Chronic opioid use is associated with altered gut microbiota and predicts readmissions in patients with cirrhosis.

BACKGROUND: Opioid use is epidemic in cirrhosis, which could precipitate hepatic encephalopathy (HE) potentially through gut dysbiosis and inflammation. AIM: To define the effect of opioids on readmissions and on gut microbiota composition and functionality. METHODS: Cohort 1 had 200 cirrhotic in-patients (with/without opioid use) followed prospectively through the index hospitalisation and 6 months post discharge. Readmissions (HE-related/unrelated) were compared between patients discharged on opioids compared to the rest, including using a multi-variable analysis. Cohort 2 consisted of 72 cirrhotics on chronic opioids who were age/model for end-stage liver disease (MELD) and prior HE-balanced with 72 cirrhotics not on opioids. Stool microbiota composition (multi-tagged sequencing), predicted functionality (PiCRUST), endotoxemia and systemic inflammation (IL-6, IL-17) were compared. RESULTS: Cohort 1: Chronic opioid use was statistically similar between those admitted with/without HE, and was judged to be an HE precipitant in <5% of cases during the index hospitalisation. Of the 144 patients alive at 6 months, 82 were readmitted. The opioid users had a significantly higher all cause (69% vs. 48%, P = 0.008), but not HE-related readmissions (30% vs. 41%, P = 0.30). On regression, opioid therapy and female gender were predictive of readmission independent of MELD score and previous HE. Cohort 2: Significant dysbiosis was noted in the opioid cohort, especially in HE+opioid patients with lower autochthonous taxa and Bacteroidaceae relative abundance. PiCRUST showed highest aromatic amino acid and endotoxin production in opioid users. Opioid users also had higher endotoxemia and IL-6 but not IL-17. CONCLUSION: Chronic opioid use in cirrhosis is associated with increased endotoxemia, dysbiosis and all-cause readmissions.

HCV eradication does not impact gut dysbiosis or systemic inflammation in cirrhotic patients.

BACKGROUND: Eradication of hepatitis C virus (HCV) is increasing but its residual impact on the pro-inflammatory milieu in cirrhosis, which is associated with gut dysbiosis, is unclear. AIM: To define the impact of sustained virological response (SVR) on gut dysbiosis and systemic inflammation in HCV cirrhosis patients. METHODS: Cirrhotic out-patients with HCV with/without SVR (achieved >1 year prior) and age-matched healthy controls underwent serum and stool collection. Serum was analysed for IL-6, TNF-α and endotoxin while stool microbiota analysis was performed using multitagged pyrosequencing. Microbial comparisons were made using UNIFRAC and cirrhosis dysbiosis ratio (lower score indicates dysbiosis). Comparisons were performed between cirrhotics with/without SVR and controls vs. cirrhotic patients. RESULTS: A total of 105 HCV cirrhotics and 45 age-matched healthy controls were enrolled. Twenty-one patients had achieved SVR using pegylated interferon + ribavrin a median of 15 months prior. No significant differences on demographics, cirrhosis severity, concomitant medications or diabetes were seen between cirrhotics with/without SVR. There was no significant difference in overall microbiota composition (UNIFRAC P = 0.3) overall or within specific microbial families (cirrhosis dysbiosis ratio median 1.3 vs. 1.0, P = 0.45) between groups with/without SVR. This also extended towards IL-6, TNF-α and endotoxin levels. Both cirrhosis groups, however, had significant dysbiosis compared to healthy controls [UNIFRAC P = 0.01, cirrhosis dysbiosis ratio (1.1 vs. 2.9, P < 0.001)] along with higher levels of endotoxin, IL-6 and TNF-α. CONCLUSIONS: Gut dysbiosis and a pro-inflammatory systemic milieu, are found in HCV cirrhosis regardless of SVR. This persistent dysbiosis could contribute towards varying rates of improvement after HCV eradication in cirrhosis.

Novel fluorescent protein from Hydnophora rigida possesses green emission.

Fluorescent proteins are a family of proteins capable of producing fluorescence at various specific wavelengths of ultra violet light. We have previously reported the identification and characterization of a novel cyan fluorescent protein (HriCFP) from a reef coral species, Hydnophora rigida. In search of new members of the diverse family of fluorescent proteins, here we report a new green fluorescent protein (HriGFP) from H. rigida. HriGFP was identified, cloned, expressed in Escherichia coli and purified to homogeneity by metal affinity and size exclusion chromatography. The dynamic light scattering and gel filtration experiments suggested the presence of monomers in solution. The peptide mass fingerprint on the purified protein established the identity of HriGFP. HriGFP had excitation peak at 507 nm and emission peak at 527 nm. HriGFP was similar to HriCFP except the last 16 amino acid sequence at the C-terminal; however, they have shown least similarity with other known fluorescent proteins. Moreover the computational model suggests that HriGFP is a globular protein which consists of 6 α-helices and 3 ß-sheets. Taken together our results suggested that HriGFP is a novel naturally occurring fluorescent protein that exists as a monomer in solution.

Randomised clinical trial: Lactobacillus GG modulates gut microbiome, metabolome and endotoxemia in patients with cirrhosis.

BACKGROUND: Safety of individual probiotic strains approved under Investigational New Drug (IND) policies in cirrhosis with minimal hepatic encephalopathy (MHE) is not clear. AIM: The primary aim of this phase I study was to evaluate the safety, tolerability of probiotic Lactobacillus GG (LGG) compared to placebo, while secondary ones were to explore its mechanism of action using cognitive, microbiome, metabolome and endotoxin analysis in MHE patients. METHODS: Cirrhotic patients with MHE patients were randomised 1:1 into LGG or placebo BID after being prescribed a standard diet and multi-vitamin regimen and were followed up for 8 weeks. Serum, urine and stool samples were collected at baseline and study end. Safety was assessed at Weeks 4 and 8. Endotoxin and systemic inflammation, microbiome using multi-tagged pyrosequencing, serum/urine metabolome were analysed between groups using correlation networks. RESULTS: Thirty MHE patients (14 LGG and 16 placebo) completed the study without any differences in serious adverse events. However, self-limited diarrhoea was more frequent in LGG patients. A standard diet was maintained and LGG batches were comparable throughout. Only in the LGG-randomised group, endotoxemia and TNF-α decreased, microbiome changed (reduced Enterobacteriaceae and increased Clostridiales Incertae Sedis XIV and Lachnospiraceae relative abundance) with changes in metabolite/microbiome correlations pertaining to amino acid, vitamin and secondary BA metabolism. No change in cognition was found. CONCLUSIONS: In this phase I study, Lactobacillus GG is safe and well-tolerated in cirrhosis and is associated with a reduction in endotoxemia and dysbiosis.

Microbial diversity and complexity in hypersaline environments: a preliminary assessment.

The microbial communities in solar salterns and a soda lake have been characterized using two techniques: BIOLOG, to estimate the metabolic potential, and amplicon length heterogeneity analysis, to estimate the molecular diversity of these communities. Both techniques demonstrated that the halophilic Bacteria and halophilic Archaea populations in the Eilat, Israel saltern are dynamic communities with extensive metabolic potentials and changing community structures. Halophilic Bacteria were detected in Mono Lake and the lower salinity ponds at the Shark Bay saltern in Western Australia, except when the crystallizer samples were stressed by exposure to Acid Green Dye #9899. At Shark Bay, halophilic Archaea were found only in the crystallizer samples. These data confirm both the metabolic diversity and the phylogenetic complexity of the microbial communities and assert the need to develop more versatile media for the cultivation of the diversity of bacteria in hypersaline environments.

A molecular reassessment of the Leptomyxid amoebae.

Leptomyxid amoebae encompass a diverse assemblage of amoeboid protists that have been implicated as encephalitis-causing agents. This characteristic is attributed to recent studies identifying new members of the Leptomyxidae, in particular, Balamuthia mandrillaris, that cause the disease. Their morphologies range from limax to plasmodial, as well as reticulated and polyaxial. Although systematic studies have identified B. mandrillaris as a new member of the Leptomyxidae, its precise placement within the leptomyxids is uncertain. To further assess the taxonomic placement of Balamuthia among the leptomyxid amoebae and to determine whether the members of the Leptomyxida form a monophyletic assemblage, we have sequenced 16S-like rRNA genes from representatives of three leptomyxid families. Our phylogenetic analyses revealed that current members of the order Leptomyxida do not constitute a monophyletic assemblage. Our analyses clearly show that Gephyramoeba, as well as Balamuthia do not belong in the order Leptomyxida. We highlight where molecular data give differing insights than taxonomic schemes based on traditional characters.

A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples.

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection of Pseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection of Pseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas 16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool for Pseudomonas population structure analyses and taxonomic confirmations.

Impact energy measurement in time-of-flight mass spectrometry with cryogenic microcalorimeters.

Time-of-flight mass spectrometry-most notably matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) spectrometry-is an important class of techniques for the study of proteins and other biomolecules. Although these techniques provide excellent performance for masses up to about 20,000 daltons, there has been limited success in achieving good mass resolution at higher masses. This is because the sensitivity of the microchannel plate (MCP) detectors used in most systems decreases rapidly with increasing particle mass, limiting the utility of MCP detectors for very large masses. It has recently been proposed that cryogenic particle detectors may provide a solution to these difficulties. Cryogenic detectors measure the thermal energy deposited by the particle impact, and thus have a sensitivity that is largely independent of particle mass. Recent experiments have demonstrated the sensitivity of cryogenic particle detectors to single biomolecules, a quantum efficiency several orders of magnitude larger than the MCP detectors, and sensitivity to masses as large as 750,000 daltons. Here we present results demonstrating an order of magnitude better energy resolution than previous measurements, allowing direct determination of particle charge state during acceleration. Although application of these detectors to practical mass spectrometry will require further development of the detectors and cryogenics, these detectors can be used to elucidate the performance-limiting processes that occur in such systems.

Sequencing homopolymer tracts and repetitive elements.

We investigated the use of Taq dye primer and Taq terminator sequencing chemistry to optimize the quality of sequence data obtained from templates containing homopolymer tracts and repetitive elements. In direct side-by-side comparisons using the Applied Biosystems Model 373A Fluorescent Sequencer, the Taq terminator sequencing chemistry gave much cleaner and more consistent results on long homopolymer tracts and dinucleotide repeats. We also investigated various thermal cycling conditions and determined that higher annealing temperatures and longer denaturation times improved the ability to sequence through these problem templates.

Large-scale genomic sequencing: optimization of genomic chemical sequencing reactions.

We have developed a streamlined, reproducible method for performing genomic chemical sequencing reactions on the genomic DNA of Mycoplasma capricolum, which has a genome size of about 750,000 base pairs and whose composition is 75% AT. The general modifications that ensure reproducibility and allow the processing of multiple samples can be widely adopted to other large-scale sequencing projects, while the specific modifications to the chemical reactions are applicable to the sequencing of other DNAs with a high AT content.

Exploring the Mycoplasma capricolum genome: a minimal cell reveals its physiology.

We report on the analysis of 214kb of the parasitic eubacterium Mycoplasma capricolum sequenced by genomic walking techniques. The 287 putative proteins detected to date represent about half of the estimated total number of 500 predicted for this organism. A large fraction of these (75%) can be assigned a likely function as a result of similarity searches. Several important features of the functional organization of this small genome are already apparent. Among these are (i) the expected relatively large number of enzymes involved in metabolic transport and activation, for efficient use of host cell nutrients; (ii) the presence of anabolic enzymes; (iii) the unexpected diversity of enzymes involved in DNA replication and repair; and (iv) a sizeable number of orthologues (82 so far) in Escherichia coli. This survey is beginning to provide a detailed view of how M. capricolum manages to maintain essential cellular processes with a genome much smaller than that of its bacterial relatives.

The genetic data environment an expandable GUI for multiple sequence analysis.

An X-Windows-based graphic user interface is presented which allows the seamless integration of numerous existing biomolecular programs into a single analysis environment. This environment is based on a core multiple sequence editor that is linked to external programs by a user-expandable menu system and is supported on Sun and DEC workstations. There is no limitation to the number of external functions that can be linked to the interface. The length and number of sequences that can be handled are limited only by the size of virtual memory present on the workstation. The sequence data itself is used as the reference point from which analysis is done, and scalable graphic views are supported. It is suggested that future software development utilizing this expandable, user-defined menu system and the I/O linkage of external programs will allow biologists to easily integrate expertise from disparate fields into a single environment.

Purification and characterization of bovine placental retinol-binding protein.

A major low mol wt acidic protein, 3B3, produced from cultures of day 29-90 bovine allantoic membranes (in the presence of [3H]leucine or [35S]methionine) and from day 29-60 allantoic fluid, has been purified. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical mol wt (23,200 +/- 900) when analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequence analysis of 3B3 isolated from allantoic fluid on the first 43 amino acids showed that 3B3 had 93% and 91% homology with rabbit and human plasma retinol-binding protein (RBP), respectively. The UV absorption spectrum and the fluorescence excitation and emission spectra of purified 3B3 from both sources indicated the presence of bound retinol. Rabbit antiserum was raised against placental RBP (3B3) isolated from allantoic membrane culture medium. Placental RBP was immunoprecipitated from radiolabeled allantois and chorion culture medium and was detected in allantoic membrane culture medium and allantoic fluid by Western blotting. These results suggested that bovine placental membranes secrete RBP into allantoic fluid and that placental RBP may play important roles in vitamin A metabolism in the developing embryo.

Phosphorylation of proteolytically-nicked rat hepatic acetyl-CoA carboxylase.

A partially-purified preparation of acetyl-CoA carboxylase was not inactivated by ATP and Mg2+ although it was phosphorylated. SDS gel electrophoresis of the phosphorylated enzyme showed phosphopeptides migrating at 140 and 40 K along with the 250 K native subunit. Phosphorylation by the catalytic subunit of cAMP-dependent protein kinase further phosphorylated an additional 120 K phosphopeptide. Neither cAMP-independent phosphorylation nor the cAMP-dependent phosphorylation of the enzyme resulted in a significant decrease in activity.

Rat-liver fatty-acid-synthesizing complex.

Under conditions favoring lipogenesis, a high-molecular-weight species of acetyl-CoA carboxylase was isolated that did not co-sediment with the in vitro polymerized enzyme. Assays for ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase indicated that all three enzymes were associated together as a high-molecular-weight complex and that under low-lipogenic conditions the level of these enzymes decreased. Phosphorylation of the isolated complex shifted it toward a lower molecular weight.