Outer Membrane Vesicle Proteome of Porphyromonas gingivalis Is Differentially Modulated Relative to the Outer Membrane in Response to Heme Availability.

Porphyromonas gingivalis is an anaerobic, Gram-negative oral pathogen associated with chronic periodontitis. P. gingivalis has an obligate requirement for heme, which it obtains from the host. Heme availability has been linked to disease initiation and progression. In this study we used continuous culture of the bacterium to determine the effect of heme limitation and excess on the P. gingivalis proteome. Four biological replicates of whole cell lysate (WCL) and outer membrane vesicle (OMV) samples were digested with trypsin and analyzed by tandem mass spectrometry and MaxQuant label-free quantification. In total, 1211 proteins were quantified, with 108 and 49 proteins significantly changing in abundance more than 1.5-fold ( p < 0.05) in the WCLs and OMVs, respectively. The proteins most upregulated in response to heme limitation were those involved in binding and transporting heme, whereas the four proteins most upregulated under the heme-excess condition constitute a putative heme efflux system. In general, the protein abundance ratios obtained for OMVs and WCLs agreed, indicating that changes to the OM protein composition are passed onto OMVs; however, 16 proteins were preferentially packaged into OMVs under one condition more than the other. In particular, moonlighting cytoplasmic proteins were preferentially associated with OMVs under heme excess.

Detecting Predeath Grief in Family Caregivers of Persons With Dementia: Validity and Utility of the Marwit-Meuser Caregiver Grief Inventory in a Multiethnic Asian Population.

Background and Objectives: Coping with predeath grief (PDG) is an unmet need in caregivers of persons with dementia (PWD). The Marwit-Meuser Caregiver Grief Inventory (MM-CGI) and its abbreviated MM-CGI-Short-Form (MM-CGI-SF) are among the few empirically developed scales that detect PDG, yet they have not been substantially validated outside United States. We evaluated the reliability and validity of the PDG scales in a multiethnic Asian population distinct from that of United States. Research Design and Methods: Family caregivers of community-dwelling PWD (n = 300) completed self-administered questionnaires containing MM-CGI and other scales of related construct. Sixty percent of the participants repeated the questionnaires 1 week later for test-retest reliability. Internal-consistency reliability was assessed by Cronbach's α, test-retest reliability by intraclass-correlation-coefficient, construct validity by Spearman's correlation-coefficient, and factorial validity by confirmatory factor analysis (CFA). Cohen's κ was used to compare the agreement between MM-CGI and a commonly-used caregiver burden scale (Zarit Burden Interview). Results: MM-CGI and MM-CGI-SF demonstrated internal-consistency reliability, test-retest reliability, construct validity, and known-group validity. In CFA, MM-CGI showed modest model-fit (comparative-fit-index, CFI = .80; Tucker-Lewis-index, TLI = .79), whereas MM-CGI-SF showed better model-fit (CFI = .91; TLI = .90). Eighty-six percent of the caregivers reported average or high levels of PDG, with 18% reporting high PDG. High scores in the caregiver burden scale only showed modest agreement with high scores in MM-CGI (κ = .47). Discussion and Implications: MM-CGI and MM-CGI-SF demonstrated adequate psychometric properties and utility, beyond that of a caregiver burden scale, in detecting high PDG in a multiethnic Asian population. They open the way for PDG intervention in clinical care, as well as further exploration in caregiver research.

Effect of azithromycin on a red complex polymicrobial biofilm.

Azithromycin has recently gained popularity for the treatment of periodontal disease, despite sparse literature supporting efficiency in treating periodontal bacterial biofilms. The aim of this study was to evaluate the effect of azithromycin on biofilms comprised of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in comparison to an amoxicillin and metronidazole combination. P. gingivalis W50, T. denticola ATCC35405, and T. forsythia ATCC43037 grown under anaerobic conditions at 37°C were aliquoted into 96-well flat-bottom plates in different combinations with addition of azithromycin or amoxicillin + metronidazole at various concentrations. For the biofilm assay, the plates were incubated at 37°C anaerobically for 48 h, after which the biofilms were stained with crystal violet and measured for absorbance at AU620. In this model, polymicrobial biofilms of P. gingivalis + T. denticola, P. gingivalis + T. forsythia, and T. denticola + T. forsythia were cultured. Combination of all three bacteria enhanced biofilm biomass. Azithromycin demonstrated a minimal biofilm inhibitory concentration (MBIC) of 10.6 mg/L, while the amoxicillin + metronidazole combination was more effective in inhibiting biofilm formation with a MBIC of 1.63 mg/L. Polymicrobial biofilm formation was demonstrated by combination of all three red complex bacteria. Azithromycin was ineffective in preventing biofilm formation within a clinically achievable concentration, whereas the combination of amoxicillin and metronidazole was more effective for this purpose.

Hemangiopericytoma of the thoracic spine: a case report.

Hemangiopericytoma (HPC) has been described to be aggressive and potentially a malignant tumour. We report a rare case of a 63-year-old Chinese male who presented with primary intradural extramedullary HPC of the thoracic spine. The main presenting complaint was gradual progression of back pain, associated with paraparesis and sensory deficit of lower limbs. He had MRI thoracolumbar with contrast which showed T9 lesion compressing on spinal cord and oedema, he was then operated upon and histopathology report confirmed a thoracic spine HPC. A T8/9 laminectomy and excision of intradural extramedullary lesion was performed, tumour section was sent for frozen section study, and more tissue was sent for paraffin studies and additional immunohistochemical staining. Surgical resection is most commonly performed, radiotherapy remains debatable. In this report, we discussed another rare case of primary spinal HPC to be added into the literature.

The potential acidogenicity of liquid breakfasts.

OBJECTIVES: To determine the potential acidogenicy of liquid breakfasts. METHODS: In vitro acid production by Streptococcus mutans was measured in the beverages at a pH of 5.5, as was the fall in pH over 10min. The buffering capacity was determined, as well as the calcium, inorganic phosphate and fluoride concentrations (total and soluble) of the beverages. Bovine milk (UHT) was used for comparison. RESULTS: The rate of acid production by S. mutans, and pH fall over 10min was greater in liquid breakfasts compared to bovine milk. All beverages except one demonstrated a significantly lower buffering capacity than bovine milk. All beverages contained significantly greater concentrations of soluble calcium than bovine milk, and all except two contained significantly more soluble inorganic phosphate. CONCLUSIONS: S. mutans was able to generate significantly more acid in the liquid breakfasts than in bovine milk, indicating these drinks may contribute to a cariogenic diet. In general, the liquid breakfasts required significantly less acid than bovine milk to reduce their pH to the approximate critical pH for enamel demineralisation. However, the liquid breakfasts also tended to contain significantly more soluble calcium and inorganic phosphate than bovine milk. CLINICAL SIGNIFICANCE: The substantial amounts and various types of sugars found within liquid breakfast beverages may result in a significant pH drop in dental plaque following consumption of these products.

Synthesis and evaluation of bisbenzylidenedioxotetrahydrothiopranones as activators of endoplasmic reticulum (ER) stress signaling pathways and apoptotic cell death in acute promyelocytic leukemic cells.

Curcumin is known to trigger ER-stress induced cell death of acute promyelocytic leukemic (APL) cells by intercepting the degradation of nuclear co-repressor (N-CoR) protein which has a key role in the pathogenesis of APL. Replacing the heptadienedione moiety of curcumin with a monocarbonyl cross-conjugated dienone embedded in a tetrahydrothiopyranone dioxide ring resulted in thiopyranone dioxides that were more resilient to hydrolysis and had greater growth inhibitory activities than curcumin on APL cells. Several members intercepted the degradation of misfolded N-CoR and triggered the signaling cascade in the unfolded protein response (UPR) which led to apoptotic cell death. Microarray analysis showed that genes involved in protein processing pathways that were germane to the activation of the UPR were preferentially up-regulated in treated APL cells, supporting the notion that the UPR was a consequential mechanistic pathway affected by thiopyranone dioxides. The Michael acceptor reactivity of the scaffold may have a role in exacerbating ER stress in APL cells.

Porphyromonas gingivalis and Treponema denticola exhibit metabolic symbioses.

Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using 13C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.

Functionalized curcumin analogs as potent modulators of the Wnt/ß-catenin signaling pathway.

Osteosarcoma is a primary bone malignancy with aggressive metastatic potential and poor prognosis rates. In our earlier work we have investigated the therapeutic potential of curcumin as an anti-invasive agent in osteosarcoma by its ability to regulate the Wnt/ß-catenin signaling pathway. However, the clinical use of curcumin is limited owing to its low potency and poor pharmacokinetic profile. In this study, an attempt was made to achieve more potent Wnt inhibitory activity in osteosarcoma cells by carrying out synthetic chemical modifications of curcumin. We synthesized a total of five series consisting of 43 curcumin analogs and screened in HEK293T cells for inhibition of ß-catenin transcriptional activity. Six promising analogs, which were 6.5- to 60-fold more potent than curcumin in inhibiting Wnt activity, were further assessed for their anti-invasive activity and Wnt inhibitory mechanisms. Western blot analysis showed disruption of ß-catenin protein nuclear translocation following treatment with analogs 2f, 3c and 4f. Using transwell assays, we also found that these compounds were more potent than 1a (curcumin) in impeding the invasion of osteosarcoma cells, possibly through suppressing MMP-9 activity. Structure-activity-relationship studies revealed that Wnt inhibitory effects could be enhanced by shortening and restraining the flexibility of the 7-carbon linker moiety connecting the terminal aromatic rings of curcumin and substituting both rings with appropriate substituents. Our results demonstrate that the synthesized curcumin analogs are more potent Wnt inhibitors in osteosarcoma cell lines as compared to parental curcumin and are good lead compounds for further development. Future in vivo tests with these compounds will define their therapeutic potentials as promising drug candidates for clinical treatment of osteosarcoma.

Infratentorial benign cystic meningioma mimicking a hemangioblastoma radiologically and a pilocytic astrocytoma intraoperatively: a case report.

INTRODUCTION: Cystic meningiomas are rare variants of meningiomas; they can pose a radiological diagnostic dilemma. CASE PRESENTATION: We present a rare case of a 30-year-old Chinese woman with a histopathological diagnosis of infratentorial cystic meningioma (World Health Organization Grade 1) in which the features in imaging modalities were suggestive of a hemangioblastoma. Intraoperatively, however, the gross macroscopic features were more in keeping with a pilocytic astrocytoma. CONCLUSION: In benign cystic meningiomas, particularly the infratentorial variety, radiological findings utilizing the various imaging modalities and intraoperative impressions may not be reflective of or in keeping with the final histopathological diagnosis.

Nfil3 is a glucocorticoid-regulated gene required for glucocorticoid-induced apoptosis in male murine T cells.

Glucocorticoids (GCs) have essential roles in the regulation of development, integrated metabolism, and immune and neurological responses, and act primarily via the glucocorticoid receptor (GR). In most cells, GC treatment results in down-regulation of GR mRNA and protein levels via negative feedback mechanisms. However, in GC-treated thymocytes, GR protein levels are maintained at a high level, increasing sensitivity of thymocytes to GCs, resulting in apoptosis termed glucocorticoid-induced cell death (GICD). CD4(+)CD8(+) double-positive thymocytes and thymic natural killer T cells in particular are highly sensitive to GICD. Although GICD is exploited via the use of synthetic GC analogues in the treatment of hematopoietic malignancies, the intracellular molecular pathway of GICD is not well understood. To explore GICD in thymocytes, the authors performed whole genome expression microarray analysis in mouse GR exon 2 null vs wild-type thymus RNA 3 hours after dexamethasone treatment. Identified and validated direct GR targets included P21 and Bim, in addition to an important transcriptional regulator Nfil3, which previously has been associated with GICD and is essential for natural killer cell development in vivo. Immunostaining of NFIL3 in whole thymus localized NFIL3 primarily to the medullary region, and double labeling colocalized NFIL3 to apoptotic cells. In silico analysis revealed a putative GC response element 5 kb upstream of the Nfil3 promoter that is strongly conserved in the rat genome and was confirmed to bind GR by chromatin immunoprecipitation. The knockdown of Nfil3 mRNA levels to 20% of normal using specific small interfering RNAs abrogated GICD, indicating that NFIL3 is required for normal GICD in CTLL-2 T cells.

Spinal cavernous malformations: magnetic resonance imaging and associated findings.

INTRODUCTION: We reviewed the clinical features, brain and spinal cord magnetic resonance (MR) imaging findings and associated abnormalities in six patients with spinal cavernous malformations (CMs). METHODS: Lesions were defined on gradient-recalled echo (GRE) images but measured on T2-weighted images performed on 1.5- and 3-tesla clinical scanners. RESULTS: Four patients had associated multiple cranial CMs and one patient had multiple spinal CMs. All spinal CMs were predominantly hypointense on GRE images, and most were predominantly hyperintense and surrounded by hypointense edge on T2-weighted images. Other associations included asymptomatic vertebral body and splenic haemangiomas. CONCLUSION: We conclude that intramedullary spinal CMs typically have 'mulberry' or 'popcorn' appearances similar to those of cranial CM. The presence of associated haemangioma or familial cranial CM syndrome on MR imaging may suggest the correct diagnosis without requiring invasive investigations.

Curcumin analogues with potent and selective anti-proliferative activity on acute promyelocytic leukemia: involvement of accumulated misfolded nuclear receptor co-repressor (N-CoR) protein as a basis for selective activity.

Curcumin arrests the proliferation of acute promyelocytic leukemia (APL) cells by stabilizing the misfolded nuclear receptor co-repressor (N-CoR) protein, thereby sensitizing APL cells to apoptosis induced by the unfolded protein response. This phenomenon was attributed to inhibition of the proteasomal and protease-induced breakdown of misfolded N-CoR by curcumin. Curcumin is, however, a modest inhibitor and affected the viability of APL cells at micromolar concentrations. Modifying curcumin at its conjugated ß-diketone linker and terminal phenyl rings yielded potent congeners with sub-micromolar growth inhibitory activities which selectively kill APL cells over non-APL leukemic and nonmalignant cells. Analogues with pronounced APL-selective anti-proliferative activities, as observed in representative dibenzylidenecyclohexanones and dibenzylidenecyclopentanones, strongly promoted the accumulation of misfolded and nonfunctional N-CoR at significantly lower concentrations than their growth inhibitory IC(50) values. These compounds also inhibited the human 20S proteasome in an enzyme-based assay, thus providing convincing support for the prevailing hypothesis that impeding the degradation of N-CoR is a key mechanistic event contributing to APL cell death.

Differential proteomic analysis of a polymicrobial biofilm.

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H2¹6O/H2¹8O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.

cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

The cAMP response element binding protein 1 (Creb1) transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/-) fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/-) fetal mice during development. Lungs of Creb1(-/-) fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/-) lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/-) lung on a Crem(-/-) genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/-) lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara), and neuroendocrine cells showed delayed onset of expression in the Creb1(-/-) lung. Finally, gene expression analyses of the E17.5 Creb1(-/-) lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

Glucocorticoids stimulate hepatic and renal catecholamine inactivation by direct rapid induction of the dopamine sulfotransferase Sult1d1.

During the stress response and metabolic fasting, glucocorticoids acting via the glucocorticoid receptor (GR) stimulate hepatic glucose production by activating specific gluconeogenic enzyme target genes. To characterize novel direct GR-regulated hepatic target genes under glucocorticoid control, we performed a whole genome gene expression microarray using dexamethasone-treated GR-null mice. Strongly induced previously characterized genes included phosphoenolpyruvate carboxykinase, serine dehydratase, tyrosine oxygenase, lipin 1, metallothionine, and cdkn1A. Novel induced genes included Ddit4, Fkbp5, Megf9, Sult1e1, and Sult1d1, and all were verified by real-time PCR. Sult1d1, a sulfotransferase, is a member of a large superfamily of detoxification enzymes and has an important role in the inactivation of endogenous dopamine-derived compounds, including the catecholamines. Treatment of primary mouse hepatocytes with dexamethasone for 6 h dramatically increased Sult1d1 mRNA levels, whereas cotreatment with RU-486, a GR antagonist, blocked induction by dexamethasone. Sult1d1 mRNA levels were also increased by dexamethasone in the kidney, a major site of Sult1d1 synthesis. Sult1d1 mRNA was localized by in situ hybridization to renal collecting ducts and was rapidly induced by glucocorticoids in renal inner medullary collecting duct (IMCD3) cells. Hepatic and renal Sult1d1 enzymatic activity was significantly induced in vivo in wild-type mice 6 h after dexamethasone treatment. Chromatin immunoprecipitation assay analysis upstream of the Sult1d1 gene promoter identified a glucocorticoid response element close to the neighboring glucocorticoid-responsive estrogen sulfotransferase Sult1e1 gene, indicating that both genes potentially share a common glucocorticoid response element. These results suggest that Sult1d1 in mice is directly induced by glucocorticoids and may attenuate elevated catecholamine activity during the stress response.

Treponema denticola biofilm-induced expression of a bacteriophage, toxin-antitoxin systems and transposases.

Treponema denticola is an oral spirochaete that has been strongly associated with chronic periodontitis. The bacterium exists as part of a dense biofilm (subgingival dental plaque) accreted to the tooth. To determine T. denticola gene products important for persistence as a biofilm we developed a continuous-culture biofilm model and conducted a genome-wide transcriptomic analysis of biofilm and planktonic cells. A total of 126 genes were differentially expressed with a fold change of 1.5 or greater. This analysis identified the upregulation of putative prophage genes in the T. denticola 35405 genome. Intact bacteriophage particles were isolated from T. denticola and circular phage DNA was detected by PCR analysis. This represents the first, to our knowledge, functional bacteriophage isolated from T. denticola, which we have designated varphitd1. In biofilm cells there was also an upregulation of genes encoding several virulence factors, toxin-antitoxin systems and a family of putative transposases. Together, these data indicate that there is a higher potential for genetic mobility in T. denticola when growing as a biofilm and that these systems are important for the biofilm persistence and therefore virulence of this bacterium.

Identification of glucocorticoid-regulated genes that control cell proliferation during murine respiratory development.

Glucocorticoids play a vital role in fetal respiratory development and act via the intracellular glucocorticoid receptor (GR) to regulate transcription of key target genes. GR-null mice die at birth due to respiratory dysfunction associated with hypercellularity and atelectasis. To identify events associated with this lung phenotype we examined perinatal cellular proliferation rates and apoptotic indices. We demonstrate that compared to wild-type controls, day 18.5 postcoitum (p.c.) GR-null mouse lungs display significantly increased cell proliferation rates (1.8-fold P < 0.05) and no change in apoptosis. To examine underlying molecular mechanisms, we compared whole genome expression profiles by microarray analysis at 18.5 days p.c. Pathways relating to cell proliferation, division and cell cycle were significantly down-regulated while pathways relating to carbohydrate metabolism, kinase activities and immune responses were significantly up-regulated. Differential levels of gene expression were verified by quantitative-RT-PCR and/or Northern analysis. Key regulators of proliferation differentially expressed in the lung of 18.5 p.c. GR-null lungs included p21 CIP1 (decreased 2.9-fold, P < 0.05), a negative regulator of the cell cycle, and Mdk (increased 6.0-fold, P < 0.05), a lung growth factor. The more under-expressed genes in 18.5 p.c. GR-null lungs included Chi3l3 (11-fold, P < 0.05), a macrophage inflammatory response gene and Ela1 (9.4-fold, P < 0.05), an extracellular matrix remodeling enzyme. Our results demonstrate that GR affects the transcriptional status of a number of regulatory processes during late fetal lung development. Amongst these processes is cell proliferation whereby GR induces expression of cell cycle repressors while suppressing induction of a well characterized cell cycle stimulator.

Gender dictates the nuclear receptor-mediated regulation of CYP3A44.

The CYP3As are broad-spectrum drug-metabolizing enzymes that are collectively responsible for more than 50% of xenobiotic metabolism. Unlike other CYP3As, murine CYP3A44 is expressed predominantly in the female liver, with much lower levels in male livers and no detectable expression in brain or kidney in either gender. In this study, we examined the role of nuclear hormone receptors in the regulation of Cyp3a44 gene expression. Interestingly, we observed differential effects of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) -mediated activation of Cyp3a44 gene expression, which was gender-specific. For example, activation of PXR by pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone (DEX) induced CYP3A44 mRNA levels in a PXR-dependent fashion in male mice, whereas no induction was detected in female mice. In contrast, PCN and DEX down-regulated CYP3A44 expression in female PXR null animals. Similar to PXR, CAR activation also showed a male-specific induction with no effect on CYP3A44 levels in females. When PXR knockout mice were challenged with the CAR activator phenobarbital, a significant up-regulation of male CYP3A44 levels was observed, whereas levels in females remained unchanged. We conclude that gender has a critical impact on PXR- and CAR-mediated effects of CYP3A44 expression.