Effects of pre-mixed hydraulic calcium silicate putties on osteogenic differentiation of human dental pulp stem cells in vitro.

OBJECTIVES: An experimental tricalcium silicate and dicalcium silicate-containing endodontic putty has been designed to overcome the issue of reduced shelf life after exposure to atmospheric moisture during repeated opening of the container for clinical retrieval. The present study examined the effects of this experimental hydraulic putty on the mineralogenic characteristics of osteogenic lineage-committed human dental pulp stem cells (hDPSCs), by comparing the cellular responses with a commercially available putty (EndoSequence BC RRM Putty). METHODS: The osteogenic potential of hDPSCs that had been exposed to the putties was examined using quantitative reverse-transcription polymerase chain reaction for osteogenic gene expressions and western blot for osteogenic protein expressions. Alkaline phosphatase activity assay and alizarin red S staining were performed to detect changes in production of the intracellular enzyme and extracellular matrix mineralization respectively. RESULTS: Osteogenic differentiation of the hDPSCs was significantly enhanced after exposure to the pre-mixed hydraulic putties, with no significant difference between these two examined putties. CONCLUSIONS: The experimental hydraulic tricalcium silicate putty enhances osteogenic differentiation of hDPSCs to the same extent as a commercially available tricalcium silicate putty. CLINICAL SIGNIFICANCE: The experimental hydraulic putty appears to be an alternative to the commercial putty when used for applications involving the regeneration of bone in endodontics. Animal models are required for validating its potential in enhancing osteogenesis in vivo.

Retrospective study on idiopathic bone cavity and its association with cementoosseous dysplasia.

OBJECTIVE: The goal of this study was to reveal clinical and pathologic findings on idiopathic bone cavity lesions (IBC). STUDY DESIGN: A retrospective analysis of 20 IBC cases diagnosed from 2004 to 2014 from a university-based maxillofacial pathology service was performed and included all pertinent clinical, histologic, and radiographic findings. RESULTS: Eleven women (age = 36 ± 12.7) and 9 men (age = 23 ± 17.9) diagnosed with IBC were selected for analysis. There was a higher African-American female predilection (40%). Thirty percent of the cases were associated with florid cementoosseous dysplasia (COD) (all middle-aged African-American women). The location of the lesions was mandibular in 85% of the patients. All symptomatic patients (25%) had concomitant COD. Only 1 patient reported previous trauma, and only 1 patient had prior orthodontic treatment. Follow-up period ranged from 1 to 8 years, with only 1 recurrence 3 years after surgery. CONCLUSIONS: The results suggest that IBC concurrent with COD may not be as rare as the literature implies. Clinicians must be attentive to this possible relationship to ensure accurate diagnosis and appropriate treatment.

Knock in of the AKT1 E17K mutation in human breast epithelial cells does not recapitulate oncogenic PIK3CA mutations.

An oncogenic mutation (G49A:E17K) in the AKT1 gene has been described recently in human breast, colon, and ovarian cancers. The low frequency of this mutation and perhaps other selective pressures have prevented the isolation of human cancer cell lines that harbor this mutation thereby limiting functional analysis. Here, we create a physiologic in vitro model to study the effects of this mutation by using somatic cell gene targeting using the nontumorigenic human breast epithelial cell line, MCF10A. Surprisingly, knock in of E17K into the AKT1 gene had minimal phenotypic consequences and importantly, did not recapitulate the biochemical and growth characteristics seen with somatic cell knock in of PIK3CA hotspot mutations. These results suggest that mutations in critical genes within the PI3-kinase (PI3K) pathway are not functionally equivalent, and that other cooperative genetic events may be necessary to achieve oncogenic PI3K pathway activation in cancers that contain the AKT1 E17K mutation.

Kaposi's sarcoma: a model of both malignancy and chronic inflammation.

Kaposi's sarcoma (KS) is a complex cancer characterized by angioproliferative multifocal tumors of the skin, mucosa and viscera. KS lesions are comprised of both distinctive spindle cells of endothelial origin and a variable inflammatory infiltrate. There are four different epidemiological forms of KS: classic (sporadic), African (endemic), AIDS-associated (epidemic), and immunosupression-associated (iatrogenic). Although these various forms of KS have different environmental and immunological components, the development of each depends upon infection with Kaposi's sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV8). KSHV encodes an arsenal of gene products that induce cellular proliferation, transformation, cell signaling, cytokine production, immune evasion, antiapoptosis and angiogenesis. Yet, KSHV alone is insufficient to give rise to KS. The exact origin of the tumor cell (spindle cell), which is generally agreed to be a type of endothelial cell, remains elusive. Current evidence supports their derivation from lymphatic endothelium. However, both lymphatic and vascular endothelial cell types can be infected by KSHV in vitro, and recent studies suggest that this virus may reprogram the target cell, thus masking the cell's true origin. It is also possible that the original target cell is an uncommitted progenitor. In addition to the potentially neoplastic spindle cells, the KS lesion also contains dendritic cells, macrophages, plasma cells and lymphocytes. The presence of this admixed immune infiltrate has led to the suggestion that KS may result from reactive hyperproliferation induced by chronic inflammation, and that it is therefore not a true neoplasm. This review details the data that support KS as a model of both oncogenesis and chronic inflammation.

[Central apnea developing during treatment with a mandibular advancement device]. / Apnées centrales induites sous traitement par orthèse d'avancée mandibulaire.

INTRODUCTION: Mandibular advancement (MA) has emerged over the last decade as an alternative solution to nasal continuous airway pressure (nCPAP) for the treatment of obstructive sleep apnea syndrome (OSAS). OBSERVATION: We report the case of a patient with history of chronic atrial fibrillation and moderate supine-dependent OSAS in whom central sleep apneas developed during treatment by a bi-bloc MA device. Central apneas increased with the level of MA and preferentially occurred in the supine position. We hypothesized that mouth opening under excessive mandibular advancement in supine position may have led to pharyngeal narrowing at the base of the tongue and potentially unstable ventilation. Sleep fragmentation that enhanced during progressive MA may also have compromised ventilatory control stability in our patient. Finally, chronic atrial fibrillation may have predisposed to central sleep apneas. CONCLUSION: Our case report highlights the importance of follow-up nocturnal recordings during progressive MA.

Morphological differences between Saturn's ultraviolet aurorae and those of Earth and Jupiter.

It has often been stated that Saturn's magnetosphere and aurorae are intermediate between those of Earth, where the dominant processes are solar wind driven, and those of Jupiter, where processes are driven by a large source of internal plasma. But this view is based on information about Saturn that is far inferior to what is now available. Here we report ultraviolet images of Saturn, which, when combined with simultaneous Cassini measurements of the solar wind and Saturn kilometric radio emission, demonstrate that its aurorae differ morphologically from those of both Earth and Jupiter. Saturn's auroral emissions vary slowly; some features appear in partial corotation whereas others are fixed to the solar wind direction; the auroral oval shifts quickly in latitude; and the aurora is often not centred on the magnetic pole nor closed on itself. In response to a large increase in solar wind dynamic pressure Saturn's aurora brightened dramatically, the brightest auroral emissions moved to higher latitudes, and the dawn side polar regions were filled with intense emissions. The brightening is reminiscent of terrestrial aurorae, but the other two variations are not. Rather than being intermediate between the Earth and Jupiter, Saturn's auroral emissions behave fundamentally differently from those at the other planets.

The PTEN tumor suppressor protein inhibits tumor necrosis factor-induced nuclear factor kappa B activity.

Nuclear factor kappaB (NF-kappaB) transcriptionally activates genes that promote immunity and cell survival. Activation of NF-kappaB is induced by an IkappaB kinase (IKK) complex that phosphorylates and promotes dissociation of IkappaB from NF-kappaB, which then translocates into the nucleus. Activation of phosphatidylinositol (PI) 3-kinase/Akt signaling by tumor necrosis factor (TNF) activates IKK and NF-kappaB. The present study shows that PTEN, a tumor suppressor that inhibits PI 3-kinase function, impairs TNF activation of Akt and the IKK complex in 293 cells. Transient expression of PTEN suppressed IKK activation and TNF-induced NF-kappaB DNA binding and transactivation. Studies were conducted with PC-3 prostate cancer cells that do not express PTEN and DU145 prostate cancer cells that express PTEN. TNF activated Akt in PC-3 cells, but not in DU145 cells, and the ability of TNF to activate NF-kappaB was blocked by pharmacological inhibition of PI 3-kinase activity in PC-3 cells, but not in DU145 cells. Expression of PTEN in PC-3 cells to a level comparable with that endogenously present in DU145 cells inhibited TNF activation of NF-kappaB. The cell type-specific ability of PTEN to negatively regulate the PI 3-kinase/AKT/NF-kappaB pathway may be important to its tumor suppressor activity.

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1.

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation.

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.

NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase.

Activation of the nuclear transcription factor NF-kappaB by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and an IKB-kinase (IKK) complex composed of IKKalpha and IKKbeta. Here we show that the Akt serine-threonine kinase is involved in the activation of NF-kappaB by tumour necrosis factor (TNF). TNF activates phosphatidylinositol-3-OH kinase (PI(3)K) and its downstream target Akt (protein kinase B). Wortmannin (a PI(3)K inhibitor), dominant-negative PI(3)K or kinase-dead Akt inhibits TNF-mediated NF-kappaB activation. Constitutively active Akt induces NF-kappaB activity and this effect is blocked by dominant-negative NIK. Conversely, NIK activates NF-kappaB and this is blocked by kinase-dead Akt. Thus, both Akt and NIK are necessary for TNF activation of NF-kappaB. Akt mediates IKKalpha phosphorylation at threonine 23. Mutation of this amino acid blocks phosphorylation by Akt or TNF and activation of NF-kappaB. These findings indicate that Akt is part of a signalling pathway that is necessary for inducing key immune and inflammatory responses.

Pyruvate and hydroxycitrate/carnitine may synergize to promote reverse electron transport in hepatocyte mitochondria, effectively 'uncoupling' the oxidation of fatty acids.

In a recent pilot study, joint administration of pyruvate, hydroxycitrate (HCA), and carnitine to obese subjects was associated with a remarkable rate of body-fat loss and thermogenesis, strongly suggestive of uncoupled fatty-acid oxidation. Hepatocytes possess an uncoupling mechanism--reverse electron transport--that enables fasting ketogenesis to proceed independent of respiratory control. Electrons entering the respiratory chain at the coenzyme Q (CoQ) level via FAD-dependent acyl coA dehydrogenase, can be driven 'up' the chain by the electrochemical proton gradient to reduce NAD+; if these electrons are then shuttled to the cytoplasm, returning to the respiratory chain at the CoQ level, the net result is heat generation at the expense of the proton gradient, enabling the uncoupled flow of electrons to oxygen. Pyruvate's bariatric utility may stem from its ability to catalyze the rapid transport of high-energy electrons from mitochondria to the cytoplasm, thus stimulating electron shuttle mechanisms. By enabling rapid mitochondrial uptake of fatty acids and thus disinhibiting hepatocyte ketogenesis, HCA/carnitine should initiate reverse electron transport: concurrent amplification of electron shuttle mechanisms by pyruvate can be expected to accelerate this reverse electron transport, thereby decreasing the electrochemical proton gradient. As a result, hepatocytes may be able to convert fatty acids to CO2 and heat with little net generation of ATP. These considerations suggest that it may be feasible to render hepatocytes functionally equivalent to activated brown fat, such that stored fat can be selectively oxidized in the absence of caloric restriction. Other measures which enhance the efficiency of hepatocyte electron shuttle mechanisms may increase the efficacy of this strategy.

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides.

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.

Expression of CD54 (intercellular adhesion molecule-1) and the beta 1 integrin CD29 is modulated by a cyclic AMP dependent pathway in activated primary rat microglial cell cultures.

Microglial cell activation plays a central role in acute and chronic inflammatory processes associated with neurodegeneration. As macrophage activation is generally associated with the up-regulation of specific surface antigens, the expression of CD54, and CD29 were evaluated on CD11b positive neonatal rat microglial cell cultures by flow cytometry. These cells when exposed to lipopolysaccharide, LPS, and gamma interferon, IFN gamma, exhibited a 2-3 fold increase in CD54 expression, an increase in CD29 and no change in CD11b. Maximal increases in CD54 and CD29 staining on CD11b positive microglial cells were apparent 20-24 h after LPS and IFN gamma while nitrite production reflecting inducible nitric oxide synthase activity, continued to increase. The increases in CD29 and CD54 staining were inhibited in a dose dependent manner by agents which increased intracellular cAMP levels including 100 microM 8-bromoadenosine 3':5'-cyclic monophosphate but not 8-bromoadenosine monophosphate, the phosphodiesterase inhibitor isobutyl methylxanthine and by direct activation of adenylate cyclase with forskolin. Concomitant with the dose dependent decreases in CD29 and CD54 staining were increases in intracellular cAMP and reduced TNF secretion. These results suggest that regulation of CD29 and CD54 expression on activated microglial cells involves a cAMP dependent pathway.

Contribution of proteases and LasR to the virulence of Pseudomonas aeruginosa during corneal infections.

The roles of the Pseudomonas aeruginosa proteases LasB (elastase) and LasA and the transcriptional activator LasR, which regulates the expression of these proteases, were evaluated in a murine model of P. aeruginosa corneal infection. In scarified corneas, P. aeruginosa PAO-A1 (LasA negative) or PAO-B1A1 (LasB and LasA negative) at a dose of 10(8) CFU per eye caused very mild or no disease following infection; however, the defect in PAO-A1 could not be complemented by supplying a functional copy of lasA either on a plasmid or inserted into the chromosome. In contrast, PAO-B1 (LasB negative) colonized the cornea and caused disease equal in severity to disease caused by the parental strain, PAO1-I. Although LasR is a known regulator of lasA expression, PAO-R1, a lasR-negative derivative of PAO1-I, was as virulent as the parental strain during corneal infection. When transcriptional fusion plasmids were used to quantify the expression of the lasB and lasA genes in P. aeruginosa PAO1-I and PAO-R1, the lasB::lacZ fusion in PAO-R1 showed only 3.5% as much activity as it did in PAO1-I, while the activity of the lasA::lacZ fusion in PAO-R1 was 27.8% of that in PAO1-I. Coadministration of 5 microg of purified LasA protease with PAO-A1 did not reconstitute a wild-type infection. This treatment produced an acute toxic reaction leading to prolonged eyelid closure without inflammatory destruction of the cornea that was similar to that observed when LasA was administered alone. These results indicate that insertional inactivation of lasA renders P. aeruginosa avirulent in a murine model of keratitis and that neither LasR nor elastase production is required for the establishment and maintenance of corneal infection. However, the lack of virulence of the LasA-deficient strains cannot be ascribed with certainty to the deficiency of LasA from the available data.

A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.

The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasAdelta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.

[Chronotolerance of corticosterone on DNA synthesis in young growing rats]. / Chronotolérance de la corticostérone sur la synthèse du DNA chez le jeune rat en croissance.

Plasma corticosterone and DNA synthesis were measured during 2 circadian periodes following the 25th day of rats. Corticosterone (5 mg/kg) injected at 05 h (1 h before the normal corticosterone bathyphase) inhibits and dissynchronizes the nycthemeral evolution of the two parameters during the two subsequent periods. The same corticosterone administration injected at 17 h (1 h before the normal corticosterone acrophase) inhibits the first DNA synthesis wave but both parameters are nycthemerally restored from the second period. In this last case, the area under the second DNA curve compensates the inhibition of the first wave. The results are discussed in the view of chronocorticotherapy recommended in patients.

Bacterial evasion of the antibody response: human IgG antibodies neutralize soluble but not bacteria-associated group B streptococcal C5a-ase.

Most strains of group B streptococci (GBS) possess an enzyme that inactivates the human anaphylatoxin C5a by cleaving a heptapeptide from the carboxyl terminus of C5a. This enzyme, called GBS C5a-ase, has been purified to homogeneity and cleaves and inactivates C5a in physiologic buffer. The enzymatic activity of soluble C5a-ase is completely inhibited, however, in the presence of plasma or serum from normal human adults. The neutralization of soluble C5a-ase by plasma and serum results largely from naturally occurring IgG antibodies directed against C5a-ase. IgG does not neutralize C5a-ase present on intact encapsulated type III GBS but does neutralize the C5a-ase activity associated with a transposon-induced mutant strain of type III GBS that lacks capsule. The location of GBS C5a-ase on the surface of encapsulated type III GBS permits the C5a-ase to inactivate C5a while evading neutralization by IgG antibodies.

The research-defining accident investigation methodology of the National Institute for Occupational Safety and Health.

The Accident Investigation Methodology (AIM), developed for the National Institute for Occupational Safety and Health through a contract effort by Safety Sciences of San Diego, California, is a composite procedure utilizing many useful concepts and approaches from existing investigative techniques in addition to innovations. The investigation procedure is oriented toward the identification of occupational accident/injury countermeasures and associated research needs. Emphasis is placed upon modern "systems theory," which considers human factors as a primarily intended for knowledgeable safety practitioners with some previous experience. Interviewing techniques and preparations for conducting an investigation are thoroughly addressed. The necessary tools and forms for actual investigation are included, as well as a sophisticated analysis procedure and coding system for the computer accessing of all relevant data.

Long-term mortality experience of steelworkers. VIII. Mortality patterns of open hearth steelworkers (a preliminary report).

The findings in this report of a deficit in mortality from cardiovascular diseases and an excess in diseases of the digestive system among open hearth workers indicate the need for further study of men working in hot environments. In future reports we hope to refine the comparisons by obtaining data which will enable classification of workers more precisely by intensity and duration of exposure within the open hearth. Of particular importance in future work are the evaluation of possible relationships between the actual levels of heat exposure and subsequent morbidity and mortality, as well as possible interactions between heat stress and physical exertion in terms of the incidence of heart disease and other select diseases.