Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes

One of the first steps in characterizing an ecosystem is to describe the organisms inhabiting it. For microbial studies, experimental limitations have hindered the ability to depict diverse communities. Here we describe oligonucleotide fingerprinting of rRNA genes (OFRG), a method that permits identification of arrayed rRNA genes (rDNA) through a series of hybridization experiments using small DNA probes. To demonstrate this strategy, we examined the bacteria inhabiting two different soils. Analysis of 1,536 rDNA clones revealed 766 clusters grouped into five major taxa: Bacillus, Actinobacteria, Proteobacteria, and two undefined assemblages. Soil-specific taxa were identified and then independently confirmed through cluster-specific PCR of the original soil DNA. Near-species-level resolution was obtained by this analysis as clones with average sequence identities of 97% were grouped in the same cluster. A comparison of these OFRG results with the results obtained in a denaturing gradient gel electrophoresis analysis of the same two soils demonstrated the significance of this methodological advance. OFRG provides a cost-effective means to extensively analyze microbial communities and should have applications in medicine, biotechnology, and ecosystem studies.     

Digestive enzyme activity and trophic behavior in two predator aquatic insects (Plecoptera, Perlidae): a comparative study

Plecoptera (Perlidae) are among the major macroinvertebrate predators in stream ecosystems and one of the insect families with lower tolerance to environmental alterations, being usually employed as bioindicators of high water ecological quality. The differences in the trophic roles of the coexisting species have been exclusively studied from their gut contents, while no data are available on the comparative digestive capacity. In the present paper, we make a comparative study of the activity of several digestive enzymes, namely proteases (at different pH), amylase, lipase, trypsin and chymotrypsin, in two species of stoneflies, Perla bipunctata and Dinocras cephalotes, which cohabit in the same stream. The study of digestive enzyme activity together with the analysis of gut contents can contribute to a better understanding of the ecology of these aquatic insects and their role in freshwater food webs. Thus, our results show that the two studied predator species inhabiting in the same stream present specializations on their feeding behaviors, facilitating their coexistence, and also differences in their capacity of use the resources. One of the main findings of this study is that D. cephalotes is able to assimilate a wider trophic resource spectrum and this could be one of the reasons why this species has a wider global distribution in all its geographical range.     

Comparative Field Summer Stress of Three Tree Species Co-occurring in Mediterranean Coastal Dunes

Chlorophyll a fluorescence, water potential (_s), and root system of Juniperus oxycedrus ssp. macrocarpa, Juniperus phoenicea ssp. turbinata, and Pinus pinea were studied in Mediterranean coastal dunes of SW Spain during summer drought and after fall rains in 1999, the driest year in the 90's. A strong and reversible depression in the photochemical efficiency of photosystem 2 of the three species was recorded, which happened concomitantly with the diurnal increase and decrease in radiation. J. phoenicea, with superficial root system, was the most affected species by summer drought. It showed high rates of down-regulation of photosynthesis by photoinhibition and positive correlation between _s and F_v/F_p, with _s lower than -7 MPa. However, it tolerated this high stress, showing a fast recovery of its physiological state after fall rains. On the other hand, J. oxycedrus and P. pinea, both with deep root systems, kept their _s values up to -3 MPa, showing lower stress during summer drought. On the other hand, J. oxycedrus and J. phoenicea were more sensible to changes in edaphic water content than P. pinea. These specific responses to summer drought would be determined by their root distributions and stomatal control of transpiration, conditioning the efficiency in getting and using the available water resources. Ecophysiological responses indicate that these species are well-adapted to long periods of drought in Mediterranean climate areas, developing different strategies: J. phoenicea tolerates high stress with a fast recovery after fall rains, while J. oxycedrus and P. pinea are less affected by summer drought since their deep root systems would allow them to reach deep water resources.     

Nymphal biology of Ephoron virgo (Olivier, 1791) (Ephemeroptera,Polymitarcyidae) in an Apenninic river (NW Italy)

Life cycle, feeding habits and nymphal density of a population of Ephoron virgo from the Bormida river (Northwestern Italy) were studied. Nymphs were present in the river from the beginning of May until August, with the highest density of nymphs recorded at the end of May–beginning of June. The life cycle was univoltine and the nymphal growth was fast. During the life cycle, an obligatory egg diapause occurs and individuals remain in this stage for up to nine months. Nymphs fed mainly on detritus, although mineral matter constituted a considerable percentage of the gut contents, predominantly in smaller nymphs.     

O-GlcNAcylation, novel post-translational modification linking myocardial metabolism and cardiomyocyte circadian clock

The cardiomyocyte circadian clock directly regulates multiple myocardial functions in a time-of-day-dependent manner, including gene expression, metabolism, contractility, and ischemic tolerance. These same biological processes are also directly influenced by modification of proteins by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc). Because the circadian clock and protein O-GlcNAcylation have common regulatory roles in the heart, we hypothesized that a relationship exists between the two. We report that total cardiac protein O-GlcNAc levels exhibit a diurnal variation in mouse hearts, peaking during the active/awake phase. Genetic ablation of the circadian clock specifically in cardiomyocytes in vivo abolishes diurnal variations in cardiac O-GlcNAc levels. These time-of-day-dependent variations appear to be mediated by clock-dependent regulation of O-GlcNAc transferase and O-GlcNAcase protein levels, glucose metabolism/uptake, and glutamine synthesis in an NAD-independent manner. We also identify the clock component Bmal1 as an O-GlcNAc-modified protein. Increasing protein O-GlcNAcylation (through pharmacological inhibition of O-GlcNAcase) results in diminished Per2 protein levels, time-of-day-dependent induction of bmal1 gene expression, and phase advances in the suprachiasmatic nucleus clock. Collectively, these data suggest that the cardiomyocyte circadian clock increases protein O-GlcNAcylation in the heart during the active/awake phase through coordinated regulation of the hexosamine biosynthetic pathway and that protein O-GlcNAcylation in turn influences the timing of the circadian clock.     

Correlative light electron ion microscopy reveals in vivo localisation of bedaquiline in Mycobacterium tuberculosis–infected lungs

Correlative light, electron, and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed correlative light, electron, and ion microscopy in tissue (CLEIMiT) and used it to identify the cell type–specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-tuberculosis (TB) drug bedaquiline (BDQ) is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.

This study uses correlative light, electron and ion microscopy (CLEIM) in vivo to reveal the intracellular fate of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis, with organelle-level resolution.     

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.     

Correction to: Jasmonate Metabolism and Its Relationship with Abscisic Acid During Strawberry Fruit Development and Ripening

Journal of Plant Growth Regulation - The original version of this article unfortunately contained some mistakes in Table 1 and it is listed below: