall survival indicated that 9 of these hub genes was correlated with poor prognosis of epithelial ovarian cancer patients.

Conclusion

The present study systematically validates the results of previous studies and fills the gap regarding a large-scale meta-analysis in the field of epithelial ovarian cancer. Furthermore, hub genes that could be used as a novel biomarkers to facilitate early diagnosis and therapeutic approaches are evaluated, providing compelling evidence for future genomic-based individualized treatment of epithelial ovarian cancer.

     

Surface roughness of orthodontic band cements with different compositions

Objectives

The present study evaluated comparatively the surface roughness of four orthodontic band cements after storage in various solutions.

Material and Methods

eight standardized cylinders were made from 4 materials: zinc phosphate cement (ZP), compomer (C), resin-modified glass ionomer cement (RMGIC) and resin cement (RC). Specimens were stored for 24 h in deionized water and immersed in saline (pH 7.0) or 0.1 M lactic acid solution (pH 4.0) for 15 days. Surface roughness readings were taken with a profilometer (Surfcorder SE1200) before and after the storage period. Data were analyzed by two-way ANOVA and Tukey''s test (comparison among cements and storage solutions) or paired t-test (comparison before and after the storage period) at 5% significance level.

Results

The values for average surface roughness were statistically different (p<0.001) among cements at both baseline and after storage. The roughness values of cements in a decreasing order were ZP>RMGIC>C>R (p<0.001). After 15 days, immersion in lactic acid solution resulted in the highest surface roughness for all cements (p<0.05), except for the RC group (p>0.05). Compared to the current threshold (0.2 µm) related to biofilm accumulation, both RC and C remained below the threshold, even after acidic challenge by immersion in lactic acid solution.

Conclusions

Storage time and immersion in lactic acid solution increased the surface roughness of the majority of the tested cements. RC presented the smoothest surface and it was not influenced by storage conditions.     

Nebivolol attenuates redox-sensitive glomerular and tubular mediated proteinuria in obese rats

Obesity and insulin resistance-related proteinuria is associated with oxidative stress and impaired tissue bioavailable nitric oxide. Recent data suggest that nicotinamide adenine dinucleotide phosphate oxidase-mediated oxidative injury to the proximal tubule, like that seen in the glomerulus, contributes to proteinuria in insulin-resistant states. The vasodilator β-blocker nebivolol reduces nicotinamide adenine dinucleotide phosphate oxidase activity, increases bioavailable nitric oxide, and improves insulin sensitivity. To test the hypothesis that a treatment strategy that reduces oxidative stress and attenuates obesity-associated increases in glomerular and proximal tubule derived protein, we treated young Zucker obese (ZO) and age-matched Zucker lean male rats with nebivolol (10 mg · kg(-1) · d(-1)) for 21 d. Compared with Zucker lean, ZO controls exhibited increased proteinuria and γ-glutamyl transpeptidase, reductions in systemic insulin sensitivity in association with increased renal renin, (pro)renin receptor, angiotensin II type 1 receptor, and mineralocorticoid receptor immunostaining, oxidative stress, and glomerular tubular structural abnormalities that were substantially improved with in vivo nebivolol treatment. Nebivolol treatment also led to improvements in glomerular podocyte foot-process effacement and improvement in podocyte-specific proteins (nephrin and synaptopodin) as well as proximal tubule-specific proteins (megalin and lysosomal-associated membrane protein-2) and proximal tubule ultrastructural remodeling in the ZO kidney. Our findings support the notion that obesity and insulin resistance lead to increased glomerulotubular oxidative stress and resultant glomerular and tubular sources of excess urine protein. Furthermore, the results of this study suggest the beneficial effect of nebivolol on proteinuria was derived from improvements in weight and insulin sensitivity and reductions in renal oxidative stress in a state of obesity and insulin resistance.     

Contribution of oxidative stress to pulmonary arterial hypertension

Recent data implicate oxidative stress as a mediator of pulmonary hypertension (PH) and of the associated pathological changes to the pulmonary vasculature and right ventricle (RV). Increases in reactive oxygen species (ROS), altered redox state, and elevated oxidant stress have been demonstrated in the lungs and RV of several animal models of PH, including chronic hypoxia, monocrotaline toxicity, caveolin-1 knock-out mouse, and the transgenic Ren2 rat which overexpresses the mouse renin gene. Generation of ROS in these models is derived mostly from the activities of the nicotinamide adenine dinucleotide phosphate oxidases, xanthine oxidase, and uncoupled endothelial nitric oxide synthase. As disease progresses circulating monocytes and bone marrow-derived monocytic progenitor cells are attracted to and accumulate in the pulmonary vasculature. Once established, these inflammatory cells generate ROS and secrete mitogenic and fibrogenic cytokines that induce cell proliferation and fibrosis in the vascular wall resulting in progressive vascular remodeling. Deficiencies in antioxidant enzymes also contribute to pulmonary hypertensive states. Current therapies were developed to improve endothelial function, reduce pulmonary artery pressure, and slow the progression of vascular remodeling in the pulmonary vasculature by targeting deficiencies in either NO (PDE-type 5 inhibition) or PGI(2) (prostacyclin analogs), or excessive synthesis of ET-1 (ET receptor blockers) with the intent to improve patient clinical status and survival. New therapies may slow disease progression to some extent, but long term management has not been achieved and mortality is still high. Although little is known concerning the effects of current pulmonary arterial hypertension treatments on RV structure and function, interest in this area is increasing. Development of therapeutic strategies that simultaneously target pathology in the pulmonary vasculature and RV may be beneficial in reducing mortality associated with RV failure.     

Clinical and radiographic evaluation of pulpotomies performed under intrapulpal injection of anaesthetic solution

AIM: The purpose of this study was to evaluate clinically and radiographically pulpotomies carried out under intrapulpal injection of anaesthetic solution. METHODOLOGY: Forty-one permanent mandibular molar teeth presenting with deep carious lesions and/or exposed pulps, with or without periapical changes on radiographic examination, were treated with pulpotomy and dressed with calcium hydroxide. The teeth were divided into three groups. Group A consisted of 15 teeth, where intrapulpal anaesthesia was administered by a slow injection of lidocaine hydrochloride 2%. Group B, with 14 teeth, where intrapulpal anaesthesia was obtained with lidocaine hydrochloride 2% with adrenaline 1:100,000. Group C consisted of 12 teeth in which anaesthesia was performed with a mandibular block using prilocaine hydrochloride 3% with felypressin 1: 100,000. Healing was evaluated using clinical and radiographic criteria: dentine barrier formation, absence of clinical symptoms and resolution of periapical involvement. RESULTS: After an observation time of 6-8 weeks (postoperative control) and 24-32 weeks (intermediate control), healing occurred in 13 teeth from group A (87%), in 11 teeth from group B (79%) and in 10 teeth from group C (83%). No statistical difference was demonstrated between the three groups (Fisher's exact test). CONCLUSIONS: Based on the methodology adopted, intrapulpal injection of anaesthetic solution did not impair healing in pulpotomized teeth.