Retinal Vessel Functionality Is Linked With ARMS2 A69S and CFH Y402H Polymorphisms and Choroidal Status in AMD Patients.

Purpose: We aimed to investigate the reactivity of retinal vessels to a flickering stimulus in patients with age-related macular degeneration (AMD) and healthy participants. We also assessed whether the parameters of retinal vessels are dependent on genetic predisposition. Methods: A total of 354 patients with AMD and 121 controls were recruited for the study. All participants underwent thorough ophthalmologic examination and static and dynamic retinal vessel analysis. AMD risk polymorphisms were genotyped in the CFH and ARMS2 genes. Results: We found no differences between the AMD group and controls in central retinal arteriolar equivalent (CRAE), central retinal venular equivalent (CRVE), arteriovenous ratio (AVR), dynamic analysis of arteries (DAAs), or dynamic analysis of veins (DAVs). Eyes with early AMD presented with significantly higher AVR values than eyes with late AMD. In the AMD group, DAA correlated positively with both choroidal thickness (Rs = 0.14, P = 0.00096) and choroidal volume (Rs = 0.23, P < 0.0001), and no such associations were observed in the controls. We found significantly lower DAA (1.47 ± 1.50) in TT homozygotes for the ARMS2 A69S polymorphism in comparison with GG homozygotes (2.38 ± 1.79) and patients with GG + GT genotypes (2.28 ± 1.84). We also observed less prominent DAV (3.24 ± 1.71) in patients with TC + CC genotypes in the CFH Y402H polymorphism compared with TT homozygotes (3.83 ± 1.68). Conclusions: Our findings suggest that retinal microcirculation appears to be associated with the genetic background, choroidal parameters, and clinical features of the patients with AMD.

Genetic factors associated with age-related macular degeneration: identification of a novel PRPH2 single nucleotide polymorphism associated with increased risk of the disease.

PURPOSE: Age-related macular degeneration (AMD) is associated with multiple environmental and genetic risk factors. Two main risk factors for AMD are variants in the CFH and ARMS2/HTRA1 genes. We investigated over 2000 variants in AMD patients and controls using high-throughput sequencing methods to search for variants associated with AMD. METHODS: A total of 296 AMD patients and 100 controls were enrolled in this study. Genetic analysis was performed with the Illumina NextSeq 500 system. RESULTS: Multivariate analysis of patients and controls, adjusted for age, sex and smoking status (pack-years), revealed that three SNPs were strong risk factors independently associated with AMD: CFH Y402H, ARMS A69S and PRPH2 c.582-67T>A (rs3818086). The TC genotype in CFH Y402H was associated with 1.90-fold higher odds, and the CC genotype was associated with 5.66-fold higher odds of AMD compared with the TT genotype. The GT genotype in ARMS A69S was associated with 2.40-fold higher odds, and the TT genotype was associated with 6.75-fold higher odds of disease compared with the GG genotype. In the case of rs3818086, the A allele could be considered a 'risk' allele, since AA + TA genotypes were associated with 2.33-fold higher odds of AMD compared with the TT genotype. CONCLUSIONS: Although PRPH2 mutations have been previously implicated in various forms of retinal degeneration, to the best of our knowledge, this study is the first to show that the rs3818086 variant increases the risk for AMD more than two times. Further studies on larger cohorts are required to elucidate how this variant affects protein structure.

A Genomic Perspective on the Potential of Wild-Type Rumen Bacterium Enterobacter sp. LU1 as an Industrial Platform for Bio-Based Succinate Production.

Enterobacter sp. LU1, a wild-type bacterium originating from goat rumen, proved to be a potential succinic acid producer in previous studies. Here, the first complete genome of this strain was obtained and analyzed from a biotechnological perspective. A hybrid sequencing approach combining short (Illumina MiSeq) and long (ONT MinION) reads allowed us to obtain a single continuous chromosome 4,636,526 bp in size, with an average 55.6% GC content that lacked plasmids. A total of 4425 genes, including 4283 protein-coding genes, 25 ribosomal RNA (rRNA)-, 84 transfer RNA (tRNA)-, and 5 non-coding RNA (ncRNA)-encoding genes and 49 pseudogenes, were predicted. It has been shown that genes involved in transport and metabolism of carbohydrates and amino acids and the transcription process constitute the major group of genes, according to the Clusters of Orthologous Groups of proteins (COGs) database. The genetic ability of the LU1 strain to metabolize a wide range of industrially relevant carbon sources has been confirmed. The genome exploration indicated that Enterobacter sp. LU1 possesses all genes that encode the enzymes involved in the glycerol metabolism pathway. It has also been shown that succinate can be produced as an end product of fermentation via the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. The transport system involved in succinate excretion into the growth medium and the genes involved in the response to osmotic and oxidative stress have also been recognized. Furthermore, three intact prophage regions ~70.3 kb, ~20.9 kb, and ~49.8 kb in length, 45 genomic islands (GIs), and two clustered regularly interspaced short palindromic repeats (CRISPR) were recognized in the genome. Sequencing and genome analysis of Enterobacter sp. LU1 confirms many earlier results based on physiological experiments and provides insight into their genetic background. All of these findings illustrate that the LU1 strain has great potential to be an efficient platform for bio-based succinate production.

Identification of Novel Molecular Markers of Human Th17 Cells.

Th17 cells are important players in host defense against pathogens such as Staphylococcus aureus, Candida albicans, and Bacillus anthracis. Th17 cell-mediated inflammation, under certain conditions in which balance in the immune system is disrupted, is the underlying pathogenic mechanism of certain autoimmune disorders, e.g., rheumatoid arthritis, Graves' disease, multiple sclerosis, and psoriasis. In the present study, using transcriptomic profiling, we selected genes and analyzed the expression of these genes to find potential novel markers of Th17 lymphocytes. We found that APOD (apolipoprotein D); C1QL1 (complement component 1, Q subcomponent-like protein 1); and CTSL (cathepsin L) are expressed at significantly higher mRNA and protein levels in Th17 cells than in the Th1, Th2, and Treg subtypes. Interestingly, these genes and the proteins they encode are well associated with the function of Th17 cells, as these cells produce inflammation, which is linked with atherosclerosis and angiogenesis. Furthermore, we found that high expression of these genes in Th17 cells is associated with the acetylation of H2BK12 within their promoters. Thus, our results provide new information regarding this cell type. Based on these results, we also hope to better identify pathological conditions of clinical significance caused by Th17 cells.

Genome analysis of a wild rumen bacterium Enterobacter aerogenes LU2 - a novel bio-based succinic acid producer.

Enterobacter aerogenes LU2 was isolated from cow rumen and recognized as a potential succinic acid producer in our previous study. Here, we present the first complete genome sequence of this new, wild strain and report its basic genetic features from a biotechnological perspective. The MinION single-molecule nanopore sequencer supported by the Illumina MiSeq platform yielded a circular 5,062,651 bp chromosome with a GC content of 55% that lacked plasmids. A total of 4,986 genes, including 4,741 protein-coding genes, 22 rRNA-, 86 tRNA-, and 10 ncRNA-encoding genes and 127 pseudogenes, were predicted. The genome features of the studied strain and other Enterobacteriaceae strains were compared. Functional studies on the genome content, metabolic pathways, growth, and carbon transport and utilization were performed. The genomic analysis indicates that succinic acid can be produced by the LU2 strain through the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. Antibiotic resistance genes were determined, and the potential for bacteriocin production was verified. Furthermore, one intact prophage region of length ~31,9 kb, 47 genomic islands (GIs) and many insertion sequences (ISs) as well as tandem repeats (TRs) were identified. No clustered regularly interspaced short palindromic repeats (CRISPRs) were found. Finally, comparative genome analysis with well-known succinic acid producers was conducted. The genome sequence illustrates that the LU2 strain has several desirable traits, which confirm its potential to be a highly efficient platform for the production of bulk chemicals.

Associations of microRNAs, Angiogenesis-Regulating Factors and CFH Y402H Polymorphism-An Attempt to Search for Systemic Biomarkers in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) remains the leading cause of blindness in elderly people, but the pathophysiology of this disease is still largely unknown. We investigated the systemic expression of angiogenesis-regulating growth factors and selected miRNAs known to regulate angiogenesis in AMD patients. We also focused on possible correlations of their expression with the presence of CFH Y402H or ARMS A69S risk variants. A total of 354 AMD patients and 121 controls were enrolled in this study. The levels of angiogenesis-regulating factors were analyzed in plasma samples using Luminex technology. The expression of selected miRNAs was analyzed in peripheral blood plasma using real-time qPCR. The genetic analysis was performed with an Illumina NextSeq500 system. AMD was an independent factor associated with lower levels of angiogenin (ß = -0.29, p < 0.001), endostatin (ß = -0.18, p < 0.001), FGF-basic (ß = -0.18, p < 0.001), PlGF (ß = -0.24, p < 0.001), miRNA-21-3p (ß = -0.13, p = 0.01) and miRNA-155-5p (ß = -0.16, p = 0.002); and with higher levels of FGF-acidic (ß = 0.11, p = 0.03), miRNA-23a-3p (ß = 0.17, p < 0.001), miRNA-126-5p (ß = 0.13, p = 0.009), miRNA-16-5p (ß = 0.40, p < 0.001), miRNA-17-3p (ß = 0.13, p = 0.01), miRNA-17-5p (ß = 0.17, p < 0.001), miRNA-223-3p (ß = 0.15, p = 0.004), and miRNA-93 (ß = 0.11, p = 0.04). The expression of analyzed miRNA molecules significantly correlated with the levels of tested angiogenesis-regulating factors and clinical parameters in AMD patients, whereas such correlations were not observed in controls. We also found an association between the CFH Y402H polymorphism and miRNA profiles, whereby TT homozygotes showed evidently higher expression of miRNA-16-5p than CC homozygotes or TC heterozygotes (p = 0.0007). Our results suggest that the balance between systemic pro- and anti-angiogenic factors and miRNAs is vital in multifactorial AMD pathogenesis.

Complete genome sequence of Lactobacillus rhamnosus Pen, a probiotic component of a medicine used in prevention of antibiotic-associated diarrhoea in children.

BACKGROUND: Lactobacillus rhamnosus Pen is a human endogenous strain with well-documented health promoting properties that is used for production of probiotics. It has a long safety history of application, and its effectiveness in the prevention of antibiotic-associated diarrhoea has also been confirmed in clinical trials. RESULTS: Here we present the complete genome sequence of L. rhamnosus Pen, which consists of a circular 2,884,4966-bp chromosome with a GC content of 46.8%. Within 2907 open reading frames (ORFs), genes involved with probiotic properties were identified. A CRISPR locus, consisting of a 1092-nt region with 16 spacers, was also detected. Finally, an intact prophage of ~ 40.7 kb, 57 ORFs, GC content 44.8% was identified. CONCLUSIONS: Genomic analysis confirmed the probiotic properties of L. rhamnosus Pen and may indicate new biotechnological applications of this industrially important strain.

Complete genome sequence of Gluconacetobacter xylinus E25 strain--valuable and effective producer of bacterial nanocellulose.

This study reports the release of complete genome sequence of the producer of bacterial nanocellulose (BNC) - Gluconacetobacter xylinus E25, a vinegar-isolated strain. Preliminary sequence analysis revealed complexity of the genome structure and familiarized genetic basis of productive properties of E25 strain. The genome consists of one chromosome and five plasmids. Whole genome sequencing has opened up new perspectives for further bioinformatics and experimental studies allowing the elucidation of molecular mechanisms responsible for regulation of production of BNC - a valuable biomaterial.

Studies on the anti-hepatitis C virus activity of newly synthesized tropolone derivatives: identification of NS3 helicase inhibitors that specifically inhibit subgenomic HCV replication.

We synthesized new tropolone derivatives substituted with cyclic amines: piperidine, piperazine or pyrrolidine. The most active anti-helicase compound (IC50=3.4 microM), 3,5,7-tri[(4'-methylpiperazin-1'-yl)methyl]tropolone (2), inhibited RNA replication by 50% at 46.9 microM (EC50) and exhibited the lowest cytotoxicity (CC50)>1 mM resulting in a selectivity index (SI=CC50/EC50)>21. The most efficient replication inhibitor, 3,5,7-tri[(4'-methylpiperidin-1'-yl)methyl]tropolone (6), inhibited RNA replication with an EC50 of 32.0 microM and a SI value of 17.4, whereas 3,5,7-tri[(3'-methylpiperidin-1'-yl)methyl]tropolone (7) exhibited a slightly lower activity with an EC50 of 35.6 microM and a SI of 9.8.

Fluorometric assay of hepatitis C virus NS3 helicase activity.

The development of techniques based on fluorescence has made it possible to create new types of assays that represent an advantageous alternative to old tests relying on radioactivity. Such a novel approach has been applied to develop a high-throughput assay to measure the helicase activity of the hepatitis C virus (HCV) NS3 protein and the inhibitory potential of several classes of compounds. The NS3 helicase is one of the most promising targets of anti-HCV-oriented screening of compounds due to the urgent need for more effective and tolerable drugs. The 96- or 384-well microplate assay that we developed is based on the use of a quenched double-stranded DNA substrate labeled with a fluorophore (Cy3 or FAM) and with a Black Hole Quencher 1 or 2. It allows for direct (real-time) measurements of substrate unwinding and inhibition of unwinding by anti-helicase compounds. After a few modifications of buffers and assay conditions this method can be applied to various variants of HCV helicase and other proteins with helicase activities.

Amidinoanthracyclines - a new group of potential anti-hepatitis C virus compounds.

Hepatitis C virus (HCV) infections represent one of the major and still unresolved health problems because current therapy is effective in only 50-80% of cases, depending on viral genotype. A large group of amidinoanthracyclines, with decreased acute toxicity and cardiotoxicity compared to the parent antibiotics, was tested in a high-throughput fluorometric HCV helicase assay. Here, we report the selection of more than 50 potent inhibitors of helicase activity that inhibit the enzyme with IC(50) values in the range of 0.03- 10 mum; four of these compounds are the most potent inhibitors of helicase activity described in the literature. The activity of these inhibitors is highly dependent on their chemical structure, mainly on the substituent at the amidino carbon atom and on the orientation of the hydroxyl group at the 4 inch position of the daunosamine moiety. The most effective compounds act not solely via intercalation into the double-stranded DNA substrate, but also compete with the enzyme for access to the substrate, impeding formation of the active helicase complex. Selected amidinoanthracyclines were tested in the subgenomic HCV replicon system. These experiments confirmed the antiviral activity of two selected inhibitors (EC(50) values below 0.2 mum with selectivity indices of 19 and 33) and proved that they may be considered as potential anti-HCV drugs.

Searching for a new anti-HCV therapy: synthesis and properties of tropolone derivatives.

Hepatitis C virus (HCV) is considered one of the most dangerous pathogens since about 3% of the world population is HCV-infected and the virus is a major cause of hepatitis, cirrhosis, and liver carcinoma. A need for a more efficient therapy prompted us to investigate new class of compounds, such as tropolone derivatives that possess antiviral, antibacterial, and antifungal activities. To synthesize bromo- and morpholinomethyl-analogues of tropolone, the previously reported methods were modified. The influence of new derivatives on the activity of the helicase and NTP-ase of HCV was investigated. The most potent inhibitory effect in the fluorometric helicase assay was exerted by 3,7-dibromo-5-morpholinomethyltropolone, for which the IC50 value was at low micromolar range. All the morpholino-derivatives had inhibitory activities higher than those of the non-modified analogues. Low toxicity in a yeast-based toxicity assay indicates that these compounds could be further modified to develop potent inhibitors of the HCV helicase and of viral replication.

A new analyzer based on pellistor sensor with neural network data postprocessing for measurement of hydrocarbons in lower explosive limit range.

We present the results of a first stage of development work on a new type of analyzer for hydrogen and C(1)-C(3) hydrocarbons concentration measurements in the lower explosive limit range, based on single pellistor sensor with artificial neural network data postprocessing.

Direct fluorometric measurement of hepatitis C virus helicase activity.

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.

Flow cytometric "rare event analysis": a standardized approach to the analysis of donor cell chimerism.

Lack of standardization of methods and detection limits contributes to the controversial results regarding microchimerism after organ transplantation and has prompted the development of a standardized, reproducible, "easy-to-use" flow cytometric method for this type of "rare event analysis".EDTA-blood of healthy, HLA-typed donors was stained simultaneously with FITC- and biotin-labeled HLA-class I antibodies (One Lambda) as well as Cy5-PE-labeled CD45 (Medac, Hamburg) according to a standard protocol and analysed on a Coulter EPICS XL Flow cytometer (FCM). An absolute range of positivity (mean MESF+/- 1 STD) was determined for 22 HLA-specific antibodies. The range of positivity ranged between 5000 and 20,000 MESF (anti-A23, 24(9) FITC) and 40,000-140,000 (anti-Bw6 FITC). The frequency of nonspecific positive signals using nonstained cells, isotype-controls and irrelevant HLA antibodies was between 0.01% and about 0.5%, in some samples up to 1.4%, with an MESF between 8000 and 150,000, thus interfering clearly with the defined positive range of most antibodies tested. Using an "HLA antibody cocktail", combining FITC- and PE-labeled antibodies for different HLA specificities and thereby creating an internal control, the identification of donor cells was improved but was only rarely applicable. Due to the lack of highly reactive antibodies, FCM analysis was not suitable for the reliable identification of very low numbers of donor hematopoetic cells despite the theoretical advantages of flow cytometric detection of rare events. The single parameter approach was hampered by a significant frequency of nonspecific positive signals, which were easily mistaken as specific (true) positive signals, whereas the multiparameter approach could only be used in selected cases.