Impact of different regenerative techniques and materials on the healing outcome of endodontic surgery: a systematic review and meta-analysis.

BACKGROUND: Regenerative techniques are increasingly applied in endodontic surgery, but different materials used in regenerative techniques may have varying impacts on wound healing. OBJECTIVES: This study evaluated the effects of different regenerative techniques and materials on the outcome of endodontic surgery. PARTICIPANTS: patients with persistent periapical lesions, treated with root-end surgery. CONTROL: endodontic surgery without the use of regenerative techniques/materials. INTERVENTION: endodontic surgery with the use of regenerative techniques/materials. OUTCOME: combined clinical and radiographic results. METHODS: PubMed, Web of Science, Embase, SinoMed and the CENTRAL Cochrane were searched up to 10th July 2020, followed by a manual search. Detailed eligibility criteria were applied. Cochrane's risk-of-bias tool 2.0 was used to assess the risk of bias of the eligible studies. Meta-analysis was conducted using RevMan software. Subgroup analyses were performed based on the regenerative materials used in endodontic surgery. RESULTS: Eleven eligible randomized controlled trials (RCTs) were included in the meta-analysis: two had a low risk of overall bias, and nine had some concerns of overall bias. Generally, the use of regenerative techniques significantly improved the outcome of endodontic surgery (risk ratio [RR]: 0.42; 95% confidence interval [CI], 0.26-0.68; P < 0.001). On subgroup analysis, the use of expanded polytetrafluoroethylene (e-PTFE) membranes alone had no added benefits (RR: 2.00; 95% CI, 0.22-18.33; P = 0.54). The application of collagen membranes or autologous platelet concentrates (APCs) alone was associated with a trend for better outcomes (RR: 0.51; 95% CI, 0.20-1.25; P = 0.14) (RR: 0.55; 95% CI, 0.18-1.71; P = 0.30). The combined use of collagen membranes and bovine-derived hydroxyapatite significantly improved the outcome (RR: 0.35; 95% CI, 0.17-0.75; P = 0.007). DISCUSSION: This systematic review evaluated the effects of collagen membranes, e-PTFE membranes, APCs and bone grafting materials, providing detailed information about the risks and benefits of using each regenerative technique/material or its combination in endodontic surgery. CONCLUSIONS: Regenerative techniques improve periapical lesion healing after endodontic surgery. The combined use of collagen membranes and bovine-derived hydroxyapatite may be beneficial as an adjunct to endodontic surgery. In contrast, the positive efficacy of e-PTFE membranes or APCs alone remains doubtful.

[Dose-response relationship between noise kurtosis and noise-induced hearing loss].

Objective: To analyse the dose-response relationships between the kurtosis metric of noise and noise-induced hearing loss (NIHL) and study the role of kurtosis in the evaluation of NIHL associated with non-Gaussian noise. Methods: From January 2012 to December 2017, a total of 1869 workers in seven manufacturing industries were selected as the study subjects. The basic data of the workers were investigated by questionnaire, personal noise waveform was collected for a long time, and pure tone hearing threshold was tested. The 8-hour continuous equivalent A sound level (L(Aeq, 8 h)) , cumulative noise exposure (CNE) and kurtosis structure indexes were calculated. The dose-response relationships between kurtosis and NIHL were analyzed by stratification analysis method, which controlled the influence of CNE, L(Aeq, 8 h), exposure duration, age and sex on hearing loss using high-frequency noise-induced permanent threshold shift (NIPTS(346)) and high-frequency noise-induced hearing loss (HFNIHL) as outcome indicators. Results: When CNE was <90 dB (A) ·year and ≥100 dB (A) ·year, NIPTS(346) in the extremely high kurtosis group was significantly greater than that in the Gaussian kurtosis, low kurtosis and medium kurtosis group (P<0.05) . In the workers exposed to L(Aeq, 8 h)<85 dB (A) and ≥94 dB (A) , NIPTS(346) in the extremely high kurtosis group was significantly greater than that in the Gaussian kurtosis group (P<0.05) . Among workers under the age of 50 or male workers, NIPTS(346) in the extremely high kurtosis group was significantly greater than that in the Gaussian kurtosis, low kurtosis and medium kurtosis group (P<0.05) . Kurtosis was positively correlated with NIPTS(346) (r=0.121, P<0.05) . When CNE was <100 dB (A) ·year, the detection rate of HFNIHL increased with the increase of kurtosis level (P<0.01) . Logistic regression analysis showed that kurtosis was an important influencing factor for HFNIHL (OR=1.321) . Conclusion: Kurtosis has a dose-response relationship with the detection rate of HFNIHL in noise exposed workers, and noise kurtosis is an influencing factor of NIHL.

[Risk factors for early rebleeding after esophageal variceal ligation in patients with liver cirrhosis].

Objective: To investigate the risk factors for early rebleeding after esophageal variceal ligation (EVL) through a multicenter retrospective study. Methods: A total of 3289 patients who were hospitalized and underwent EVL in 17 upper second-class hospitals or hospitals of higher classes from January 1999 to May 2015 were collected and screened according to the exclusion criteria. A total of 2531 patients were screened out, and a retrospective analysis was performed for their clinical data including age, sex, endoscopic findings, and results of laboratory examination (liver function, biochemical results, routine blood test, and coagulation function) to collect related data. According to the presence or absence of rebleeding within 1 month after EVL, the patients were divided into rebleeding group and non-rebleeding group. SPSS22.0 software was used for independent t-test and one-way analysis of variance, and P < 0.05 was considered statistically significant. Results: In the 2531 patients who underwent EVL, the rate of early rebleeding after EVL was 6.6%, and the mortality rate was 12.0%. The results showed that sex (P = 0.014), number of veins with varices (P = 0.203), prothrombin time (P = 0.001), prothrombin activity (P = 0.014), albumin (P = 0), total bilirubin (P = 0.011), aspartate aminotransferase (P = 0.004), white blood cell count (P = 0.342), hepatic encephalopathy (P = 0.021), ascites (P = 0.027), Child-Pugh class (P = 0), Child-Pugh score (P = 0), glue injection for gastric varices (P = 0.521), gastric varices (P = 0.32), shunt (P = 0.174), number of ligation points (P = 0.001), number of ligation times (P = 0.024), number of times of hematemesis before treatment (P = 0), number of times of tarry stool (P = 0.008), and volume of blood in hematemesis before treatment (P = 0) were risk factors for early rebleeding after EVL. The regression analysis showed that male sex, a Child-Pugh score of >7.2, and volume of blood in hematemesis before treatment were independent risk factors for early rebleeding after EVL, while an albumin concentration of > 31.5 g/L was the protective factor. Conclusion: EVL has a good therapeutic effect in esophageal variceal rebleeding. Among all the factors analyzed, male sex, a Child-Pugh score of > 7.2, and volume of blood in hematemesis before treatment are independent risk factors for early rebleeding after EVL, and an albumin concentration of > 31.5 g/L is a protective factor.

First Report of Pantoea anthophila Causing Soft Rot Disease in Clausena lansium (Wampee) in China.

Clausena lansium, also known as wampee (Clausena wampi), is a plant species native to China, Vietnam, the Philippines, Malaysia, and Indonesia, where it is widely cultivated, and also grown in India, Sri Lanka, Queensland, Florida, and Hawaii, but less frequently (3). The fruit can be consumed fresh or made into juice, jam, or succade. In summer to fall 2014, a soft rot disease was found in a wampee planting region in Yunan County, Guangdong Province, China. On Sept. 18, we collected diseased samples from a wampee orchard with about 20% disease incidence. The infected fruit initially showed pinpoint spots on the peel, water-soaked lesions, and light to dark brown discoloration. Spots expanded in 2 days, and tissues collapsed after 5 days. Severely affected fruit showed cracking or nonodorous decay. Five diseased samples were collected, and causal agents were isolated from symptomatic tissues 1 cm under the peel after surface sterilization in 0.3% NaOCl for 10 min and rinsing in sterile water three times. Tissues were placed on a Luria Bertani (LB) plate for culture. Ten representative isolates were selected for further characterization. No colony was isolated from healthy tissues. Colonies were round, smooth, with irregular edges, and produced a yellow pigment in culture. Biolog identification (Version 4.20.05) showed that all strains were gram negative, negative for indole production, and utilized glucose, maltose, trehalose, sucrose, D-lactose, and pectin but not sorbitol or gelatin. The isolates were identified as Pantoea agglomerans (SIM 0.69). Multilocus sequence analysis (MLSA) was conducted for rapid classification of the strains. Sequences of atpD, gyrB, infB, and rpoB were amplified using corresponding primers (2). All sequences of the 10 isolates were identical in each gene. BLASTn was performed, and maximum likelihood trees based on the concatenated nucleotide sequences of the four genes were constructed using MEGA6. Bootstrap values after 1,000 replicates were expressed as percentages. Results showed that the tested strain named CL1 was most homologous to P. anthophila, with 98% identity for atpD (KM521543), 100% for gyrB (KM521544), infB (KM521545), and rpoB (KM521546). The 16S rRNA sequence (KM521542) amplified by primers 27f and 1492r shared 99% identity with that of P. anthophila M19_2C (JN644500). P. anthophila was previously reclassified from P. agglomerans (3); therefore, we suggest naming this wampee pathogen P. anthophila. Subsequently, 10 wampee fruits were injected with 20 µl of bacterial suspension (1 × 108 CFU/ml) of strains CL1 and CL2, respectively, and another 10 were injected with 20 µl of LB medium as controls, all kept at 28°C for 4 days. Symptoms similar to those of natural infections were observed on inoculated fruits but not on the negative controls. Bacteria were isolated from diseased tissues and further identified as P. anthophila by gyrB sequencing. P. anthophila was reported to naturally infect balsam and marigold (1,2). To our knowledge, this is the first report of P. anthophila naturally causing soft rot disease and cracking on C. lansium (wampee). References: (1) C. Brady et al. Syst. Appl. Microbiol. 31:447, 2008. (2) C. Brady et al. Int. J. Syst. Evol. Microbiol. 59:2339, 2009. (3) J. Morton. Fruits of Warm Climates. Echo Point Books & Media, Miami, FL, 1987.

Genetic diversity and relationships among accessions of five crested wheatgrass species (Poaceae: Agropyron) based on gliadin analysis.

Agropyron Gaertn. is the most important genus in Triticeae (Poaceae), which includes many forage grasses with high economic value. The genetic diversity and relationships of 36 accessions from five crested wheatgrass species were analyzed by gliadin markers. A total of 54 product bands were detected after acid polyacrylamide gel electrophoresis (A-PAGE), of which 100% were polymorphic. The genetic similarity coefficient based on Nei-Li's method ranged from 0.065 to 0.755 with an average of 0.451. The Shannon diversity information index showed that there was a high level of genetic diversity among the accessions. An unweighted pair group method with arithmetic average (UPGMA) dendrogram was constructed based on the Nei-Li's genetic similarity coefficients, which showed the phylogenetic relationships among accessions of different species. Analysis of molecular variance (AMOVA) showed that the proportion of variance explained by inter- and intraspecific variance was 9.34 and 90.66%, respectively, which revealed that the genetic variations within species were higher than the variations among species. Based on pairwise genetic distances (ΦST) among species, the cluster analysis indicated that A. mongolicum had a low-affinity relationship with other species, while A. fragile showed a close relationship with A. cristatum ssp pectinatum. Finally, the implications of the results for the taxonomy of Agropyron were discussed.

First Report of Bacterial Foot Rot of Rice Caused by a Dickeya zeae in China.

A bacterial disease of rice, bacterial foot rot, was found in Guangdong Province, China in September 2011, with an incidence about 10%. The typical symptom was a dark brown decay of the tillers. In the early stages of the disease, a brown sheath rot seemed to spread from the ligulae regions. The lesions quickly extended down to the nodes, culms, and finally to the crowns. Neighboring tillers of the same crown were invaded systemically, causing foot rot symptoms. A soft rot with an unpleasant odor developed in young tissues of infected tillers. In the advanced stage, many tillers decayed, so that entire diseased plants could easily be pulled from the soil. Six diseased samples were collected and bacteria were isolated from the edge of symptomatic tissues, after samples were sterilized in 0.3% NaOCl for 10 min, rinsed in sterile water three times, and placed on nutrient agar (beef extract 3 g, yeast extract 1 g, peptone 5 g, glucose 10 g, agar 16 g, distilled water 1 L, pH 6.8 to 7.0). For identification, a total of 12 representative isolates were selected. All strains were Gram negative, grew at 37°C, were positive for indole production, and utilized malonate, glucose, and sucrose, but not glucopyranoside, trehalose, or palatinose. Biolog identification (Version 4.20.05, Hayward, CA) identified isolate EC1 as Pectobacterium chrysanthemi (SIM 0.827), which has since been transferred to genus Dickeya. PCR was used to amplify the 16S rDNA gene with primers 27f and 1492r, the dnaX gene with primers dnaXf and dnaXr (2), and the gyrB gene with primers gyrBf1 (5'-ATGTCGAATTCTTATGACTCCTC-3') and gyrB-r1 (5'-TCARATATCRATATTCGCYGCTTTC-3'), which were designed based on published gyrB gene sequences of genus Dickeya. A BLASTn search of all three loci [16S rDNA (JQ284040), dnaX (JQ284041), and gyrB (JQ284042)] revealed that EC1 had 100% sequence identify to Dickeya zeae [16S rDNA (AB713560), dnaX (AB713593), gyrB (AB713635)]. Pathogenicity tests were conducted by injecting 10 rice seedlings with 100 µl of the bacterial suspension (1 × 108 CFU/ml) in the stem base, and an additional 10 rice seedlings were injected with 100 µl of sterile water as negative controls. Inoculations were carried out in a greenhouse at 28 to 32°C and 90% relative humidity. Foot rot symptoms identical to those described above were observed after 7 days on inoculated plants, but not on the negative controls. The bacterium was reisolated from the lesions and had 100% sequence identity for all three loci to EC1. Previously, similar symptoms were reported on rice in Guangdong province of China, and the causal agent was identified as Erwinia chrysanthemi (1). To our knowledge, this is the first report of D. zeae causing foot rot disease on rice in China. References: (1) Q. G. Liu et al. J. South China Agric. Univ. 18:128, 1997. (2) M. Slawiak et al. Eur. J. Plant Pathol. 125:245, 2009.

First Report of a Soft Rot of Philodendron 'Con-go' in China Caused by Dickeya dieffenbachiae.

Philodendron is a popular foliage plant cultivated in interiorscapes of homes, offices, and malls throughout China. A severe outbreak of a soft rot of Philodendron 'Con-go' occurred in Guangzhou, China from 2010 to 2011. The disease was characterized by leaf infections starting as pinpoint spots that are water soaked and yellow to pale brown. The lesions are sometimes surrounded by a diffuse yellow halo. When the humidity is high and temperatures are warm to hot, the spots expand rapidly, becoming slimy, irregular, and sunken with light tan centers, darker brown borders, and diffused yellow margins and may involve the entire leaf in a few days. An invasion of the midrib and larger veins by the causal bacterium often results in advancement into the petiole and stem. A survey of three areas of production of Philodendron 'Con-go' (5 ha) in Guangzhou revealed that 91% of the fields were affected at an incidence ranging from 15 to 30%. Of 41 bacterial isolates obtained from lesions, three were selected randomly for further characterization. All strains were gram negative, negative for oxidase and positive for catalase and tryptophanase (indole production), and utilized citrate, tartrate, malonate, glucose, sucrose, fructose, and maltose but not glucopyranoside, trehalose, or palatinose. Biolog analysis (version 4.20.05, Hayward, CA) identified the isolates as Pectobacterium chrysanthemi (SIM 0.804 to 0.914). According to Samson et al. (1), it was renamed as a Dickeya sp. PCR was performed on the 16S rDNA gene with primers 27f and 1495r (3) and 1,423 bp of the 16S rDNA gene (GenBank No. JN709491) showed 99% identity to P. chrysanthemi (GenBank No. AF373202), and 98% to Dickeya dieffenbachiae (GenBank No. JF311644). Additionally, the gyrB gene was amplified with primers gyrB-f1 (5'-atgtcgaattcttatgactcctc-3') and gyrB-r1 (5'-tcaratatcratattcgcygctttc-3') designed based on all the submitted gyrB gene sequences of Dickeya spp. The dnaX gene was amplified with primers dnaXf and dnaXr (2). The products were sequenced and phylogeny analyses were performed by means of MEGA 5.05. Results showed that the gyrB and the dnaX genes of the strains were 98% homologous to those of D. dieffenbachiae (GenBank Nos. JF311652 and GQ904757). Therefore, on the basis of phylogenetic trees of the 16S rDNA, gyrB, and dnaX gene sequences, the bacterial isolate named PC1 is related to D. dieffenbachiae (100% bootstrap values). Pathogenicity of each of the three strains on Philodendron 'Con-go' was confirmed by injecting 60 50-day-old seedlings each with 0.1 ml of the isolate suspension (108 CFU/ml) into the leaves. Another 60 were injected with sterile water to serve as the control treatment. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 32°C day and 28°C night temperatures with high humidity. After 72 h, all the injected plants started to show symptoms similar to those observed on field plants, but no symptoms appeared on the control plants. The reisolates were identical to the inoculated strains in biochemical characteristics. Bacteria characteristic of the inoculated strains were not reisolated from the control plants. To our knowledge, this is the first report of D. dieffenbachiae causing soft rot of Philodendron 'Con-go' in China. References: (1) R. Samson et al. Evol. Microbiol. 55:1415, 2005. (2) M. Slawiak et al. Eur. J. Plant Pathol. 125:245, 2009. (3) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

First Report of a Soft Rot of Phalaenopsis aphrodita Caused by Dickeya dieffenbachiae in China.

Phalaenopsis orchids, originally from tropical Asia, are mainly planted in Thailand, Singapore, Malaysia, the Philippines, and Taiwan and have gained popularity from consumers all over the world. The cultivation area of Phalaenopsis orchids has been rising and large-scale bases have been established in mainland China, especially South China because of suitable environmental conditions. In September 2011, a soft rot of Phalaenopsis aphrodita was found in a Phalaenopsis planting base in Guangzhou with an incidence of ~15%. Infected plants initially showed water-soaked, pale-to-dark brown pinpoint spots on leaves that were sometimes surrounded by a yellow halo. Spots expanded rapidly with rising humidity and temperatures, and in a few days, severely extended over the blade with a light tan color and darker brown border. Lesions decayed with odorous fumes and tissues collapsed with inclusions exuding. The bacterium advanced to the stem and pedicle. Finally, leaves became papery dry and the pedicles lodged. Six diseased samples were collected, and bacteria were isolated from the edge of symptomatic tissues after sterilization in 0.3% NaOCl for 10 min, rinsing in sterile water three times, and placing on nutrient agar for culture. Twelve representative isolates were selected for further characterization. All strains were gram negative, grew at 37°C, were positive for indole production, and utilized malonate, glucose, and sucrose but not glucopyranoside, trehalose, or palatinose. Biolog identification (version 4.20.05, Hayward, CA) was performed and Pectobacterium chrysanthemi (SIM 0.868) was confirmed for the tested isolates (transfer to genus Dickeya). PCR was used to amplify the 16S rDNAgene with primers 27f and 1492r, dnaX gene with primers dnaXf and dnaXr (3), and gyrB gene with primers gyrBf (5'-GAAGGYAAAVTKCATCGTCAGG-3') and gyrB-r1 (5'-TCARATATCRATATTCGCYGCTTTC-3') designed on the basis of the published gyrB gene sequences of genus Dickeya. BLASTn was performed online, and phylogeny trees (100% bootstrap values) were created by means of MEGA 5.05 for these gene sequences, respectively. Results commonly showed that the representative tested strain, PA1, was most homologous to Dickeya dieffenbachiae with 98% identity for 16S rDNA(JN940859), 97% for dnaX (JN989971), and 96% for gyrB (JN971031). Thus, we recommend calling this isolate D. dieffenbachiae PA1. Pathogenicity tests were conducted by injecting 10 P. aphrodita seedlings with 100 µl of the bacterial suspension (1 × 108 CFU/ml) and another 10 were injected with 100 µl of sterile water as controls. Plants were inoculated in a greenhouse at 28 to 32°C and 90% relative humidity. Soft rot symptoms were observed after 2 days on the inoculated plants, but not on the control ones. The bacterium was isolated from the lesions and demonstrated identity to the inoculated plant by the 16S rDNA sequence comparison. Previously, similar diseases of P. amabilis were reported in Tangshan, Jiangsu, Zhejiang, and Wuhan and causal agents were identified as Erwinia spp. (2), Pseudomonas grimontii (1), E. chrysanthemi, and E. carotovora subsp. carovora (4). To our knowledge, this is the first report of D. dieffenbachiae causing soft rot disease on P. aphrodita in China. References: (1) X. L. Chu and B. Yang. Acta Phytopathol. Sin. 40:90, 2010. (2) Y. M. Li et al. J. Beijing Agric. Coll. 19:41, 2004. (3) M. Slawiak et al. Eur. J. Plant Pathol. 125:245, 2009. (4) Z. Y. Wu et al. J. Zhejiang For. Coll. 27:635, 2010.

Gene expression analysis in the human hypothalamus in depression by laser microdissection and real-time PCR: the presence of multiple receptor imbalances.

Hyperactivity of corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus (PVN) of the hypothalamus is a prominent feature in depression and may be important in the etiology of this disease. The activity of the CRF neurons in the stress response is modulated by a number of factors that stimulate or inhibit CRF expression, including (1) corticosteroid receptors and their chaperones, heat shock proteins 70 and 90, (2) sex hormone receptors, (3) CRF receptors 1 (CRFR1) and 2, (4) cytokines interleukin 1-beta and tumor necrosis factor-alpha, (5) neuropeptides and receptors, vasopressin (AVP), AVP receptor 1a (AVPR1A) and oxytocin and (6) transcription factor cAMP-response element-binding protein. We hypothesized that, in depression, the transcript levels of those genes that are involved in the activation of the hypothalamo-pituitary-adrenal (HPA) axis are upregulated, whereas the transcript levels of the genes involved in the inhibition of the HPA axis are downregulated. We performed laser microdissection and real-time PCR in the PVN and as a control in the supraoptic nucleus. Snap-frozen post-mortem hypothalami of seven depressed and seven matched controls were used. We found significantly increased CRF mRNA levels in the PVN of the depressed patients. This was accompanied by a significantly increased expression of four genes that are involved in the activation of CRF neurons, that is, CRFR1, estrogen receptor-alpha, AVPR1A and mineralocorticoid receptor, while the expression of the androgen receptor mRNA involved in the inhibition of CRF neurons was decreased significantly. These findings raise the possibility that a disturbed balance in the production of receptors may contribute to the activation of the HPA axis in depression.

Decreased cerebral blood flow velocity in apolipoprotein E epsilon4 allele carriers with mild cognitive impairment.

The aim of this study was to investigate the changes in cerebral blood flow velocity (CBFV) and its relation to apolipoprotein E (apoE) epsilon4 allele in mild cognitive impairment (MCI). Thirty MCI and 30 controls were assessed using Mini-Mental State Examination (MMSE) and Cambridge Cognitive Examination-Chinese version (CAMCOG-C), and then insonated in the anterior (ACA), middle (MCA) and basilar (BA) cerebral arteries using transcranial Doppler ultrasonography. Compared with controls, MCI showed significant decreases in the mean (V(m)), systolic (V(s)) and diastolic (V(d)) CBFV, bilaterally in MCA and ACA (P < 0.05-0.001), but not in BA. Compared with 17 apoE epsilon4 allele non-carriers, 13 carriers in MCI showed significant CBFV decreases, bilaterally in MCA (P < 0.05-0.001). Our findings, the decreased CBFV in apoE epsilon4 allele carriers with MCI, suggest that a large sample and longitudinal study in CBFV and cognitive changes may have the implications on early diagnosis of Alzheimer's disease.

Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferator-activated receptor gamma-dependent repression of cyclin D1.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARgamma exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARgamma ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARgamma ligands on cellular proliferation. Recent findings that PPARgamma ligands convey PPARgamma-independent activities through IkappaB kinase (IKK) raises important questions about the specific mechanisms through which PPARgamma ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARgamma. Herein PPARgamma, liganded by either natural (15d-PGJ(2) and PGD(2)) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ(2) was not observed in PPARgamma-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ(2). Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARgamma but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARgamma involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ(2) enhanced recruitment of p300 to PPARgamma but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.

Alterations in arginine vasopressin neurons in the suprachiasmatic nucleus in depression.

BACKGROUND: Circadian rhythm disturbances are frequently found in depressed subjects. Although it has been presumed that these disturbances may reflect a disorder of the circadian pacemaker, this has never been established. The suprachiasmatic nucleus (SCN) is the pacemaker of the circadian timing system in mammals, and arginine vasopressin (AVP) is one of its major neuropeptides. As peptide content is often taken as a measure for activity, we hypothesized that a decreased number of AVP-immunoreactive (AVP-IR) neurons and amount of AVP-messenger RNA (mRNA) would be present in the SCN of depressed subjects. METHODS: Brains of 11 subjects suffering from major depression (8 cases) and bipolar disorder (3 cases), and of 11 controls, matched for sex, age, and clock time at death, were collected. The number of AVP-IR neurons in the SCN was determined by means of a digitizer (CalComp Inc, Reading, England). The amount of AVP-mRNA expression in the SCN was quantified with the Interaktive Bild Analyse System image analysis system (Kontron, Munich, Germany). RESULTS: In depressed subjects, the number of AVP-IR neurons in the SCN was more than one and a half times higher than in controls, while the total masked area of silver grains, as an estimate of the amount of AVP-mRNA, was about one half that of controls. CONCLUSIONS: Contrary to our hypothesis, an increase in the number of AVP-IR neurons in the SCN in depression was found, together with an expected decrease in AVP-mRNA. These findings suggest that, in depressed patients, both the synthesis and release of AVP in the SCN is reduced, resulting in an impaired functional ability. A disbalance between AVP production and transport needs further investigation in future studies.

DNA microarray: a high throughput approach for methylation detection.

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.

Decreased vasopressin gene expression in the biological clock of Alzheimer disease patients with and without depression.

Circadian rhythm disturbances are frequently present in Alzheimer disease (AD). In the present study, we investigated the expression of vasopressin (AVP) mRNA in the human suprachiasmatic nucleus (SCN). The in situ hybridization procedure on formalin-fixed paraffin-embedded material was improved to such a degree that we could, for the first time, visualize AVP mRNA expressing neurons in the human SCN and carry out quantitative measurements. The total amount of AVP mRNA expressed as masked silver grains in the SCN was 3 times lower in AD patients (n = 14; 2,135 +/- 597 microm2) than in age- and time-of-death-matched controls (n = 11; 6,667 +/- 1466 microm2) (p = 0.003). No significant difference was found in the amount of AVP mRNA between AD patients with depression (n = 7) and without depression (n = 7) (2,985 +/-1103 microm2 and 1,285 +/- 298 microm2, respectively; p = 0.38). In addition, the human SCN AVP mRNA expressing neurons showed a marked day-night difference in controls under 80 years of age. The amount of AVP mRNA was more than 3 times higher during the daytime (9,028 +/- 1709 microm2, n = 7) than at night (2,536 +/- 740 microm2, n = 4; p = 0.02), whereas no clear diurnal rhythm of AVP mRNA in the SCN was observed in AD patients. There was no relationship between the amount of AVP mRNA in the SCN and age at onset of dementia, duration of AD and the neuropathological changes in the cerebral cortex. These findings suggest that the neurobiological basis of the circadian rhythm disturbances that are responsible for behavioral rhythm disorders is located in the SCN. It also explains the beneficial effects of light therapy on nightly restlessness in AD patients.

Decreased melatonin levels in postmortem cerebrospinal fluid in relation to aging, Alzheimer's disease, and apolipoprotein E-epsilon4/4 genotype.

Sleep disruption, nightly restlessness, sundowning, and other circadian disturbances are frequently seen in Alzheimer's disease (AD) patients. Changes in the suprachiasmatic nucleus and pineal gland are thought to be the biological basis for these behavioral disturbances. Melatonin is the main endocrine message for circadian rhythmicity from the pineal. To determine whether melatonin production was affected in AD, melatonin levels were determined in the cerebrospinal fluid (CSF) of 85 patients with AD (mean age, 75 +/- 1.1 yr) and in 82 age-matched controls (mean age, 76 +/- 1.4 yr). Ventricular postmortem CSF was collected from clinically and neuropathologically well defined AD patients and from control subjects without primary neurological or psychiatric disease. In old control subjects (>80 yr of age), CSF melatonin levels were half of those in control subjects of 41-80 yr of age [176 +/- 58 (n = 29) and 330 +/- 66 (n = 53) pg/mL, respectively; P = 0.016]. We did not find a diurnal rhythm in CSF melatonin levels in control subjects. In AD patients the CSF melatonin levels were only one fifth (55 +/- 7 pg/mL) of those in control subjects (273 +/- 47 pg/mL; P = 0.0001). There was no difference in the CSF melatonin levels between the presenile (42 +/- 11 pg/mL; n = 21) and the senile (59 +/- 8 pg/mL; n = 64; P = 0.35) AD patients. The melatonin level in AD patients expressing apolipoprotein E-epsilon3/4 (71 +/- 11 pg/mL) was significantly higher than that in patients expressing apolipoprotein E-epsilon4/4 (32 +/- 8 pg/ml; P = 0.02). In the AD patients no significant correlation was observed between age of onset or duration of AD and CSF melatonin levels. In the present study, a dramatic decrease in the CSF melatonin levels was found in old control subjects and even more so in AD patients. Whether supplementation of melatonin may indeed improve behavioral disturbances in AD patients should be investigated.

Male-to-female transsexuals have female neuron numbers in a limbic nucleus.

Transsexuals experience themselves as being of the opposite sex, despite having the biological characteristics of one sex. A crucial question resulting from a previous brain study in male-to-female transsexuals was whether the reported difference according to gender identity in the central part of the bed nucleus of the stria terminalis (BSTc) was based on a neuronal difference in the BSTc itself or just a reflection of a difference in vasoactive intestinal polypeptide innervation from the amygdala, which was used as a marker. Therefore, we determined in 42 subjects the number of somatostatin-expressing neurons in the BSTc in relation to sex, sexual orientation, gender identity, and past or present hormonal status. Regardless of sexual orientation, men had almost twice as many somatostatin neurons as women (P < 0.006). The number of neurons in the BSTc of male-to-female transsexuals was similar to that of the females (P = 0.83). In contrast, the neuron number of a female-to-male transsexual was found to be in the male range. Hormone treatment or sex hormone level variations in adulthood did not seem to have influenced BSTc neuron numbers. The present findings of somatostatin neuronal sex differences in the BSTc and its sex reversal in the transsexual brain clearly support the paradigm that in transsexuals sexual differentiation of the brain and genitals may go into opposite directions and point to a neurobiological basis of gender identity disorder.

VIP neurons in the human SCN in relation to sex, age, and Alzheimer's disease.

The brains of 46 control subjects and 21 Alzheimer's disease (AD) patients were studied to determine whether there are age-related or AD-related changes in the vasoactive intestinal polypeptide (VIP) neuron population of the human suprachiasmatic nucleus (SCN). The number of VIP expressing neurons in the SCN of females, ranging in age from 10-91 years, did not change during normal aging. In males, however, the number of VIP neurons in the SCN was highest in the young subjects (10-40 years of age), after which, a dramatic decrease occurred in middle-aged subjects. This resulted in an age-dependent sex difference in the VIP cell population of the SCN: young males had twice as many VIP expressing SCN neurons as young females, whereas in the middle-aged groups, the females had twice as many VIP SCN neurons as the males. A significant decrease in the number of VIP expressing neurons in the SCN was found in female presenile AD patients, i.e., those younger than 65 years.

ERK signalling in metastatic human MDA-MB-231 breast carcinoma cells is adapted to obtain high urokinase expression and rapid cell proliferation.

Increased urokinase plasminogen activator (u-PA) production is associated with tumor invasion and metastasis in several malignancies, including breast cancer. The mechanisms underlying constitutive u-PA expression are not well understood. We examined the relationship between the signal strength of the ERK pathway and the level of u-PA expression in the metastatic human breast cancer cell line MDA-MB-231. Treatment with the MEK1 inhibitor PD98059 resulted in decreased ERK1/2 phosphorylation and decreased u-PA mRNA and protein expression. Inhibition of ERK1/2 activity also led to decreased cell proliferation and to decreased cyclin D1 expression. Less than 5% of total ERK1/2 was phosphorylated in exponentially growing MDA-MB-231 cells, and ERK1/2 activity could be stimulated by okadaic acid. Okadaic acid did not stimulate u-PA expression, but induced strong expression of the cdk-inhibitor p21Cip1. These findings suggest that ERK1/2 signaling is tuned to a level which results in high u-PA expression and rapid cell proliferation.

Induction of experimental autoimmune neuritis in CD4-8-C57BL/6J mice.

The C57BL/6J mice strain is known to be reputedly resistant to induction of experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. Here we describe the induction of EAN in mice of the C57BL/6J background by transfer into naive syngeneic recipients bovine peripheral nerve myelin (BPM)-primed donor lymph node cells that had been stimulated in vitro with the bovine peripheral nervous system (PNS) myelin P2 protein peptide 57-81 followed by challenge with BPM, Freund's complete adjuvant and pertussis toxin. EAN was more severe, both clinically and histologically, and accompanied by extensive infiltration of inflammatory cells and demyelination in peripheral nerves when examined on day 30 after transfer of primed T cells from CD4- 8- mice into identical naive hosts than after transfer of cells from primed wild type, CD4-/- or CD8-/- mice to corresponding recipient animals. EAN in CD4-8- mice was also associated with elevated numbers of P2 peptide-reactive interferon-y (TFN-gamma) secreting cells and alphabeta T cells were present in lymph nodes and spleens. The data suggest that PNS myelin activated T cells from an EAN-resistant mice strain are capable of homing to the PNS. The expanded CD4-8- alphabeta T cells may have helper and effector functions, related to initiation of EAN in the CD4-8- mice. Lack of CD4+ and CD8+ expressing cells does not prevent the initiation of an autoimmune disease.

Biological rhythms in the human life cycle and their relationship to functional changes in the suprachiasmatic nucleus.

Biological rhythms play a prominent role in the human life cycle. The endogenous rhythms are entrained by the environment and have an astronomical counterpart which is obvious for daily, monthly, and yearly rhythms, and may possibly also be present in weekly rhythms. Circadian rhythms are present in, e.g. testosterone levels, spontaneous birth, strokes, and death from cardiovascular causes. Circaseptan rhythms are present in, e.g. spontaneous birth, 17-ketosteroid levels, myocardial infarctions, and strokes. The relationship of these rhythms with the suprachiasmatic nucleus (SCN) has not yet been established. Circatrigintan rhythms, such as the menstrual cycle, have so far not been associated with the SCN. Circannual rhythms are present in, e.g. mood, suicides, reproduction, birth weight, sleep and season of birth of psychiatric patients. The human SCN shows strong circadian and circannual fluctuations in the number of neurons expressing vasopressin. The vasopressin and VIP cell population of the SCN develop late, i.e. for a major part postnatally. After the age of 50 the amplitudes of circadian and circannual fluctuations of the vasopressin cell numbers are reduced whereas the number of vasopressin expressing neurons decreases after the age of 80 and do so even more and earlier in Alzheimer's disease. Sex differences are present in the shape of the vasopressin subnucleus of the SCN and in the vasoactive intestinal polypeptide (VIP) cell number. The sex differences in the SCN, the doubling of the number of vasopressin neurons in the SCN of homosexual men, and a variety of animal experimental observations indicate that the SCN is involved in sexual behavior and reproduction. The exact role of the SCN in these processes is subject to current research.