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Background The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. Results A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 uM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. Conclusions The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6% - 28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.
Summary The basal transcription apparatus of archaea is well characterized. However, much less is known about the mechanisms of transcription termination and translation initation. Recently, experimental determination of the 5´-ends of ten transcripts from Pyrobaculum aerophilum revealed that these are devoid of a 5´-UTR. Bioinformatic analysis indicated that many transcripts of other archaeal species might also be leaderless. The´-ends and 3´-ends of 40 transcripts of two haloarchaeal species, Halobacterium salinarum and Haloferax volcanii, have been determined. They were used to characterize the lengths of 5´-UTRs and 3´-UTRs and to deduce consensus sequence-elements for transcription and translation. The experimental approach was complemented with a bioinformatics analysis of the H. salinarum genome sequence. Furthermore, the influence of selected 5´-UTRs and 3´-UTRs on transcript stability and translational efficiency in vivo was characterized using a newly established reporter gene system, gene fusions, and real-time PCR. Consensus sequences for basal promoter elements could be refined and a novel element was discovered. A consensus motif probably important for transcriptional termination was established. All 40 haloarchaeal transcripts analyzed had a 3´-UTR (average size 57 nt), and their 3´-ends were not posttranscriptionally modified. Experimental data and genome analyses revealed that the majority of haloarchaeal transcripts are leaderless, indicating that this is the predominant mode for translation initiation in haloarchaea. Surprisingly, the 5´-UTRs of most leadered transcripts did not contain a Shine-Dalgarno (SD) sequence. A genome analysis indicated that less than 10% of all genes are preceded by a SD sequence and even most proximal genes in operons lack a SD sequence. Seven different leadered transcripts devoid of a SD sequence were efficiently translated in vivo, including artificial 5´-UTRs of random sequences. Thus, an interaction of the 5´-UTRs of these leadered transcripts with the 16S rRNA could be excluded. Taken together, either a scanning mechanism similar to the mechanism of translation initiation operating in eukaryotes or a novel mechanism must operate on most leadered haloarchaeal transcripts. Author Summary Expression of the information encoded in the genome of an organism into its phenotype involves transcription of the DNA into messenger RNAs and translation of mRNAs into proteins. The textbook view is that an mRNA consists of an untranslated region (5´-UTR), an open reading frame encoding the protein, and another untranslated region (3´-UTR). We have determined the 5´-ends and the 3´-ends of 40 mRNAs of two haloarchaeal species and used this dataset to gain information about nucleotide elements important for transcription and translation. Two thirds of the mRNAs were devoid of a 5´-UTR, and therefore the major pathway for translation initiation in haloarchaea involves so-called leaderless transcripts. Very unexpectedly, most leadered mRNAs were found to be devoid of a sequence motif believed to be essential for translation initiation in bacteria and archaea (Shine-Dalgarno sequence). A bioinformatic genome analysis revealed that less than 10% of the genes contain a Shine-Dalgarno sequence. mRNAs lacking this motif were efficiently translated in vivo, including mRNAs with artificial 5´-UTRs of total random sequence. Thus, translation initiation on these mRNAs either involves a scanning mechanism similar to the mechanism operating in eukaryotes or a totally novel mechanism operating at least in haloarchaea.
Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168. To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.
Background and aims: Spontaneous bacterial peritonitis (SBP) is a severe complication of decompensated cirrhosis. The prevalence of multidrug-resistant organisms (MDROs) in patients with cirrhosis is increasing. Identification of patients at risk for SBP due to MDROs (ie, SBP with the evidence of MDROs or Stenotrophomonas maltophilia in ascitic culture, MDRO-SBP) is crucial to the early adaptation of antibiotic treatment in such patients. We therefore investigated whether MDROs found in ascitic cultures can also be found in specimens determined by noninvasive screening procedures.
Patients and methods: This retrospective study was conducted at the liver center of the University Hospital Frankfurt, Germany. Between 2011 and 2016, patients with cirrhosis were included upon diagnosis of SBP and sample collection of aerobic/anaerobic ascitic cultures. Furthermore, the performance of at least one complete MDRO screening was mandatory for study inclusion.
Results: Of 133 patients diagnosed with SBP, 75 (56.4%) had culture-positive SBP and 22 (16.5%) had MDRO-SBP. Multidrug-resistant Escherichia coli (10/22; 45.5%) and vancomycin-resistant enterococci (7/22; 36.4%) resembled the major causatives of MDRO-SBP. Rectal swabs identified MDROs in 17 of 22 patients (77.3%) who developed MDRO-SBP with a time-dependent sensitivity of 77% and 87% after 30 and 90 days upon testing, while negative predictive value was 83% and 76%, respectively. The majority of patients were included from intensive care unit or intermediate care unit.
Conclusion: MDRO screening may serve as a noninvasive diagnostic tool to identify patients at risk for MDRO-SBP. Patients with decompensated cirrhosis should be screened for MDROs from the first day of inpatient treatment onward.
Background: Different parameters have been determined for prediction of treatment outcome in hepatitis c virus genotype 1 infected patients undergoing pegylated interferon, ribavirin combination therapy. Results on the importance of vitamin D levels are conflicting. In the present study, a comprehensive analysis of vitamin D levels before and during therapy together with single nucleotide polymorphisms involved in vitamin D metabolism in the context of other known treatment predictors has been performed.
Methods: In a well characterized prospective cohort of 398 genotype 1 infected patients treated with pegylated interferon-α and ribavirin for 24–72 weeks (INDIV-2 study) 25-OH-vitamin D levels and different single nucleotide polymorphisms were analyzed together with known biochemical parameters for a correlation with virologic treatment outcome.
Results: Fluctuations of more than 5 (10) ng/ml in 25-OH-vitamin D-levels have been observed in 66 (39) % of patients during the course of antiviral therapy and neither pretreatment nor under treatment 25-OH-vitamin D-levels were associated with treatment outcome. The DHCR7-TT-polymorphism within the 7-dehydrocholesterol-reductase showed a significant association (P = 0.031) to sustained viral response in univariate analysis. Among numerous further parameters analyzed we found that age (OR = 1.028, CI = 1.002–1.056, P = 0.035), cholesterol (OR = 0.983, CI = 0.975–0.991, P<0.001), ferritin (OR = 1.002, CI = 1.000–1.004, P = 0.033), gGT (OR = 1.467, CI = 1.073–2.006, P = 0.016) and IL28B-genotype (OR = 2.442, CI = 1.271–4.695, P = 0.007) constituted the strongest predictors of treatment response.
Conclusions: While 25-OH-vitamin D-levels levels show considerable variations during the long-lasting course of antiviral therapy they do not show any significant association to treatment outcome in genotype 1 infected patients.
Background: Vitamin D is required to maintain the integrity of the intestinal barrier and inhibits inflammatory signaling pathways.
Objective: Vitamin D deficiency might be involved in cirrhosis-associated systemic inflammation and risk of hepatic decompensation in patients with liver cirrhosis.
Methods: Outpatients of the Hepatology Unit of the University Hospital Frankfurt with advanced liver fibrosis and cirrhosis were prospectively enrolled. 25-hydroxyvitamin D (25(OH)D3) serum concentrations were quantified and associated with markers of systemic inflammation / intestinal bacterial translocation and hepatic decompensation.
Results: A total of 338 patients with advanced liver fibrosis or cirrhosis were included. Of those, 51 patients (15%) were hospitalized due to hepatic decompensation during follow-up. Overall, 72 patients (21%) had severe vitamin D deficiency. However, patients receiving vitamin D supplements had significantly higher 25(OH)D3 serum levels compared to patients without supplements (37 ng/mL vs. 16 ng/ml, P<0.0001). Uni- and multivariate analyses revealed an independent association of severe vitamin D deficiency with the risk of hepatic decompensation during follow-up (multivariate P = 0.012; OR = 3.25, 95% CI = 1.30–8.2), together with MELD score, low hemoglobin concentration, low coffee consumption, and presence of diabetes. Of note, serum levels of C-reactive protein, IL-6 and soluble CD14 were significantly higher in patients with versus without severe vitamin D deficiency, and serum levels of soluble CD14 levels declined in patients with de novo supplementation of vitamin D (median 2.15 vs. 1.87 ng/mL, P = 0.002).
Conclusions: In this prospective cohort study, baseline vitamin D levels were inversely associated with liver-cirrhosis related systemic inflammation and the risk of hepatic decompensation.
Background: To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C.
Methodology/Principal Findings: Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D3 (25[OH]D3) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D3<20 ng/mL) during all seasons, but 25(OH)D3 serum levels were not associated with treatment outcome.
Conclusions/Significance: Our study suggests a role of bioactive vitamin D (1,25[OH]2D3, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D3 is not a suitable predictor of treatment outcome.
Opportunities to treat infection with hepatitis C virus (HCV) are evolving rapidly. From the introduction of interferon-α monotherapy in 1992 to the approval of telaprevir- and boceprevir-based triple therapies with pegylated interferon-α and ribavirin in 2011, the chances of curing patients infected with HCV genotype 1 have improved from <10% to approximately 70%. Significant further improvements are on the horizon, which may well cure virtually all hepatitis C patients with an all-oral, interferon-free regimen in the very near future. These exciting developments are reviewed in the present article.
It is long known that Kasugamycin inhibits translation of canonical transcripts containing a 5’-UTR with a Shine Dalgarno (SD) motif, but not that of leaderless transcripts. To gain a global overview of the influence of Kasugamycin on translation efficiencies, the changes of the translatome of Escherichia coli induced by a 10 minutes Kasugamycin treatment were quantified. The effect of Kasugamycin differed widely, 102 transcripts were at least twofold more sensitive to Kasugamycin than average, and 137 transcripts were at least twofold more resistant, and there was a more than 100-fold difference between the most resistant and the most sensitive transcript. The 5’-ends of 19 transcripts were determined from treated and untreated cultures, but Kasugamycin resistance did neither correlate with the presence or absence of a SD motif, nor with differences in 5’-UTR lengths or GC content. RNA Structure Logos were generated for the 102 Kasugamycin-sensitive and for the 137 resistant transcripts. For both groups a short Shine Dalgarno (SD) motif was retrieved, but no specific motifs associated with resistance or sensitivity could be found. Notably, this was also true for the region -3 to -1 upstream of the start codon and the presence of an extended SD motif, which had been proposed to result in Kasugamycin resistance. Comparison of the translatome results with the database RegulonDB showed that the transcript with the highest resistance was leaderless, but no further leaderless transcripts were among the resistant transcripts. Unexpectedly, it was found that translational coupling might be a novel feature that is associated with Kasugamycin resistance. Taken together, Kasugamycin has a profound effect on translational efficiencies of E. coli transcripts, but the mechanism of action is different than previously described.