Despite the increasing interest towards the biological role of L-ergothioneine, little is known about the serum concentrations of this unusual aminothiol in older adults. We addressed this issue in a representative sample of community-dwelling middle-aged and older adults.Body mass index, estimated glomerular filtration rate, serum concentrations of L-ergothioneine, taurine, homocysteine, cysteine, glutathione, cysteinylglycine, and glutamylcysteine were evaluated in 439 subjects (age 55–85 years) randomly selected from the Hunter Community Study.Median L-ergothioneine concentration in the entire cohort was 1.01 IQR 0.78–1.33 µmol/L. Concentrations were not affected by gender (P = 0.41) or by presence of chronic medical conditions (P = 0.15). By considering only healthy subjects, we defined a reference interval for L-ergothioneine serum concentrations from 0.36 (90% CI 0.31–0.44) to 3.08 (90% CI 2.45–3.76) µmol/L. Using stepwise multiple linear regression analysis L-ergothioneine was negatively correlated with age  and with glutamylcysteine concentrations .A thorough analysis of serum L-ergothioneine concentrations was performed in a large group of community-dwelling middle-aged and older adults. Reference intervals were established. Age and glutamylcysteine were independently negatively associated with L-ergothioneine serum concentration. It is generally accepted that damage caused by free radicals is a hallmark of ageing with potential clinical consequences Early studies showed that humans and other vertebrates are unable to biosynthesize ERT, and that in mammals it is acquired solely by dietary means The knowledge regarding tissue ERT concentration distribution at population level is limited to one study in 400 healthy male individuals Given the availability of this new sensitive assay method, we studied the distribution of serum ERT concentrations in an established epidemiological cohort of human ageing, the Hunter Community Study (HCS) Participants were a representative sample of the Hunter Community Study (HCS), a population-based cohort study to assess the impact of clinical, genetic, biochemical, socioeconomic, and behavioural factors on human ageing Blood was collected into tubes without anticoagulant and centrifuged at 4°C and 3,000×g for 10 min to separate serum, which was stored for three years at −80°C before analysis. There was no previous freeze-thaw cycles prior to analysis. Serum ERT concentration was measured by capillary electrophoresis-laser-induced fluorescence method as recently described by Sotgia et al. This study examined the association between serum ERT with age, gender, body mass index (BMI), estimated glomerular filtration rate (eGFR), and the thiols homocysteine (Hcy), cysteine (Cys), glutathione (GSH), cysteinylglycine (CysGly), glutamylcysteine (GluCys), and taurine (Tau). Body mass index was calculated using height and weight measurements obtained at baseline and eGFR was calculated using the Modification of Diet in Renal Disease formula Statistical analyses were performed using MedCalc for Windows, version 12.5 64 bit  and SPSS for Windows, version 14.0 32 bit . Data were tested for normality of distribution using the Kolmogorov-Smirnov test. Data were presented as either mean±SD or median and interquartile range (IQR) as appropriate. Non-normally distributed variables were log10-transformed prior to being used with parametric tests, and the normal distribution of the residuals was checked to assess the goodness of fit of the transformations. Differences between groups were compared by non-parametric Mann-Whitney U Test or by independent t-test as appropriate. Spearman rank correlation coefficients were used to characterise associations between serum ERT concentration and continuous clinical, biochemical and demographic variables. Partial correlation and stepwise multiple linear regression analysis was used to assess the contribution of different variables to serum ERT concentration. Multicollinearity was tested by measuring the tolerance and the variance inflation factor values for each analysis. A 2-sided P value of 0.05 indicated statistical significance.Demographic, clinical, and biochemical characteristics of the study sample are outlined in Those with at least one chronic medical condition were classified as unhealthy (N = 359) while those without were classified as healthy (N = 80). The presence of a chronic medical condition was not associated with a significant difference in ERT serum concentrations . In the subgroups obtained by a further split according to the gender, ERT serum concentration was slightly higher in males than females but the difference was not statistically significant both in healthy (P = 0.99) and unhealthy (P = 0.41) groups . In viewTo detect potentially confounding variables, we performed a partial correlation analysis by using gender and healthy state as covariates and age, BMI, eGFR, Tau, Hcy, GSH, Cys, CysGly, and GluCys as covariates or as variables. This analysis confirmed only the statistically significant negative association of ERT serum concentration with age  and with GluCys . When redundant, non-significant variables were removed by using stepwise multiple regression analysis (Forward+Backward method), independent and stronger associations with age  and with GluCys  were observed . The gooLow-molecular-weight thiols have been extensively investigated in older adults Despite the analogies suggested by some authors with GSH, e.g. the antioxidant activity and the similarities between intracellular and extracellular concentrations, no independent association was found in this study between ERT and GSH. Similarly, no independent association was observed with Tau, the other antioxidant considered in the study.Although bivariate analysis showed associations between ERT serum concentration and other aminothiols , stepwise regression only showed independent negative associations with GluCys. Age was the only other variable showing independent negative associations with ERT serum concentrations. Although ERT concentration reduces with age, and indeed the median age in the unhealthy group was higher vs. healthy subjects (66.0±7.7 years vs. 63.5±6.1 years), ERT concentrations are unlikely to serve as an index of health status. The ERT concentrations between two groups were, in fact, not significantly different, and almost all ERT values in unhealthy subjects (∼98%) lie within the reference interval (0.36–3.08 µmol/L). The decreased levels of ERT with age could reflect either a modification of dietary habits or alterations in the expression of the SLC22A4 gene.The negative association between ERT and GluCys is not easy to explain, also in view of the lack of a clear relation with GSH. GluCys, the immediate precursor of GSH, is synthesised by the enzyme glutamylcysteine synthetase in the γ-glutamyl cycle. The activity of glutamylcysteine synthetase is limited by availability of Cys and glutamate, and is inhibited by GSH through a feedback mechanism


Objectives. β2-adrenergic agonists, such as clenbuterol, have been shown to promote the hypertrophy of healthy skeletal muscles and to ameliorate muscle wasting in a few pathological conditions in both animals and humans. We intended to investigate the clinical efficacy of clenbuterol on attenuating denervation-induced muscle atrophy. Methods. A double-blind, placebo-controlled, parallel, and randomized trial was employed. 71 patients, suffering from brachial plexus injuries, were given either clenbuterol  or placebo for 3 months. Before and at the end of the study, patients were given physical examinations, biopsies of biceps brachii, electromyograms (EMGs), and other laboratory tests. Results. Compared with placebo treatment, clenbuterol significantly mitigated the decreases in cross-sectional areas of type I and II muscle fibers and alleviated the reduction in fibrillation potential amplitudes, without any adverse effects. Conclusions. Clenbuterol safely ameliorated denervated muscle atrophy in this cohort; thus larger clinical studies are encouraged for this or other β2 agonists on denervation-induced muscle atrophy. This is attributable to their roles in controlling protein synthesis and degradation, via Gectively . β2 agong models .β2 agonists, such as clenbuterol, increase lean body mass and enhance skeletal muscle functions [μg/day) promotes myocardial recovery in patients with myocardial unloading atrophy resulting from applications of left ventricular assist devices [In patients with immobilization conditions or muscular dystrophy, unctions –11. In a devices –14.β2 agonists, clenbuterol, a long-acting and functionally selective agent, is the most effective drug in preventing muscle wasting, with the least adverse effects [β2 agonists' roles in promoting hypertrophy of intact skeletal and cardiac muscles, and in preventing muscle wasting in preclinical studies, we hypothesized that clenbuterol may mitigate denervated muscle atrophy in humans. The current pilot trial tested clenbuterol's effects on retarding denervation-induced muscle atrophy in patients.In comparison with other  effects , 15–18.  effects . Based oβ-blockers for other diseases; (4) patients who had experienced serious adverse events from other β-agonists before; (5) patients, who were not strictly compliant in taking trial drugs and completing the adjunctive therapies as above, were not analyzed; (6) patients, who did not revisit at their scheduled time, were not analyzed.71 patients were recruited to this study at the Hand Surgery Department of Huashan Hospital, Fudan University in Shanghai. Entering criteria: (1) patients had loss of innervation in the biceps brachii for more than one month, and diagnosis was achieved through the combination of history, physical examinations, electromyogram (EMG), and examinations during surgeries. (2) The loss of innervation must have resulted solely from traumatic injuries to the brachial plexus, namely, C5/6, C5/6/7, or C5-T1 injuries, and not from other causes, such as virus infections or autoimmune diseases. (3) Besides the trial, all studied patients had phrenic nerves transferred to musculocutaneous nerves, in addition to various other nerve transfers. (4) All patients were routinely given oral Vitamin B6 (10 mg tid) and Dibazolum (10 mg tid) to facilitate nerve regeneration during the trial period. (5) The participating patients had no central nervous system injuries or other diseases and were not in any other trials. (6) All patients signed the informed consents. Excluding criteria: (1) ischemia or traumatic compressions occurring directly on the biceps brachii or musculocutaneous nerves; (2) pregnant or breast feeding women; (3) patients on μg/10 mL in sealed glass tubes by pharmacists. A total of 20,000 tubes of clenbuterol or placebo were made, respectively. Shelf stability at 4°C was tested to guarantee the constancy of the active pharmacological ingredient over the entire study period. Each patient was prescribed 200 tubes of clenbuterol or placebo and was asked to take the medicine orally 60 μg twice per day for 3 months (~2 μg/kg/day), without food interference. Significant muscle atrophy was observed during 3-month period after denervation [Patients were recruited from 5 therapeutic teams led by independent investigators at the department. They were labeled with case numbers and randomly assigned into clenbuterol or placebo groups. The codes were stored separately. It was only referred to by the organizer at the end of the study or when a severe adverse event occurred. Neither the therapeutic team members nor patients knew which group a patient was assigned to. The EMG examination staffs and the processor/evaluator of the biopsy samples were masked from treatment allocations. Clenbuterol was formulated as 60 Before the trial, each patient was admitted and given a set of laboratory evaluations including a 12-lead electrocardiogram, chest X-ray, pulmonary function test, and several liver function studies. Blood urea nitrogen, creatinine, bleeding time, prothrombin time, glucose, calcium, phosphorus, electrolytes, and complete blood counts with differentials were also recorded. By the end of the trial, all patients repeated the above examinations regardless of their groups.At the commencement and conclusion of the trial, each patient was given EMG examinations with a Dantec Neuromatic-2000 M (Denmark) electromyography in an air-conditioned room, after adapting to a constant temperature for 10 minutes. Patients were given a full set of conventional EMG examinations on each nerve of both upper extremities, including sensory and motor nerve conduction speeds, maximum and evoked motor unit action potentials (MUAPs), and somatosensory evoked potentials (SEPs) at different segments of the brachial plexus and terminal branches. The results were compared between both sides by EMG professionals. If there were any doubts about the EMG diagnosis, an intraoperational EMG examination was conducted after exposing the root, trunk, cord, or some proximal branches of the brachial plexus. If no MUAP or SEP was detected, fibrillation potential amplitudes were then recorded, via a concentric needle electrode inserted perpendicularly into at least three spots around the midpoints of the biceps brachii. Only the one with the largest amplitude and biphasic or triphasic shape was analyzed when several fibrillation firings were recorded, because the largest amplitudes best reflect the sizes of muscle fibers closest to the needle tip . The comμm thick fresh cryostat sections were made on a cryostat microtome at −20°C and mounted on slides. Myofibrillar adenosine triphosphatase staining at either pH 4.6 or 9.4 was performed on each sample to show fiber types [All 71 patients were given muscle biopsies at the midpoints of the biceps brachii during nerve repair operations right before the start of the trial. Biopsy samples were covered with talc powder and snap frozen in isopentane precooled in liquid nitrogen. 10 er types . Fiber cP < 0.05 was taken as statistical significance.All patient information and examinations were put into SPSS software. The changes in fibrillation potential amplitudes and cross-sectional areas over the trial period in each patient were used for statistical analysis. 35 patients were randomized into clenbuterol group, and 36 patients were allocated into placebo group. Poor compliance (not taking medicine as prescribed and delayed followup visit) and lost to follow-up accounted 19 cases in clenbuteral or placebo group, respectively. One patient in the clenbuterol group showed signs of reinnervation of the biceps brachii (M2) 3 months after the phrenic nerve transfer. This was determined through physical and EMG examinations. 15 patients in clenbuteral group and 17 patients in the placebo group completely obeyed the guidelines of the 3-month trial with no indications of reinnervation of their biceps brachii . Among tμV in the amplitudes of fibrillation potentials, while patients accepting the placebo had a 4-time greater decrease over the same period of time  of one patient recorded newly occurring sinus arrhythmia and bradycardia after 3 months of clenbuterol administration. He had no discomfort complaints. Six patients in the clenbuterol group had EKG abnormalities before enrollment, namely, sinus arrhythmia (2 cases), sinus bradycardia (1 case), incomplete right bundle branch block (1 case), right ventricular high voltage (1 case), and counterclockwise rotation of the heart (1 case). They all had no corresponding symptoms and required no treatments per consulting with cardiologists. Clenbuterol treatment for 3 months did not change their EKG presences. Seven patients in the placebo group recorded slight abnormalities in EKG (4 cases), glucose (2 cases), and creatinine (1 case) by the end of the study. There were no abnormalities in lung, liver functions, bleeding time, prothrombin time, electrolytes, or complete blood counts. No complaints of muscle tremors, tachycardia, dizziness, appetite changes, or diarrhea were reported over the trial period in either group.In the present study, we were able to recruit a special cohort of patients in their twenties. This helped to minimize the effects of aging on muscle atrophy and eliminate this potential confounding factor in the study, because spontaneous denervation occurs with aging , 24. Allμg/day attenuated denervation-induced muscle atrophy in humans. It is reported that clenbuterol, at incremental doses from 120 to 720 μg/day over 12 weeks, increases the mass and strength of the healthy skeletal muscles in man [μg/day of clenbuterol is believed to be a reasonably high dosage for the denervated muscles of patients. In the present study, the changes of fiber sizes and fibrillation potentials were used to evaluate the efficacy, aiding the avoidance of confounding factors resulting from variations in basal levels between individuals. Hence, it seems that fewer patients were required to reach a conclusion than the number needed by a study limited to just comparing endpoints.This is the first clinical study showing that clenbuterol at 120 s in man , 14. Anis in man . TherefoIn animal and large population studies, fibrillation potential amplitudes reflect fiber sizes of denervated muscles , 21, 26.β1 [Reinnervation of muscle fibers, such as via axon regeneration following surgical repairs, often halts muscle atrophy and restores muscle functions . Understβ1 , 28, 29.β-adrenoceptor subtypes , with about a 10-fold greater proportion of the β2-adrenoceptor isoform than β1 or β3 receptors [β1-adrenoceptors than β2 [β2 agonists to affect skeletal rather than myocardial muscles. Indeed, proper dosages of β2 agonists, such as clenbuterol, separate the effects on different types of muscles in patients [β2 agonists, clenbuterol appears to have the safest cardiac profile. To elaborate, clenbuterol given at 1000 μg/kg/day, caused no myocardial hypertrophy in rats, but rather skeletal muscle hypertrophy [μg/kg/day) in animals can induce cardiac hypertrophy, that is still physiological in its functions, structure, and gene expressions, it is ultimately free of pathological changes [μg/day) promotes cardiac recovery in patients with left ventricular unloading atrophy [Skeletal muscle contains all three eceptors , 31. Thepatients –11. Amonertrophy . Even th changes , 17, 33. atrophy –14. μg/kg/day, attenuated denervated muscle atrophy without affecting the heart or causing myocyte death [μg/kg/day in rats is equivalent to 1 μg/kg/day in humans, a dose safely used in asthma treatments [μg/day (~2 μg/kg/day for a 60 kg person). It was well tolerated and not associated with any obvious discomfort, except for one patient with transient nervousness. The newly occurring sinus bradycardia after the clenbuterol trial seemed not be relevant to the activation of the β1/2 agonist, which usually leads to tachycardia. Moreover, clenbuterol at the present dose did not exacerbate preexisting minor EKG abnormalities. This is consistent with previous reports that the effects of clenbuterol on the heart are observable at a dose of up to 2100 μg/day in combination therapy for patients using left ventricular assist device. Even at those doses, no severe adverse effects were encountered but tremors and muscle cramps [Physiological dose of clenbuterol in rats, 10 te death , 34. Thaeatments , 35. Thee cramps , 13. In This pilot single-center study was small in scale. The efficacy would be more apparent in a larger patient population with either gradual increments of clenbuterol doses, or an on and off schedule to avoid the occurrence of receptor desensitization. Recent animal studies also show that clenbuterol is neuroprotective and promotes axon regeneration , 28, 29.


Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type  and T3SS-deficient  strains in exponential and stationary phases of growth.ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described  and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected.Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent  Aeromonas salmonicida subsp. salmonicida, a gram-negative bacterium, is the etiologic agent of furunculosis, a frequent and major pathogen of fisheries worldwide which is generating significant economic losses related to deficits in zootechnical profits and the intensive use of antibiotics ). This effector disrupts cytoskeletal binding sites on the inner surface of host membranes, causes plasma membrane blebbing and probably contributes to cell death by facilitating lysis , P. luminescens plu4750) and ASA_0010 (homologuous to V. parahaemolyticus VopS [VP1686]) [A. salmonicida A449 [Our study did not detect T3SS effectors AopX (homologuous to VP1686]) , suggestida A449  and alsoA. salmonicida SNs and detailed in the first part of this work were not due to cell lysis.From these results we concluded that our MS analysis localized 100% of T3SS components that are structurally linked to the bacteria and associated to pellets  or T3 secreted and associated to SNs  with effectiveness and accuracy. These results also support the idea that highly conserved cytoplasmic proteins unexpectedly present in ΔascV knock-out mutation induced a strong down-regulation of the expression of many T3SS genes. AopD, AopB, AopH, AscV, Ati2, AcrV, AopO and AexT were the most abundant T3SS proteins present in the GP wt pellet , and significantly lower in mutant pellets in comparison to wt , but clearly to a lower extent than in wt SNs . Another possibility is that two mechanisms of effectors/translocators secretion operate in parallel along phases of growth, the first would be actively dependent on intact T3SSs while the second, clearly less efficient, would explain the progressive accumulation of effectors/translocators in the extracellular medium of the mutant strain. The alternative secretion of T3SS effectors through classical/unclassical pathways has never been described contrary to major constituents of the OM ring which are transported to the periplasm by the Sec-dependent secretion pathway [Shewanella[Yersinia surface without physical association with the injectisome [The mutant strain might continue to release these T3SS components in SNs, either from the resting structural T3SS components or by an alternative secretion pathway. Recent publications argue that the T3SS arose from an exaptation of the flagellum, i.e. the recruitment of part of the flagellum structure for the evolution of the new protein delivery function ,34 and, utations . Howeverpneumonia. While n pathway . Further pathway  and OMVs pathway  has rarehewanella, but Groectisome . While tA. salmonicida secretion systems (T1- to T6SS), flagella , pili , it was clear that the T3SS was the most expressed system by A. salmonicida  were either not expressed at all or were expressed to a lower level, suggesting that they could be impaired by mutations similar to the ones observed in the reference A449 strain [When we compared the total LFQ values of 9 strain .A. salmonicida T3SS effectors and T3-independent virulence factors. We targeted wt secreted proteins with PMSS values over 25, a PMSS or LFQ intensity 4-fold increased in the wt SN, and a PEP value inferior to 10-8 or equal to zero , and two proteins without a predicted motif for T3 secretion were shown to have homologues that are T3 secreted in other bacteria (Pta and ASA_P5G088) . Foror EF-Tu ,51. It iYersinia T3SS and enhanced the ability of the bacterium to withstand the killing activities of murine macrophages [Salmonella enterica and Dickeya dadantii, PNPase downregulated the transcription of T3SS genes [Polynucleotide phosphorylase PNPase has pleiotropic roles in bacteria such as degrading mRNA (degradosome) and mediating post-transcriptional regulation . Howeverrophages . In SalmSS genes ,55.A. salmonicida were targeted by our screening as putative T3SS effectors. In Salmonella, a Pta mutant showed that this enzyme was associated to virulence [E. coli Pta  might be secreted by the T3SS [V. parahaemolyticus (35% and 38% of similarity with VP1677 and VP1678) were T3 secreted [Although they did not have the N-terminal motif for T3-secretion, the phosphate acetyl transferase  and the putative α/β hydrolase ASA_P5G088 of irulence , and a rthe T3SS . Interessecreted .ΔascV mutant SNs, did not have any predicted signal for a secretion system and were not characterized as T3SS effectors in other bacteria . AcnB and WecB have homologous proteins that have been associated to the virulence in other bacteria  in the literature, and certain conserved proteins are homologous to virulent toxins, adhesins and enzymes identified in other bacteria , elastase AhpB and toxic extracellular endopeptidase AsaP1 genes are impaired by deletions and insertion elements. According to these observations, we did not detect any polypeptides for these proteins in our MS experiments, suggesting that they would be also disrupted in our A. salmonicida strain. Furthermore, the insecticidal cytolytic delta-endotoxin (ASA_2128), putative RTX toxins , a secreted metalloprotease (ASA_1723) and the pullulanase PulA were not identified, and their expression might be induced in the host. Finally, 15 prophage proteins were identified in pellets (12% of prophage genes detected in the genome of the reference strain A449) and only one (ASA_2013) was detected in SNs, but without any significant differences between the wt and mutant strains.In the genome of A. salmonicida secretomes from wt and T3SS-deficient strains is a powerful method that gave us the opportunity (i) to characterize the full in vitro repertoire of T3SS effectors represented mainly by AopH, Ati2, AexT, AopP, AopO, AopN and ExsE, (ii) to identify new putative virulence factors that are secreted in the extracellular medium or might be translocated into the host cell by the T3SS or alternative mechanisms, and (iii) to confirm that A. salmonicida secreted toxins, adhesins and enzymes that have been described until now and are additionally found in this study are secreted to a higher extent in the extremely low-virulent ΔascV mutant.The comparison by high-throughput proteomics of ascV in this study) can induce the down-regulation of several other genes (only associated to the T3SS in our study), not necessary transcriptionally linked in the same operon. To respect the molecular Koch’s postulates, we can conclude from this study that each work investigating phenotypic characters by site-directed mutagenesis should ideally be completed by a larger analysis studying the impact of the mutation over the global gene expression.Our results also clearly show that the deletion of one gene  that might be necessary to survive in new environments [A. salmonicida (15% of specific genes) than in genetic regions common to all Aeromonas species (8% of common genes). Further proteomics studies will be necessary in order to confirm the in vivo A. salmonicida secretome.Due to the fact that we studied in vitro secretomes, T3SS effectors that we have found might be considered as the first line of weapons that ronments . InteresAeromonas salmonicida wt and ΔascV mutant strains used in this study were characterized in a previous work [A. salmonicida wt cultures into a maximum T3SS activation state, we used JF2267 strain which was freshly reisolated from an experimentally infected dead fish (JF5054). This re-isolated strain was highly virulent, since intraperitoneal inoculation of only 500 cfu per fish was sufficient to induce 70 to 80% of mortality in challenge assays [ΔascV mutant strain JF2747 is considered to have extremely low-virulence because 105 cfu/fish induced no mortality [8 cfu/fish induced a weak mortality of only 20%.ous work . To get ortality , and 108ΔascV A. salmonicida, 50 ml of TSB medium were inoculated with 109 bacteria and cultivated at 18°C under shaking (160 rpm) in the presence of protease inhibitors . The bacterial growth was stopped during the exponential phase of growth (DO600 = ~1.5) and the stationary phase (DO600 >2.0). Supernatants were separated from bacterial pellets by centrifugation  and filtration through a 0.22 μM Acrodisc filter . The bacterial pellets were resuspended in 10 ml of PBS, and 250 μL of these solutions were mixed with 250 μL of SDS loading buffer and heated at 100°C for 5 min. To precipitate proteins from supernatants, 12.5 ml of 100% ice-cold trichloroacetic acid were added to the solutions , then immediately vortexed and incubated overnight on ice. Supernatants were removed and brown protein pellets were suspended and washed several times by centrifugation in ice-cold 100% acetone in 2 ml Eppendorf tubes (low binding protein). Finally, the pellets were dried, diluted in 250 μL of SDS loading buffer (~200 times concentration) and heated at 100°C for 5 min. Proteins were separated in non-adjacent wells (to avoid well to well contamination) on 15% acrylamide SDS-PAGE gels and stained with Coomassie. One run for each of the eight biological conditions  was completely sliced from the stacking gel to the buffer front in 20 to 25 bands, and each band was cut into small (~1 mm3) cubes for protein in-gel digestion and MS analysis, as described elsewhere [To precipitate and concentrate proteins from the supernatant of wt and lsewhere ,67. Pepthttp://proteomecentral.proteomexchange.org) via the PRIDE partner repository [The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium  is a label-free technique that assumes ideal scoring for proteins as the summative of the identification scores of their constituent peptides freed upon digestion. A higher score represents a more abundant protein [LC-MS/MS data interpretation was made against the current UniProtKB database release (2012_06) of all known  protein . The Eas protein  was used protein . The obt protein , and its protein  allowed http://www.cbs.dtu.dk/services/SignalP/) [http://www.cbs.dtu.dk/services/TatP/) [http://gecco.org.chemie.uni-frankfurt.de/T3SS_prediction/T3SS_prediction.html) [http://www.effectors.org/) [A. salmonicida ABC transporters was provided by ABCdb (https://www-abcdb.biotoul.fr/) [http://phast.wishartlab.com/index.html) [Detection of signal sequences for secretion was carried out using the SignalP 4.1 server  , TatP 1.s/TatP/)  and the on.html)  and Effers.org/) . The lisoul.fr/)  and prohex.html) .APC: Antigen presenting cell; CTL: Cytotoxic T lymphocytes; GP: Exponential phase of growth; LFQ: Label-free quantitation; PMSS: Peptide-matching score summation; SN: Supernatant; SP: Stationary phase of growth; T3SS: Type-three secretion system; wt: wild-type.The authors have declared that no competing interests exist.PVB conceived of the study, carried out the experiments, analyzed data from MS, performed bioinformatic analyses and drafted the manuscript. SB-L and MH performed MS experiments and interpretation of MS data. JF helped to draft the manuscript. Authors read and approved the final manuscript.Table: PMSS, LFQ values, ratios, PEP values, subcellular localization, secretion system signals for each protein identified in SNs and pellets of wt and mutant strains in GP and SP. Column B: Proteins names. Red = T3SS components; dark red = other virulence factors ; light red = putative secondary virulence factors; yellow = proteins specific of JF2267 or B526; mauve = multidrud resistance-associated proteins; orange = ABC transporters; light green = proteins associated to flagella, pili, T4SS; dark blue = phage proteins; light blue = cytoplasmic moonlighting proteins present in SNs; grey = T5SS; light pink T6SS, pink: transposases. Column E: A449 Loci. Grey = genes conserved among Aeromonas sp.; white = genes shared with at least one other Aeromonas species; green = genes specific of A. salmonicida; yellow = genes specific of A. salmonicida JF2267 and B526; pink = transposases.Click here for fileFigure: most abundant proteins in A. salmonicida supernatants of wt and mutant strains in GP and SP. The diagram represents the most abundant proteins secreted by A. salmonicida . Below the name of the protein circles represent T3SS components (red), other virulence factors  (pink), putative secondary virulence factors (yellow) and cytoplasmic proteins with putative moonlighting activity (green).Click here for fileFigure: most abundant proteins in A. salmonicida pellets of wt and mutant strains in GP and SP. The diagram represents the most abundant proteins detected in A. salmonicida pellets . Below the name of the protein circles represent T3SS components (red), other virulence factors  (pink), putative secondary virulence factors (yellow) and secreted cytoplasmic proteins with putative moonlighting activity (green).Click here for fileFigure: confirmation by western blotting of the difference in quantity observed between the pellets of the wt and the ΔascV mutant in the GP for AopD, AcrV and AexT.Click here for fileTable: A. salmonicida secreted proteins that have homologues in other bacteria with a putative role in virulence.Click here for file


Many cases which were recognized as “idiopathic” are now known to have an infectious etiology. Here, we present a proof-of-concept study to examine the microbial link between intra-amniotic infection (IAI) and early-onset neonatal sepsis (EONS).1) Group 1– neonatal blood culture-positive EONS (n = 6). 2) Group 2– neonatal blood culture-negative presumed EONS with positive IAI (n = 16). 3) Group 3– neonatal blood culture-negative presumed EONS with no IAI (n = 7); 4) Group 4– no EONS or IAI (n = 7). In addition, samples from term healthy deliveries (n = 8) served as technical controls. A total of 31 species (15 non-redundant) were identified in AF, of which only 1/3 were cultivated. Significantly fewer microorganisms were detected in CB, with a total of 18 species (7 non-redundant) identified, of which only 2  were cultivated. Of those, Bergeyella, Fusobacterium nucleatum, and Sneathia sanguinegens had not been detected in EONS before. The novel species identified in AF by PCR include Peptoniphilus harei and Lachnospiraceae sp. The majority (72%) of CB species were also detected in the matching AF, with E. coli and F. nucleatum as the most prevalent. The 16S rRNA sequences of paired AF and CB were 99.9–100% identical, while no identical sequences were found between different pregnancies.Using culture independent methods, we analyzed paired amniotic fluid (AF) and cord blood (CB) samples from 36 singleton pregnancies complicated by preterm birth (PTB), IAI, and/or EONS. PTB cases were grouped as F. nucleatum should be placed on the same importance scale as E. coli.Previously unrecognized, uncultivated or difficult-to-cultivate species are implicated in EONS. Microbial species in paired AF and CB likely share the same infectious origin. Given its prevalence in EONS,  Studies of microbes have traditionally focused on bacterial species isolated in culture. Because a large proportion of bacteria are uncultivated, traditional microbiological cultures provide an incomplete picture of the human microbiome Fusobacterium nucleatum, Leptotrichia (Sneathia), Bergeyella, Peptostreptococcus, Bacteroides and a species of the order Clostridiales. Interestingly, women with positive microbial cultures and/or PCR results often delivered neonates with early-onset neonatal sepsis (EONS) We were among the first to employ metagenomic approaches to evaluate amniotic fluid (AF) microbial diversity in pregnancies complicated by preterm birth (PTB) Streptococcus agalactiae  and Escherichia coliEONS (sepsis in the first 3 days of life) remains a major cause of neonatal morbidity and mortality The current view is that EONS is a syndrome with at least 3 pathogenic variants Metagenomics has played a pivotal role in characterizing pathogenic microbiota and is becoming the new “gold standard” for the diagnosis of infectious diseases We studied paired AF-CB samples retrieved from 44 women pregnant with singletons. The research protocol was approved by the Human Research Protection Program Review Board at Yale University and Internal Review Board at Case Western Reserve University. All patients provided written informed consent.Group 1: premature newborns with neonatal blood culture “confirmed” EONS : 25 Group 2: premature newborns with negative neonatal blood culture but “presumed” EONS and positive IAI  from September 2004 to February 2009 who had paired AF and CB available for analysis (n = 161). To avoid selection bias, cases fulfilling the clinical group requirements were selected consecutively based on the availability of at least 1 ml umbilical vein CB for DNA extraction and other research analyses. The study groups were designed using the results of AF and neonatal blood bacterial cultures as reported by YNHH microbiology laboratory. The following groups were studied: th percentile), and anhydramnios were excluded. A diagnosis of preterm premature rupture of the membranes (PPROM) was established following clinical evaluation of the patient by sterile speculum exam, complemented by a “nitrazine”, “ferning”, and “amnio-dye” tests 3), trans-abdominal uterine tenderness and/or foul smelling AF Gestational age was established based on the last menstrual period and/or ultrasound evaluation. Term delivery was defined as birth at GA ≥37 weeks. Preterm labor was defined as presence of regular uterine contractions, advanced cervical dilatation (≥3 centimeters) or effacement at GA<37 weeks Groups 1–4 presented with symptoms of preterm labor or PPROM had amniocentesis to rule-out IAI. Following amniocentesis, each woman was followed prospectively till delivery. Corticosteroids for lung maturity were administrated if patients were ≤34 weeks GA or <32 weeks for PPROM cases. Digital exams were not permitted, unless clinically indicated. Antibiotic prophylaxis was indicated for extending latency period or for GBS as appropriate and in accordance to the American College of Obstetrics and Gynecology Group 5 were healthy women without any significant past medical history undergoing planned Cesarean delivery in the absence of labor. Indications for abdominal delivery included prior uterine surgery or breech presentation. All infants were appropriately grown for GA and had reassuring fetal heart rate patterns prior to surgery. The decisions for amniocentesis or indicated delivery including Cesarean were made independent of the research protocol.All mothers of the 36 premature newborns in Groups 1–4) were admitted in YNHH Neonatal Intensive Care Unit (NICU) and underwent clinical and laboratory evaluations of EONS within 2-hours post-delivery 3; absolute band count >1,500 cells/mm3; immature/total neutrophil (I:T) ratio >0.16; platelet count <150,000 cells/mm3 as previously described Group 5) displayed signs or symptoms of sepsis and were all discharged home at 48–72 hours of life.All premature neonates (Groups 1–4) or at the time of Cesarean delivery after direct visualization of the intact amniotic bag through the uterine incision (Group 5). AF of Groups 1–4 was sent to YNHH biochemical and microbiological laboratories for determination of glucose concentration, lactate dehydrogenase (LDH) activity, white (WBC) and red (RBC) blood cell counts and traditional Gram stain as rapid indicators to rule-out or confirm IAI. Results of these tests were used for clinical antepartum management as described above. A glucose level of ≤15 mg/dL AF was retrieved under sterile conditions by trans-abdominal amniocentesis  and Columbia CNA agar, Ureaplasma broth and agar. To identify anaerobic bacteria the biological specimens were cultured at 37°C in an anaerobic chamber . After 5 days of culturing, the results of the microbiological tests were reported as final. Morphology of the bacterial colonies, medium reaction, Gram stain and the VITEK 2 automated card system  were used for bacterial identification. PCR was employed for separation among Ureaplasma species. For neonatal blood cultures, as per YNHH NICU protocol, 1 mL of neonatal blood was collected in aseptically from 2 separate sites within 2 hours of birth, prior to antibiotic treatment. Detection of early neonatal bloodstream infections was performed using an automated instrument , which detects viable microorganisms within pediatric blood culture bottles .AF was cultured for aerobic and anaerobic bacteria, GTTTGATCCTGGCTCAG) and 1512R (TACCTTGTTACGACTT) were used for PCR amplification of the 16S rRNA genes as previously described Panctomycetales, Thermotoga, Fervidobacterium and some spirochetes F. nucleatum 12230 as a testing strain, the limit of detection has improved from 1×106 CFU reported in our previous study 4 CFU (data not shown). As expected, not all species were detected with the same sensitivity. For example, the limit of detection for Gardnerella vaginalis was 1×106 CFU, possibly due to two mismatches near the 5′ end in A17F (data not shown). All PCR products were observed by 1% agarose gel electrophoresis. Negative controls were included with each PCR reaction set to control for environmental or human contamination.DNA was extracted from 1 ml AF or CB serum as previously described GTAAAACGACGGCCAGTG) and M13 reverse (GGAAACAGCTATGACCATG). The sequences were assembled and aligned using VectorNTI (Invitrogen). A local alignment algorithm (BLAST) was used against NCBI GenBank and Human Oral Microbiome Database  to assign each sequence to a species based on the generally accepted >97% sequence similarity with the reference strain To identify the species amplified by PCR and to ensure that the PCR amplicons were indeed bacterial rRNA genes rather than artifacts, the PCR products were cloned into the pCR8 vector . From each PCR positive sample, a total of 10–20 random clones were selected for full-length 16S rRNA gene sequence analysis  using universal primers M13 forward  software. Kruskal-Wallis ANOVA on Ranks followed by Dunn’s tests (to adjust for multiple comparisons) or Student t-tests were used as appropriate. Comparisons between proportions were done with Chi-square or Fisher’s exact tests. For each common species identified in AF and CB from the same pregnancy, Cohen’s Kappa coefficient was used to assess the co-occurrence, which was classified into the following categories: excellent (Kappa >0.8), good (0.6–0.8), moderate (0.4–0.6), fair (0.2–0.4), low (0–0.2), or none (0 or below). A Groups 1–4) are presented in Group 5 was not considered for this comparative analysis on the basis of the normal pregnancy course with term delivery and the a priori designation of the samples as technical controls. Groups 1 and 2 had lower GA at amniocentesis and at delivery, and delivered neonates with lower birth weights. The amniocentesis-to-delivery interval of women with IAI (Group 2) was shorter. The results of the clinical laboratory tests used for case management are presented in Group 1), 5 neonates were delivered in the setting of a positive AF culture result. An advanced degree of histological chorioamnionitis was seen for Groups 1 and 2. Higher degrees of neutropenia  were observed for Group 1 neonates.The demographic, clinical and outcome characteristics of pregnancies resulting in PTB  cases in Group 2 , with 11 cases (55%) mono-microbial  AF samples, all in  1 and 2  and 5. I6% AF samicrobial , 7 casesicrobial  and 5. NUreaplasma parvum and Escherichia coli (n = 5 each), Fusobacterium nucleatum (n = 4), Bergeyella sp., Peptoniphilus indolicus, and Sneathia sanguinegens (n = 2 each), and Bacteroides stercoris, Bacteroides ureolyticus, Haemophilus influenzae, Klebsiella pneumonia, Lachnospiraceae sp., Listeria monocytogenes, Mycoplasma hominis, Prevotella bivia, Streptococcus agalactiae (GBS), Streptococcus anginosus, Streptococcus mitis, and Streptococcus pneumoniae (n = 1 each) :  1 each) .Group 1 . The PCR-positive CB cases were identified in Groups 1 (3/6) (confirmed EONS), 2 (8/16) (presumed EONS with positive IAI), and 3 (2/8) (presumed EONS with negative IAI), but not in Groups 4 or 5 (no EONS). Eight out of thirteen (62%) of the PCR-positive CB cases were mono-microbial (E. coli and F. nucleatum (n = 5 each), S. sanguinegens, GBS, U. parvum (n = 2 each), Bergeyella sp. and M. hominis (n = 1 each)  .E. coli in case 1-02, F. nucleatum in case 2-04, and S. sanguinegens in cases 3-06 and 3-07, the majority of the species  identified in CB was also detected in AF (E. coli and case 1-04: GBS) when both culture-dependent and independent results were taken into consideration. As several possible intersections among biological compartments lacked shared species, the complete 4-way Venn diagram with the number of species for each possible intersection is shown in To investigate the level of non-random association between bacterial species encountered in AF and CB, we performed an overlap analysis. In ed in AF  and 6. FE. coli, F. nucleatum, and M. hominis , followed by Bergeyella and GBS , and U. parvum   . No agresmatches . The limIn AF, the culture-dependent method reported 7 species which were not confirmed by PCR: Lactobacillus, Klebsiella pneumoniae, Ureaplasma, Mycoplasma, Streptococcus anginosus, Peptostreptococcus asaccharolyticus, and Bacteroides stercoris . For PepIn the group of newborns with positive blood cultures, out of the total 6 species cultivated from neonatal blood, 4 were not identified by PCR: E. coli (n = 2), Streptococcus agalactiae (n = 1) and Staphylococcus coagulase-negative (n = 1). However, among all samples of CB in this study, PCR identified 16 species that were not found by cultures. Of these, F. nucleatum (n = 5), S. sanguinegens and U. parvum (n = 2 each), Bergeyella sp. and M. hominis (n = 1 each) were detected in CB only by culture-independent methods.There is a paucity of metagenomic studies in human pregnancy, and in particular in fetal and neonatal septic conditions related to IAI. In this study we evaluated for the first time bacterial diversity in AF and CB in parallel by using culture-dependent and independent techniques. Culture-dependent methods more often suggested a single-species profile in AF or neonatal blood. Conversely, by 16S-rRNA sequencing, most AF infections were found to be poly-microbial with 15 distinctive species identified as potential etiologic agents of IAI. The same technique indicated that the majority of CB infections were, in contrast, mono-microbial. Only 7 distinct species were identified in CB as potential etiologic agents of EONS. Collectively, these results suggest that the microbial diversity of AF and CB differ with respect to specific bacterial species and with the detection method. The reduced microbial diversity observed in CB compared to AF, both by culture and by PCR, indicates that only a limited number of species could invade into the fetal compartment. Alternatively, this discrepancy could be due to administration of antibiotics before and/or during labor as AF was collected prior to the use of antibiotics while CB was collected after delivery.Bergeyella sp., an uncultivated oral species, had never been associated with pregnancy complications. In the current study, Bergeyella was once again identified by the culture-independent methods, further validating the involvement of this uncultivated species in IAI. A novel discovery of the current research was identification of two new species, Lachnospiraceae sp., and P. harei, in AF. Neither had been linked to IAI before. We anticipate that many more species will be discovered to be involved in pregnancy complications using the culture-independent technologies.Using culture-independent methods, we previously identified significantly more microbial species in AF than the traditional culturing methods Klebsiella pneumoniae (Staphylococcus Peptoniphilus asaccharolyticus vs. Peptoniphilus harei). These disagreements are likely due to taxonomical or typing errors due to limitations of the culturing method. P. asaccharolyticus (formerly known as Peptostreptococcus asaccharolyticus) is a Gram positive anaerobic coccus known to have the same phenotypical features as P. harei and its identity is often mistaken by cultures P. harei nor Sneathia spp are included on the VITEK2 card.The culture-dependent and independent methods complement, but cannot entirely replace, each other. As reported in our previous study Using conventional culturing methods, only 3 commons species are shared between AF and neonatal blood. When the analysis was performed on paired AF-CB samples using culture-independent methods, the number was significantly increased to 13 species. This suggests that fetal exposure in the setting of IAI may be incomplete based on microbial cultures alone. The high sequence-similarity levels (99.9-100%) among the 13 species shared between AF and CB argue against randomness as reason for their co-occurrence.Bergeyella sp., E. coli, F. nucleatum, M. hominis, GBS and U. parvum, are more likely to breech the fetal barrier than others. In particular, F. nucleatum, E. coli and M. hominis emerged as the most “transmissible” species of IAI and hence of EONS-I based on their Kappa scores. In comparison, U. parvum, one of the most prevalent species detected in AF, was only detected in 2 of 5 matching CB samples. Bergeyella was detected in one of the 2 matching CB samples, while no agreement between AF and CB was observed for S. sanguinegens. Interestingly, F. nucleatum and GBS were detected in more cases of CB than AF. These results indicate that not all infections in CB are related to those in AF.Our understanding of the extent and identity of the AF pathogens that gain access to the fetal compartment is incomplete. The use of metagenomics techniques allowed us to appreciate for the first time the microbial differences and similarities between AF and CB as two distinct yet unquestionably linked compartments. Our results suggest that only some bacteria, i.e. F. nucleatum, a common oral anaerobe, as one of the most prevalent etiological agents of EONS-I is novel. While F. nucleatum has been frequently detected in IAI F. nucleatum may be due to its ability to invade epithelial and endothelial cells F. nucleatum colonizes the mouse placenta by crossing the endothelium, followed by induction of Toll-like receptor 4-mediated inflammatory responses leading to fetal loss F. nucleatum requires FadA, a filamentous adhesin exposed on the surface of the bacteria F. nucleatum to cross the placental barrier and to breach into the fetal compartment. Impairment of the endothelial permeability by FadA also allows the penetration by other bacterial species, such as E. coliF. nucleatum is often found in mixed infections. The species co-identified with F. nucleatum in this study, such as Bergeyella sp., Lachnospiraceae, S. sanguinegens, and S. mitis, are all common oral species F. nucleatum from the oral cavity. This observation is consistent with our previous report of co-translocation of diverse oral species from the dental plaque and saliva to the murine placentas The finding of One major limitation of the study is the small sample size of the groups, which reflect the real-life situation in any NICU in the world, where the number of culture-positive cases of EONS are rare to detect, despite best practice in sample collection and blood culture. In the absence of a positive culture, the duration of treatment of the babies becomes dependent upon subjective clinical criteria and hematological criteria-based scoring systems. The hematological criteria used in the present manuscript have been validated in multiple previous studies Groups 2 and 3). In the current study the neonatal blood was not available for PCR analysis, and CB was used instead. Several possible scenarios can explain why bacterial DNA was detected in CB by PCR in a significant portion , although not in all newborns with “presumed” EONS. Similar to AF, a significant limitation of culture is the small number of microbiological cultivation protocols restricted to a few known pathogens Bergeyella sp., F. nucleatum, and Leptotrichia (Sneathia). Bacterial killing by appropriate immune defense mechanisms and/or use of antenatal antibiotics represent additional explanations given that microbial cultures will only identify viable and multiplying species whereas PCR may amplify DNA of both live and dead bacteria. Presence of bacterial DNA in CB of 43% of newborns with presumed EONS is of significant clinical relevance because even if the identified species are no longer viable, such microbes may still represent etiologic agents of the EONS syndrome (EONS-II variant). Bacterial PAMPs, including free DNA of dead bacteria, can actively engage fetal Toll-like receptors initiating an inflammatory cascade in neonates with “presumed” EONS Culture “confirmed” or “presumed” EONS are a frequent encounter in pregnancies complicated by IAI F. nucleatum at the same level on the importance scale with E. coli. A better understanding of IAI and of EONS variants from etiologic and pathogenic standpoints is critical for development of targeted treatments to improve neonatal outcome.This study demonstrates the following: (1) the profile of etiologic agents responsible for EONS is greater than currently appreciated by culture; (2) bacteria in AF are more likely than others to breech the fetal barrier, underscoring IAI as a possible ascendant of EONS; and (3) among the newly identified etiologic agents of EONS-I, our study places


Garcinia lucida with the minimal inhibitory concentrations (MIC) varying from 128 to 512 μg/mL, Garcinia kola (MIC of 256 to 1024 μg/mL), and Picralima nitida (MIC of 128 to 1024 μg/mL) were active on all the twenty-nine studied bacteria including MDR phenotypes. The association of phenylalanine arginine β-naphthylamide (PAβN or efflux pumps inhibitor) to different extracts did not modify their activities. At the concentration of MIC/2 and MIC/5, the extracts of P. nitida and G. kola improved the antibacterial activities of some commonly used antibiotics suggesting their synergistic effects with the tested antibiotics. The results of this study suggest that the tested plant extracts and mostly those from P. nitida, G. lucida and G. kola could be used alone or in association with common antibiotics in the fight of bacterial infections involving MDR strains.The present work was designed to assess the antibacterial properties of the methanol extracts of some Cameroonian medicinal plants and the effect of their associations with currently used antibiotics on multidrug resistant (MDR) Gram-negative bacteria overexpressing active efflux pumps. The antibacterial activities of twelve methanol extracts of medicinal plants were evaluated using broth microdilution. The results of this test showed that three extracts  Beauv.) Schott and Endl. (Sterculiaceae), Buchholzia coriacea Engl. (Capparidaceae), Garcinia kola Heckel (Guttifeare), and Garcinia lucida Vesque (Guttifeare), the seeds and fruits of Picralima nitida; the potential of the extract from the above plant extracts to increase the activity of some antibiotics on MDR bacteria was also investigated as well as the role of bacterial efflux pumps in the resistance to the tested plant extracts.Bacterial infections are responsible for 90% of infections found in health care services. The emergence of MDR bacterial strains appears as the major cause of treatment failure . Among t strains . Efflux Citrus medica, the bulbs of Allium sativum and Allium cepa, the seeds of Carica papaya, Cola acuminata, Buchholzia coriacea, Garcinia kola, and Garcinia lucida, the seeds and fruits of Picralima nitida. The plants were identified by Mr. Tadjouteu Fulbert (Botanist) of the National Herbarium  where voucher specimens were deposited under a reference number  from each plant was extracted with methanol (MeOH) for 48 h at room temperature. The extract was then concentrated under reduced pressure to give a residue that constituted the crude extract. They were then kept under 4°C until further use.The presence of major secondary metabolite classes, namely, alkaloids, flavonoids, phenols, saponins, tannins, anthocyanins, anthraquinones, sterol, and triterpenes was determined using common phytochemical methods as described by Harborne . p-Iodonitrotetrazolium chloride (INT) and phenylalanine arginine β-naphthylamide (PAβN) were used as microbial growth indicator and efflux pumps inhibitor (EPI), respectively.Ciprofloxacin (CIP), chloramphenicol (CHL), streptomycin (STR), tetracycline (TET), norfloxacin (NFX), cloxacillin (CLX), ampicillin (AMP), erythromycin (ERY), kanamycin (KAN), and cefepim (CEF)  were used as reference antibiotics. Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumonia, and Enterobacter cloacae (The studied microorganisms include references (from the American Type Culture Collection) and clinical (Laboratory collection) strains of  cloacae . They weμL) of inoculum (1.5 × 106 CFU/mL) prepared in MHB was then added. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a shaker and incubated at 37°C for 18 hrs. The final concentration of DMSO was lower than 2.5% and does not affect the microbial growth. Wells containing MHB, 100 μL of inoculums, and DMSO at a final concentration of 2.5% served as a negative control. Ciprofloxacin was used as reference antibiotic. The MICs of samples were detected after 18 hrs of incubation at 37°C, following addition (40 μL) of 0.2 mg/mL INT and incubation at 37°C for 30 minutes [The respective MICs of samples on the studied bacteria were determined by using rapid INT colorimetric assay . BrieflyβN at 30 μg/mL final concentration. Two of the best extracts, those from seeds of Garcinia kola and Picralima nitida fruits were also tested in association with antibiotics at MIC/2 and MIC/5. These concentrations were selected following a preliminary assay on one of the tested MDR bacteria, P. aeruginosa PA124 . All assays were performed in triplicate and repeated thrice. Fractional inhibitory concentration (FIC) was calculated as the ratio of MICAntibiotic  in  combination/MICAntibiotic  alone and the interpretation made as follows: synergistic (FIC ≤ 0.5), indifferent (0.5 < FIC < 4), or antagonistic (FIC ≥ 4) [Samples were tested alone and then, in the presence of PAThe results of qualitative analysis showed that each plant contains various phytochemicals compounds such as alkaloids, anthocyanins, anthraquinons, flavonoids, phenols, saponins, tannins, and triterpenes as shown in βN on a panel of Gram-negative bacteria by the microdilution method. Results summarized in Garcinia lucida (MIC ranged from 128 to 512 μg/mL), Garcinia kola (MIC from 128 to 1024 μg/mL), and the fruits of Picralima nitida (MIC from 256 to 1024 μg/mL). The antibacterial activities of these plant species were recorded against all the 29 studied microorganisms. Other extracts exhibited weak activities against a limited number of strains studied.Extracts were tested for their antibacterial activities alone and in combination with PAβN, an EPI. Preliminary tests showed that PAβN did not have any antibacterial activity at 30 μg/mL. The association of the PAβN with the extracts reduced the MIC values of some of the extracts on some tested bacteria  in the presence of PAbacteria . However P. aeruginosa PA124 was used to find the appropriate subinhibitory concentration of the antibiotic-crude extract to be tested on other bacteria strains. The association of the extracts of P. nitida and G. kola reduced the MIC of ten antibiotics  at MIC/2 and/or MIC/5 explaining the use of such concentrations. The associations of the extracts of P. nitida fruits and G. kola with antibiotics did not show any case of antagonism (FIC ≥ 4) meanwhile indifference was observed in some cases of the associations of the extracts with FEP, CLX, and AMP  were associated with antibiotics with the aim to evaluate the possible synergistic effects of their associations. A preliminary study using P. aeruginosa PA124, one of the ten MDR bacteria used in this paper, was carried out with ten antibiotics  to select the appropriate sub-inhibitory concentrations of the extract to be used. The results  allowed the selection of G. kola, G. lucida and their MIC/2 and MIC/5 as the sub-inhibitory concentrations. No antagonistic effect (FIC ≥ 4) was observed between extracts and antibiotics meanwhile indifference was observed in the case of CLX, FEP, AMP, which are β-lactams acting on the synthesis of the bacteria cell wall [P. nitida extract meanwhile G. kola extract also increased the activity of NOR, TET (on 100%), ERY, and CIP (on 70%). Plant can be considered as an efflux pumps inhibitor if a synergistic effect with antibiotics is induced on more than 70% bacteria expressing active efflux pumps [P. nitida and G. kola probably contain compounds that can acts as EPI. The results of the present paper corroborate with those of Iwu et al. [G. kola extract and gatifloxacin  against Bacillus subtilis and the proportions of G. kola/gatifloxacin  against Staphylococcus aureus. Three of the most active plants extracts (ell wall  (Tables ell wall . It is well wall . The MDRell wall . At MIC/ux pumps . Therefod CIP on 0%. PlantG. Lucida, G. Kola, and P. Nitida extracts in the treatment of bacterial infections involving MDR phenotypes. In addition, the extracts of these plants could be used in association with common antibiotics to combat multidrug resistant pathogens.The overall results of the present work provide baseline information for the possible use of the studied plants and mostly


To examine the link between increased body mass index and benign prostatic hyperplasia (BPH) related complications, namely: acute urinary retention (AUR), Bladder stones and Bladder diverticula.Patients: We reviewed the medical records of BPH patients who underwent prostatectomy over three years period from 2010–2012. Prostatectomy was either done in the gold standard transurethral resection of the prostate (TURP) or using green light laser selective photo-vaporization (PVP). Age, PSA, Prostate Volume (PV) as measured by ultrasonography, patients’ weight & height, BPH related complications mainly AUR, bladder stones, and bladder diverticula were all taken in account. The BMI was calculated as weight in kg divided by square height in meters. The BMI was divided in 4 categories according to WHO classification: underweight if BMI ≤18.50 kg/m2; normal weight if BMI = 18.50-24.9 kg/m2; overweight: BMI ≥25 kg/m2; and obese BMI if ≥30 kg/m2. Statistical analysis: done using the SPSS package version 16. Chi-square test was used for comparison between groups where p-value was considered significant if <0.05 and ANOVA test was used for comparison between multiple variables.197 patients were included in this study, of those 95(48%) underwent TURP and 102(52%) underwent PVP. The two groups were found to be similar in mean age and PSA, and significantly different in prostate volume and operating room (O.R.) time.Although the mean weight for patients undergoing prostatectomy in this study was in the overweight range, yet only 59/197(30%) patients with increased BMI presented with AUR. In this particular cohort of patients undergoing prostatectomy there was no significant differences in the development of AUR, bladder stone and diverticulum formation between patients with increased BMI (overweight and obese) and those with normal BMI. Further studies are recommended to explore the influence of increased BMI on BPH-related complications. Obesity has recently been declared to be associated with several urological diseases  or using green light laser selective photo-vaporization (PVP). Age, PSA, Prostate Volume (PV) as measured by ultrasonography, patients’ weight & height, BPH related complications mainly AUR, bladder stones, and bladder diverticulum were all taken in account. The BMI was calculated as weight in kg ÷ square height in meter. The BMI was divided in 4 categories according to WHO classification: underweight if BMI ≤18.50 kg/m2; normal weight if BMI = 18.50-24.9 kg/m2; overweight: BMI ≥25 kg/m2; and obese BMI if ≥30 kg/m2. All participants signed an informed consent prior to inclusion in the study. The study was approved by the Institution’s Unit of Biomedical Ethics- Research Committee.Statistical Analysis: Was done using the SPSS package version 16. Chi-square test was used for comparison between groups where p-value was considered significant if <0.05 and ANOVA test was used for comparison between multiple variables.197patients were included in this study, of those 9548%) underwent TURP and 102(52%) underwent PVP. The two groups were found to be matched in mean age and PSA, and significantly different in prostate volume and operating room (O.R.) time. The results are summarized in Tables 8% underwEvidence elicited from the current study did not demonstrate an increase in the risk of developing BPH-related complications with an increased BMI. No significant differences were noted in the development of AUR, bladder stone and diverticulum formation; between patients with increased BMI (overweight and obese) and those with normal BMI. A relationship between higher BMIs and elevated PSA, poorer uroflow, increased incidence of retention, and larger prostate volume were expected to be seen, however our study demonstrated a relationship that was NOT statistically significant.The metabolic syndrome is common in Arabian Gulf countries especially Saudi Arabia. It comprises a number of disorders—including insulin resistance, hypertension and central abdominal obesity—that all act as risk factors for cardiovascular diseases. Accumulating evidence now exists to link urological diseases to the metabolic syndrome  as this is a leading cause of death. Furthermore, an association between benign prostatic hyperplasia and primary hypertension was reported  patients with increased BMI presented with AUR. In this particular cohort of patients undergoing prostatectomy there was no significant differences in the development of AUR, bladder stone and diverticulum formation between patients with increased BMI (overweight and obese) and those with normal BMI. Further studies are recommended to explore the influence of increased BMI on BPH-related complications. Subsequently, active life style and weight reduction can be discussed with patients with symptomatic BPH within the context of benefit not only in improving LUTS, and slowing the growth of the prostate but also to improve response to medical therapy and prevent BPH related complications.


Progressive familial intrahepatic cholestasis (PFIC) type 2 results from a mutation in the bile salt exporter pump, impeding bile acid transport. Patients usually present with jaundice, pruritus, growth failure, and fat soluble vitamin deficiencies. We present two patients diagnosed with PFIC type 2 due to severe coagulopathy and bleeding without jaundice. Escherichia coli, and this was continued for a full seven-day course.Case  A is a healthy 5-month-old female who presented to a local hospital with scratching to the point of bleeding and ecchymoses on her abdomen, back, and legs. She had no history of jaundice since birth and jaundice was not noted at several doctors' visits prior to this presentation. Physical examination showed neither icterus nor hepatosplenomegaly. Initial labs demonstrated AST 223 U/L, ALT 334 U/L, total bilirubin 3.4 mg/dL, direct bilirubin 2.8 mg/dL, GGT 33 U/L, partial thromboplastin time (PTT) 90.8 seconds, prothrombin time (PT) > 120.0 seconds, and INR > 13.7. She received a one-time intravenous vitamin K with repeat INR of 1.0 and then was admitted for further evaluation. Of note, she was treated with IV cefotaxime at the outside hospital due to a positive urine culture forCholestasis persisted with a peak direct bilirubin of 7.5 mg/dL. An abdominal ultrasound showed normal hepatic echotexture and biliary system without focal lesions. Infectious work-up included negative serologies for CMV, EBV, HIV, HSV, HCV, and HBV. As vitamin K deficiency was thought to be from cholestasis related malabsorption, fat soluble vitamin studies were performed and revealed a vitamin D 25-OH) level of <5 ng/mL, normal vitamin A, and an alpha-tocopherol level of 0.8 mg/L. Total serum bile acids were elevated at 205.3 umol/L . Percutaneous liver biopsy revealed mild chronic portal inflammation, periportal fibrosis, ballooning hepatocytes, significant cholestasis, and early bile duct loss with ductular proliferation  and percutaneous liver biopsy. The ERCP was normal. Liver biopsy showed marked canalicular and hepatocellular cholestasis, with mild to moderate portal and lobular fibrosis including BSEP staining , with 9% of patients presenting with manifestations of vitamin deficiency [Patients with PFIC type 2 can be diagnosed based on mutations in the ABCB11 gene, which encodes the BSEP protein. To date, there are more than 100 mutations that have been discovered in the ABCB11 gene, leading to a 70% reduction or complete absence of bile salts removed from the liver . Two of  ABCB11, has also been reported by Strautnieks et al. [In Case A the first mutation is c.3692 G>A in exon 27 . The pres et al. . In thatIn Case B, his first mutation is 890 A>G in exon 9 . The preThere are technical challenges to the exploration of the mRNA consequences of the splice site mutations by sequencing the corresponding cDNA. Since expression of BSEP protein in extrahepatic tissues is low, the only way to get sufficient mRNA is sampling the explanted liver and conserving the RNAse inhibitors immediately at the time of surgery, which was not feasible in both cases. We can assume that Case B must have similar mutations as his sister whose mRNA sequencing was performed and published by Scheimann et al. . On the In this report we conclude that patients who present with cholestasis and significant coagulopathy that respond to vitamin K should have congenital liver diseases such as PFIC included in their differential diagnosis though cholestasis may not be pronounced as a common clue in the diagnosis. Genetic analysis for mutations in the ABCB11 gene can aid in diagnosis after ruling out common causes of cholestasis. Further study in larger cohorts of PFIC type 2 populations may reveal more specific genotype-phenotype correlations.


HIV-associated cognitive impairment has declined since the introduction of combination antiretroviral treatment (cART). However, milder forms of cognitive impairment persist. Inflammation in the cerebrospinal fluid (CSF) has been associated with cognitive impairment, and CSF neurofilament light chain protein (NFL) and CSF neopterin concentrations are increased in those patients. Microbial translocation in HIV infection has been suggested to contribute to chronic inflammation, and lipopolysaccharide (LPS) and soluble CD14 (sCD14) are markers of microbial translocation and the resulting monocyte activation, respectively. We hypothesised that microbial translocation contributes to inflammation and axonal damage in the central nervous system (CNS) in untreated HIV infection.We analyzed paired samples of plasma and CSF from 62 HIV-infected, untreated patients without cognitive symptoms from Sahlgrenska University Hospital, Gothenburg, Sweden. Measurements of neopterin and NFL in CSF were available from previous studies. Plasma and CSF sCD14 was measured using ELISA , and plasma and CSF LPS was measured using LAL colorimetric assay . Univariate and multivariate regression analyses were performed.r = 0.31, P = 0.015), and plasma sCD14 was associated with CSF sCD14 . Furthermore, CSF sCD14 was associated with NFL  and neopterin  in CSF. LPS was not detectable in CSF. In a multivariate regression model CSF sCD14 remained associated with NFL and neopterin after adjusting for age, CD4+ cell count, and HIV RNA in CSF.LPS in plasma was associated with plasma sCD14 (In a group of untreated, HIV-infected patients LPS was associated with sCD14 in plasma, and plasma sCD14 was associated CSF sCD14. CSF sCD14 were associated with markers of CNS inflammation and axonal damage. This suggest that microbial translocation might be a driver of systemic and CNS inflammation. However, LPS was not detectable in the CSF, and since sCD14 is a marker of monocyte activation sCD14 may be increased due to other causes than microbial translocation. Further studies regarding cognitive impairment and biomarkers are warranted to fully understand causality. Prior to introduction of combination antiretroviral treatment (cART) a large proportion of HIV-infected patients developed severe neurocognitive disorders , 2. WithA number of studies suggest that cognitive impairment may be caused by inflammation in the central nervous system (CNS) –8. Thus,Systemic inflammation in HIV-infected patients has been reported repeatedly –20 and iWe hypothesised that microbial translocation contributes to inflammation in the CNS of HIV-infected patients. We therefore aimed to investigate if markers of microbial translocation (LPS) and monocyte activation (sCD14) were detectable in CSF and to determine possible associations between these markers and markers of inflammation and axonal damage in the CNS  in untreated HIV-infected patients.Since 1985, HIV-infected patients in Gothenburg, Sweden have been included in a longitudinal study that includes serial sampling of CSF, plasma, and serum. This cohort has previously been described in detail . Lumbar From this cohort 20 untreated HIV-infected patients without CNS opportunistic infection but with pleocytosis the CSF (>5 cells/μL) indicating HIV-induced inflammation was included in a pilot study in order to determine if it was possible to detect sCD14 and LPS in CSF during HIV infection. The 20 participants from the pilot study did not participate in the following analyses.Afterwards, 62 untreated, HIV-infected patients without cognitive symptoms or CNS opportunistic infections with samples collected between 2004 and 2014 were selected for further analyses. Untreated patients were selected to avoid confounding effects of cART on microbial translocation and inflammation. 30 patients had pleocytosis.n = 31, CD4+ cell count range 380–1400, median 530 cells/μL) or CD4+ cell count <200 cells/μL . Groups were matched on gender, age, and CSF leucocyte count. The selection was done randomly from the list of patients fulfilling the inclusion criteria  and was performed in accordance with the Helsinki Declaration. All blood and CSF samples were obtained after informed consent of subjects under the IRB-approved protocol.CSF NFL was measured using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA)  as previously described , 30. CSFValid measurement of neopterin in plasma and CSF, NFL and leucocyte count in CSF were available in 60, 61, 46, and 60 patients, respectively.LPS in plasma and CSF was analysed by the Limulus amebocyte lysate colorimetric assay  according to the manufacturer’s instructions and with the following modifications: Samples were diluted 10-fold to avoid interference with background colour, and preheated to 68 °C for 12 min prior to analysis to dissolve immune complexes, as previously described , 33.Soluble CD14 in plasma and CSF was analysed using ELISA according to the manufacturer’s instructions .P value ≤0.05 was considered significant. When comparing groups, the Student T-test and Mann Whitney U-test was used as appropriate. Uni- and multivariate linear regression models were used to assess associations and adjusted for possible confounders. When appropriate data were log-transformed (ln). In order to investigate possible associations between microbial translocation and inflammation in the CSF, multivariate linear regression models were performed with NFL and neopterin as the dependent variables, and sCD14 in the CSF was added as an independent variable. The model was adjusted for age, HIV RNA in the CSF, and CD4+ cell count. Since patients were included in two CD4+ cell count groups, CD4+ cell count was added to the model as both a continuous variable and as a nominal variable. Only the continuous variable was significant and therefore used in the final model. The analyses were performed using IBM SPSS 22.0 .Data are given as median (interquartile range (IQR)). A The pilot study included 20 untreated, asymptomatic, HIV-infected patients with CSF pleocytosis as a sign of HIV-induced inflammation. Both LPS and sCD14 was detectable in all plasma samples. However, LPS was not detectable in any CSF samples whereas CSF-sCD14 was detectable in all CSF samples (data not shown). Hence, only sCD14 was measured in CSF in the subsequent analyses.Samples from 62 untreated, HIV-infected patients without clinical evidence of impaired cognitive function and with CD4+ cell count either above 350 or below 200 cells/μL were selected for further analysis. Characteristics of the study population are shown in Table Valid measurement of LPS in plasma and sCD14 in plasma was obtained in all 62 participants whereas sCD14 in CSF was obtained in 61 participants.P = 0.042), whereas there were no differences in age, gender, CD4+ cell count, or plasma HIV RNA between these two groups. However, when dividing the patients into two groups with sCD14 in CSF above or below median, no differences were found in regard to age, gender, CD4+ cell count, plasma HIV RNA, plasma LPS, or CFS HIV RNA .Patients with plasma sCD14 above median had higher plasma LPS compared to patients with sCD14 below median ). Furthermore, concentration of sCD14 in CSF was 308 (IQR 196–385) in the group with CD4+ cell count <200 vs. 201 (IQR 90–317) in the group with CD4+ cell count >350 (P = 0.015). Leucocyte cell counts in CSF were median 3 cells/μL (IQR 1–11) and 5 cells/μL (IQR 4–9) (P = 0.406) in the group with CD4+ cell count <200 vs. the group with CD4+ cell count >350. Concentrations of CSF sCD14 was associated with CSF leucocyte cell count in a univariate analysis .Increased concentrations of sCD14 in plasma was found in patients with CD4+ cell count <200 compared to patients with CD4+ cell count >350 (median of 2579 ng/mL (IQR 2061–3641) vs. 2357 ng/mL (IQR 1174–2959) , as well as borderline significant association with CSF sCD14 . Furthermore, plasma sCD14 was associated with CSF sCD14 . Importantly, CSF sCD14 was associated with a higher concentration of both NFL  and neopterin in the CSF  and neopterin (P = 0.002) when adjusted for age, CD4+ cell count, and HIV RNA in CSF. In line with previous studies, higher age (P = 0.001) and lower CD4+ cell count (P = 0.001) were associated with higher NFL in multivariable analysis [P < 0.001) and lower CD4+ cell count (P < 0.001) were associated with higher neopterin. When LPS was added to the model, no association was found with NFL or neopterin.In a multivariate linear regression model, CSF sCD14 remained associated with NFL  . NeopterSeveral studies have shown that markers of microbial translocation are associated with cognitive impairment in HIV-infected patients , 52–54. Guidelines to assess cognitive function are based on the use of neuropsychological test . Studies3CSK4, polyI:C, and CpG DNA, cross the gastrointestinal mucosa and may activate the immune system and drive inflammation [This study was limited by limited sample size and the cross-sectional design. Furthermore, measuring microbial translocation using LPS is challenging since the method is sensitive to both time and contamination . Howeverammation , 51. Recammation . A diffeSignificant associations between sCD14 in CSF and markers of CNS inflammation and axonal damage were found suggesting that monocyte activation, possibly driven by microbial translocation, may contribute to CNS inflammation. Further investigations into the pathogenesis of cognitive impairment in HIV infection, as well as potential biomarkers are needed, in order to increase the understanding and supply reliable methods of diagnosing these conditions.The study was approved by the Regional Ethical Review Board in Gothenburg (Ö 588–01) and was performed in accordance with the Helsinki Declaration. All blood and CSF samples were obtained after informed consent of subjects under the IRB-approved protocol.Not applicable.All data are available upon request.


Aim. The galactose single-point (GSP) test assesses functioning liver mass by measuring the galactose concentration in the blood 1 hour after its administration. The purpose of this study was to investigate the impact of hemodialysis (HD) on short-term and long-term liver function by use of GSP test. Methods. Seventy-four patients on maintenance HD  with a mean time on HD of 60.77 ± 48.31 months were studied. The GSP values were compared in two groups: (1) before and after single session HD, and (2) after one year of maintenance HD. Results. Among the 74 HD patient, only the post-HD Cr levels and years on dialysis were significantly correlated with GSP values . 14 of 74 patients were selected for GSP evaluation before and after a single HD session, and the hepatic clearance of galactose was similar . GSP values decreased from 420.20 ± 175.26 g/mL to 383.40 ± 153.97 g/mL after 1 year maintenance HD in other 15 patients . Conclusions. Patients on maintenance HD for several years may experience improvement of their liver function. However, a single HD session does not affect liver function significantly as assessed by the GSP test. Since the metabolism of galactose is dependent on liver blood flow and hepatic functional mass, further studies are needed. Liver disease is a substantial contributor to the development of end-stage renal disease (ESRD). Patients with ESRD, especially those on hemodialysis (HD), are at high risk of hepatic viral infection. Traditional hepatic function tests including indicators for hepatocellular injury (aminotransferase levels), biliary tract injury , and functional synthesis  are routinely checked in patients on HD . NonetheCertain measurements are used to determine the amount of liver cells indirectly by measuring metabolic function or drug clearance as a substitute for quantitative liver function. The galactose single point (GSP) test is one novel method of assessing residual liver function. Galactose is a C-4 epimer of glucose and is catalyzed to glucose-1-phosphate by galactokinase, the rate-limiting step of metabolism for galactose exclusively in the liver , 6. The n = 74; 46 men and 28 women; mean age, 60.38 ± 11.86 yr) who underwent maintenance HD therapy at two nephrology units in different hospitals were prospectively investigated. All of the patients were informed about the study design and gave written consent to join the study. Patients were included in the study if they were more than 18 years of age, had stable, chronic renal failure , and underwent maintenance HD three times per week. Patients with galactose intolerance (based on a questionnaire) or serious liver disease  were excluded from this study. The study protocol was in accordance with the principles of the Helsinki Declaration and was approved by the Human and Ethics Committees of the respective institutions.Patients with ESRD (Kt/V), normalized protein catabolic rate (nPCR), alanine transaminase (ALT), aspartate aminotransferase (AST), albumin, glucose, uric acid, and alkaline phosphatase (Alk-P). Serum urea levels were recorded pre- and postdialysis for the analysis of single-pool Kt/V, parameter of adequeacy for dialysis . The nPCR, as the parameter of dietary protein intake, was calculated from monthly kinetic modeling sessions by applying the 2-blood urea nitrogen (BUN) method for the predialysis BUN level, and an estimate of the equilibrated postdialysis BUN level was obtained using the Daugirdas-Schniditz rate equation [All patients underwent conventional HD using a low-flux polysulfone dialyzer. The following baseline parameters were recorded on the day of blood sampling: body weight , body mass index, mean blood pressure, hematocrit (Hct), fractional clearance of urea  was infused intravenously at a dosage of 0.5 g/kg of body weight, and the infusion time was limited to 3 to 5 minutes. Blood sampling was performed 60 minutes after infusion. All 74 patients underwent predialysis GSP testing. Fourteen of the 74 patients were selected for evaluation of postdialysis GSP data after the HD session on the next week in order to avoid effect of residual galactose of pre-HD infusion. Postdialysis GSP was corrected according to postdialysis hematocrit. Another 15 of the 74 patients were selected for analysis of postdialysis GSP decreases after one year of maintenance HD.Blood for each galactose test was withdrawn 60 minutes after galactose infusion; blood samples were obtained by venipuncture 60 min after injection. Predialysis GSP testing was performed before hemodialysis. Postdialysis GSP testing was done after HD was completed. During the HD session, each patient was in bed resting. Blood samples were kept in an ice bath until measured by an enzymatic method. A colorimetric galactose dehydrogenase (GADH) method was used to determine galactose levels using a modification of the neonatal screening test . The normal concentration range of the calibration curve was 50–1000 mg/mL. Same-day variation was evaluated by the standard deviation and percentage coefficient of variation (CV) for each concentration. A maximum 10% CV was permitted. The slope and intercept of the calibration curves were also checked on a daily basis .E = N − B − T, where N is the total number of individuals or units in the study (minus1), B is the blocking component representing environmental effects allowed for in the design (minus1), T is the treatment component corresponding to the number of treatment groups (including control group) being used or the number of questions being asked (minus1), and E is the degrees of freedom of the error component and should be somewhere between 10 and 20. In our study, the study using subjects is planned with two groups (pre-HD versus post-HD) (T = 1), with 15 numbers per group, making 30 subjects total (N = 29), without any further stratifications (B = 0); then, E would equal 28, which is above the cutoff of 20, indicating that our samples size may be large enough.The sample size used in a study is determined based on Mead's resource equation. t-tests to compare the differences in GSP levels before and after a single session of HD and after one year of maintenance HD. Statistical significance was defined as P < 0.05. All statistical analyses were conducted using SigmaStat for Windows, version 2.03 .Data are presented as means ± standard deviations. We correlated predialysis GSP levels with baseline liver function and biochemical parameters using Pearson product-moment correlation. In selecting patients who received more than one GSP sampling, we used Student's paired The geographic and demographic results of the 74 patients are shown in Kt/V) among the 74 HD patients are given in r = 0.280, P < 0.05 and r = −0.240, P < 0.05, resp.). Dialysis adequacy (Kt/V) had no correlation with GSP level. r = −0.240, P = 0.004).The correlations between GSP values and BUN, Cr, AST, ALT, albumin, uric acid, years on HD, and adequacy of dialysis (P = 0.49). r = 0.851, P < 0.05).P < 0.05).Kt/V) greater than the National Kidney Foundation Kidney Disease Outcomes Quality Initiative guidelines [We notice that one HD session did not result in changes in liver function compared to baseline as evaluated by GSP testing. Additionally, the galactose level seems to be correlated inversely with years of maintenance HD. Long-term HD of at least one year improved galactose metabolism (GSP testing). Hence, maintenance HD could have beneficial effects on liver function as assessed by GSP testing. We compared pre- and post-HD GSP levels in 14 patients after a single HD session, which did not affect GSP levels significantly. A single HD treatment did not have an immediate impact on hepatic metabolism of galactose. These patients had dialysis adequacy , a potent mitogen for hepatocytes, is released from peripheral blood mononuclear cells and mesenchymal cells after acute hepatic injury . It acceThe correlation between galactose metabolism and hepatic blood flow in patients under HD is complicated. Studies in rats with glycerol-induced acute renal failure showed an initial decrease in cardiac output and liver blood flow during the first 12 hours after induction followed by an increase in 24 to 48 hours as measured using radioactive microspheres . In end-There are still several limitations in our study. Although we found that duration of hemodialysis may influence the GSP level, we could not get more case numbers because the procedure was time-consuming and galactose infusion might induce discomfort for some patients. Besides, since hepatic blood flow may influence the metabolism of galactose, further image studies such as Doppler scan on hepatic blood flow during hemodialysis or PET scan may be helpful. Since other cytokines such as HGF may be activated in HD patients, further studies for interaction between cytokines may help us understand the hepatic clearance in HD patients.In conclusion, patients on maintenance HD for several years experience improvement of liver function. However, a single HD session does not appear to affect liver function significantly. Since the metabolism of galactose is dependent on liver blood flow and hepatic functional mass, whether the blood flow to the liver is slightly increased after HD, which may result in improved liver function as reflected in the GSP test, deserves further study.