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Acute liver damage (ALD) is associated with high-dose intravenous (iv) glucocorticoid (GC) (ivGC) pulse therapy in ~1 % of patients for Graves’ orbitopathy (GO). It has been proposed that statins may increase the risk of ALD. Here we investigated the frequency of ALD according to the assumption of statins in a large retrospective cohort study. We studied 1076 consecutive patients with GO given ivGC. ALD was defined as an increase in alanine aminotransferase ≥300 U/l. At the time of ivGC, 62 patients were taking statins and 1014 were not. The frequency of ALD has been reported to be 1.2 cases/100,000 statins users and 1300/100,000 in GO patients given ivGC. Thus, the expected frequency of ALD in patients given both statins and ivGC is 1560/100,000. Transferring these data to our series, one would have expected at least 0.96 cases of ALD (~one case), in the 62 patients given both ivGC and statins. However, no cases of ALD were observed in patients given statins, and the previously reported 14 cases of ALD in this series were seen in patients who were not taking statins. The lack of observation of cases of ALD in patients given ivGC and statins is quite reassuring. Although caution should be applied to any patient candidate to ivGC treatment and this should be particularly accurate in patients given statins, our findings somehow justify the use of ivGC in patients under statins, although further studies in larger cohorts are needed to confirm our conclusions.

ABSTRACTThe Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a new quantitative electrochemiluminescence immunoassay which uses onboard dilution and a simple algorithm to determine HBsAg levels expressed in international units (IU)/ml (standardized against the World Health Organization [WHO] Second International Standard). This study evaluated its performance using routine serum samples from a wide range of HBsAg carriers and patients with chronic hepatitis B (CHB). HBsAg levels were measured in serum samples collected independently by five centers in Europe, Australia, and Asia. Serial dilution analyses were performed to assess the recommended dilution algorithm and determine the assay range free of hook effect. Assay precision was also established. Following assessment of serial dilutions (1:100 to 1:1,000,000) of the 611 samples analyzed, 70.0% and 85.6% of samples tested with analyzers incorporating 1:100 (Elecsys 2010 and cobas e 411) and 1:400 (Modular Analytics E170) onboard dilution, respectively, fell within the linear range of the assay, providing a final result on the first test. No high-dose hook effect was seen up to the maximum HBsAg serum level tested (870,000 IU/ml) using the dilution algorithm. HBsAg levels were reliably determined across all hepatitis B virus (HBV) genotypes, phases of HBV infection, and stages of disease tested. Precision was high across all analyzers (% coefficient of variation [CV], 1.4 to 9.6; HBsAg concentrations, 0.1 to 37,300 IU/ml). The Elecsys HBsAg II quantitative assay accurately and reliably quantifies HBsAg in routine clinical samples. Onboard dilution minimizes retesting and reduces the potential for error.

ABSTRACTPolymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 103genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B.

We report a case of acute hepatitis of autoimmune origin which occurred in a 43-yr-old woman during iv glucocorticoid (GC) pulse therapy for Graves’ ophthalmopathy (GO). Prior to therapy, liver function tests were normal with no previous history of liver disorders or conditions predisposing to GC-associated liver damage. After the administration of a 4.7-g cumulative dose of methylprednisolone acetate, there was a marked increase of liver enzymes, prompting immediate discontinuation of iv GC. Nevertheless, liver enzymes increased further, reaching a peak 45 days later, with values 30- to 50-fold greater than those prior to therapy, associated with evidence of impaired liver function. Liver biopsy showed a marked lymphocytic infiltration, likely indicating an autoimmune hepatitis. Based on the assumption that following GC-induced immune suppression, autoimmune hepatitis might have been precipitated by sudden re-activation of the immune system during interpulse periods, we treated the patient with im and then oral GC, in order to re-induce immune suppression. Within three days from re-institution of GC therapy, there was a marked reduction of liver enzymes and amelioration of liver function. Complete normalization was achieved two months later, while the patient was still receiving a low maintenance dose of oral prednisone.

The aim of antiviral therapy of chronic hepatitis B is to control Hepatitis B Virus (HBV) replication and to cure liver disease avoiding the progression of chronic hepatitis to cirrhosis and the end stage complications of cirrhosis. HBeAg / anti-HBe seroconversion is the hallmark of response in hepatitis B “e” antigen (HBeAg) positive patients. In the patients with antibody against HBeAg (anti-HBe positive) the combination of HBV DNA and anti-HBc IgM tests provides adequate diagnostic accuracy. Patients with biochemical and / or histological disease activity are eligible to therapy. The drug choice is based on age, disease severity, risk of complications, side effects and compliance, particularly in anti-HBe positive patients where prolonged treatment is needed. Interferon (5-6 MU daily or 9-10 MU thrice weekly for 4-6 months) is the first line therapy for HBeAg positive patients and (5-6 MU thrice weekly for 12-24 months) for anti-HBe positive patients. When IFN is contraindicated or ineffective, Lamivudine (100 mg) or Adefovir Dipivoxil (10 mg) are given as long as 4-6 months after HBeAg / anti-HBe seroconversion or for long-term treatments in HBeAg positive non-responders and anti-HBe positive patients. Patients with more advanced forms of cirrhosis and portal hypertension are to be treated within liver transplantation programs. Fifteen to 30 of treated patients achieve sustained response and more than 60 of them experience long-term disease remission during therapy. In perspectives, currently available molecular and immunologic tools and modelling of viral dynamics will help to address the therapy issue with more complex, efficacious and individually tailored treatment schedules.

Summary The apparent shear strength of rock discontinuities is considerably smaller than that of small scale samples. At the same time, the sliding behavior is characterized, in situ, by marked instabilities, with the typical features of Critical Phenomena. Contact mechanics permits to calculate normal and tangential forces at any point, and to follow the stick-slip transition for arbitrary loading histories. On the other hand, the above aspects are not captured by the classical theories, including those based on roughness indices. We argue that the multiscale topology of contact domains plays a fundamental role in determining the behavior of rock joints. In particular, experiments and numerical simulations show that these domains are lacunar sets with fractal dimension smaller than 2.0. This provides peculiar scaling of normal and tangential pressures at the interface, and the consequent size-dependence of the apparent friction coefficient. Moreover, we implement Renormalization Group to determine the critical point (e.g. the critical shear force) when rock sliding occurs. We show that the critical force is less than the one predicted by the classical Coulomb’s theory, and that it depends on the specimen size and on the topology of the interface. The same reasoning can be extended to other phenomena, e.g., to the rupture of brittle materials.

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A simple and quick procedure has been developed for the on-line preparation of a one phase emulsion in a flow injection (FI) system for chromium determination by electrothermal atomic absorption spectrometry (ETAAS) with Zeeman-effect background correction. The entire FI system was controlled by a computer, independent of the spectrometer. A 1-ml plug of sample solution was injected into the carrier stream of hexane and was subsequently mixed with streams of 3.8% (m/v) sodium chloride, 5.0% (v/v) sodium dodecylsulfate (SDS) and 5.0% (v/v) of sec-butanol. The flow rates of the carrier, sodium chloride, SDS and sec-butanol streams were 1.0, 0.5, 0.5 and 0.2 ml min⁻¹, respectively. The sonication of the flowing solutions improved the stability of the emulsion and provided quantitative recoveries. An emulsified sub-sample of 200-µl was collected in a capillary of a sampling arm assembly. Thereafter, 20 µl of this sample solution were introduced by means of positive displacement with air through a time-based solenoid injector (TBSI) in the graphite tube atomizer. The furnace program included two drying steps (at 110 and 130 °C) to minimize the sputtering of the sample in the atomizer. In all instances, the pyrolysis, atomization and cleaning temperatures of 1600, 2400 and 2500 °C were used to obtain good peak profiles with low background signals without the addition of any chemical modifier. The method provides a linear range for chromium from 7.0 to 50 µg l⁻¹ and a limit of detection of 4.0 µg l⁻¹, which corresponds to 6.0 ng g⁻¹. The results were precise (0.6–0.8% RSD) and the recovery values found in the analysis of spiked samples ranged from 99.2 to 102.2%. The agreement between the observed and certified values obtained from two NIST Standard Certified Materials was good. The method described is satisfactory for determining chromium in new and used lubricating oil samples.

A method for the determination of different mercury species in whole blood is described. Inorganic mercury (InHg) was determined in 2 ml of standard solutions or blood samples using head space (HS) injection coupled to atomic absorption spectrometry (AAS) after treatment with concentrated sulfuric and tin(II) chloride as a reductant agent in a closed HS vial. After stirring, the InHg was converted to elementary mercury and carried with a nitrogen flow through a quartz cell heated at 200 °C and the absorbance signal was evaluated by AAS. For the determination of methylmercury (MeHg), 2 ml of a standard solution or a blood sample were treated with 10 mg of iodoacetic acid and 0.4 ml of concentrated H2SO4. Then, the MeHg species were HS-injected into a gas chromatograph (GC), separated on a semicapillary column (AT-1000) with a flow of helium, then carried to the quartz cell heated at 1000 °C and detected by AAS. The high content of salts in blood samples, where sodium chloride is the major component (0.14 mol l–1), affected the gas–liquid distribution coefficient of both mercury species in the HS vial. A linear calibration graph was obtained in the ranges 1–20 and 1–125 µg Hg l–1 added as InHg and MeHg, respectively. The detection limits for InHg and MeHg were 0.6 and 0.2 µg Hg l–1, respectively. The relative standard deviations for eleven independent measurements were 5% for both mercury species. Recovery values ranging from 98 to 106% for InHg and from 95 to 105% for MeHg and from 93 to 95% for ethylmercury (EtHg) were obtained. The accuracy of the proposed method was also established by the analysis of certified whole blood samples for InHg and MeHg. No difference between the sum of these two species determined by our procedure and the recommended total mercury concentrations in the certified samples was observed. Results for the determination of MeHg and InHg in 30 controls and 30 dentists are presented to illustrate the practical utility of the proposed method.

Summary. Allograft infection in hepatitis B surface antigen (HBsAg)‐positive patients undergoing liver transplant (OLT) is still significant, despite post‐transplant prophylaxis with high doses of immunoglobulin to HBsAg. Baseline status and post‐OLT levels of viraemia and wild‐type and hepatitis B e antigen (HBeAg)‐negative hepatitis B virus (HBV) were correlated with the clinical course of 16 consecutive HBsAg carriers, positive for hepatitis B e antibody, with hepatocellular carcinoma who underwent OLT and received permanent post‐OLT prophylaxis with antibody to HBsAg (HBsAb). Fourteen patients had less than 103HBV genome equivalents ml‐1(eqml‐1) at baseline and remained HBV free after a median of 36 months following OLT. Two patients with mean pre‐OLT viraemia higher than 105genome eq ml‐1and prevalent HBeAg‐negative HBV viraemia before OLT suffered a severe graft hepatitis. Interferon‐α2b (3 MU m‐2per day) was able to reduce viraemia in both patients and to revert the clinical course of the infection in one, who remained infection‐free 22 months after IFN treatment. Fourteen patients had less than 103HBV genome eq ml‐1at baseline and remained HBV free, after a median of 36 months following OLT, with permanent HBsAb immunoprophylaxis. These observations suggest that the quantitative analysis of HBV pre‐OLT viraemia levels may provide a very useful tool for predicting the ideal time of liver replacement. Clinical trials on the use of antiviral drugs capable of inhibiting HBV serum levels before liver transplantation should be pursued on this premise.

The possibility of hepatitis B virus (HBV) infection in HBsAg-negative patients has been shown. However, an “inapparent” coinfection by HBV in hepatitis C virus (HCV)-positive patients generally is not taken into account in clinical practice. Mechanisms responsible for resistance to interferon (IFN) have not been completely clarified. The aim of this study was to investigate whether an “inapparent” coinfection by HBV in anti-HCV-positive chronic liver disease patients may influence IFN response. Fourteen anti-HCV positive, HBsAg-negative but serum HBV DNA-positive patients by PCR and 111 anti-HCV-positive, HBsAg-negative and HBV DNA (PCR)-negative patients with chronic hepatitis were treated with 3 MU of recombinant α-2a IFN 3 times weekly for 12 months. Serum HBV DNA and HCV RNA were determined before treatment, after 6–12 months and in coincidence with ALT flare-up by PCR. HBV PCR was performed using primers specific for the S region of the HBV genome and HCV PCR with primers localised in the 5′NC region of HCV genome. IgM anti-HBc was tested using IMx Core-M Abbott assay. By the end of treatment, ALT values had become normal in 4/14 HBV DNA-positive patients (28%), but all “responders” (4/4) relapsed between 2 and 5 months after therapy. All but one patient were HCV RNA-positive before treatment, 6 were also both HBV DNA and HCV RNA-positive during ALT flare-ups. In 5 patients, only HBV DNA and in 3 patients, only HCV RNA was detected when transaminase values increased. All patients remained HBsAg-negative and anti-HCV-positive. IgM anti-HBc was detected both before treatment and during ALT elevation in 3 patients and only during ALT relapse in 3 others. Of the 111 anti-HCV positive, HBsAg-negative and HBV DNA (PCR)-negative patients with chronic hepatitis, a biochemical response to IFN treatment was observed in 54% of the cases. Relapse of ALT values was observed in 47% of the cases during a follow-up of 1 year after treatment. “Inapparent” HBV/HCV coinfection may be implicated in cases of resistance to IFN treatment. In addition, HBV replication may persist in patients in whom HCV replication was inhibited by IFN treatment. The pathogenic role of HBV in liver disease was confirmed by detection of IgM anti-HBc in some cases; the appearance of these antibodies only after IFN treatment suggests that IFN may exert a selective role in favour of HBV. Further studies will show the effect of different treatment schedules. HBV DNA and/or IgM anti-HBc detection with very sensitive methods may be important both as a prognostic factor and as a tool for better understanding interviral relationships and mechanisms involved in multiple hepatitis virus infections.

Summary. The intriguing co‐infection of two flaviviruses (GBV‐A and GBV‐B) in tamarins and the recent discovery of another flavivirus (GBV‐C/HGV) in humans raises the question of the relations between hepatitis C virus (HCV) and GBV‐C/HGV. To address this issue the sera of 285 patients with liver disease (102 patients with cryptogenic and 183 with known forms of chronic liver disease) and 19 patients without liver disease were tested for HGV‐RNA. GBV‐C/HGV‐RNA was detected by RT‐PCR using primers encompassing 5'NC and NS5 regions and hybridization with specific biotinilated and radiolabelled probes. GBV‐C/HGV RNA was found in 11 of 20 (55%) acute hepatitis C patients, in 13 of 117 (11.1%) patients with chronic hepatitis C, in 11 of 27 patients with a liver transplant (40.7%), one of 19 (5.3%) patients with chronic HBV infection, 15 out of 102 (14.7%) patients with cryptogenic liver disease and two out of 19 patients with inflammatory bowel disease. In cryptogenic patients, elevated serum gammaglutamyl transpeptidase (GGT, higher than twice the normal values) and alkaline phosphatase (ALP, above normal values) levels were significantly associated with GBV‐C/HGV‐RNA infection (P<0.001). In conclusion GBV‐C/HGV appears to be transmitted in humans by blood exposure and to be associated with liver disease in HCV co‐infected patients and in a minority of patients with cryptogenic disease. The virus is only occasionally pathogenic for the liver and when liver damage is present; the association with the combined elevation of GGT and APH serum levels might represent a specific feature of the liver tropism of the agent.

Summary. Clinico‐epidemiological data show that the most severe forms of hepatitis C virus (HCV) associated liver disease occur in patients with multifactorial liver damage. We found that the prevalence of hepatitis B virus (HBV) markers in anti‐HCV positive patients with cirrhosis complicated by hepatocellular carcinoma (HCC) is higher than in cirrhotics with comparable age and disease history, but without HCC. HBV can persist in integrated forms in HBsAg negative, anti‐HBc positive individuals and we may speculate that in such patients concurrent liver pathogens, as HCV, could cause HCC more easily than in patients without previous exposure to HBV. Analysing the relations between age at HCC diagnosis and the different risk factors in consecutive HCC patients we found that patients with single hepatitis virus infections (HBsAg and/or anti‐HCV positive) were of an older median age than patients with multiple hepatitis virus infections. We also studied patients with compensated cirrhosis and hepatitis virus infections, untreated or treated with interferon‐alpha. The independent effect of treatment was analysed by matching groups with regard to all the other significant HCC risk factors. The overall relative HCC risk was three times higher (risk ratio 3.1) in untreated vstreated anti‐HCV positive patients and more than six times higher (risk ratio 6.2) in untreated vstreated anti‐HCV positive/anti‐HBc negative patients. The difference between treated and untreated patients was not statistically significant in hepatitis B surface antigen carriers and in anti‐HCV positive/anti‐HBc positive patients. The evidence that HBV coinfection may worsen the course of liver cirrhosis in patients with chronic hepatitis C is intriguing, but it has important practical consequences. It warrants the identification of high risk patients with chronic hepatitis C who need to be treated as early as possible and patients who can still benefit from interferon‐alpha therapy once cirrhosis has already been diagnosed.

The paper describes the development of an automatic on-line column pre-concentration technique using a time based-flow injection atomic absorption spectrometry system. A manifold incorporating a micro-column containing 25 mg of Dowex 50W-X8 was used with a time-based injector for the pre-concentration and determination of copper in natural and drinking waters. The system features depend on the alternate positions of a solenoid valve. The 3σ detection limits, enrichment factors, sampling frequency, relative standard deviations and linear calibration graphs were, respectively, in the range 0.6-1.5 μg/l, 25-60, 15-30 measurements/h, 1.0-3.1% and 1-65 μg/ml for pre-concentration times of 1 min. The procedure was successfully applied to a range of water samples and the accuracy was assessed through recovery experiments, the analysis of certified reference water samples and by independent analysis by atomic absorption spectrometry with electrothermal atomization.

Abstract: Antimony(III) and antimony(V) species have been selectively determined in liver tissues by optimizing the acidic conditions for the evolution of stibine using the reduction with sodium borohydride. The results show that a response for Sb(III) of 0.5 to 20 mgrg l–1 was selectively obtained from samples in a 1 mol l–1 acetic acid medium. The best response for total antimony from 1 to 20 mgrg l–1 is obtained after sample treatment with a 0.5 mol l–1 sulfuric acid and 10% w/v potassium iodide. Microwave digestion has been necessary to release quantitatively antimony species from sample slurries. The amount of Sb(V) was calculated from the difference between the value for total antimony and Sb(III) concentrations. A relative standard deviation from 2.9 to 3.1% and a detection limit of 0.15 and 0.10 mgrg l–1 for Sb(III) and total Sb has been obtained. The average accuracy exceeded 95% in all cases comparing the results obtained from recovery studies, electrothermal atomic absorption spectrometry and the analysis of certified reference materials.

Using an oligonucleotide hybridization assay, we studied the clinical implication of wild-type hepatitis B virus (HBV) and a HBV mutant that is unable to secrete hepatitis B e antigen (HBeAg) because of a translational defect due to a stop codon in the pre-C region in 106 hepatitis B surface antigen-positive patients with chronic hepatitis B. Wild-type HBV was detected in 31 of 42 (73.8%) HBeAg-positive patients, whereas a mixed viral population was present in 10 (23.8%). Significant differences in the severity and outcome of liver disease were not observed in the two groups of patients. However, the emergence of HBeAg-minus HBV in wild-type HBV carriers was associated with an exacerbation of liver disease and was followed by the presence of antibodies against HBeAg (anti-HBe) in serum in 50% of the cases. In 61 of 64 (95.3%) anti-HBe-positive patients, HBeAg-minus HBV was the predominant virus: HBeAg-minus HBV was detected in 42 patients (65.6%), whereas both wild-type and HBeAg-minus HBV were present in 19 (29.7%). HBeAg-minus HBV was associated with a course of hepatitis characterized by flare-ups of liver cell necrosis interspersed with periods of asymptomatic HBV carriage (P less than 0.01). These data support the hypothesis that genetic heterogeneity of HBV significantly influences the course and outcome of chronic hepatitis B. Wild-type HBV secreting HBeAg induces immunologic tolerance and causes chronic infection. HBeAg-minus HBV might be unable to induce chronic infection without the helper function of wild-type HBV, but it appears to be more pathogenic. Once chronic infection is established, HBeAg-minus HBV variants may prevail and displace wild-type virus.

The adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4+ T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCR) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCR identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCR transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein NS4 is highly immunogenic for intrahepatic CD4+ T cells primed by HCV in vivo, and that there can be compartmentalization of some NS4-specific CD4+ T cells to the liver of patients with chronic hepatitis C.

A factorial composite design has been employed for a rapid exploration of the effect of some parameters on the stability of spiked methylmercury iodide solutions. The analysis has been performed by capillary gas chromatography with electron-capture detection. The results obtained by the factorial and the univariate methods are in a good agreement. Although, the factorial design does not allow a discrimination of the significance among the same kind of factors, it greatly reduces the number of experiments when the different variables are investigated.

Prebiotics, defined as dietary fibers that can benefit the host by selectively stimulating the growth and activity of beneficial intestinal bacteria, have gained increased attention in companion animal nutrition. However, limited information is available on prebiotic supplementation and canine GI microbiota using high-throughput molecular techniques. Thus, the aim of this study was to evaluate the effects of increasing dietary concentrations of a yeast culture product (YCP) on fecal microbiota of adult healthy dogs. Eighteen dogs (15.8 ± 7.37 kg mean BW; 3.7 ± 1.62 yr mean age) were randomly assigned to 1 of 3 treatments: control diet (CO), CO supplemented 0.3% YCP (YCP-0.3), and CO supplemented 0.6% YCP (YCP-0.6). The experimental period consisted of 21 d of diet adaptation, followed by 3 d of fresh fecal collection. Three fecal samples were collected from each dog within 15 minutes of defecation and stored at -80°C. Fecal genomic DNA was extracted using Mo-Bio PowerSoil kits. The V4 region of the 16S rRNA gene was amplified and sequenced using Fluidigm specific primers and MiSeq (v3 reagents). High quality sequences were analyzed using QIIME 1.9.1 and mixed procedure of SAS, with P≤ 0.05 considered as statistically significant, whereas a P≤ 0.10 was considered as a trend. A total of 917,433 reads were obtained. An even sampling depth of sequences per sample (16,300 reads) was used for assessing alpha- and beta-diversity. YCP supplementation did not affect fecal microbial alpha- and beta-diversities. Firmicutes and Fusobacteria made up about 85–88% of bacterial sequences, with Bacteroidetes, Proteobacteria, Actinobacteria, and Deferribacteres contributing to only about 8–12%, 1–3%, 0.1–2%, and <0.02% of sequences, respectively. The predominant fecal bacterial genera were Clostridium(18–15%), Fusobacterium(16–25%), and Blautia(7–11%). Supplementation of YCP-0.6 tended to increase the relative abundance of Collinsellaand Slackiabut tended to decrease (P< 0.10) fecal Coprobacillus. In conclusion, supplementation of 0.6% of YCP resulted in modest changes in fecal microbial communities of healthy adult dogs. Further studies should evaluate higher doses of YCP supplementation and its effects on fecal microbiota and gut health of dogs suffering from gastrointestinal disorders.

The paper describes the development of an automatic on-line column pre-concentration technique using a time based-flow injection atomic absorption spectrometry system. A manifold incorporating a micro-column containing 25 mg of Dowex 50W-X8 was used with a time-based injector for the pre-concentration and determination of copper in natural and drinking waters. The system features depend on the alternate positions of a solenoid valve. The 3s detection limits, enrichment factors, sampling frequency, relative standard deviations and linear calibration graphs were, respectively, in the range 0.6-1.5 µg/l, 25-60, 15-30 measurements/h, 1.0-3.1% and 1-65 µg/ml for pre-concentration times of 1 min. The procedure was successfully applied to a range of water samples and the accuracy was assessed through recovery experiments, the analysis of certified reference water samples and by independent analysis by atomic absorption spectrometry with electrothermal atomization.

The paper describes the development of an automatic on-line column pre-concentration technique using a time based-flow injection atomic absorption spectrometry system. A manifold incorporating a micro-column containing 25 mg of Dowex 50W-X8 was used with a time-based injector for the pre-concentration and determination of copper in natural and drinking waters. The system features depend on the alternate positions of a solenoid valve. The 3σ detection limits, enrichment factors, sampling frequency, relative standard deviations and linear calibration graphs were, respectively, in the range 0.6-1.5 μg/l, 25-60, 15-30 measurements/h, 1.0-3.1% and 1-65 μg/ml for pre-concentration times of 1 min. The procedure was successfully applied to a range of water samples and the accuracy was assessed through recovery experiments, the analysis of certified reference water samples and by independent analysis by atomic absorption spectrometry with electrothermal atomization.