A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.     

Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.     

Systematics and species-specific response to pH of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora,Hypotricha) from acid mining lakes

We investigated the morphology, phylogeny of the 18S rDNA, and pH response of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) isolated from two chemically similar acid mining lakes (pH ～ 2.6) located at Langau, Austria, and in Lusatia, Germany. Oxytricha acidotolerans sp. nov. from Langau has 18 frontal-ventral-transverse cirri but a very indistinct kinety 3 fragmentation so that the assignment to Oxytricha is uncertain. The somewhat smaller species from Lusatia has a highly variable cirral pattern and the dorsal kineties arranged in the Urosomoida pattern and is, therefore, preliminary designated as Urosomoida sp. The pH response was measured as ciliate growth rates in laboratory experiments at pH ranging from 2.5 to 7.0. Our hypothesis was that the shape of the pH reaction norm would not differ between these closely related (3% difference in their SSU rDNA) species. Results revealed a broad pH niche for O. acidotolerans, with growth rates peaking at moderately acidic conditions (pH 5.2). Cyst formation was positively and linearly related to pH. Urosomoida sp. was more sensitive to pH and did not survive at circumneutral pH. Accordingly, we reject our hypothesis that similar habitats would harbour ciliate species with virtually identical pH reaction norm.     

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.     

Multiple Single-Cell Genomes Provide Insight into Functions of Uncultured Deltaproteobacteria in the Human Oral Cavity

Despite a long history of investigation, many bacteria associated with the human oral cavity have yet to be cultured. Studies that correlate the presence or abundance of uncultured species with oral health or disease highlight the importance of these community members. Thus, we sequenced several single-cell genomic amplicons from Desulfobulbus and Desulfovibrio (class Deltaproteobacteria) to better understand their function within the human oral community and their association with periodontitis, as well as other systemic diseases. Genomic data from oral Desulfobulbus and Desulfovibrio species were compared to other available deltaproteobacterial genomes, including from a subset of host-associated species. While both groups share a large number of genes with other environmental Deltaproteobacteria genomes, they encode a wide array of unique genes that appear to function in survival in a host environment. Many of these genes are similar to virulence and host adaptation factors of known human pathogens, suggesting that the oral Deltaproteobacteria have the potential to play a role in the etiology of periodontal disease.     

Hearing in Cichlid Fishes under Noise Conditions

Background

Hearing thresholds of fishes are typically acquired under laboratory conditions. This does not reflect the situation in natural habitats, where ambient noise may mask their hearing sensitivities. In the current study we investigate hearing in terms of sound pressure (SPL) and particle acceleration levels (PAL) of two cichlid species within the naturally occurring range of noise levels. This enabled us to determine whether species with and without hearing specializations are differently affected by noise.

Methodology/Principal Findings

We investigated auditory sensitivities in the orange chromide Etroplus maculatus, which possesses anterior swim bladder extensions, and the slender lionhead cichlid Steatocranus tinanti, in which the swim bladder is much smaller and lacks extensions. E. maculatus was tested between 0.2 and 3kHz and S. tinanti between 0.1 and 0.5 kHz using the auditory evoked potential (AEP) recording technique. In both species, SPL and PAL audiograms were determined in the presence of quiet laboratory conditions (baseline) and continuous white noise of 110 and 130 dB RMS. Baseline thresholds showed greatest hearing sensitivity around 0.5 kHz (SPL) and 0.2 kHz (PAL) in E. maculatus and 0.2 kHz in S. tinanti. White noise of 110 dB elevated the thresholds by 0–11 dB (SPL) and 7–11 dB (PAL) in E. maculatus and by 1–2 dB (SPL) and by 1–4 dB (PAL) in S. tinanti. White noise of 130 dB elevated hearing thresholds by 13–29 dB (SPL) and 26–32 dB (PAL) in E. maculatus and 6–16 dB (SPL) and 6–19 dB (PAL) in S. tinanti.

Conclusions

Our data showed for the first time for SPL and PAL thresholds that the specialized species was masked by different noise regimes at almost all frequencies, whereas the non-specialized species was much less affected. This indicates that noise can limit sound detection and acoustic orientation differently within a single fish family.     

Characterization of a Novel Porcine Parvovirus Tentatively Designated PPV5

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1–67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%–85.1% for ORF1 and 54.0%–54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca²⁺ binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A₂ (PLA₂) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs.     

Altered Visual and Feet Proprioceptive Feedbacks during Quiet Standing Increase Postural Sway in Patients with Severe Knee Osteoarthritis

Objective

The objective was to investigate how postural control in knee osteoarthritis (KOA) patients, with different structural severities and pain levels, is reorganized under different sensory conditions.

Methods

Forty-two obese patients (BMI range from 30.1 to 48.7 kg*m⁻², age range from 50 to 74 years) with KOA were evaluated. One minute of quiet standing was assessed on a force platform during 4 different sensory conditions, applied 3 times at random: Eyes open (EO) and eyes closed (EC) standing on firm and soft (foam) surfaces (EO-soft and EC-soft). Centre of pressure (Cop) standard deviation, speed, range and Cop mean position in both directions (anterior-posterior and medial-lateral) were extracted from the force platform data. Structural disease severity was assessed from semiflexed standing radiographs and graded by the Kellgren and Lawrence (KL) score. Pain intensity immediately before the measurements was assessed by numeric rating scale (range: 0–10).

Results

The patients were divided into “less severe” (KL 1 and 2, n = 24) and “severe” (KL>2, n = 18) group. The CoP range in the medial-lateral direction was larger in the severe group when compared with the less severe group during EC-soft condition (P<0.01). Positive correlation between pain intensity and postural sway (range in medial-lateral direction) was found during EC condition, indicating that the higher the pain intensity, the less effective is the postural control applied to restore an equilibrium position while standing without visual information.

Conclusion

The results support that: (i) the postural reorganization under manipulation of the different sensory information is worse in obese KOA patients with severe degeneration and/or high pain intensity when compared with less impaired patients, and (ii) higher pain intensity is related to worse body balance in obese KOA patients.     

Phylogeny and physiology of candidate phylum ‘Atribacteria' (OP9/JS1) inferred from cultivation-independent genomics

The ‘Atribacteria'' is a candidate phylum in the Bacteria recently proposed to include members of the OP9 and JS1 lineages. OP9 and JS1 are globally distributed, and in some cases abundant, in anaerobic marine sediments, geothermal environments, anaerobic digesters and reactors and petroleum reservoirs. However, the monophyly of OP9 and JS1 has been questioned and their physiology and ecology remain largely enigmatic due to a lack of cultivated representatives. Here cultivation-independent genomic approaches were used to provide a first comprehensive view of the phylogeny, conserved genomic features and metabolic potential of members of this ubiquitous candidate phylum. Previously available and heretofore unpublished OP9 and JS1 single-cell genomic data sets were used as recruitment platforms for the reconstruction of atribacterial metagenome bins from a terephthalate-degrading reactor biofilm and from the monimolimnion of meromictic Sakinaw Lake. The single-cell genomes and metagenome bins together comprise six species- to genus-level groups that represent most major lineages within OP9 and JS1. Phylogenomic analyses of these combined data sets confirmed the monophyly of the ‘Atribacteria'' inclusive of OP9 and JS1. Additional conserved features within the ‘Atribacteria'' were identified, including a gene cluster encoding putative bacterial microcompartments that may be involved in aldehyde and sugar metabolism, energy conservation and carbon storage. Comparative analysis of the metabolic potential inferred from these data sets revealed that members of the ‘Atribacteria'' are likely to be heterotrophic anaerobes that lack respiratory capacity, with some lineages predicted to specialize in either primary fermentation of carbohydrates or secondary fermentation of organic acids, such as propionate.