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Competing Interests: Conflict of interest statement. None declared.

Background and Aims: Fatigue is a common symptom of primary biliary cirrhosis (PBC), and is associated with an impaired quality of life., Study Question: No studies have assessed the use of modafinil in fatigue related to PBC in a controlled manner., Study Design, Measures, and Outcomes: A randomized, double-blind, placebo-controlled study was conducted to determine the safety and efficacy of modafinil for the treatment of fatigue in PBC. Forty patients were randomized to modafinil (n = 20) or placebo (n = 20) for 12 weeks. A verbal report of fatigue for at least 6 months was required for enrollment. Modafinil was administered at 100 mg by mouth once daily; a change by 50 mg every 2 weeks (maximum: 200 mg once daily) was allowed, depending on the subject's response to treatment. The primary outcome was defined as a ≥50% improvement in fatigue severity [quantified by the Fisk Fatigue Impact Scale (FFIS)] after 12 weeks of treatment, compared with baseline values., Results: Thirty-three PBC patients completed the study. After 12 weeks of therapy, only 5 patients had a ≥50% reduction in FFIS scores: 3 patients (17.6%) in the modafinil arm and 2 (12.5%) in the placebo arm (P = 1.00). Change in median FFIS score was not statistically different between patients in the 2 treatment groups (P = 0.36). Modafinil was associated with minimal adverse events (headaches, diarrhea, and rash)., Conclusions: In patients with PBC who have fatigue, treatment with modafinil for 12 weeks was safe and fairly well tolerated; however, it did not result in beneficial effects on fatigue compared with patients treated with placebo (CONSORT Table 1). ClinicalTrials.gov identifier NCT00943176.

Background: Emerging data suggest that oral antibiotics may have therapeutic effects in primary sclerosing cholangitis (PSC), but published studies are limited., Aims: To investigate the safety and efficacy of oral vancomycin and metronidazole in patients with PSC., Methods: Thirty-five patients with PSC were randomised in a double-blind manner into four groups: vancomycin 125 mg or 250 mg four times/day, or metronidazole 250 mg or 500 mg three times/day for 12 weeks. The primary endpoint was decrease in alkaline phosphatase (ALK) at 12 weeks. Secondary end points included serum bilirubin and Mayo PSC risk score; pruritus; and adverse effects (AEs). Nonparametric tests were used for analysis., Results: The primary endpoint was reached in the low-dose (-43% change in ALK, P = 0.03) and high-dose (-40%, P = 0.02) vancomycin groups, with two patients in the former experiencing ALK normalisation. Bilirubin decreased significantly in the low-dose metronidazole group (-20%, P = 0.03) and trended towards significance in the low-dose vancomycin group (-33%, P = 0.06). Mayo PSC risk score decreased significantly in the low-dose vancomycin (-0.55, P = 0.02) and low-dose metronidazole group (-0.16, P = 0.03). Pruritus decreased significantly in the high-dose metronidazole group (-3.4, P = 0.03). AEs led to medication discontinuation in six patients, four of whom were receiving metronidazole., Conclusions: Both vancomycin and metronidazole demonstrated efficacy; however, only patients in the vancomycin groups reached the primary endpoint, and with less adverse effects. Larger, longer-term studies are needed to further examine the safety and efficacy of antibiotics as a potential treatment for patients with primary sclerosing cholangitis (clinicaltrials.gov NCT01085760)., (© 2013 Blackwell Publishing Ltd.)

In Arabidopsis thaliana, XIPOTL1 encodes a phosphoethanolamine N-methyltransferase with a central role in phosphatidylcholine biosynthesis via the methylation pathway. To gain further insights into the mechanisms that regulate XIPOTL1 expression, the effect of upstream open reading frame 30 (uORF30) on the translation of the major ORF (mORF) in the presence or absence of endogenous choline (Cho) or phosphocholine (PCho) was analysed in Arabidopsis seedlings. Dose-response assays with Cho or PCho revealed that both metabolites at physiological concentrations are able to induce the translational repression of a mORF located downstream of the intact uORF30, without significantly altering its mRNA levels. PCho profiles showed a correlation between increased endogenous PCho levels and translation efficiency of a uORF30-containing mORF, while no correlation was detectable with Cho levels. Enhanced expression of a uORF30-containing mORF and decreased PCho levels were observed in the xipotl1 mutant background relative to wild type, suggesting that PCho is the true mediator of uORF30-driven translational repression. In Arabidopsis, endogenous PCho content increases during plant development and affects root meristem size, cell division, and cell elongation. Because XIPOTL1 is preferentially expressed in Arabidopsis root tips, higher PCho levels are found in roots than shoots, and there is a higher sensitivity of this tissue to translational uORF30-mediated control, it is proposed that root tips are the main site for PCho biosynthesis in Arabidopsis.

Upstream open reading frames (uORFs) are common in eukaryotic transcripts, but those that encode conserved peptides occur in less than 1% of transcripts. The peptides encoded by three plant conserved peptide uORF (CPuORF) families are known to control translation of the downstream ORF in response to a small signal molecule (sucrose, polyamines, and phosphocholine). In flowering plants, transcription factors are statistically over-represented among genes that possess CPuORFs, and in general it appeared that many CPuORF genes also had other regulatory functions, though the significance of this suggestion was uncertain (Hayden and Jorgensen, 2007). Five years later the literature provides much more information on the functions of many CPuORF genes. Here we reassess the functions of 27 known CPuORF gene families and find that 22 of these families play a variety of different regulatory roles, from transcriptional control to protein turnover, and from small signal molecules to signal transduction kinases. Clearly then, there is indeed a strong association of CPuORFs with regulatory genes. In addition, 16 of these families play key roles in a variety of different biological processes. Most strikingly, the core sucrose response network includes three different CPuORFs, creating the potential for sophisticated balancing of the network in response to three different molecular inputs. We propose that the function of most CPuORFs is to modulate translation of a downstream major ORF (mORF) in response to a signal molecule recognized by the conserved peptide and that because the mORFs of CPuORF genes generally encode regulatory proteins, many of them centrally important in the biology of plants, CPuORFs play key roles in balancing such regulatory networks.

Background: Limited data are available regarding the serum lipids in primary sclerosing cholangitis., Aims: To determine the lipid levels in patients with primary sclerosing cholangitis., Methods: We monitored the serum lipid levels annually for up to 6 years in 157 patients included in three previous trials of ursodeoxycholic acid., Results: The baseline lipid values were: total cholesterol=207 mg/dL (127-433); high-density lipoprotein=56 mg/dL (26-132); low-density lipoprotein=129 mg/dL (48-334); triglycerides=102 mg/dL (41-698). Cirrhotic stage was associated with lower levels of total cholesterol (186 mg/dL vs. 217 mg/dL, p=.02). A significant correlation between the liver biochemistries and total and low-density lipoprotein cholesterol levels was observed. Ursodeoxycholic acid, as compared to placebo, significantly decreased total (-27 mg/dL vs. 22 mg/dL, p=.0004) and low-density lipoprotein cholesterol (-24 mg/dL vs. 17 mg/dL, p=.0001). After extended follow-up, small changes in the lipid levels were noticed. The incidence of coronary artery disease was 4%., Conclusions: Our findings suggest that the lipid levels in primary sclerosing cholangitis are often above levels where treatment with lipid-lowering agents is recommended. However, primary sclerosing cholangitis patients seem to have no elevated risk for cardiovascular events. The correlation of total and low-density lipoprotein cholesterol with liver biochemistries implies that mechanisms linked to cholestasis may regulate cholesterol metabolism., (Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

The perspective presented here is that modern genetics is at a similar stage of development as were early formulations of quantum mechanics theory in the 1920s and that in 2010 we are at the dawn of a new revolution in genetics that promises to enrich and deepen our understanding of the gene and the genome. The interrelationships and interdependence of two views of the gene - the molecular biological view and the epigenetic view - are explored, and it is argued that the classical molecular biological view is incomplete without incorporation of the epigenetic perspective and that in a sense the molecular biological view has been evolving to include the epigenetic view. Intriguingly, this evolution of the molecular view toward the broader and more inclusive epigenetic view of the gene has an intriguing, if not precise, parallel in the evolution of concepts of atomic physics from Newtonian mechanics to quantum mechanics that are interesting to consider.

Primary biliary cirrhosis (PBC) is a chronic, autoimmune, cholestatic liver disease with a slowly progressive course. Without treatment, most patients eventually develop fibrosis of the liver and may need liver transplantation in the late stage of disease. Fatigue and pruritus are the most common symptoms of PBC, but the majority of patients are asymptomatic at first presentation. There is no specific treatment for fatigue in PBC, but modafinil has shown some potential beneficial effects, such as increased energy levels and decreased total sleep time. This Review article discusses the natural history and the measurement of fatigue in patients with PBC. The central and the peripheral mechanisms that have been suggested for the pathogenesis of fatigue in PBC are also discussed and treatment options are reviewed.

A novel technique is described that targets specific populations of transcripts for homology-based gene silencing using transitive RNAi. This approach is designed to target a subset of the transcriptome in order to identify genes involved in a particular localized process, such as photosynthesis. As a proof-of-concept approach, mesophyll cells from Arabidopsis thaliana were laser-microdissected from whole leaves to generate a focused cDNA library that was bi-directionally cloned into a transitive RNAi vector that had been designed to induce silencing of homologous, endogenous genes. Approximately 15% of the transformant plants identified from both sense and antisense libraries exhibited visible phenotypes indicative of photosynthetic defects. Amplification from the genome and sequencing of cDNA inserts identified candidate genes underlying the phenotypes. For 10 of 11 such mutants, re-transformation with an RNAi construct corresponding to the candidate gene recapitulated the original mutant phenotype, and reduction of corresponding endogene transcripts was confirmed. In addition, one of the re-transformed transgenes also silenced transcripts of closely related family members, thereby demonstrating the utility of this approach for mutagenesis of redundant gene functions. Preliminary results using tissue-specific transitive RNAi forward mutagenesis of the Arabidopsis vegetative shoot apical meristem demonstrate the broad applicability of this forward mutagenesis technique for a variety of plant cell types.

Blockade of angiotensin II synthesis attenuates hepatic fibrosis in different experimental models of chronic liver injury. We evaluated the safety and efficacy of moexipril, an angiotensin-converting enzyme inhibitor, in patients with primary biliary cirrhosis (PBC) who have had a suboptimal response to ursodeoxycholic acid (UDCA). Twenty PBC patients on UDCA (13-15 mg/kg/day) therapy with an elevation of serum alkaline phosphatase at least twice the upper limit of normal were treated with oral moexipril 15 mg/day for one year. No significant changes in serum alkaline phosphatase (379 +/- 32 vs. 379 +/- 51), bilirubin (0.8 +/- 0.1 vs. 0.9 +/- 0.1), aspartate aminotransferase (60 +/- 8 vs. 63 +/- 9), and Mayo risk score (3.55 +/- 0.2 vs. 3.62 +/- 0.2) was associated with the treatment. Fatigue and health-related quality of life scores during treatment demonstrated a trend toward improvement. Moexipril was not clinically beneficial to PBC patients responding suboptimally to UDCA.

Objectives: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of young adults that is associated with significant morbidity and mortality. No effective medical therapy is available. Minocycline has been found to exert biological effects independent of its antimicrobial properties, including anti-inflammatory activities such as inhibition of inducible nitric oxide synthase, upregulation of interleukin 10, and direct suppressive effect on B- and T-cell function. Minocycline may also inhibit cell death pathways by reducing both proapoptotic and proinflammatory enzyme activation. We sought to investigate the safety and efficacy of minocycline among patients with PSC., Methods: We evaluated the efficacy of minocycline in patients with PSC in a pilot study. Sixteen patients with PSC were enrolled. Minocycline, 100 mg orally twice daily, was given for 1 year., Results: A statistically significant improvement in serum alkaline phosphatase activity (330 U/l vs. 265 U/l, P=0.04) and Mayo risk score (0.55 vs. 0.02, P=0.05) occurred with treatment. Serum bilirubin and albumin remained essentially unchanged while on treatment., Conclusions: The results of this pilot study indicate that minocycline is reasonably well tolerated and potentially effective in patients with PSC. These findings might be explained by the anti-inflammatory and antiapoptotic properties of minocycline. Though the data presented are too preliminary to support the clinical use of minocycline in the treatment of PSC at this time, its use should be further investigated.

No effective medical therapy is available for patients with primary sclerosing cholangitis (PSC). We evaluated the safety and estimated the efficacy of silymarin in patients with PSC in a pilot study. Thirty patients with PSC were enrolled. Silymarin, 140 mg orally three times daily, was given for 1 year. A statistically significant improvement in serum alkaline phosphatase activity (1131 +/- 216 vs. 861 +/- 139, P = 0.007), and aspartate aminotransferase (AST) levels (116 +/- 15 vs. 83 +/- 11, P = 0.01) occurred with treatment. Serum bilirubin levels were not significantly affected by the treatment, while serum albumin and the Mayo risk score remained essentially unchanged. Overall, 34% of patients had a positive response to silymarin as defined by > or =50% improvement or normal status in liver tests. The results of this pilot study warrant further evaluation of silymarin in patients with PSC in a large-scale, controlled trial.

Background: Upstream open reading frames (uORFs) can mediate translational control over the largest, or major ORF (mORF) in response to starvation, polyamine concentrations, and sucrose concentrations. One plant uORF with conserved peptide sequences has been shown to exert this control in an amino acid sequence-dependent manner but generally it is not clear what kinds of genes are regulated, or how extensively this mechanism is invoked in a given genome., Results: By comparing full-length cDNA sequences from Arabidopsis and rice we identified 26 distinct homology groups of conserved peptide uORFs, only three of which have been reported previously. Pairwise Ka/Ks analysis showed that purifying selection had acted on nearly all conserved peptide uORFs and their associated mORFs. Functions of predicted mORF proteins could be inferred for 16 homology groups and many of these proteins appear to have a regulatory function, including 6 transcription factors, 5 signal transduction factors, 3 developmental signal molecules, a homolog of translation initiation factor eIF5, and a RING finger protein. Transcription factors are clearly overrepresented in this data set when compared to the frequency calculated for the entire genome (p = 1.2 x 10(-7)). Duplicate gene pairs arising from a whole genome duplication (ohnologs) with a conserved uORF are much more likely to have been retained in Arabidopsis (Arabidopsis thaliana) than are ohnologs of other genes (39% vs 14% of ancestral genes, p = 5 x 10(-3)). Two uORF groups were found in animals, indicating an ancient origin of these putative regulatory elements., Conclusion: Conservation of uORF amino acid sequence, association with homologous mORFs over long evolutionary time periods, preferential retention after whole genome duplications, and preferential association with mORFs coding for transcription factors suggest that the conserved peptide uORFs identified in this study are strong candidates for translational controllers of regulatory genes.

Sequence analysis of left and right border integration sites of independent, single-copy T-DNA inserts in Arabidopsis thaliana revealed three previously unrecognized concomitants of T-DNA integration. First, genomic pre-insertion sites shared sequence similarity not only with the T-DNA left and right border regions, as was previously reported, but also at high frequency with the inverted complement of the T-DNA right border region. Second, palindromic sequences were frequently found to overlap or lie adjacent to genomic target sites, suggesting a high recombinogenic potential for palindromic elements during T-DNA integration and a possible role during the primary contact between the T-DNA and the target DNA. Third, "filler" DNA sequences between genomic pre-insertion site DNA and T-DNA often derive from sequences in the T-DNA left and right border regions that are clustered around palindromic sequences in these T-DNA regions, suggesting that these palindromic elements are "hot spots" for filler DNA formation. The discovery of inverted sequence similarities at the right border suggests a previously unrecognized mode of T-DNA integration that involves heteroduplex formation at both T-DNA borders and with opposite strands of the target DNA. Scanning for sequence similarities in both direct and inverted orientation may increase the probability and/or effectiveness of anchoring the T-DNA to the target DNA. Variations on this scheme may also account for inversion events at the target site of T-DNA integration and inverted T-DNA repeat formation, common sequence organization patterns associated with T-DNA integration.

Background: Results from a pilot investigation with tacrolimus for primary sclerosing cholangitis (PSC) demonstrated biochemical improvement without excessive drug toxicity. To date, no confirmatory study has been performed., Aims: We sought to determine the safety and efficacy of tacrolimus in PSC., Methods: An open-label, phase II study of tacrolimus 0.05 mg/kg twice daily for 1 year was performed. Target whole-blood concentrations ranged between 3 and 7 ng/ml., Results: A total of 16 patients were enrolled. The median age was 50 years (range, 28-68), with 31% being women. The median serum alkaline phosphatase was 903 U/l, AST 88 U/l, total bilirubin 0.9 mg/dl, and albumin 3.8 g/dl. Based primarily on drug-related adverse events, only eight (50%) patients completed 1 year of therapy. After 1 year of therapy, however, significant improvements in median serum alkaline phosphatase (903 vs. 483, P=0.0001) and AST levels (88 vs. 78, P=0.002) were observed in these patients. The median tacrolimus level in patients completing 1 year of therapy was 4.0 ng/ml. Drug-related adverse events, however, were responsible for 31% of participants withdrawing from the study., Conclusions: Despite significant improvements in serum alkaline phosphatase, oral tacrolimus is poorly tolerated in patients with PSC.

Cucurbita moschata, a cucurbit species responsive to inductive short-day (SD) photoperiods, and Zucchini yellow mosaic virus (ZYMV) were used to test whether long-distance movement of FLOWERING LOCUS T (FT) mRNA or FT is required for floral induction. Ectopic expression of FT by ZYMV was highly effective in mediating floral induction of long-day (LD)-treated plants. Moreover, the infection zone of ZYMV was far removed from floral meristems, suggesting that FT transcripts do not function as the florigenic signal in this system. Heterografting demonstrated efficient transmission of a florigenic signal from flowering Cucurbita maxima stocks to LD-grown C. moschata scions. Real-time RT-PCR performed on phloem sap collected from C. maxima stocks detected no FT transcripts, whereas mass spectrometry of phloem sap proteins revealed the presence of Cm-FTL1 and Cm-FTL2. Importantly, studies on LD- and SD-treated C. moschata plants established that Cmo-FTL1 and Cmo-FTL2 are regulated by photoperiod at the level of movement into the phloem and not by transcription. Finally, mass spectrometry of florally induced heterografted C. moschata scions revealed that C. maxima FT, but not FT mRNA, crossed the graft union in the phloem translocation stream. Collectively, these studies are consistent with FT functioning as a component of the florigenic signaling system in the cucurbits.

Fatigue is a common symptom in primary biliary cirrhosis (PBC). In animal models of cholestasis, abnormalities in serotonin neurotransmission are observed with fatigue. The role of selective serotonin reuptake inhibitors in fatigue-related PBC, however, is unknown. A double-blind, placebo-controlled study design was conducted to determine the safety and efficacy of fluoxetine for the treatment of fatigue in PBC. Patients were randomized to fluoxetine, 20 mg daily, or matched placebo for 8 weeks' duration. Fatigue was assessed by the Fisk Fatigue Impact Scale (FFIS). The primary study endpoint was a > or =50% reduction in overall FFIS score at the end of treatment. Health-related quality of life (HRQL) was assessed as a secondary endpoint. Among 220 consecutively screened patients, only 18 (9%) eligible individuals were randomized to fluoxetine (n=10) or placebo (n=8) for 8 weeks. Baseline variables including median FFIS scores (52 vs 42; P=0.21) were similar between treatment arms (P > 0.05). After 8 weeks of therapy, no statistically significant change in median FFIS score was observed in the fluoxetine group. Median FFIS score in the placebo group was reduced (42 to 28), but not statistically significant. No difference in HRQL was observed between treatment arms after 8 weeks. Fourteen (78%) patients completed therapy, while four (22%) individuals withdrew from the trial. Three of the four patients had drug-related adverse events with fluoxetine. In this study, fluoxetine did not improve fatigue in PBC and was associated with adverse events.

Plasmodesmata are intercellular organelles in plants that allow the passage of molecules between plant cells. Movement through plasmodesmata may allow transcription factors expressed in one cell to move into adjacent cells, thereby regulating gene expression non-cell autonomously. The two animations illustrate (i) movement of a protein through an individual plasmodesma and (ii) an experiment to detect the movement of the transcription factor through plasmodesmata from the L1 layer of a plant meristem into the L2 and L3 layers. These two animations would be useful in teaching plant biology or plant development or a cell biology class discussing mechanisms of intercellular transport.

We describe features of RNA silencing and associated epigenetic imprints that illustrate potential roles for RNA interference (RNAi) in maintenance and transmission of epigenetic states between cells, throughout a plant, and perhaps even across sexual generations. Three types of transgenes can trigger RNAi of homologous endogenous plant genes: (1) "sense" transgenes that overexpress translatable transcripts, (2) inverted repeat (IR) transgenes that produce double-stranded RNA (dsRNA), and (3) antisense transgenes. Each mode of RNAi produces a different characteristic developmental silencing pattern. Single-copy transgenes are sufficient for sense-RNAi and antisense-RNAi, but not inverted repeat-RNAi. A single premature termination codon dramatically attenuates sense-RNAi, but it has no effect on antisense or inverted repeat-RNAi. We report here that antisense transgenes altered by removal of nonsense codons generate silencing patterns characteristic of sense-RNAi. Duplication of a sense overexpression transgene results in two types of epigenetic events: (1) complete loss of silencing and (2) altered developmental pattern of silencing. We also report that duplicating only the transgene promoter results in complete loss of silencing, whereas duplicating only transcribed sequences produces the second class, which are vein-based patterns. We infer that the latter class is due to systemic RNA silencing signals that interact with certain epigenetic states of the transgene to imprint it with information generated at a distance elsewhere in the plant.

Summary: Errors are prevalent in cDNA sequences but the extent to which sequence collections differ in frequencies and types of errors has not been investigated systematically. cDNA quality control, or cQC, was developed to evaluate the quality of cDNA sequence collections and to revise those sequences that differ from a higher quality genomic sequence. After removing rRNA, vector, bacterial insertion sequence and chimeric cDNA contaminants, small-scale nucleotide discrepancies were found in 51% of cDNA sequences from one Arabidopsis cDNA collection, 89% from a second Arabidopsis collection and 75% from a rice collection. These errors created premature termination codons in 4 and 42% of cDNA sequences in the respective Arabidopsis collections and in 7% of the rice cDNA sequences.

Bone loss is a well-recognized complication of primary biliary cirrhosis (PBC). Although it has been suggested that alendronate might improve bone mineral density (BMD) in PBC, no randomized placebo-controlled trial has been conducted. The primary aim of this study was to compare the effects of alendronate versus placebo on BMD and biochemical measurements of bone turnover in patients with PBC-associated bone loss. We conducted a double-blinded, randomized, placebo-controlled trial. Patients with a PBC and BMD t score of less than -1.5 were randomized to receive 70 mg per week of alendronate or placebo over 1 year. BMD of the lumbar spine and proximal femur were measured at entry and at 1 year. Changes from baseline in BMD and biochemical measurements of bone turnover were assessed. Thirty-four patients were enrolled. Seventeen patients were randomized to each arm. After 1 year, a significantly larger improvement (P = .005) in spine BMD was observed in the alendronate group (0.09 +/- 0.03 g/cm2 SD from baseline) compared with the placebo group (-0.003 +/- 0.02 g/cm2 SD from baseline). A larger improvement (P = .046) was also observed in the femoral BMD of alendronate patients versus placebo. BMD changes were independent of concomitant estrogen therapy. The rate of adverse effects was similar in both groups. In conclusion, in patients with PBC-related bone loss, alendronate significantly improves BMD compared with placebo. Although in this study oral alendronate appears to be well tolerated in patients with PBC, larger studies are needed to formally evaluate safety.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown etiology. Despite advances in understanding the pathophysiology underlying this disorder, no effective medical therapy has been identified for halting disease progression. The aim of this investigation was to determine the safety and estimated efficacy of mycophenolate mofetil (MMF) for the treatment of PSC. Thirty patients with PSC received MMF 1 g daily to a maximum of 3 g daily for 1 yr. Liver tests were determined at 3-month intervals with the Mayo risk score calculated at baseline and at the end of therapy. Twenty-three (77%) patients completed 1 yr of therapy. Significant but clinically marginal improvement in serum alkaline phosphatase level after 1 yr of therapy was observed (1135 +/- 581 U/L vs 912 +/- 463 U/L, p= 0.02). No other significant changes in liver biochemistries or Mayo risk score was observed. Seven patients (23%) discontinued MMF due to adverse events possibly related to therapy. Adverse reactions resolved spontaneously or with dose reduction in 10 (33%) patients. One patient developed pancreatitis, bacterial cholangitis, and sepsis during the eighth month of MMF therapy. No patient developed cytopenia on therapy. In conclusion, MMF does not appear to have clinically important benefits for PSC despite being tolerated by most patients. The results of this pilot study do not support further study of MMF as a single agent in the treatment of PSC.

Background & Aims: Despite evidence for therapeutic efficacy with ursodeoxycholic acid (UDCA) in primary biliary cirrhosis (PBC), only 30-50% of patients achieve complete biochemical remission within 1 year of therapy. Mycophenolate mofetil (MMF) is an immunosuppressive medication that inhibits T and B lymphocyte proliferation. The aim of this investigation was to determine the safety and estimated efficacy of MMF in patients with PBC., Methods: Twenty-five patients with incomplete responses to UDCA (defined as persistent elevation of serum alkaline phosphatase > or =2 times the upper limit of normal) received MMF 1 g daily to a maximum of 3 g daily with UDCA (13-15 mg/kg per day) for 1 year. Liver biochemistries were determined at 3-month intervals with Mayo Risk Score calculated at baseline and end of therapy., Results: Nineteen (76%) patients completed 1 year of therapy. Despite improvements in serum alkaline phosphatase (920 +/- 308 vs. 709 +/- 242 IU/L, P = 0.001) and AST (65 +/- 31 vs. 51 +/- 19 IU/L, P = 0.007) levels, these findings were clinically insignificant. Exploratory analysis revealed a strong correlation between advanced PBC defined by higher Mayo Risk Score and reduction in serum alkaline phosphatase levels (r = -0.74, P = 0.006). Six patients (24%) did not complete therapy; adverse drug events were responsible for study withdrawal in 3 individuals. Adverse reactions that resolved spontaneously or by dose reduction occurred in 13 patients., Conclusions: MMF is not associated with important clinical benefits in PBC based on the results of this pilot investigation.

RNA interference (RNAi) can be used to study gene function by effecting degradation of the targeted transcript. However, the effectiveness of transgene-induced RNAi among multiple target genes has not been compared systematically. To this end, we developed a relative quantitative RT-PCR protocol that allows use of a single internal standard over a wide range of target gene expression levels. Using this method in an analysis of transgenic Arabidopsis thaliana RNAi lines targeting 25 different endogenes revealed that independent, homozygous, single-copy (sc) T4 lines targeting the same gene generally reduce transcript levels to the same extent, whereas multi-copy RNAi lines differed in the degree of target reduction and never exceeded the effect of sc transgenes. The maximal reduction of target transcript levels varied among targets. These observations suggest that each target sequence possesses an inherent degree of susceptibility to dsRNA-mediated degradation.

Nonalcoholic fatty liver disease is a condition gaining increasing recognition as a cause of cirrhosis and end-stage liver disease. The condition appears identical to alcoholic liver disease histologically, yet occurs in patients with negligible alcohol intake. Nonalcoholic fatty liver disease covers a spectrum of diseases ranging from simple fatty deposition in the liver to fat and inflammation and finally to fibrosis and cirrhosis. Conditions most frequently found in association with nonalcoholic fatty liver disease include obesity, Type 2 diabetes, and hyperlipidemia. Although the exact etiology of nonalcoholic fatty liver disease is not clear, insulin resistance is thought to play an important factor. Patients typically present with asymptomatic serum aminotransferase elevations of 2-3 times normal. Symptoms may include fatigue and abdominal pain. The clinical course is difficult to predict due to a lack of research in the natural history of the disease. It is known a percentage of patients progress to end-stage liver disease and may require liver transplantation. No medical treatment has been found to be totally effective. Patients who are overweight or obese should be encouraged in gradual weight reduction that has been associated with improvement in liver test abnormalities.

The natural history of pruritus in primary biliary cirrhosis (PBC) remains poorly defined. The aim of this investigation was to evaluate outcomes of pruritus in clinical trials for ursodeoxycholic acid (UDCA). In a UDCA-placebo trial begun in 1988 (n = 180), a 55% prevalence rate for pruritus was observed. Serum alkaline phosphatase level and Mayo risk score were independent risk factors for pruritus (P < 0.0001). Among placebo-treated patients (n = 91), the annual risks for development or improvement/resolution of pruritus were 27% and 23%, respectively. For UDCA-treated patients (n = 89), a trend toward improvement in pruritus was observed after 1 year compared to placebo (30% vs. 23%, P = 0.08) but not at 2 or 3 years. In a 3-dose UDCA trial begun in 1995 (n = 155), the overall prevalence of pruritus was significantly lower at 37% when compared to UDCA-placebo participants (P < 0.001). Baseline serum alkaline phosphatase level and Mayo risk score were again independent risk factors for pruritus (P < 0.0001). Among 13 (3.9%) patients with refractory pruritus, symptom resolution (n = 5) or improvement (n = 8) was associated with the use of oral rifampin (300 or 600 mg daily). Two patients treated with rifampin developed biochemical evidence for hepatotoxicity necessitating drug withdrawal. Although less prevalent among recently diagnosed individuals, more than one third of PBC patients develop pruritus. No significant risk reduction in developing pruritus with UDCA therapy was observed compared to placebo-treated patients. The long-term administration of rifampin for refractory pruritus is associated with occasional hepatotoxicity.

Sequence similarity and profile searching tools were used to analyze the genome sequences of Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster for genes encoding three families of histone deacetylase (HDAC) proteins and three families of histone acetyltransferase (HAT) proteins. Plants, animals and fungi were found to have a single member of each of three subfamilies of the GNAT family of HATs, suggesting conservation of these functions. However, major differences were found with respect to sizes of gene families and multi-domain protein structures within other families of HATs and HDACs, indicating substantial evolutionary diversification. Phylogenetic analysis identified a new class of HDACs within the RPD3/HDA1 family that is represented only in plants and animals. A similar analysis of the plant-specific HD2 family of HDACs suggests a duplication event early in dicot evolution, followed by further diversification in the lineage leading to Arabidopsis. Of three major classes of SIR2-type HDACs that are found in animals, fungi have representatives only in one class, whereas plants have representatives only in the other two. Plants possess five CREB-binding protein (CBP)-type HATs compared with one to two in animals and none in fungi. Domain and phylogenetic analyses of the CBP family proteins showed that this family has evolved three distinct types of CBPs in plants. The domain architecture of CBP and TAF(II)250 families of HATs show significant differences between plants and animals, most notably with respect to bromodomain occurrence and their number. Bromodomain-containing proteins in Arabidopsis differ strikingly from animal bromodomain proteins with respect to the numbers of bromodomains and the other types of domains that are present. The substantial diversification of HATs and HDACs that has occurred since the divergence of plants, animals and fungi suggests a surprising degree of evolutionary plasticity and functional diversification in these core chromatin components.

Our aim was to evaluate the safety and assess the efficacy of pirfenidone, an antifibrotic drug, in patients with primary sclerosing cholangitis (PSC). Twenty-four patients with PSC were enrolled in this pilot study. Oral pirfenidone, 2400 mg daily, was given for one year. Liver biochemistries were determined at three-month intervals. The Mayo risk score was calculated, and liver biopsy and endoscopic cholangiography were performed at entry and at one year of treatment. No significant changes in liver biochemistries were noted at the end of the treatment period or at any of the three-month intervals. The Mayo risk score did not change significantly, and no significant changes were noted in the degree of inflammation, fibrosis, histologic stage of disease, or cholangiographic findings at the end of the treatment period. Adverse events occurred in 20/24 (83%) patients, but disappeared shortly after pirfenidone was discontinued. Pirfenidone did not benefit patients with PSC, and it was frequently associated with adverse events. The results of this pilot study discourage further trials of pirfenidone in patients with PSC.

Transgenes designed to overexpress anthocyanin genes An6 (encoding dihydroflavonol-4-reductase) or Hf1 (encoding flavonoid-3',5'-hydroxylase) in Petunia hybrida L. produced flower colour phenotypes similar to those caused by sense cosuppression of chalcone synthase (Chs) genes. However, unlike Chs, sense cosuppression of An6 and Hf1 resulted in female infertility in transgenotes exhibiting complete phenotypic suppression of anthocyanins. Female sterility appeared to be due to embryo abortion, with discolouration of ovules first appearing about 4 d post-fertilization, followed by gradual collapse of the ovule. Pollen from cosuppressed, female-sterile transgenotes placed on wild-type stigmas produced normal seed set, indicating that sterility of cosuppressed plants was maternally controlled. We suggest an hypothesis that cosuppression of An6 and Hf1 leads to accumulation of dihydroflavonols in the seed coat, a maternal tissue, and that this accumulation inhibits embryo growth, either directly or indirectly. In this hypothesis, direct inhibition of embryo growth would require that dihydroflavonols diffuse from the seed coat into the embryo and act there, whereas indirect inhibition would require that dihydroflavonols interfere with some capacity of the seed coat to promote embryo growth.

Objective: The aim of this study was to assess the safety and efficacy of high-dose ursodeoxycholic acid (UDCA, 28-32 mg/kg/day) in patients with primary biliary cirrhosis (PBC) who had shown an incomplete response to the standard dose (13-15 mg/kg/day)., Methods: A total of 25 patients with PBC who had been on UDCA (13-15 mg/kg/day) therapy for 24-141 months and had shown persistent elevation of ALP activity at least two times the upper limit of normal were enrolled. The dose of UDCA was increased to 30 (28-32) mg/kg/day and given for 1 yr., Results: A significant but marginal improvement in serum ALP activity (707+/-52 vs 571+/-32, p = 0.001) was noted at 1 yr of treatment with high-dose UDCA. However, levels of total bilirubin (1.1+/-0.2 vs 1.0+/-0.2, p = 0.1), AST (58+/-9 vs 54+/-1, p = 0.1), albumin (4.1+/-0.7 vs 4.0+/-0.08, p = 0.1), or Mayo risk score (4.13+/-0.3 vs 4.12+/-0.3, p = 0.2) remained essentially unchanged. Normalization of liver tests did not occur in any patient, and adverse events were not recorded in any case., Conclusions: Although UDCA at a dose of 28-32 mg/kg/day is well tolerated, this dosage does not seem to benefit most patients with PBC responding incompletely to a dose of 13-15 mg/kg/day. The results of this pilot study would seem to discourage further controlled trials of high-dose UDCA in suboptimal responders to the standard dose of UDCA.

Objectives: No effective therapy currently exists for patients with nonalcoholic steatohepatitis (NASH). Betaine, a naturally occurring metabolite of choline, has been shown to raise S-adenosylmethionine (SAM) levels that may in turn play a role in decreasing hepatic steatosis. Our aim was to determine the safety and effects of betaine on liver biochemistries and histological markers of disease activity in patients with NASH., Methods: Ten adult patients with NASH were enrolled. Patients received betaine anhydrous for oral solution (Cystadane) in two divided doses daily for 12 months. Seven out of 10 patients completed 1 yr of treatment with betaine., Results: A significant improvement in serum levels of aspartate aminotransferase (p = 0.02) and ALAT (p = 0.007) occurred during treatment. Aminotransferases normalized in three of seven patients, decreased by >50% in three of seven patients, and remained unchanged in one patient when compared to baseline values. A marked improvement in serum levels of aminotransferases (ALT -39%; AST -38%) also occurred during treatment in those patients who did not complete 1 yr of treatment. Similarly, a marked improvement in the degree of steatosis, necroinflammatory grade, and stage of fibrosis was noted at 1 yr of treatment with betaine. Transitory GI adverse events that did not require any dose reduction or discontinuation of betaine occurred in four patients., Conclusions: Betaine is a safe and well tolerated drug that leads to a significant biochemical and histological improvement in patients with NASH. This novel agent deserves further evaluation in a randomized, placebo-controlled trial.

Objectives: To assess the tolerability and efficacy of high-dose (25-30 mg/kg per day) ursodeoxycholic acid (UDCA) in patients with primary sclerosing cholangitis (PSC)., Methods: Thirty patients with PSC were enrolled in this pilot study and treated for 1 yr. Changes in the Mayo risk score at 1 yr of treatment and projected survival at 4 yr were compared with that observed in patients randomized to placebo (n = 52) or UDCA (n = 53) at a dose of 13-15 mg/kg per day., Results: A marked improvement in serum alkaline phosphatase activity (1265+/-172 vs 693+/-110 U/L, p < 0.001), AST (161+/-037 vs 77+/-13 U/L, p = 0.001), albumin (4.0+/-0.1 vs 4.2+/-0.1 g/dl, p = 0.03), and total bilirubin (1.6+/-0.3 vs 1.3+/-0.2 mg/dl, p = 0.1) occurred at 1 yr of therapy with high-dose UDCA. Changes in the Mayo risk score after 1 yr of treatment were significantly different among the three groups (p < 0.001), and these changes would be translated into a significantly different expected survival at 4 yr (p = 0.05). This expected survival at 4 yr was significantly different between placebo and the dose of 25-30 mg/kg per day (p = 0.04), but not between placebo and the dose of 13-15 mg/kg per day (p = 0.4). High-dose UDCA was well tolerated., Conclusions: UDCA at a dose of 25-30 mg/kg per day may be of benefit for patients with PSC, and this regimen deserves further evaluation in a long-term, randomized, placebo-controlled trial.

Background/aim: Osteoporosis is a common complication of primary biliary cirrhosis but there is no accepted therapy for the osteoporosis. In this randomized controlled trial, we compared the effects of etidronate to placebo on the treatment of osteoporosis associated with primary biliary cirrhosis., Methods: Sixty-seven patients with primary biliary cirrhosis and osteopenia, defined by bone mineral density criteria (T-score < -2.0) were enrolled. Measurements of the lumbar spine and proximal femur, as well as x-rays of the lumbar spine, were obtained. Patients received cyclical etidronate 400 mg/day for 14 days every 3 months for at least 1 year. Supplemental calcium was administered on the days patients did not receive etidronate., Results: Of the 67 patients entered, 60 completed at least 1 year of therapy. There was no significant difference in changes in bone density at either the lumbar spine or femur in patients receiving etidronate when compared to placebo. Fractures occurred in eight patients, four receiving etidronate. Etidronate therapy was associated with a significant reduction in markers of bone turnover compared to placebo. These changes did not correlate with changes in bone density., Conclusions: Cyclical etidronate administered with supplemental calcium did not significantly improve bone density in patients with primary biliary cirrhosis.

Cell fate in plants is predominantly determined by position during development. Jorgensen discusses a new mechanism by which cells can regulate gene expression in neighboring cells. Several lines of evidence suggest that transcription factors expressed in one cell can traffic through plasmodesmata to regulate gene expression in adjacent cells that are not expressing the transcription factor themselves. In plants, some transcription factors appear to be intercellular signaling molecules, as well as intracellular regulators of gene expression.

Ursodeoxycholic acid (UDCA) is a safe and effective medical therapy for most patients with primary biliary cirrhosis (PBC), but some patients show an incomplete response. Silymarin is a potent antioxidant with immunomodulatory and antifibrotic properties. The aim of this study was to evaluate the safety and assess the efficacy of silymarin in patients with PBC who had shown a suboptimal response to UDCA. Twenty-seven patients with PBC who had been on UDCA (13-15 mg/kg/day) therapy for 7 to 221 months and had shown a persistent elevation of alkaline phosphatase activity at least 2 times the upper limit of normal for more than 6 months were enrolled. Oral silymarin, 140 mg 3 times daily was given for 1 year, and patients continued on the same dosage of UDCA. No significant changes in serum alkaline phosphatase activity (897 +/- 84 vs. 876 +/- 95, P =.5), total bilirubin (0.9 +/- 0.1 vs. 1 +/- 0.1, P =.07), aspartate transaminase (AST) (58 +/- 5 vs. 56 +/- 6, P =.4), albumin (4.0 +/-.06 vs. 4.1 +/-.06, P =.4), or Mayo risk score (3.82 +/- 0.2 vs. 3.88 +/- 0.2, P =.4) were noted after 1 year of treatment with combination therapy. Transitory gastrointestinal adverse events occurred in 2 patients. In conclusion, although silymarin was well tolerated, this medication did not provide benefit to patients with PBC responding suboptimally to UDCA. The results of this pilot study would seem to discourage further controlled trials of silymarin in patients with PBC.

Objective: This study was designed to evaluate the safety and estimate the efficacy of oral budesonide in patients with primary sclerosing cholangitis (PSC)., Methods: Twenty-one patients with PSC were treated with 9 mg daily of oral budesonide for 1 yr., Results: Significant, but marginally important, improvement in serum alkaline phosphatase (1,235 +/- 190 vs 951 +/-206 U/L, p = 0.003) and AST levels (119 +/- 14 vs 103 +/- 19 U/L, p = 0.02) was noted at the end of the treatment period. Serum bilirubin levels increased significantly in the 18 patients who completed 1 yr of treatment (1.1 +/- 0.1 vs 1.4 +/- 0.3, p = 0.01) and no significant changes in liver tests were noted 3 months after budesonide was discontinued. The Mayo risk score did not change significantly, and although a significant improvement in the degree of portal inflammation was noted at the end of the treatment period, the degree of fibrosis and stage of disease were not significantly affected. There was a marked loss of bone mass of the femoral neck (0.851 +/- 0.02 vs 0.826 +/- 0.02 g/cm2, p = 0.002) and lumbar spine (1.042 +/- 0.02 vs 1.029 +/- 0.02 g/cm2, p = 0.09) at 1 yr of treatment with budesonide. Two patients required evaluation for liver transplantation during treatment, and two patients developed cosmetic side effects., Conclusions: Oral budesonide appears to be of minimal, if any, benefit and it is associated with a significant worsening of osteoporosis in patients with PSC.