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.

Novel fluorescent protein from Hydnophora rigida possess cyano emission.

Currently, a broad diversity of fluorescent proteins among marine organisms range from cyano-red emissions. Fluorescent proteins differ in their DNA sequences from green fluorescent protein (GFP). We identified cDNA encoding the gene of a new protein from the reef coral Hydnophora rigida of the Merulinidae family. Both the spectral properties and putative primary sequence of the protein has been determined. The cloned cDNA encode peptide we call HriCFP is comprised of 134 amino acids. It has characteristics of a cyano fluorescent protein (HriCFP) and its sequence is markedly different from known GFP from the hydroid jellyfish Aequorea victoria. HriCFP was cloned, expressed, purified and exist as monomer. The peptide mass finger print on the purified protein confirmed identity of HriCFP.

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.

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.

Tumor markers: the potential of "omics" approach.

Tumor markers are the molecules that indicate the presence or prognosis of malignancy. Most often, tumor markers are produced by the cancer tissue itself. Many of them could be secreted into the body fluids in small quantities. Thus, tumor markers could be useful for early diagnostics of primary tumors and relapsed disease, as well as for determining tumor prognosis and predicting likely response of the tumor to therapy. Tumor markers are part of the clinical routine. Nevertheless, lack of sensitivity and specificity precludes routine usage of single tumor markers in population-based screening. Shortcomings of single tumor markers could be solved by parallel evaluation of multiple tumor markers that can perform with required certainty. Genome and proteome-wide approaches currently lead to identification and initial characterization of hundreds new tumor marker candidates. Most prominent of such methods are serological analyses of recombinant cDNA expression libraries (SEREX), 2-dimensional polyacrylamide gel electrophoresis, mass spectrometry, as well as protein and DNA microarrays. Last but not the least is a computational approach allowing high-throughput detection of tumor marker candidate genes in publicly available datasets. Listed approaches are critically discussed in this review as well as the most crucial tumor-related findings. Finally, a perspective on the future of tumor markers in the tailored medicine is given.

A population survey of members of the phylum Bacteroidetes isolated from salt marsh sediments along the east coast of the United States.

The population diversity of cultured isolates of the phylum Bacteroidetes was investigated from salt-marsh sediments. A total of 44 isolates that belonged to this phylum were isolated either from high-dilution plates or from end-dilution most-probable-number (MPN) tubes. The majority of the isolates came from Virginia, with others isolated from salt marshes in Delaware and North Carolina. All the isolates were aerobic Gram-negative, catalase positive small rods that formed uniform colonies; most had either yellow or orange pigmentation. Riboprinting of 40 isolates revealed they were genotypically diverse, consisting of 33 different riboprint patterns; there were four riboprint groups with two or more members. The isolates could be divided into 23 different fatty acid methyl ester (FAME) profiles at the species level with 14 of the profiles being unique to single isolates. One group of 10 isolates was closely related, suggesting this group may be well adapted for life in salt marshes. Thirteen of the isolates were selected for sequencing of the small-subunit ribosomal RNA gene representing a diverse group of isolates that fell within the classes Sphingobacteria and Flavobacteria. Only one of the isolates was >97% similar at the 16S rDNA to a described species of Cytophaga marinoflava; the other isolates were 94 to 96.5% related to undescribed isolates mostly within the class Flavobacteria. There was good concordance between the FAME dendrogram and a phylogenetic tree based on comparison of 16S sequences. There were no obvious temporal or spatial distribution patterns to the isolates, suggesting that this group of bacteria is inherently diverse.