Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

Observations of terrestrial locomotion in wild Polypterus senegalus from Lake Albert,Uganda

Polypterids, the most basal actinopterygians, are a group of fish long-considered living fossils and holding a key position for understanding fish and tetrapod evolution. Knowledge of the natural history of Polypterus is limited, their having been studied in little detail since the early 1900s. The locomotory habits of wild Polypterus senegalus from Lake Albert, Uganda, were investigated in 2014. High-speed videography demonstrated the capability of large Polypterus to move overland successfully. Contrary to previous evidence, field observations found that terrestrial locomotion in Polypterus is not inherently restricted by body size. Evidence that Polypterus exhibit this behaviour as part of their natural life history can be found in the existence of environmental challenges and the presence of adaptations for amphibious life.     

Comprehensive Investigation of Marine Actinobacteria Associated with the Sponge Halichondria panicea

Representatives of Actinobacteria were isolated from the marine sponge Halichondria panicea collected from the Baltic Sea (Germany). For the first time, a comprehensive investigation was performed with regard to phylogenetic strain identification, secondary metabolite profiling, bioactivity determination, and genetic exploration of biosynthetic genes, especially concerning the relationships of the abundance of biosynthesis gene fragments to the number and diversity of produced secondary metabolites. All strains were phylogenetically identified by 16S rRNA gene sequence analyses and were found to belong to the genera Actinoalloteichus, Micrococcus, Micromonospora, Nocardiopsis, and Streptomyces. Secondary metabolite profiles of 46 actinobacterial strains were evaluated, 122 different substances were identified, and 88 so far unidentified compounds were detected. The extracts from most of the cultures showed biological activities. In addition, the presence of biosynthesis genes encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in 30 strains was established. It was shown that strains in which either PKS or NRPS genes were identified produced a significantly higher number of metabolites and exhibited a larger number of unidentified, possibly new metabolites than other strains. Therefore, the presence of PKS and NRPS genes is a good indicator for the selection of strains to isolate new natural products.Sponges are multicellular invertebrates and sessile filter feeders which are abundant in the oceans as well as in freshwater habitats (41). They gained great interest due to their association with a wide variety of microorganisms. These microorganisms are known to be a rich source of secondary metabolites (108), which exhibit a broad range of bioactivities such as inhibition of enzyme activities and cell division and antiviral, antimicrobial, anti-inflammatory, antitumor, cytotoxic, and cardiovascular properties (77).Numerous studies concerning specific aspects of sponge-bacterium associations were accomplished using distinct methods for the evaluation of the microbial diversity (mostly molecular approaches) or the bioactivities (culture-dependent methods) or biosynthetic aspects (chemical analyses and molecular approaches) of secondary metabolites of the associated bacteria (19, 47, 51, 54, 110, 122, 126). So far, there is less comprehensive information about the integration of this knowledge into concepts for sponge-bacterium interactions based on small molecules.We focused on Actinobacteria associated with Halichondria panicea Pallas (Porifera, Demospongiae, Halichondriida, Halichondriidae), a sponge species living in coastal habitats worldwide (9). Previous work demonstrated a phylogenetically diverse array of bacterial groups present in this sponge: representatives of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Cytophaga/Flavobacteria, the Deinococcus group, low-G+C-content Gram-positive bacteria, Actinobacteria, and Planctomycetales were identified by means of a genetic approach (47, 122). Among these, though representing only 3 to 20% of the sponge-associated bacterial community (41, 47, 103), Actinobacteria are the most promising bacterial group regarding secondary metabolite production. Members of this phylum account for approximately half of the bioactive secondary metabolites that have so far been discovered in bacteria (64). Although the majority of secondary metabolite-producing Actinobacteria originate from terrestrial habitats (101), recent studies of marine Actinobacteria have revealed many new chemical entities and bioactive metabolites (13, 30, 50, 100). Among these, only a few substances were isolated from Actinobacteria associated with H. panicea (85, 123), e.g., the antimicrobially active substances 2,4,4′-trichloro-2′-hydroxydiphenylether and acyl-1-(acyl-6′-mannobiosyl)-3-glycerol produced by Micrococcus luteus (17). By combining data about the phylogenetic characterization of the Actinobacteria associated with H. panicea, their biosynthetic potential for secondary metabolite production, and their chemical profiles, we present comprehensive insights into a great variety of produced natural products as well as their bioactivities. By means of these results, we attempt to close the gap of knowledge about Actinobacteria associated with H. panicea and discuss the biological roles of identified small molecules in the sponge-associated community.     

ACPA fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression

Introduction

Autoantibodies against citrullinated peptides/proteins (ACPA) are found in approximately 75% of the sera of patients with rheumatoid arthritis (RA). The RA-specific ACPA are frequently present prior to disease onset and their presence associates with a more erosive disease course. ACPA can therefore be used to aid the diagnosis and prognosis of RA. Recently, it became clear that ACPA are very heterogeneous, both in an individual patient and among different patients. The aim of this study was to investigate whether clinically meaningful ACPA profiles exist in early RA patients.

Methods

Twenty citrullinated peptides and the corresponding non-citrullinated control peptides were immobilized on microarray sensor chips. Sera from 374 early arthritis patients were analyzed by surface plasmon resonance imaging (iSPR) of biomolecular interactions on the sensor chip.

Results

Cluster analysis of the reactivities with the citrullinated peptides, after subtraction of the reactivities with the corresponding control peptides confirmed the heterogeneity of the ACPA response in RA and revealed 12 distinct ACPA profiles. The association of the 5 most frequent profiles with clinical features at diagnosis and during the disease course was examined, showing no statistically significant associations.

Conclusions

Compared to the detection of ACPA in RA sera by CCP-based assays, ACPA profiling in early arthritis patients did not reveal associations with disease activity and progression scores.     

Genetic characterization of Strongyloides spp. from captive, semi-captive and wild Bornean orangutans (Pongo pygmaeus) in Central and East Kalimantan, Borneo, Indonesia

Orangutans (Pongo spp.), Asia's only great apes, are threatened in their survival due to habitat loss, hunting and infections. Nematodes of the genus Strongyloides may represent a severe cause of death in wild and captive individuals. In order to better understand which Strongyloides species/subspecies infect orangutans under different conditions, larvae were isolated from fecal material collected in Indonesia from 9 captive, 2 semi-captive and 9 wild individuals, 18 captive groups of Bornean orangutans and from 1 human working with wild orangutans. Genotyping was done at the genomic rDNA locus (part of the 18S rRNA gene and internal transcribed spacer 1, ITS1) by sequencing amplicons. Thirty isolates, including the one from the human, could be identified as S. fuelleborni fuelleborni with 18S rRNA gene identities of 98·5-100%, with a corresponding published sequence. The ITS1 sequences could be determined for 17 of these isolates revealing a huge variability and 2 main clusters without obvious pattern with regard to attributes of the hosts. The ITS1 amplicons of 2 isolates were cloned and sequenced, revealing considerable variability indicative of mixed infections. One isolate from a captive individual was identified as S. stercoralis (18S rRNA) and showed 99% identity (ITS1) with S. stercoralis sequences from geographically distinct locations and host species. The findings are significant with regard to the zoonotic nature of these parasites and might contribute to the conservation of remaining orangutan populations.     

Development and Validation of a Fast and Optimized Screening Method for Enhanced Production of Secondary Metabolites Using the Marine Scopulariopsis brevicaulis Strain LF580 Producing Anti-Cancer Active Scopularide A and B

Natural compounds from marine fungi are an excellent source for the discovery and development of new drug leads. The distinct activity profiles of the two cyclodepsipeptides scopularide A and B against cancer cell lines set their marine producer strain Scopulariopsis brevicaulis LF580 into the focus of the EU project MARINE FUNGI. One of the main goals was the development of a sustainable biotechnological production process for these compounds. The secondary metabolite production of strain LF580 was optimized by random mutagenesis employing UV radiation. For a fast and reliable detection of the intracellular secondary metabolite production level, a miniaturized bioactivity-independent screening method was developed, as the random mutagenesis yielded a large number of mutants to be analysed quantitatively and none of the existing hyphenated bioassay-dependent screening systems could be applied. The method includes decreased cultivation volume, a fast extraction procedure as well as an optimized LC-MS analysis. We show that deviation could be specifically reduced at each step of the process: The measuring deviation during the analysis could be minimized to 5% and technical deviation occurring in the downstream part to 10–15%. Biological variation during the cultivation process still has the major influence on the overall variation. However, the approach led to a 10-fold reduction of time and similar effects on costs and effort compared to standard reference screening methods. The method was applied to screen the UV-mutants library of Scopulariopsis brevicaulis LF580. For validation purposes, the occurring variations in the miniaturized scale were compared to those in the classical Erlenmeyer flask scale. This proof of concept was performed using the wild type strain and 23 randomly selected mutant strains. One specific mutant strain with an enhanced production behavior could be obtained.     

Thyroid disease is a favorable prognostic factor in achieving sustained virologic response in chronic hepatitis C undergoing combination therapy: A nested case control study

Background

Interferon-α in combination with ribavirin is the current gold standard for treatment of chronic hepatitis C. It is unknown if the development of autoimmune thyroid disease (TD) during treatment confers an improved chance of achieving sustained virologic response. The aim of this study is to assess the chance of achieving sustained virologic response (SVR) in patients who developed TD during treatment when compared with those who did not.

Methods

We performed a tertiary hospital-based retrospective nested case-control analysis of 19 patients treated for hepatitis C who developed thyroid disease, and 76 controls (matched for age, weight, gender, cirrhosis and aminotransferase levels) who did not develop TD during treatment. Multivariate logistic-regression models were used to compare cases and controls.

Results

The development of TD was associated with a high likelihood of achieving SVR (odds ratio, 6.0; 95% confidence interval, 1.5 to 24.6) for the pooled group containing all genotypes. The likelihood of achieving SVR was increased in individuals with genotype 1 HCV infection who developed TD (odds ratio, 5.2; 95% confidence interval, 1.2 to 22.3), and all genotype 3 patients who developed TD achieved SVR.

Conclusions

Development of TD during treatment for hepatitis C infection is associated with a significantly increased chance of achieving SVR. The pathophysiogical mechanisms for this observation remain to be determined.

Trial Registration

The Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRB12610000830099     

Evaluation of stability and size distribution of sunflower oil-coated micro bubbles for localized drug delivery

Background

In recent years magnesium alloys have been intensively investigated as potential resorbable materials with appropriate mechanical and corrosion properties. Particularly in orthopedic research magnesium is interesting because of its mechanical properties close to those of natural bone, the prevention of both stress shielding and removal of the implant after surgery.

Methods

ZEK100 plates were examined in this in vitro study with Hank's Balanced Salt Solution under physiological conditions with a constant laminar flow rate. After 14, 28 and 42 days of immersion the ZEK100 plates were mechanically tested via four point bending test. The surfaces of the immersed specimens were characterized by SEM, EDX and XRD.

Results

The four point bending test displayed an increased bending strength after 6 weeks immersion compared to the 2 week group and 4 week group. The characterization of the surface revealed the presence of high amounts of O, P and Ca on the surface and small Mg content. This indicates the precipitation of calcium phosphates with low solubility on the surface of the ZEK100 plates.

Conclusions

The results of the present in vitro study indicate that ZEK100 is a potential candidate for degradable orthopedic implants. Further investigations are needed to examine the degradation behavior.     

Induction of long-term B-cell depletion in refractory rheumatoid arthritis patients preferentially affects autoreactive more than protective humoral immunity

Introduction

B-cell depletion has become a common treatment strategy in anti-TNF-refractory rheumatoid arthritis (RA). Although the exact mechanism of how B-cell depletion leads to clinical amelioration in RA remains to be elucidated, repetitive treatment with B-cell-depleting agents leading to long-term B-cell depletion has been reported to be beneficial. The latter has led to the hypothesis that the beneficial effects of B-cell depletion might act through their influence on pathogenic autoreactive plasma cells.

Methods

In this study, we investigated the effects of a fixed retreatment regimen with anti-CD20 mAbs on the humoral (auto)immune system in a cohort of therapy-refractory RA patients.

Results

Fixed retreatment led to long-term B-cell depletion in peripheral blood, bone marrow and, to a lesser extent, synovium. Also, pathologic autoantibody secretion (that is, anticitrullinated peptide antibodies (ACPAs)) was more profoundly affected by long-term depletion than by physiological protective antibody secretion (that is, against measles, mumps and rubella). This was further illustrated by a significantly shorter estimated life span of ACPA-IgG secretion compared to total IgG secretion as well as protective antibody secretion.

Conclusion

By studying plasma cell function during an extensive 2-year period of B-cell depletion, autoantibody secretion was significantly shorter-lived than physiologically protective antibody secretion. This suggests that the longevity of autoreactive plasma cells is different from protective long-lived plasma cells and might indicate a therapeutic window for therapies that target plasma cells.