Gynura bicolor (Compositae) which is widely used by the locals as natural remedies in folk medicine has limited scientific studies to ensure its efficacy and nontoxicity. The current study reports the total phenolic content, antioxidant capacity, cytotoxicity, and acute oral toxicity of crude methanol and its fractionated extracts  of G. bicolor leaves. Five human colon cancer cell lines , one human breast adenocarcinoma cell line (MCF7), and one human normal colon cell line (CCD-18Co) were used to evaluate the cytotoxicity of G. bicolor. The present findings had clearly demonstrated that ethyl acetate extract of G. bicolor with the highest total phenolic content among the extracts showed the strongest antioxidant activity , possessed cytotoxicity, and induced apoptotic and necrotic cell death, especially towards the HCT 116 and HCT-15 colon cancer cells. The acute oral toxicity study indicated that methanol extract of G. bicolor has negligible level of toxicity when administered orally and has been regarded as safe in experimental rats. The findings of the current study clearly established the chemoprevention potential of G. bicolor and thus provide scientific validation on the therapeutic claims of G. bicolor. Gynura bicolor, locally known as “Hong Feng Cai” (Chinese) and “Sambung Nyawa Ungu” , belongs to the botanical family of Compositae. The leaves of G. bicolor distinctively show reddish purple color on the abaxial side and green color on the adaxial side. The native geographic distribution of G. bicolor occurs in China, Taiwan, Myanmar, and Thailand. The G. bicolor is consumed as a culinary cooked vegetable and believed to confer a wide range of benefits such as anticancer, anti-inflammatory, and possibly antihypertensive effects. G. bicolor is also used for post-labor recovery, blood circulation improvement, treatment of dysmenorrhea, hemoptysis, and diabetes by the locals [Plants have been used to maintain human health for a very long time. It is well known that some of the plant species possess interesting bioactivities with potential therapeutic applications which can benefit us. e locals –3.G. bicolor is widely used in Malaysia, there are no recorded data of locally grown G. bicolor species. Scientific studies on the use of locally grown G. bicolor to ensure its efficacy and nontoxicity have not been carried out. Several phytochemical and biological reports showed that G. bicolor cultivated in Japan, China, and Taiwan has antihyperglycemic effect [G. bicolor leaves [Although c effect , 4, antic effect , and antc effect  and coulc effect . Chemicar leaves , 8–10.G. bicolor leaves. The crude methanol and its fractionated extracts  were firstly prepared prior to the bioactivity assessments. Folin-Ciocalteu's method was used for the measurement of total phenolic content of extracts, while two established testing systems were carried out to evaluate the antioxidant capacity, namely, DPPH radical scavenging assay and metal chelating assay. These antioxidant assays have been widely used to determine the antioxidant capacities as these assays require standard equipments and deliver reproducible fast results. More than one antioxidant assays were performed in the present study because a single antioxidant assay is not sufficient to measure the various modes of action of antioxidants in a test sample. As G. bicolor is believed to have anticancer properties especially against colon cancer by the locals, the cytotoxic activities of G. bicolor extracts were investigated against four human colon adenocarcinoma cell lines with varying molecular characteristics , a human colon carcinoma cell line (HCT 116), a human breast adenocarcinoma cell line (MCF7), and a human normal colon cell line (CCD-18Co). There is limited information regarding the cytotoxic activity of G. bicolor on human cancer cell lines. To the best of our knowledge, this is the first report on the cytotoxic effect of G. bicolor on colon and breast adenocarcinoma cell lines. The cells induced with cytotoxic extract were then subjected to AO (acridine orange)/EB (ethidium bromide) staining and annexin-V/PI (propidium iodide) flow cytometry in order to assess the cell death and morphological changes. In view of the increasing popular consumption of G. bicolor, acute oral toxicity assay was carried out in the present study to ensure that G. bicolor is safe for human consumption. Hence, the resulting information will certainly provide some scientific validation on the traditional use of locally grown G. bicolor leaves.Oxidative stress plays an important role in carcinogenesis as free radicals could cause various modifications to biological macromolecules like DNA, protein, and lipid and could increase the risk of mutagenesis . The precis-platin, carboxymethyl cellulose, EDTA (ethylenediaminetetraacetic acid), and DMSO (dimethyl sulfoxide) were purchased from Sigma-Aldrich Company. Fetal bovine serum, penicillin/streptomycin (100X), amphotericin B (250 μg/mL), and sodium pyruvate (100 mM) were from PAA Laboratories. Methanol, hexane, and ethyl acetate were purchased from Fisher Scientific Company. FITC Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences Company.Gallic acid, BHA (butylated hydroxyanisole), DPPH , potassium ferricyanide, Folin-Ciocalteu's phenol reagent, AO (acridine orange), EB (ethidium bromide), MTT (methylthiazolyldiphenyl-tetrazolium bromide), RPMI 1640 medium, McCoy's 5A medium, EMEM , sodium bicarbonate, G. bicolor were collected from Seremban, Negeri Sembilan, Malaysia, in February 2011. The identification of the plant species was done by Dr. Yong Kien Thai at the Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia. A voucher specimen (herbarium no. KLU47744) was deposited at the herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.Fresh leaves of G. bicolor (8049.20 g) were washed, dried, and ground to fine powder . The powder was extracted with methanol at room temperature yielding a dark green crude methanol extract . The crude methanol extract (61.10 g) was further extracted with hexane to obtain hexane-soluble extract  and hexane-insoluble residue. Hexane-insoluble residue was further partitioned with ethyl acetate-water (1 : 1) to yield ethyl acetate  and water  extracts. The percentage yield of fractionated extracts was based on the weight of crude methanol extract (61.10 g) used for fractionation. All of the extracts  were kept in the dark at 4°C for no more than one week prior to evaluation of total phenolic content, antioxidant effect, cytotoxic activity, and acute oral toxicity.Briefly, the fresh leaves of G. bicolor extracts was determined by Folin-Ciocalteu's method that was modified to accommodate the 96-well plate as described previously by Sulaiman and Ooi [μL of Folin-Ciocalteu's reagent was firstly added to 10 μL of extract solution (concentrations ranged 4–20 mg/mL) in each well of 96-well plate. The mixture was then incubated at room temperature for five minutes before adding 25 μL of 20% (w/v) sodium carbonate to the mixture. This was then followed by addition of distilled water to make a final volume of 200 μL per well. The reaction mixture was further incubated for 30 minutes at room temperature before the absorbance reading taken at 760 nm using a microplate reader (Thermo Scientific Multiskan GO). A standard curve was plotted using gallic acid (0–1000 mg/L). BHA was used as the positive reference standard in the present study. All of the extracts and positive reference standard were assayed in triplicate. The results were expressed as milligram of gallic acid equivalents per gram of extract (mg of GAEs/g of extract).The phenolic content of  and Ooi . BrieflyG. bicolor extracts was measured according to the method described by Sulaiman and Ooi [μL of DPPH solution (0.3 mM) was added to 50 μL of extract (at various concentrations) in each well of 96-well plate before incubation for 30 minutes at room temperature. As for the blank, 50 μL of distilled water or methanol was added to the DPPH solution instead of the extract. The absorbance value was taken at 515 nm using a microplate reader (Thermo Scientific Multiskan GO) after incubation. BHA was used as positive reference standard in the present study. All of the extracts and positive reference standard were assayed in triplicate. The scavenging ability of the extract was expressed as IC50 value, which is the concentration at which 50% of DPPH radicals were scavenged. The IC50 value was obtained by extrapolating from the graph of DPPH radical scavenging activity (%) versus concentration of extract.DPPH radical scavenging activity of  and Ooi . BrieflyμL of extract (at various concentrations) was incubated with 5 μL of ferrous chloride hexahydrate (2 mM) and 130 μL of deionized water in each well of a 96-well plate for 5 minutes at room temperature. The reaction was initiated by the addition of 15 μL ferrozine (5 mM). After reaction mixture reached equilibrium, the absorbance value was measured at 562 nm using a microplate reader (Thermo Scientific Multiskan GO). EDTA was used as positive reference standard for the experiment. All of the extracts and positive reference standard were assayed in triplicate. The chelating effect of the extracts was expressed as IC50, which is the concentration at which 50% of metal ions were chelated. The lower IC50 value indicates the stronger metal chelating ability of the extract.The metal chelating assay was performed in 96-well plate based on protocol described by Sulaiman and Ooi . Briefly2 incubator.Human colon adenocarcinoma , colon carcinoma (HCT 116), breast adenocarcinoma (MCF7), and human normal colon (CCD-18Co) cell lines were purchased from American Type Culture Collection . HCT-15, SW480, and MCF7 cells were maintained in RPMI 1640 medium; HT-29 and HCT 116 cells in McCoy's 5A medium; Caco-2 and CCD-18Co cells in EMEM medium. All media were supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (100X), and 0.5% amphotericin B except for EMEM medium that was supplemented with additional 1% sodium pyruvate. Caco-2 cells were maintained in EMEM medium which was supplemented with 20% fetal bovine serum instead of 10%. The cells were cultured at 37°C in COμg/mL) of the extract. The final concentration of DMSO in each well was 0.5%. Untreated cells were used as negative controls. Following 24, 48, and 72 hours of incubation, 20 μL of MTT (5 mg/mL) was added into each well and further incubated for another four hours. The medium was then removed and replaced with DMSO. The absorbance was measured at 570 nm with 650 nm as background using a microplate reader (Thermo Scientific Multiskan GO). All of the extracts were assayed in triplicate. Cis-platin was used as a positive reference standard. IC50 value is the concentration of extract or positive reference standard that inhibits 50% of the cells growth.The MTT cytotoxicity assay was carried out as described previously by Mosmann . All of 5 cells/well) for 24 hours before treatment with 20 μg/mL of cytotoxic ethyl acetate extract. Untreated cells were used as negative controls. After incubation of 24 hours, 160 μL of AO/EB solution (one part of 100 μg/mL AO and one part of 100 μg/mL EB in PBS) was loaded into each well which contained 2 mL of medium. The cells were immediately visualised under inverted fluorescence microscope (Olympus IX73) with blue excitation mirror unit at 200x magnification. Cis-platin (20 μg/mL) was used as positive reference in the present study.Briefly, HCT 116 cells were seeded into 6-well plate (1 × 105 cells) were seeded in 60 mm petri dish. After 24 hours, HCT 116 cells were treated with 20 μg/mL of cytotoxic ethyl acetate extract. Untreated cells were used as negative controls. After 24 hours, cells were harvested and stained with FITC annexin-V and PI following instruction of the FITC Annexin V Apoptosis Detection Kit I. Cells treated with 20 μg/mL of cis-platin were used as positive controls. A total of 10,000 cells were analyzed by flow cytometer (BD FACSCanto II) using FACSDiva software.Briefly, HCT 116 cells  which were obtained from the Laboratory Animal Center, Faculty of Medicine, University of Malaya. The weight variation in the rats did not exceed ±20% SD of the mean weight. The animal research protocol was approved by the Institutional Animal Care and Use Committee, University of Malaya (UM IACUC) before commencing the study ).G. bicolor was conducted using the procedure described by OECD guideline 423 [The acute oral toxicity of line 423  with somP < 0.05). The IC50 values for cytotoxic activity were obtained by nonlinear regression using GraphPad Prism statistical software.The antioxidant data in the present study were subjected to one-way analysis of variance (ANOVA), and the significance of the difference between the means was determined by Duncan's multiple-range tests at 95% least significant difference  was translated into total phenolic content ) using the gallic acid calibration plot with the following formula: total phenolic content = (y − 0.0496)/0.001; R2 = 0.9855. Total phenolic content of ethyl acetate extract was significantly higher than those of other extracts (P < 0.05). The total phenolic content of G. bicolor extracts in descending order was ethyl acetate > hexane > methanol > water, as shown in G. bicolor leaves in previous studies reported by Lu et al. [Phenolic compounds such as phenolic acids, flavonoids, and tannins are the major determinants of antioxidant potentials in foods, and they could be a natural source of antioxidants, antimutagenic, and antitumour activities . In the u et al.  and Shim y − 0.046/0.001; 50 value indicates stronger ability of the extract to act as DPPH scavenger as lesser scavengers were required to achieve 50% scavenging reaction.Radical scavenging activity is very important due to the deleterious role of free radicals in food and biological systems . DPPH raG. bicolor extracts were investigated through the free radical scavenging activity via their reactions with the stable DPPH radicals (G. bicolor in DPPH assay as DPPH scavenging activity of ethyl acetate extract was significantly higher than those of other extracts (P < 0.05). The descending order of DPPH scavenging activity of G. bicolor extracts was ethyl acetate > methanol > hexane > water. The ethyl acetate extract exhibited the lowest IC50 value (0.53 mg/mL) followed by methanol, hexane, and water extracts with IC50 values of 4.93, 11.15, and 13.57 mg/mL, respectively. This indicates that the ethyl acetate extract may contain antioxidative substances which have hydrogen-donating activity to scavenge DPPH radicals and are able to terminate the chain reaction of free radicals.In the present study, radicals . Ethyl aG. bicolor extracts was investigated by assessing the ability of the antioxidants to compete with the indicator ferrozine to complex with ferrous ion (Fe2+) in solution. Metal chelating activity of ethyl acetate extract was significantly higher than those of other extracts (P < 0.05). The metal chelating activity of G. bicolor extracts in descending order was ethyl acetate > methanol > hexane > water, as shown in 50 values in the range of 3.80–4.90 mg/mL, while water extract on the other hand showed inhibitory activity only at 28.37 mg/mL. All of the extracts showed low metal chelating capability as compared to EDTA, which was the positive reference standard. This could be possibly due to lack of chemical compounds that could act as metal chelators in the G. bicolor extracts.The metal ion chelating ability plays a significant role in the antioxidant mechanism because it prevents oxyradical generation and the consequent oxidative damage . In the G. bicolor extracts was evaluated against four human colon adenocarcinoma cell lines with varying molecular characteristics , one colon carcinoma cell line (HCT 116), and one human breast adenocarcinoma cell line (MCF7) using the MTT cytotoxicity assay which measures the mitochondrial activity in viable cells. The human normal colon cell line (CCD-18Co) was used to determine the specificity for cancerous cells. According to the United States National Cancer Institute Plant Screening Program, a plant extract is considered to possess cytotoxicity if the IC50 value is 20 μg/mL or less after incubation between 48 and 72 hours [It is generally known that the genetic background of cell lines could affect the sensitivity of anticancer agent. In the present study, the cytotoxic activity of line CCD-Co was usG. bicolor extracts against the selected human cell lines, expressed as IC50 value averaged from three experiments, are summarized in 50 values > 100 μg/mL against all the tested human cell lines), except that ethyl acetate extract with cytotoxic sensitivity differed between the various human cancer cell lines. Among the tested cells, cytotoxicity of ethyl acetate extract was found to be most active against HCT 116 and HCT-15 cells, with the IC50 values of 16.0 and 12.8 μg/mL after 24 hours of incubation; 20.9 and 18.7 μg/mL after 48 hours of incubation, respectively. HCT 116 and HCT-15 are the colon cancer cell lines which have wild-type p53, and both are MMR-deficient, while HT-29, SW480, and Caco-2 cells have mutant p53 and are MMR-proficient [The results of cytotoxicity screening of oficient . Based o50 values obtained were increased over time . The cytotoxic effect of ethyl acetate extract was reduced when the treatment duration increased. The tested carcinoma cell lines seem to be able to overcome the treatment inhibitory effect. This can be explained by the possibility that the ethyl acetate extract may contain some chemical constituents which may repair the cell mechanism in order to increase cell growth. Another explanation could be due to the stability of the active chemical constituents in ethyl acetate extract by which the chemical constituents may be degraded over time.Generally, the tested human cancer cell lines exhibited time-dependent response, by which IC50 > 100 μg/mL). This indicated that ethyl acetate extract was selectively toxic against cancerous cell lines. Thus, the active chemical constituents in the ethyl acetate extract may lead to valuable constituents that may have the ability to kill cancerous cells but exert no damage to normal cells. The cytotoxic effect of ethyl acetate extract may be due to the presence of anthocyanins and flavonols which had been identified in previous studies [G. bicolor leaves [G. bicolor leaves by Lu et al. [Gynura species, cytotoxic cerebroside (gynuraoside) which had been identified in ethyl acetate extract of G. divaricata [G. bicolor as well and thus contributed to the cytotoxic activity of the G. bicolor ethyl acetate extract.As shown in  studies , 7, 9. Ar leaves . Few flau et al.  were repu et al. –24. As cvaricata  may be pIn cell death induction, apoptosis is usually more desired than necrosis as apoptosis is a normal well-regulated process and does not often result in inflammation, while necrotic cell death is caused by external injury and almost always results in inflammation . HoweverG. bicolor. AO (acridine orange) can penetrate viable and nonviable cells and makes the nuclei appear green, while nonviable cells which lost the membrane integrity will take up EB (ethidium bromide) that makes the nuclei appear red. Hence, viable cells will have green organized structure nuclei; early apoptotic cells will have green nuclei with condensed or fragmented chromatin; late apoptotic cells will have orange to red nuclei with condensed or fragmented chromatin (EB overwhelms AO); necrotic cells will show orange to red organized structure nuclei.In the present study, AO/EB staining was used to assess the type of cell death and morphological changes induced by cytotoxic ethyl acetate extract of G. bicolor. The ethyl acetate extract-treated HCT 116 cells were rounding up compared with untreated control cells. Chromatin condensation is one of the hallmarks of apoptosis. Green and red nuclei with condensed chromatin were observed in HCT 116 cells after 20 μg/mL of ethyl acetate extract treatment, indicating that ethyl acetate could induce HCT 116 cells to undergo early and late apoptosis, whilst red nuclei with noncondensed chromatin observed in As shown in G. bicolor, flow cytometry was used to analyze FITC annexin-V and PI stained cells as it can quantitatively determine the percentage of cells within a population that are undergoing early and late apoptosis or necrosis. During early apoptosis, PS (phosphatidylserine) is translocated from inner to outer leaflet of plasma membrane. The exposed PS could be detected by FITC conjugated annexin-V. In the event of late apoptosis and necrosis, loss of membrane integrity could be detected by PI.In order to further asses and confirm the type of cell death induced by cytotoxic ethyl acetate extract of μg/mL of ethyl acetate extract, HCT 116 cells demonstrated an increase of necrotic cells  from 0.3% (untreated control) to 57.2%. Cells stained with both annexin-V and PI , which represented later stage of apoptosis, were also found to increase from 4.3% (untreated control) to 10.5% after treatment with the ethyl acetate extract. Taken together, the results clearly showed that the ethyl acetate extract appeared to induce late apoptosis and necrosis in HCT 116 cells.According to G. bicolor methanol extract as human consumption. As shown in G. bicolor extracts did not show cytotoxic activity against the normal cell line (IC50 > 100 μg/mL). If this occurs in vivo as well, the use of G. bicolor as natural remedies would have scientific support. Throughout the 14 days of observation period after oral administration, there were no deaths reported and no changes observed on the outer appearance  of treated rats which were dosed at 300, 2000, and 5000 mg of crude methanol extract per kilogram of body weight. The treated rats also did not show any sign of toxicity  or behavioral changes (hyperactivity and hypoactivity). The overall condition of treated rats was similar to that of control rats.Acute oral toxicity test was carried out in the present study to evaluate the safety of G. bicolor methanol extract was classified in category 5 according to Globally Harmonized Classification System for Chemical Substances and Mixtures (GHS) of OECD as test sample in category 5 is of relatively low acute toxicity and the expected LD50 is more than 5000 mg/kg. This is in agreement with Kennedy et al. [50 more than 5000 mg/kg is considered as nontoxic. The current result of acute oral toxicity is in agreement with that of no in vitro toxicity against the normal cell line and confirmed the safety of the G. bicolor for consumption. As compared to other Gynura species, the ethanol extract of G. procumbens also showed no toxicity to experimental rats dosed at 2 g/kg and 5 g/kg [As shown in y et al.  who statd 5 g/kg .G. bicolor with the highest total phenolic content among the extracts generally showed the strong ability in scavenging DPPH radicals and metal chelating activity. The current study had also clearly demonstrated for the first time that the ethyl acetate extract of G. bicolor strongly possessed cytotoxic activity against the human cancer cell lines and induced apoptotic and necrotic cell death, especially towards the HCT 116 and HCT-15 colon cancer cells. The acute oral toxicity indicated that G. bicolor has negligible level of toxicity when administered orally and has been regarded as safe in experimental rats. Further work is now on the way to investigate the bioactive chemical constituents in ethyl acetate extract and mechanism of cell death in order to provide more convincing evidence on the traditional usage of G. bicolor. The present research findings have evidently met the objectives of the study. The ethyl acetate extract of


Tuberculosis (TB) is still endemic in many developed countries. Involvement of the hand and wrist at presentation is extremely rare, and the diagnosis is often missed. A 57 years old male presented with swelling over the left wrist since 3 years Three swellings over dorsal aspect of the left wrist Soft in consistency Non tender Non compressible Mobile at right angles to the plane of the wrist joint. ESR: 45 mm in 1 hr and rest blood investigations were normal. Ultrsonography showed giant cell tumor of Extensor Digitorum sheath. X-ray: soft tissue swelling and MRI was suggestive of extensor tendon sheath extraskeletal synovial Koch's, or giant cell tumor of tendon sheath. Excision of swelling was planned and intraoperatively, rice bodies were seen inside it. Histopathological examination showed caseous necrosis with granuloma formation. Patient was put on DOT1 therapy. Tuberculous tenosynovitis was first described by Acrel in 1777. Rice bodies occurring in joints affected by tuberculosis were first described in 1895 by Reise. Rice bodies will be diagnosed on plain radiographs when mineralization occurs. More than 50% of cases recur within 1 year of treatment. The currently recommended 6-month course is often adequate with extensive curettage lavage and synovectomy should be performed. Surgery is essential, but the extent of surgical debridement is still debatable. The surgeon has to be aware of the significance of loose bodies when performing routine excision of innocuous looking wrist ganglia. Tuberculosis (TB) is still endemic in many developed countries. Involvement of the hand and wrist at presentation is extremely rare, and the diagnosis is often missed. Extrapulmonary tuberculosis involvement of the musculoskeletal system is uncommon, accounting for only 10% of tuberculosis (TB) cases.57-years-old male presented with swelling over the left wrist since 3 years. No history of trauma, fever. Past history: no history of tuberculosis, diabetes, and hypertension. Pulse-78 beat/min BP-128/70 mmhg.No pallor, cyanosis, clubbing, edema, and lymphadenopathyThree swellings over dorsal aspect of the left wrist: soft in consistency, nontender, noncompressible, and mobile at right angles to the plane of the wrist joint. Respiratory system: AEBE. Cardiovascular system: S1 S2 present. Abdominal examination: NAD. Central nervous system: NAD.  Hb: 12 gm%, TLC: 8600/cmm, P: 65%, L: 20%, M: 03%, and E: 01% ESR: 45 mm in 1 hr, Bsl (R): 95 mg%, BT: 01 min 5 sec, and CT: 04 min 10 sec, BUN: 25 mg%, S.creat: 0.8 mg%, and USG: Giant-cell tumor of Extensor Digitorum sheath.  X-ray: soft tissue swelling, MRI: extensor tendon sheath, extraskeletal synovial Koch's, or giant cell tumor of tendon sheath.Histopathological examination showed caseous necrosis with granuloma formation. Patient was started on DOTS Category I and stitches were removed on postoperative day 8.Tuberculous tenosynovitis was first described by Acrel in 1777 . Rice boTuberculous tenosynovitis of wrist is rare and treatment comprises of excision of lesion and antituberculous chemotherapy will minimize recurrence of disease. The surgeon has to be aware of the significance of loose bodies when performing routine excision of innocuous looking wrist ganglia.


This study aimed to identify possible socio-behavioral correlates of ECC focusing 6–36 months old children and their caretakers.Early childhood caries (ECC) is a serious problem that has remained unexplored in sub-Saharan AfricaCross sectional studies were conducted in a high fluoride rural area, Manyara, Tanzania and a low fluoride urban area, Kampala, Uganda. Totals of 1221 and 816 child - caretaker pairs attending health care facilities for growth monitoring were recruited in Manyara and Kampala, respectively. All caretakers completed face to face interviews at the health care facility. Children underwent oral clinical examination whereby ECC and Enamel hypoplasia were recorded using the dmft (WHO 1997) and the DDE index (FDI 1992).The prevalence of ECC was 3.7% in Manyara and 17.6% in Kampala. According to multiple logistic regression analyses, received oral health information from health worker was the strongest determinant of ECC in Manyara, adjusted OR 0.3, 95% CI 0.09 – 0.93. In Kampala, visible plaque, high sugar intake and presence of enamel hypoplasia associated with ECC, adjusted ORs 2.8 (95% CI 1.61- 4.95), 3.0 (95% CI 1.39 – 6.34) and 2.3 (95% CI 1.36 - 3.95).Oral health education aimed at caretakers of 6–36 months, including health care workers’ information regarding the detrimental consequences for oral health of frequent sugar consumption and poor oral hygiene is important for prevention of ECC in Tanzania and Uganda. Early childhood caries (ECC) is a serious oral health problem, especially in socially disadvantaged populations -4. AccorWhereas studies conducted in developed countries report a substantial reduction in the prevalence of dental caries in the permanent dentition of school aged children, there are no conclusive evidence regarding the trends in the primary dentition . A comprA recently published multi-level conceptual model, incorporating influences of ECC exerted at the individual, family and community level suggests that both social- and behavioral change is important in the prevention of this oral disease . Some stIn sub-Saharan Africa, globalization has been linked with change from a traditional- to a western-style diet. Between 1991 and 2005 per capita sugar consumption (kilogram’s per year) in Tanzania and Uganda increased reportedly from 4.4 to 7.2 and from 2.4 to 9.7 . Sugar cDetailed information regarding the prevalence and influencing factors of ECC provides a valuable tool in the planning, implementation and evaluation of oral health promotion programs. Such evidence is rare when it comes to infants in Tanzania and Uganda. Focusing 6–36 month olds and their caretakers from high- and low fluoridated areas of Tanzania and Uganda, this study aimed to assess the frequency and socio-behavioral distribution of infants’ ECC and sugar consumption.2 and has a population of 1,037,605 of whom 18% are children below 5 years of age. The region is populated with a predominately poor rural population with a literacy rate of 73% [,2 total population 237,280), Hanang  and Babati , constituted the study areas [Recruitment of children and their primary caretakers took place at Haydom Lutheran Hospital (HLH) and in its 20 mobile outreach community service sites in Mbulu, Hanang and Babati districts of Manyara region, northern Tanzania, from August 2010 to January 2011. Manyara region covers an area of 50,836 kme of 73% . It is ady areas . The disdy areas . The RCHdy areas . During 2 and has a population of 1.2 million of whom 18% are under 5 years. Kampala has an overall literacy rate of 75% [2, total population in 2008 300,000) and Makindye , constituted the study areas. The districts have drinking water with fluoride content about 0.3 mgF/L [A cross-sectional Reproductive and Child Health (RCH)-based study was conducted in Kampala district, Uganda, from June to October, 2007. Kampala covers an area of 197 kme of 75% . It is a.3 mgF/L . One non.3 mgF/L .In Tanzania, permission was granted by Medical Research Coordinating Committee of Ministry of Health and Social Welfare and the Ethical Research Committee in Norway (REK VEST). In Uganda, permission was given by The Ethical Committee of Uganda National Council of Science and Technology, Research and Publication Committee at Makerere University. Informed written consent was obtained from participating caregivers in both recruitment sites. When the caregivers could not read and write, verbal consents were obtained.Clinical examinations were carried out by trained and calibrated dentists , whereas trained assistants recorded the observations. Calibration exercises were carried out according to the guidelines on training and calibration published by British Association of the study of Community Dentistry (BASCD) . ChildreSugar consumption was used as both dependent and independent variable in the analyses and was assessed by asking “have you ever given child (Name) glucose water, sugar water, milk tea with sugar, black tea with sugar, sweet potatoes, sugared soda, biscuits/cakes/ice-cream, and sweets/toffees/chocolate in the past 24 hours? Responses were given as (0) No and (1) Yes. A sum score was constructed  and dichotomised using the median split into 0 = low intake and 1 = high intake. Teething symptoms was assessed by asking “Did (Name) experience the following symptoms  during his/her tooth eruption? Responses was given as (0) No and (1) Yes. A sum score was created (range 0–8) and dichotomized yielding 0 = less symptoms (score ≤3) and 1 = more symptoms (score >3). Dental awareness was assessed by asking, “Have you ever received information from health worker/radio on how to care for your own teeth and gums?” Responses were given as (0) No and (1) Yes. Current breastfeeding were assessed in terms of yes (1) and no (0). A measure of breastfeeding duration was achieved by asking mothers who confirmed breastfeeding “For how long have you breastfed”. Socio-demographic variables were assessed in terms of age and sex. Mother’s and fathers education were assessed by asking; “What is the highest level of school you/father of the child have attended? Responses were given as (0) No formal education, (1) Did not complete primary school, (2) Completed primary school, (3) Secondary, (4) Completed Secondary, (5) College/University. A dummy variable was constructed 0 = lower education and 1 = at least primary education. Family wealth was assessed as an indicator of socio-economic status according to a standard approach in equity analysis [st quartile (least poor) to the 4th quartile (poorest).An interview schedule was constructed in English and translated into Luganda and Kiswahili, the main languages in Kampala and Manyara. Kiswahili is the national official language in Tanzania spoken proficiently by almost 95% of Tanzanians. Single words of the Kiswahili interview was translated into Iraque, Datoga, Nyaturu and Nyisanzu languages, when deemed necessary during the interview. The interview schedule was translated in several steps; from English into local languages by bi-lingual Kiswahili/English and Luganda/English professionals, and then back translated to English by independent translators. Project staff at both sites reviewed the interview schedule for semantic, experiential and conceptual equivalence to the original version. Sensitivity to culture and selection of appropriate words were considered. The interview schedule was piloted among caregivers of primary school children to evaluate the quality of the translations in terms of comprehensibility, readability and relevance to assess face validity. At both sites, a slightly modified interview schedule was administered in the field by trained locally recruited research assistants. The interviews were performed in face to face interviews with primary caretakers before their children underwent oral clinical examination. analysis . DurablePredictive Analytics SoftWare (PASW) version 18.0 was used for data analysis. Design effect was adjusted using generalized linear models for binominal distributions in STATA (version 10.0). Univariate analyses were performed by use of chi-square statistics. Multiple variable analyses were performed using logistic regression with odds ratios (OR) and 95% confidence intervals (CI) and Poisson regression with rate ratios (RR). Since using dummy variables runs the risk of loosing information, results from logistic regression analyses were checked using Poisson regression with count variables.In Manyara, the agreement between the examiner and the gold standard on the DDE- score was Cohen’s kappa 0.82. Duplicate examinations were performed with 80 child-caregiver pairs 3 weeks apart. In Kampala, duplicate examinations involved 20 child-caregiver pairs 3 weeks apart. Test-retest reliability of caregiver’s report on child’s sugar intake, in terms of Intra class correlation coefficients (ICC) was 0.96 (95% CI 0.94 – 0.98) in Manyara. Corresponding figure in Kampala was 0.80 (95% CI 0.59 – 0.91). In Manyara, intra-examiner reliability in terms of Cohen’s kappa for dental caries and enamel hypoplasia ranged from 0.85 to 1.0 and from 0.91 to 0.97, respectively. In Kampala, Cohen’s kappa for both dental caries and enamel hypoplasia ranged from 0.8 -1.0. Thus, acceptable levels of intra-examiner agreement (kappa > 0.60) was obtained at both study sites .A total of 816  and 1221  caregiver/child pairs participated in Kampala and Manyara, respectively. Table Table All socio-demographic-, behavioral-, and clinical variables that were statistically significantly associated with ECC and the sum score of sugar consumption in univariate analyses (P < 0.05) were included in multiple variable logistic regression analyses and Poisson regression analyses Table . AccordiAs shown in Table This is one of the first population based studies to systematically investigate correlates of ECC and sugar consumption among infants 6–36 months old residing in rural high fluoridated- and urban low fluoridated regions of Tanzania and Uganda. Thus, the present study provides information about age groups that have not been covered by the national oral health surveys in Tanzania and Uganda. Information about the prevalence of ECC in the general pediatric population of sub-Saharan Africa is scarce and the Kampala and Manyara region have been surveyed to a very limited extent ,6. Many The prevalence of ECC in Manyara 3.7%) and Kampala 17.6%), reflected neglect of infants’ oral health, suggesting that caries might be a public health problem in the two areas . Althoug%, reflec.7% and KAs depicted in Figures Consistent with a number of studies, but at odds with others, the present study revealed a positive association between sugar consumption and ECC in Manyara and Kampala ,47. A prPoor oral hygiene practices have been found to be strongly associated with the prevalence of ECC ,50. In KPrevious studies have shown that socio-demographic factors such as parental education and household income have a direct influence on ECC ,6,47. TrIn sum, this study showed that ECC is present shortly after tooth eruption among infants in both high and low fluoridated areas of Manyara and Kampala. Moreover, this study confirmed the importance of dietary habits and oral hygiene for the ECC development, thus extending findings from developed countries to 6–36 month old children in Tanzania and Uganda. According to the present study, lack of information from health workers, high sugary food and beverage intake, presence of visible plaque and presence of enamel hypoplasia were risk indicators for ECC. ECC and sugar consumption were less prevalent in breastfed children, whereas duration of breastfeeding was longer in children with and without ECC. Thus the role of breastfeeding in protection of ECC remains equivocal. Sugar consumption was determined by low household income, experiences of teething symptoms and health information received by radio and health care workers. Future studies should assess risk indicators using longitudinal analyses. The findings presented can be utilized in the planning of oral health promotion programs that should start during pregnancy and the first months after birth in order to optimize ECC prevention in infants and youngsters.The authors declare that they have no competing interests.RM: principal investigator, designed the study, collected the data (Manyara site), performed the statistical analyses, and wrote the manuscript. AB: participated in the design of the study and provide valuable guidance in the data collection and has been actively involved statistical analyses and writing the manuscript. KM: provide valuable guidance in statistical analyses. JK collected data . AN: main supervisor, designed the study, guided the statistical analyses and writing the manuscript. All authors have read and approved the final manuscript.RM: PhD candidate, University of Bergen. AB: Professor, Department of Clinical Dentistry, University of Bergen, Norway. KM: PhD, Muhimbili University of Health and Allied Sciences, Dar Es salaam, Tanzania. AN: DDS PhD, Professor, Department of Clinical Dentistry, Community Dentistry, and Centre for International Health, University of Bergen, Norway.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/12/24/prepub


H. pylori eradication failures as an alternative to bismuth based quadruple therapy. Methods. Design: prospective single-center study. Patients in whom a first eradication trial with omeprazole/esomeprazole, clarithromycin plus amoxicillin or tinidazole/metronidazole had failed were included. H. pylori status: established by histology, rapide urease test and polymerase chain reaction. Intervention: esomeprazole 20 mg, ciprofloxacin 500 mg, and metronidazole 500 mg, administered together before breakfast and dinner for 10 days. Susceptibility testing was performed by the Epsilometer test. Ciprofloxacin resistance was defined as a MIC of ≥1 μg/mL. Eradication was established by a negative 13C-UBT and 4–6 weeks post-therapy. Efficacy and side effects were determined. Results. 34 patients were enrolled, 32 completed the study. Compliance was excellent (100%). Side effects were mild. Ciprofloxacin-based therapy cured 65% (22/34) of patients by intention to treat and 69% (22/32) per protocol analysis. The prevalence of ciprofloxacin resistance was 8%. Conclusions. The effectiveness of ciprofloxacin-based therapy was greatly reduced despite the high prevalence of ciprofloxacin sensitive H. pylori strains. Bismuth based quadruple therapy still remain the best choice as a “rescue” regimen in our region.The aim of our study was to evaluate the efficacy and tolerability of a ciprofloxacin-based regimen for  Helicobacter pylori infection is the main cause of gastroduodenal diseases. Infected individuals develop chronic active gastritis that in a subset of patients might progress to sever complications. The cure of H. pylori infection changes the natural history of the gastritis. Antibiotic combination therapy for H. pylori eradication is now the standard of care for patients positive for the infection. Guidelines for treatment regimens have been proposed by the European Helicobacter pylori Study Group [dy Group .Proton pump inhibitors (PPIs-)clarithromycin-amoxicillin or metronidazole treatment is the recommended first choice treatment in populations with less than 15 to 20% clarithromycin resistance. In populations with less than 40% metronidazole resistance, PPI-clarithromycin-metronidazole is preferable. Quadruple treatments are alternative first choice treatments. Bismuth-containing quadruple treatments remain the best second choice regimen, if available.H. pylori treatment in Sardinia [Previous studies demonstrated that in northern Sardinia, 1-week triple therapies consisting of a PPI and metronidazole and clarithromycin or amoxicillin have produced lower than acceptable cure rates, ranging from 55% to 57% . ResponsOther combinations using different medications have been proposed as “rescue” therapies including therapy with fluoroquinolones –9 with aH. pylori infection after a treatment failure we evaluated the efficacy and tolerability of a Ciprofloxacin-based regimen.In order to offer an alternative to our patients with H. pylori infection. This study was a prospective, single center trial for treatment of consecutive patients in whom a first eradication trial with omeprazole/esomeprazole, clarithromycin, and amoxicillin (14 patients) or omeprazole/esomeprazole, clarithromycin, and tinidazole/metronidazole (20 patients), had failed to eradicate  Patients who received bismuth compounds, antisecretory drugs, or antibiotics during the 4 weeks before endoscopy were excluded. Other exclusion criteria included pregnancy or lactation; regular use of nonsteroidal anti-inflammatory drugs including acetylsalicylic acid and/or corticosteroids, malignancy, severe liver, heart, kidney or endocrine diseases, and any other clinically significant medical condition, alcohol abuse, drug addiction, history of allergy to any of the drug used in the study. Written informed consent was obtained from all participants, and the study was conducted according to the guidelines of the local ethics committee.H. pylori infection was defined as a positive rapid urease test , the presence of H. pylori on histological examination, and by polymerase chain reaction (PCR) analysis. Posttreatment success was defined by a negative 13C-UBT 4-5 weeks after completing therapy. Pretreatment antrum, one from the angulus and one from the gastric corpus for histology. Biopsy specimens were immediately fixed in 10% buffered formalin and subsequently stained with hematoxylin and eosin and with Giemsa to assess the presence of H. pylori.  Two biopsy specimens were taken from the  The 13C-UBT was performed according to a standardized protocol, the sensitivity, and specificity of which have been reported to be of 95% . All bre2  and 100% relative humidity. The identity of H. pylori was confirmed by colony appearance, Gram stain, and positive biochemical tests . The minimum inhibitory concentration (MIC) was determined by the E-test  following the manufacturer's instructions in Columbia agar plates (90 mm) containing 5% sheep blood as previously described [E-test strips were aseptically placed onto dried surfaces of the inoculated plates. Test results were read after incubation at 37°C for 72 h in a standard atmosphere. MICs were determined by the intercept of the elliptical zone of inhibition with the graded E-test strip. The breakpoints used to define resistant H. pylori were for amoxicillin ≥8 μg/mL, clarithromycin ≥2 μg/mL, metronidazole ≥2 μg/mL, and for tetracycline ≥2 μg/mL, respectively [One antral biopsy was immediately transported at room temperature in a Portagerm pylori  for culture. Biopsies were streaked on Columbia agar plates (bioMerieux) and were incubated for 3–5 days at 37°C, 12% COμg/mL [The cutoff concentrations used to define ciprofloxacin resistance was ≥1 μg/mL , 18. Posantrum and one from the corpus using the QIAamp Tissue kit , according to the manufacturer's instructions. Genomic DNA was extracted from gastric biopsies collected one from the H. pylori vacA gene (VAG-F: 5′-CAA TCT GTC CAA TCA AGC GAG-3′ and VAG-R: 5′ GCG TCA AAA TAA TTC CAA GG-3′) with a reported sensitivity of 98.4% [m1 and 645-bp product for m2 from the middle region of the vacA gene [A set of primers specific for the of 98.4%  was usedacA gene .The intervention consisted of esomeprazole 20 mg, plus ciprofloxacin 500 mg, and metronidazole 500 mg bid. The choice of ciprofloxacin was based on the fact that the efficacy of ciprofloxacin-based regimen was evaluated in a small number of studies –15 and n The patients were evaluated for compliance at followup, after completing treatment, by a physician, both by direct questioning and by pill count and were scored according to the number of days the patient took the study medication. All patients were interviewed for possible side effects by questionnaire. Side effects were graded as mild (did not limit daily activities), moderate (limited daily activities to some extent), or severe .H. pylori eradication efficacy was performed on an “intention-to-treat” (ITT) basis including all eligible patients enrolled in the study and on a “per-protocol” (PP) basis excluding patients lost to the followup. For quantitative variables, percentage and 95% confidence interval (CI) were calculated. Analysis of A total of 34 consecutive patients fulfilling the inclusion criteria were enrolled in the study . H. pylori eradication status was assessed after therapy. Two patients were lost to followup.No one patient had peptic ulcer disease, gastric, nor duodenal erosions. All patients had experienced dyspeptic symptoms. Thirty-two patients took medications as prescribed andH. pylori infection by ITT analysis and 69%  by PP analysis  of patients with analysis . PatientOverall tolerability was good. Excellent compliance (100%) was achieved in all cases. No one pill was returned by the patients. Data on side effects were available for all 32 treated patients. No one patient complained severe side effects. Fourteen patients (9 women) developed mild side effects such as fatigue, diarrhea, nausea, vomiting, or stomachache, making working more difficult in the final treatment days.H. pylori resistant to ciprofloxacin, and amoxicillin, one H. pylori isolate (4%) was resistant to clarithromycin and 3 (12%) to metronidazole. All strains were susceptible to tetracycline  harbored acycline .vacA gene in all gastric samples. In 29 cases the genomic fragment of vacA m2 subtype, more prevalent in our region [vacA m1 subtype. DNA analysis by PCR amplified the middle region of r region , 21, wasH. pylori infection remains a challenge. Quadruple therapy consisting of a PPI combined with bismuth (525 mg four times daily) and two antibiotics  given for 10 to 14 days has been generally used as the optimal second-line therapy after proton pump inhibitor clarithromycin amoxicillin failure and has been the recommended rescue regimen in several guidelines. The introduction of combination capsules containing bismuth-quadruple therapy has led to a resurgence in interest in bismuth-containing regimens as appropriate first-line therapy [Despite the large number of studies, identifing an optimal therapeutic regimen for  therapy –24.Quadruple therapy is especially appropriate as initial therapy in areas in which the prevalence of resistance to clarithromycin or metronidazole is greater than 20% or in patients with recent or repeated exposure to clarithromycin or metronidazole for any reason . We prevH. pylori infection after a treatment failure.In order to offer an alternative to quadruple regimen to our patients we evaluated the efficacy and tolerability of a ciprofloxacin-based regimen in patients with H. pylori infections when first- and second-line therapies have failed [H. pylori and is highly active in vitro in eradicating the organism [Ciprofloxacin belongs to the fluoroquinolone group of antibiotics that are generally used as part of rescue therapy for treating e failed , 27. Rese failed . In addiorganism , and higorganism .H. pylori because of a reduced antibiotic activity in an acid pH environment due to increasing ionization [in vivo activity, ciprofloxacin was administered with acid-suppressing drug such as omeprazole and cure rates of 70% were obtained [In small early trials ciprofloxacin failed to show efficacy against nization , 32. Basobtained . Similarobtained . In Iranobtained . In the obtained .H. pylori infection by ITT analysis and 69% (22/32) per PP analysis beside the low rate of ciprofloxacin resistance among strains (2 out 26).In our study, ciprofloxacin-based therapy cured 65% (22/34) of patients with H. pylori to ciprofloxacin in our country; however, primary resistance to levofloxacin in Italy has been demonstrated to be low [There are no data about resistance of o be low . The liko be low .H. pylori eradication [In the other hand Güzelbulut et al. have shown that in Turkey, a modified sequential therapy regimen, consisting of PPI plus amoxicillin (5 days) followed by PPI plus levofloxacin (5 days) was not superior (ITT: 67%) over standard sequential therapy for dication .We do not have a convincing explanation about the numerous factors that could have affected eradication rates in our study. We may suppose that the drug formulation used, chemical properties of the drug, its stability at varying pH, concentrations of active drug in the gastric tissue, frequency, and timing of drug or the regimen combination could have interfered in the final efficacy of ciprofloxacin-based treatment.In conclusion, based on the results of our study we have to consider ciprofloxacin-based treatment a nonacceptable option as a “rescue” treatment in our region.


AbstractMichanthidium Urban is described and figured from Sergipe and Bahia States, northeastern Brazil. An identification key, illustrations, and a distribution map for the three species of the genus are presented. The male genitalia of Michanthidium almeidai sp. n. and Michanthidium albitarse are illustrated and compared for the first time.A new species of  Michanthidium Urban, 1995 is an exclusively Neotropical anthidiine genus, according to the classification proposed by Hypanthidioides Moure, 1947, along with nine other subgenera. Two species of Michanthidium, Michanthidium  sakagamii (Urban)and Michanthidium  ferrugineum (Urban) were originally described in Gnathanthidium Urban, 1993 and subsequently renamed by Gnathanthidium PasPageBreakteels, 1969. In a recent paper, Hypanthidioides (Michanthidium) albitarsis  as the oldest name, resulting in Michanthidium sakagamii as a junior synonym.The species of the genus can be recognized by the following combination of characters present in both sexes: presence of a juxtantennal carina; strong hooked hairs on the underside of the labial palpus and throughout the length of the galeal blade; omaulus with short carina dorsally; pronotal lobe with curved lamellae in profile view; and base of propodeum with row of lateral pits. Additionally, males are characterized by the basal tooth of mandibles separated from the middle tooth by a broad concave margin; fifth sternum with lateral spine-like projections and the presence of arolia. The females are distinguished by the mandibles with smooth apical margin between two small apical teeth and the basal angle, and the external surface with slender carinae not reaching basal half of mandible.Michanthidium ferrugineum  from Argentina (Tucuman) and Michanthidium albitarse  from Brazil  and Argentina (Misiones). The new species, described here is the first record of the genus from northeastern Brazil  and Coleção Entomológica do Departamento de Zoologia, Universidade Federal de Minas Gerais, Belo Horizonte (DZMG).Urban & Parizotto sp. n.urn:lsid:zoobank.org:act:39581B7E-DF5E-4987-876D-A2C6B6D24B57http://species-id.net/wiki/Michanthidium_almeidaiPageBreakmaculae. Male with light yellow bands in all terga, laterally wider; distal tergum bilobed with a rounded median emargination . One male paratype: same data as holotype, except by “25.10.2005, “L 172 P1190 / Schultesia guianensis” (DZUP). One male paratype: BRAZIL, Bahia. “Lençois, BA / BRASIL 08/01/1997 / E. A. B. Almeida” (DZMG).Type material. Holotype female. BRAZIL, Sergipe. “Canindé do The specific epithet is homage to our friend and bee specialist, Dr. Eduardo Andrade Botelho de Almeida, professor at the Universidade de São Paulo, Ribeirão Preto (FFCLRP).The paratype male from Bahia State is probably a teneral specimen judging the pale coloration of legs and sterna. The paratype from Sergipe State has the sterna black with light yellow bands and tibiae and femora with black areas. The wings and antennae are partially damaged.Larocanthidium Urban, 1997, although in Michanthidium those hairs are also present throughout the length of the galeal blade in both sexes. This morphology suggests a special floral relationship (Michanthidium species. Michanthidium ferrugineum were collected on flowers of Cuphea sp. (Lythraceae). The holotype female of Michanthidium almeidai sp. n. was collected on Scoparia dulcis Linnaeus (Scrophulariaceae) and the paratype male from Canindé de São Francisco on Schultesia guianensis (Aublet) Malme (Gentianaceae). Although these are only isolated records, they could be a first step to understanding specialization in these bees.The curved hairs on the underside of the labial palpi are shared with tionship  although


Oedura lesueurii) are a primary prey for the endangered broad-headed snake , which is restricted to sandstone habitats in southeastern Australia. We sequenced the ND2 gene from 179 velvet geckos, to clarify the lizards’ phylogeographic history and landscape genetics. We also analysed 260 records from a longterm (3-year) capture-mark-recapture program at three sites, to evaluate dispersal rates of geckos as a function of locality, sex and body size.To conserve critically endangered predators, we also need to conserve the prey species upon which they depend. Velvet geckos (The genetic analyses revealed three ancient lineages in the north, south and centre of the species’ current range. Estimates of gene flow suggest low dispersal rates, constrained by the availability of contiguous rocky habitat. Mark-recapture records confirm that these lizards are highly sedentary, with most animals moving < 30 m from their original capture site even over multi-year periods.The low vagility of these lizards suggests that they will be slow to colonise vacant habitat patches; and hence, efforts to restore degraded habitats for broad-headed snakes may need to include translocation of lizards. To conserve an endangered species, we need to provide suitable habitat, shelter, prey items, and other resources (see e.g.-4). PreyHoplocephalus bungaroides, Elapidae) is a small elapid snake restricted to rocky areas (sandstone plateaux) within a 200 km radius of Sydney, in south-eastern Australia)han 3 Ma, and theeen them,34.O. lesueurii have two main implications. The first is that this gecko will be slow to recolonise any local areas from which it is extirpated . Thus, habitat suitability for the endangered broad-headed snake may be spatially heterogeneous as a result of relatively ancient local events that reduced gecko numbers. Second, the low dispersal rates of the geckos need to be considered in any management plan that includes the restoration of degraded habitat previously hosting H. bungaroides. The poor dispersal capacity of O. lesueurii  likely We collected tissue samples from 179 geckos representing 20 populations in southeastern Australia  were performed with the primer pairs AT4882 /ND2R2  was useWe performed ML heuristic searches and bootstrap analyses (1000 replicates) with phyml and we sST, taking into account haplotype frequencies and the genetic distance between haplotypes, in Arlequin 3.0) and (2) the split between Pseudothecadactylus and the New Caledonian Rhacodactylus chahoua and the remaining members of the Diplodactylidae (60.3 Ma [95% HPD: 41.5-79.2]).We used two secondary calibration points from a robust phylogeny focusing on Australasian geckos: (1) TheThe analysis was performed with two independent chains and 20 million generations; chains were sampled every 1000 generations with a burn-in of 2 million generations. Additional simulations were run with the same dataset and the same models, but strictly based on a rate of divergence of 1.3% derived from numerous studies as e.g. Zamudio & Greene’s study onO. lesueurii using GPS co-ordinates imported into ArcGIS 10.0[We conducted mark-recapture surveys on velvet geckos by turning rocks and measuring, individually marking (by toe-clipping) and releasing any geckos found. These studies were conducted in and around Morton National Park (Morton) on a monthly basis between March 2007 and October 2009, and in Dharawal Conservation area  and Yengo and Wollemi National Park  from March 2008 until November 2010. We classified geckos as adult males if they were > 40 mm snout-vent length (SVL) with overt hemipenial bulges; adult females if they were > 40 mm SVL and without such bulges; and juveniles if they were < 40 mm SVL. We determined the distance between rocks used by individual GIS 10.0.The authors declare that they have no competing interests.SD, BC, DP, JW and RS contributed with the conceptual development of the work and the writing of the manuscript. SD, DP, BC, and JW collected samples and data in the field. SD, BC, DP carried out the analyses. All authors read and approved the final version of the manuscript. Maximum parsimony (MP) consensus tree (50% majority rule).Click here for file Pairwise ΦST values between populations and p-values.Click here for file


Piper longum root aqueous extract (PlrAqe) in streptozotocin (STZ) induced diabetic rats.The available drugs for diabetes, Insulin or Oral hypoglycemic agents have one or more side effects. Search for new antidiabetic drugs with minimal or no side effects from medicinal plants is a challenge according to WHO recommendations. In this aspect, the present study was undertaken to evaluate the antihyperglycemic and antihyperlipidemic effects of Diabetes was induced in male Wister albino rats by intraperitoneal administration of STZ (50 mg/kg.b.w). Fasting blood glucose (FBG) levels were measured by glucose-oxidase & peroxidase reactive strips. Serum biochemical parameters such as glycosylated hemoglobin (HbA1c), total cholesterol (TC), triglycerides (TG), very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol were estimated. The activities of liver and kidney functional markers were measured. The statistical analysis of results was carried out using Student t-test and one-way analysis (ANOVA) followed by DMRT.During the short term study the aqueous extract at a dosage of 200 mg/kg.b.w was found to possess significant antidiabetic activity after 6 h of the treatment. The administration of aqueous extract at the same dose for 30 days in STZ induced diabetic rats resulted in a significant decrease in FBG levels with the corrections of diabetic dyslipidemia compared to untreated diabetic rats. There was a significant decrease in the activities of liver and renal functional markers in diabetic treated rats compared to untreated diabetic rats indicating the protective role of the aqueous extract against liver and kidney damage and its non-toxic property.From the above results it is concluded that the plant extract is capable of managing hyperglycemia and complications of diabetes in STZ induced diabetic rats. Hence this plant may be considered as one of the potential sources for the isolation of new oral anti hypoglycemic agent(s). Galega officinalis led to the discovery and synthesis of metformin [Piper Longum is one of those plants used by tribes to treat diabetes, digestive disorders, obesity etc.Diabetes mellitus (DM) is a chronic metabolic disorder characterized by high levels of glucose in the blood due to the impaired secretion of insulin or insulin insensitivity . DM affeetformin . Therefoetformin . In pursPiper longum belongs to family Piperaceae. It grows all over India, in evergreen forests and is cultivated in Assam, Tamil Nadu and Andhra Pradesh. It is a small shrub with a large woody root and numerous creeping, jointed stems, thickened at the nodes. The leaves are alternate, spreading, without stipules and blade varying greatly in size. The fruit, commonly known as pippali and its root, called as pippali mula or modi are used for medicinal purpose. The fruits contain 1% volatile oil, resin, a waxy alkaloid, a terpenoid substance and alkaloids piperine and piperlongumine [Piper longum. Plants of this genus such as Piper betle, Piper nigrum and Piper sarmentosum have been reported for their antidiabetic activities [Piper longum in Streptozotocin (STZ) induced diabetic rats.ongumine . There ativities -12. HencPiper longum (PL), were purchased from the local market and identified by the Botanist, Department of Botany, S.V.University, Tirupati. Voucher specimens (Herbarium Accession Number 713) were deposited in the herbarium, Department of Botany, S.V. University, Tirupati.Dry roots of Hexane, ethyl acetate and methanol extracts were prepared by successive solvent extraction of PL root powder in soxhlet apparatus at 68°C-70°C. The filtrates obtained were distilled and concentrated under reduced pressure at low temperature (40°C to 45°C) in Buchi rotavapor R-200 and finally freeze dried. The yields of the hexane, ethyl acetate and methanol extracts were 38%, 15% and 21% w/w respectively. To prepare aqueous extract the root powder was soaked in distilled water in a glass jar for 48h at room temperature and the solvent was filtered. This was repeated 3–4 times until the filtrate gave no coloration. The filtrate was concentrated to dryness under reduced pressure in Buchi Rotavapor R-200 and finally freeze dried. The yield of the extract was 22% (w/w). All the extracts were stored at 0°C in airtight containers until needed for further studies.Diabetes was induced in male Wistar albino rats aged 2–3 months (180–200 g body weight) by intraperitoneal administration of STZ (single dose of 50 mg/kg b.w.) dissolved in freshly prepared 0.01M citrate buffer, pH 4.5 . After 7This study was approved by Institute’s Animal Ethics Committee vide Resolution no: 08/2011-20129(i)/a/CPCSEA/IAEC/SVU/CHA-SAN/dt.25.09.2011.The animals were divided into six groups and each group consisted of six rats:Group 1: Untreated normal ratsGroup 2: Untreated diabetic ratsGroup 3: Diabetic rats treated with 200 mg Plr.hexane extract /kg b.w.Group 4: Diabetic rats treated with 200 mg Plr.ethylacetate extract /kg b.w.Group 5: Diabetic rats treated with 200 mg Plr.methanolic extract/kg b.w.Group 6: Diabetic rats treated with 200 mg Plr. Aqueous extract /kg b.w.After an overnight fast the diabetic treated rat groups received the ethyl acetate, methanol, aqueous extracts (dissolved in 1 ml of distilled water) and hexane extract (dissolved in 1 ml of 5% Tween 80) by gastric intubation using a force feeding needle. Untreated normal and diabetic rats were fed distilled water alone. Blood samples were collected from the tail vein at 0, 1, 2, 3, 4, 5 and 6 h after the administration of Plr extracts and blood glucose levels were determined by using glucose oxidase–peroxidise reactive strips.The animals were divided into 9 groups and each group consisted of six rats:Group 1: Untreated normal ratsGroup 2: Untreated diabetic ratsGroup 3: Normal rats treated with 200 mg PlrAqe/kg b.w.Group 4: Normal rats treated with 300 mg PlrAqe/kg b.w.Group 5: Normal rats treated with 400 mg PlrAqe/kg b.w.Group 6: Diabetic rats treated with 200 mg PlrAqe/kg b.w.Group 7: Diabetic rats treated with 300 mg PlrAqe/kg b.w.Group 8: Diabetic rats treated with 400 mg PlrAqe/kg b.w.Group 9: Diabetic rats treated with 0.02 g glibenclamide/kg b.w.After an overnight fast diabetic treated groups received PlrAqe suspended in distilled water in respective doses, where as untreated normal and diabetic rat groups were fed with distilled water alone with force feeding needle. Blood samples were collected from the tail vein at 0, 1, 2, 3, 4, 5 and 6 h after the administration of PlrAqe and blood glucose levels were determined by using glucose oxidase–peroxidase reactive strips.Phytochemical analysis was carried out in the PlrAqe by different methods of phytochemical analysis .The rats were divided into three groups, with 6 animals (n = 6) in each group.Group 1: Normal untreated ratsGroup 2: Normal rats treated with 0.02 g glibenclamide/kg b.wGroup 3: Normal rats treated with 200 mg PlrAqe/kg b.w.After an overnight fast group 2 & Group 3 rats were fed with glibenclamide and PlrAqe respectively. Normal untreated rats (group 1) were fed with distilled water alone. Thereafter, following 30 min of post extract and drug administration all the animals were fed with glucose (2 g/kg.b.wt). Blood samples were collected from tail veins prior to dosing and after 30, 60, 90 and 120 min of glucose administration. FBG levels were analyzed using glucose-oxidase-peroxidase reactive strips  .The rats were divided into 5 groups and each group consisted of 6 rats.Group 1: Normal untreated rats.Group 2: Normal rats treated with 200 mg PlrAqe /kg b.w/day.Group 3: Diabetic untreated rats.Group 4: Diabetic rats treated with 200 mg PlrAqe /kg b.w/day.Group 5: Diabetic rats treated with 0.02g of glibenclamide/kg b.w/day.th, 20th and 30th days of the treatment and fasting blood glucose levels were estimated. All the five groups of rats were sacrificed on the 30th day after an overnight fast, by anesthetizing with anesthetic ether and further by cervical dislocation and then blood, liver and kidney were collected and immediately stored at -20°C till further analysis. Body weights of all the animals were recorded prior to the treatment and sacrifice.PlrAqe or glibenclamide was administered to the rats every day morning for 30 days by gastric intubation using oral gavage. Blood samples were collected from tail veins before the start of the treatment and on 10Estimation of blood glucose was carried out by glucose oxidase–peroxidase method . The estThe results were expressed as mean ± S.D. The statistical analysis of results was carried out using Student t-test and one-way analysis (ANOVA) followed by DMRT.The effects of hexane, ethyl acetate, methanolic and aqueous extracts of Plr on the fasting blood glucose levels of diabetic rats are given in Table Table Phytochemical analysis revealed the presence of glycosides, alkaloids and carbohydrates in PlrAqe.In this study the FBG levels of all groups of animals were estimated from 0 min to 120 min. In all groups the FBG levels were raised at 60 min (due to glucose load) but after that there was a significant decrease in the FBG levels of group II and III when compared to group I. The results are depicted in Figure 1C levels of the diabetic untreated group were significantly higher than those in normal control group (11.18 ± 2.0% and 5.73 ± 0.47% respectively). Treatment with the PlrAqe in diabetic rats reduced the HbA1C to a significant level (7.1 ± 0.46%), indicating a significant improvement in glycemic control in diabetic rats upon treatment. The body weights of normal, normal treated, diabetic treated and glibenclamide treated group, increased significantly by +25.8 g, +31.6 g, +24.1 g and +21.2 g respectively, whereas the body weights of diabetic control group decreased by −33.3 g (Table PlrAqe exhibited significant antihyperglycemic activity after 30 days of treatment in diabetic rats. It produced 66.7% (P < 0.001) fall in FBG levels compared to the initial FBG levels prior to the treatment. No significant effect was observed in normoglycemic rats. Glibenclamide produced 33% decrease in FBG levels in diabetic rats. At the end of 30 days treatment, the HbAFigure Figure Piper longum roots to treat the diabetes mellitus [Piper longum to treat DM. Earlier, Shanmugam Manoharan et al. demonstrated that the ethanolic extract of dried fruits of Piper longum has potent antihyperglycemic and antilipidperoxidative activity in alloxan induced diabetic rats [Piper longum for evaluating antihyperglycemic and antihyperlipidemic activity since the phytochemical constituents of fruits and roots are different. In our study STZ was used to induce diabetes mellitus in rats. At low dose, STZ (50 mg/kg b.w) partially destructs the beta cells resulting in insufficient insulin secretion causing type 2 diabetes [Sheshachala forest , which lie geographically in the South Eastern Ghats are known for the rich heritage of flora where the tribes use mellitus . Hence otic rats . In the diabetes . It is wdiabetes , is oftediabetes ,33.During the short term study the aqueous and methanolic extracts produced significant antihyperglycemic activity at a dosage of 200 mg/kg b.w in diabetic treated rats. Aqueous extract is more potent than methonolic extract. The hexane and ethylacetate extracts did not show significant antihyperglycemic activity, may be due to the lack of phytochemical constituents like alkaloids and glycosides which are present in methanolic and aqueous extracts.During the dose dependent study, the aqueous extract at a dosage of 200 mg/kg b.w has produced maximum antihyperglycemic activity in diabetic rats. The higher doses of PlrAqe (300 & 400 mg/kg b.w) could not produce the predictable higher antihyperglycemic effect due to the presence of some other substances in the aqueous extract, which interfere with the antihyperglycemic effect . So, theThe oral glucose tolerance test also confirmed blood glucose lowering activity of PlrAqe. The onset of antihyperglycemic action was observed from 60 min of the treatment and a steady state increase in the action continued up to 120 min. The PlrAqe may be involved in enhancement of glucose utilization, so blood glucose levels were significantly decreased in glucose loaded rats.et.al[et.al[Piper betle leaves and Piper nigrum seeds respectively for 30 days.The loss in body weights observed in STZ induced diabetic rat group (after a period of 30 days) may be due to muscle wasting and loss of tissue proteins upon induction of diabetes with STZ ,36. The et.al and Kale.al[et.al reportedPiper betle which belongs to same genus has been reported to have the ability to reduce HbA1C levels in diabetic rats [HbA1c is used as a marker for estimating the degree of protein glycation in diabetes mellitus. HbA1c was found to increase in patients with diabetes mellitus and the amount of increase is directly proportional to the fasting blood glucose level . In diabtic rats .Diabetes mellitus is usually associated with prominent levels of serum lipids and such an increase causes the risk factor for coronary heart diseases . A varieUnder hyperglycemic condition disturbances in carbohydrate, lipid and protein metabolisms together with oxidative stress are likely to affect hepatic and renal functions. Hence our study was also focused to know the protective activity of PlrAqe against hepatic and renal damage caused by diabetes. In the present study serum enzymes such as SGOT, SGPT and ALP were used in the evaluation of hepatic damage Figure . In diabIn our study elevated levels of serum urea and creatinine were observed in diabetic untreated rats, which are considered as significant markers of renal dysfunction . NegativPiper longum by traditional healers in the treatment of diabetes. However the nature of the active principle(s) responsible for all these positive effects requires further investigation.All these beneficial effects of PlrAqe are especially hopeful in preventing hyperglycemia, cardiovascular, hepatic and renal diseases. In conclusion, this study has undoubtedly provided scientific confirmation and evidence for the safe use of the roots of Piper longum root aqueous extract; STZ: Streptozotocin; FBG: Fasting blood glucose; HbA1c: Glycosylated hemoglobin; TC: Total cholesterol; TG: Triglycerides; VLDL: Very low density lipoprotein; LDL: Low density lipoproteins; HDL: High density lipoproteins; Plr: Piper longum root; OGTT: Oral glucose tolerance test.PlrAqe: All authors are in agreement with the content of the manuscript and authors do not have any conflict of interest.Piper longum roots to the completion of the study. RBK participated in planning the experiments, evaluation of anti hyperglycaemic activity and statistical analysis. SS, TKT and MVJK maintained the animals and helped in estimation of lipid profiles, liver & renal functional markers. CA designed the whole study, analyzed & interpreted the results and corrected the manuscript. All authors read and approved the final manuscript.SAN has made significant contribution throughout the study starting from collection of The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/37/prepub


Research investigating animal models of FXS, characterized by limited or lack of fragile X mental retardation protein, (FMRP), has linked brain dysfunction to deficits in the cholinergic and glutamatergic systems. Thus, we sought to examine The study participants included 18 adolescents and young adults with FXS, and a comparison group of 18 individuals without FXS matched for age, sex and general intellectual functioning. Proton magnetic resonance spectroscopy (MRS) was used to assess neurometabolite levels in the caudate nucleus, a region known to be greatly enlarged and involved in abnormal brain circuitry in individuals with FXS. A general linear model framework was used to compare group differences in metabolite concentration.P = 0.027) and in glutamate + glutamine (P = 0.032) in the caudate nucleus of individuals with FXS, relative to individuals in the comparison group.We observed a decrease in choline (This study provides evidence of metabolite differences in the caudate nucleus, a brain region of potential importance to our understanding of the neural deficits underlying FXS. These metabolic differences may be related to aberrant receptor signaling seen in animal models. Furthermore, identification of the specific neurometabolites involved in FXS dysfunction could provide critical biomarkers for the design and efficacy tracking of disease-specific pharmacological treatments. FMR1) gene promoter region and reduced production of the fragile X mental retardation protein (FMRP). Reduced FMRP expression has been linked to increased density of immature dendritic spines and abnormal dendritic morphology in humans with FXS [Fmr1-knockout (KO) mouse) [Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, affects approximately 1 in 5,000 males and 1 in 10,000 females , hypermewith FXS  and mous) mouse) . FMRP is) mouse) .Reduced FMRP results in a constellation of behavioral and cognitive impairments, including specific weaknesses in social cognition, communication and executive function -9, in adFmr1-KO mouse results in amplified signaling through specific G protein coupled receptors (GPCRs) – group I metabotropic glutamate receptors (mGluR1 and mGluR5) [Fmr1-KO mice, and subsequently result in reduction of some maladaptive behaviors associated with FXS [Other neuroimaging research implicates specific neurotransmitter systems involving choline, glutamine and gamma-aminobutyric acid (GABA) ,16. Rese mGluR5)  and musc mGluR5) . Potentiwith FXS ,19. Idenin vivo investigation of neurometabolite levels in males with FXS reported reduced choline/creatine ratios in bilateral dorsolateral prefrontal cortex [To date, examination of specific brain systems in humans with FXS is very limited. One l cortex , an intel cortex .The present study sought to examine neurometabolite levels in a broader sample of individuals with FXS, including both females and males, to address the hypothesis that similar neurometabolic profiles are present in both sexes. Females, like males with fragile X syndrome, have reduced FMRP, and disadvantageous cognitive and behavioral symptoms, albeit to a lesser degree than their male counterparts . Furtherin vivo single-voxel proton magnetic resonance spectroscopy (MRS) and included N-acetylaspartate (NAA), creatine, choline, myo-inositol, glutamate, and glutamine + glutamate (Glx). We hypothesized that individuals with FXS, including females, would display lower levels of choline and glutamate-related metabolites relative to the comparison group.We examined the caudate nucleus because previous evidence has indicated this region’s importance to our understanding of the neurobiological basis of FXS -12,14,24FMR1 mutation on DNA testing utilizing standard Southern blot techniques; mean age = 20.79 years, SD = 3.38, 18 females), and a comparison group of 24 individuals without FXS (confirmed via genetic screening [Ps >0.10). Potential participants in the comparison group were excluded for any other known genetic condition, premature birth, low birth weight, or a history of severe psychiatric, neurological or medical disorder. Participants were free from magnetic resonance imaging (MRI) contraindications. Participants were recruited across the USA and Canada through advertisements, referrals, word of mouth and from our database. Participants and/or their parents gave written informed consent and assent to participate in the study. All protocols were approved by the Institutional Review Board at Stanford University, CA, USA.The participants included 27 adolescents and young adults with FXS  stimulants; 2) selective serotonin reuptake inhibitors (SSRIs); and 3) atypical antipsychotics, anticonvulsants and other drugs affecting neurological functioning. The association of medication class with metabolite concentration was assessed via multiple regression, separately in each participant group. One participant in the FXS group was taking the acetylcholinesterase inhibitor donepezil. Given this drug’s intended effect on the cholinergic system we considered this participant a potential outlier for all subsequent analyses.General intellectual function was assessed via the Wechsler Abbreviated Scale of Intelligence (age 17 years or older)  or the WParticipants were scanned on a 3 Tesla MRI Scanner  at the Lucas Center for Neuroimaging, Stanford University, using one of two custom single-channel quadrature head coils (one head coil was decommissioned midway through the study). A T2-weighted gradient echo spiral pulse sequence  was used to produce an image on which a 1.5 cm isotropic voxel was prescribed, encompassing as much of the right caudate head as possible  [myo-inositol (3.58 ppm), glutamate (2.36 ppm) and Glx (3.78 ppm)  . The res 0.8 ms) . MetabolPs >0.10), and all subsequent reporting includes only this final sample  of the NAA peak was equal to or greater than 15 and the spectral line width was less than 20 Hz. Nine spectra from participants in the FXS group and seven spectra from participants in the comparison group were eliminated, resulting in a final sample size of 18 participants with FXS  and 18 participants in the comparison group . These smaller groups did not differ in age, sex or general intelligence (http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/) were used to segment each T2-weighted image, and calculate the percentage of each tissue type  within each MRS voxel.The coordinate location of the MRS voxel was used to prescribe a corresponding 1.5 cm isotropic voxel on each participant’s T2-weighted anatomical image. Brain extraction and segmentation tools from the Oxford Centre for Functional MRI of the Brain , head coil type was related to within group metabolite concentration and was therefore added as a covariate in analyses of metabolite concentration.Our primary analysis compares metabolite ratios relative to creatine (3.03 ppm), but we also report absolute values as a secondary analysis. A general linear model framework was employed to evaluate group differences in metabolite concentrations and performance on cognitive/behavioral assessments. Our primary goal was to compare group differences in choline and glutamate-related metabolites; comparisons of NAA and P = 0.015) and a correspondingly smaller proportion of CSF (P = 0.035) for individuals with FXS relative to individuals in the comparison group; white matter proportions did not differ . The model assessing group differences in metabolite concentration ratios included group (FXS versus comparison) as the independent variable, metabolite concentration ratio as the dependent variable, and head coil type and grey matter percentage as covariates. Due to the high variability in absolute metabolite values we used the non-parametric Mann–Whitney U test, and thus were unable to account for head coil type and grey matter percentage as covariates at the group level for these absolute values, but metabolite concentrations were normalized for voxel tissue fractions at the individual level. We assessed within group relationships between metabolite concentration ratios and cognitive/behavioral assessment scores with two-tailed Pearson’s correlations.The MRS voxel contained a greater proportion of grey matter  and Glx/creatine ratios (P = 0.032) in the FXS group relative to the comparison group (Table P = 0.082); glutamate/creatine and myo-inositol/creatine concentrations did not differ (Ps >0.10). The pattern of results and significance did not change with the individual taking donepezil excluded: the FXS group displayed reduced choline/creatine (P = 0.015) and Glx/creatine (P = 0.025); NAA/creatine, glutamate/creatine and myo-inositol/creatine levels did not differ (Ps >0.10). When comparing absolute values we observed reduced choline (P = 0.010), Glx (P = 0.031) and NAA (P = 0.012) in the FXS group relative to the comparison group. Glutamate, myo-inositol and creatine concentrations did not differ (Ps >0.10).We observed reduced choline/creatine ratios P = 0.07 and Glxs P = 0.02 in the Ps >0.10). For females only, the FXS group displayed significantly lower choline/creatine (P = 0.027) and Glx/creatine (P = 0.043) levels relative to the comparison group. Statistical analyses were not undertaken for male subgroups due to sample size , although effect sizes for between group differences in choline/creatine and Glx/creatine levels were similar to those for females .Choline/creatine and Glx/creatine ratios were also compared between female only subgroups , which did not differ in age or general intellectual functioning (Ps >0.10). Group comparisons of metabolite concentration were repeated including only medication-free individuals . Choline/creatine and Glx/creatine levels were lower for the FXS group, but the differences did not reach significance (Ps >0.10).Within group analysis of medication effects on each metabolite ratio indicated that metabolite concentration was not significantly related to medication status in either group ; results did not change when excluding the participant taking donepezil (Ps >0.10).As an exploratory analysis we examined within group correlations between metabolites for which we found a significant group difference – choline/creatine and Glx/creatine – age, and cognitive/behavioral scores. There were no significant correlations within either group  for biomarkers of FXS based on neuroimaging data. JLB, EWS, EMQ, MR, SP, DS, DM, MG and AAL reported no biomedical financial interests or potential conflicts of interest.JLB carried out the statistical analysis and drafted the manuscript. ALR conceived of the study, participated in its design and coordination, and helped to draft the manuscript. EWS, EMQ, MR, SP and AAL recruited participants, collected the data, and helped to analyze the data and draft the manuscript. DS, DM and MG contributed to MRS pulse sequence design, spectroscopy data analysis and interpretation. All authors read and approved the final manuscript.


Objective. Determine the potential of procalcitonin (PCT) to predict neurological outcome after hypothermia treatment following cardiac arrest. Methods. Retrospective analysis of patient data over a 2-year period. Mortality and neurological outcome of survivors were determined 6 months after cardiac arrest using the Cerebral Performance Category (CPC) score. Results. Data from 53 consecutive patients were analyzed. Median age was 63 (54–71) and 79% were male. Twenty-seven patients had good outcome (CPC ≤ 2) whereas 26 had severe neurological sequelae or died (CPC 3–5). At 48 h, after regaining normothermia, PCT was significantly higher in patients with bad outcome compared to those with good outcome: 3.38 (1.10–24.48) versus 0.28 (0–0.75) ng/mL (P < 0.001). PCT values correlated with bad neurological outcome  and predicted outcome with an area under the curve of 0.84 (95% CI 0.73–0.96). A cutoff point of 1 ng/mL provided a sensitivity of 85% and a specificity of 81%. Above a PCT level of 16 ng/mL, no patient regained consciousness. PCT provided an additive value over simplified acute physiology score II. Conclusions. PCT might be an ancillary marker for outcome prediction after cardiac arrest treated by induced hypothermia.  Successful cardiopulmonary resuscitation after sudden cardiac arrest (CA) is followed by the early development of a systemic inflammatory response syndrome, characterized by an increase of serum markers of inflammation including C-reactive protein (CRP), tumor-necrosis factor alpha, interleukin-6, and procalcitonin (PCT) . In patients with sepsis, PCT has been shown to be a specific marker of bacterial infection, a valuable tool to guide antibiotic therapy, and a predictor of sepsis-associated mortality –4. In coMild therapeutic hypothermia (MTH) has been shown to improve neurologic outcome after CA in several studies but may modulate the serum PCT concentration and its utility as an outcome-predictive marker –11. PCT This cohort study was conducted in an 18-bed mixed intensive care unit (ICU) of an academic tertiary care centre linked to the national institute for cardiosurgery and interventional cardiology of Luxembourg. This investigation was approved by the National Ethics Committee of Luxembourg and written informed consent from the next of kin of each patient was obtained prior to study inclusion.All adult patients (age >18 years) with primary cardiac arrest and successful resuscitation with an initial Glasgow coma score (GCS) <8 were eligible for the study. Tracheal intubation, mechanical ventilation, and sedation were mandatory for every patient. Patients with cardiac arrest following trauma, patients with known severe neurological disorders or stroke, head injury, or patients with end-stage noncardiac disease were excluded.Prior to the transfer to our ICU patients were subjected to coronary intervention according to published international guidelines . InductiArterial blood samples were collected 48 h after cardiac arrest for measurement of procalcitonin (PCT), C-reactive protein (CRP), and white blood cells (WBC). The samples were immediately processed in the central hospital laboratory.No decision to withdraw or withhold treatment in patients remaining unconscious was taken until sedation was completely stopped for at least 5 days. Thereafter, therapeutic limitations were implemented for patients presenting one or more predefined signs of bad neurological outcome: fixed pupils, seizures, no motor response, absence of somatosensory evoked potentials, and major signs of hypoxic brain damage on CT-Scan or MRI .All pre- and intrahospital data were collected retrospectively from the files of the patients and were recorded according to the Utstein-style template . Only paPCT was measured using an electrochimiluminescent immuno assay  on the Cobas e601 analyzer . A Modular analytics P module  was used to determine CRP by immunoturbidimetry using 3rd-generation CRP Tina-quant reagent . The functional sensitivity for PCT and CRP testing was 0.09 ng/mL, respectively, 0.6 mg/L. WBC was determined automatically by the Advia 2120 Haematology system . Only values from samples free of haemolysis were used for this study.3), purulent tracheal aspiration, and microbacterial confirmation of tracheal aspirates.Pneumonia was defined as new or progressive infiltrate on the chest X-ray, leucocytosis  with need for vasopressor despite fluid therapy. In contrast to current guidelines, body temperature and respiratory rate were not included in the definition, since all patients were mechanically ventilated and subjected to MTH.Sepsis was defined as heart rate >100/min, WBC >12000 cells/mmCardiogenic shock was defined as a systolic blood pressure <90 mmHg despite fluid replacement, the need for inotropes, vasopressors, and the need for intra-aortic balloon pump (IABP) without evidence of infection.Neurological outcome was assessed by the Cerebral Performance Category (CPC) score by physicians unaware of the blood tests and implicated neither in the treatment of the patient nor in this study . The CPCPrimary end point of this study was the correlation of PCT values at 48 hours after CA with neurological outcome at 6 months.Secondary endpoints were the correlation of CRP and WBC values at 48 h after CA with neurological outcome at 6 months.t-test for data following a Gaussian distribution and Mann-Whitney test for non-gaussian data. Fisher's exact test was used for categorical variables. Correlation between outcome and PCT, CRP, and WBC was estimated using the Spearman test. The area under the receiver operating characteristic curves (AUC) was used for prediction capacity evaluation. Multiple logistic regression models were implemented to investigate the predictive value of multiple clinical parameters. A P value <0.05 was considered significant. Continuous variables are expressed as median and interquartile range (IQR) and categorical variables are expressed as count and proportions, unless stated otherwise. All datasets were subjected to the Shapiro-Wilk normality test before statistical analysis. Comparisons between 2 groups of continuous variables were performed using http://protein.bio.puc.cl/star.html) software.Statistical analysis was performed using SigmaPlot  and StAR . Between April 2008 and June 2010, 68 consecutive adult patients subjected to MTH after successful cardiopulmonary resuscitation were admitted to our ICU. Five patients died during the first 48 h after admission, nine patients did not have the full dataset available, and one patient had no informed consent signed. Therefore, 53 patients were included in the analysis. The median age was 63 (54–71) years and 42 patients (79%) were male. Cardiac arrest was caused by a cardiac aetiology in 43 (81%), by respiratory failure in 5 (9.5%) and by other nontraumatic causes in 5 (9.5%) patients. The initial rhythm was ventricular fibrillation or pulseless ventricular tachycardia in 35 (66%) patients and asystole or pulseless electric activity in 18 (34%) patients. Out of hospital cardiac arrest (OHCA) occurred in 40 (75%) patients and in hospital cardiac arrest (IHCA) in 13 (25%) patients. Patients were dichotomised into two groups according to their CPC score 6 months after CA. Patients were considered having a good neurological outcome when CPC was 1-2 while the patients in the group with bad neurological outcome displayed a CPC score of 3–5.Demographic and Utstein-style data are presented in P = 0.15), respectively, sepsis versus no sepsis (1.18 (0.17–78.05) ng/mL versus 0.85 (0.19–3.75) ng/mL, P = 0.59).The incidence of pneumonia and sepsis was similar in both groups. Nevertheless, antibiotic use was higher in survivors than in nonsurvivors. There was no significant difference in PCT at 48 h in patients with pneumonia versus no pneumonia (0.70 (0–1.71) ng/mL versus 1.30 (0.22–8.31) ng/mL, P < 0.001). There was a significant correlation between PCT values and bad neurological outcome .PCT, CRP, and WBC levels at 48 hours after cardiac arrest are displayed in P < 0.0001; A receiver operating characteristic curve (ROC) analysis revealed a capacity of PCT to predict neurological outcome with an AUC of 0.84 . PCT as well-predicted outcome (P = 0.03), and provided an additive value over SAPS II as showed by an increase of R2 values from 0.29 to 0.36 when PCT was added over SAPS II prediction.Finally, we investigated the predictive value of PCT in relationship with other clinical parameters such as simplified acute physiology score II (SAPS II), cardiogenic shock, and time to return of spontaneous circulation (ROSC), all known to affect outcome after cardiac arrest. Using multiple logistic regression models, we observed that SAPS II was the best predictor of outcome  and perhaps death. Therefore, PCT might be a marker of the severity of the postcardiac arrest syndrome , 5, 27. Neurological outcome in our study was evaluated at 6 months to assure a stable neurological situation and avoid underestimation of the patients' recovery . In factOur study clearly has some limitations. As a single centre retrospective trial it has to deal with all inherent risks of bias of this study type. Although a sample size calculation has been done, we must acknowledge that the sample size of our study population might still be too small to be definitively conclusive. Our findings and hypothesis clearly require to be verified in a larger, multicentre and investigator-blinded trial.We included IHCA and OHCA as well as all types of initial rhythms as we think that a prognostic marker after cardiac arrest should neither be influenced by the location of cardiac arrest nor the initial rhythm to be universally used.We only studied PCT values at 48 hours after CA, as this was part of the blood sampling protocol of the study. As no PCT values before or beyond this time point were available, we might have missed other time points with significant differences between groups to predict outcome or potential effects of MTH , 6.In this small study of 53 cardiac arrest patients treated with MTH, we found that PCT, measured 48 hours after cardiac arrest, might be an ancillary predictor for the 6-month outcome after successful resuscitation. A cutoff value above 1 ng/mL predicted bad outcome with an accuracy of 84%. Above 16 ng/mL, PCT predicted bad outcome with a 100% sensitivity (95% CI: 0.87–1.00). Sequential measurements of PCT in larger multicentre trials are required to confirm these findings.