Reengineering Rate-Limiting, Millisecond Enzyme Motions by Introduction of an Unnatural Amino Acid

Rate-limiting millisecond motions in wild-type (WT) Ribonuclease A (RNase A) are modulated by histidine 48. Here, we incorporate an unnatural amino acid, thia-methylimidazole, at this site (H48C-4MI) to investigate the effects of a single residue on protein motions over multiple timescales and on enzyme catalytic turnover. Molecular dynamics simulations reveal that H48C-4MI retains some crucial WT-like hydrogen bonding interactions but the extent of protein-wide correlated motions in the nanosecond regime is decreased relative to WT. NMR Carr-Purcell-Meiboom-Gill relaxation dispersion experiments demonstrate that millisecond conformational motions in H48C-4MI are present over a similar pH range compared to WT. Furthermore, incorporation of this nonnatural amino acid allows retention of WT-like catalytic activity over the full pH range. These studies demonstrate that the complexity of the protein energy landscape during the catalytic cycle can be maintained using unnatural amino acids, which may prove useful in enzyme design efforts.     

Plasma-initiated graft polymerization as an immobilization platform for metal free Russian propolis ethanol extracts designed specifically for biomaterials

The antibacterial and anti-biofilm activities of propolis have been intensively reported. However, the application of this folk remedy as a means to prevent biomedical implant contamination has yet to be completely evaluated. In response to the significant resistant and infectious attributes of biofilms, biomaterials engineered to possess specific chemical and physical properties were immobilized with metal free Russian propolis ethanol extracts (MFRPEE), a known antibacterial agent. The results obtained from this study begin to examine the application of MFRPEE as a novel alternative method for the prevention of medical and biomedical implant infections. When constructed under specific experimental conditions, immobilized biomaterials showed excellent stability when subjected to simulated body fluid and fetal bovine serum. The ability of immobilized biomaterials to specifically target pathogens (both Gram-positive and Gram-negative biofilm forming bacteria), while promoting tissue cell growth, renders these biomaterials as potential candidates for clinical applications.     

Soil Carbon Turnover Measurement by Physical Fractionation at a Forest-to-Pasture Chronosequence in the Brazilian Amazon

The effect of conversion from forest-to-pasture upon soil carbon stocks has been intensively discussed, but few studies focus on how this land-use change affects carbon (C) distribution across soil fractions in the Amazon basin. We investigated this in the 20 cm depth along a chronosequence of sites from native forest to three successively older pastures. We performed a physicochemical fractionation of bulk soil samples to better understand the mechanisms by which soil C is stabilized and evaluate the contribution of each C fraction to total soil C. Additionally, we used a two-pool model to estimate the mean residence time (MRT) for the slow and active pool C in each fraction. Soil C increased with conversion from forest-to-pasture in the particulate organic matter (>250 μm), microaggregate (53–250 μm), and d-clay (<2 μm) fractions. The microaggregate comprised the highest soil C content after the conversion from forest-to-pasture. The C content of the d-silt fraction decreased with time since conversion to pasture. Forest-derived C remained in all fractions with the highest concentration in the finest fractions, with the largest proportion of forest-derived soil C associated with clay minerals. Results from this work indicate that microaggregate formation is sensitive to changes in management and might serve as an indicator for management-induced soil carbon changes, and the soil C changes in the fractions are dependent on soil texture.     

Arterially perfused neurosphere-derived cells distribute outside the ischemic core in a model of transient focal ischemia and reperfusion in vitro

BACKGROUND: Treatment with neural stem cells represents a potential strategy to improve functional recovery of post-ischemic cerebral injury. The potential benefit of such treatment in acute phases of human ischemic stroke depends on the therapeutic viability of a systemic vascular delivery route. In spite of the large number of reports on the beneficial effects of intracerebral stem cells injection in experimental stroke, very few studies demonstrated the effectiveness of the systemic intravenous delivery approach. METODOLOGY/PRINCIPAL FINDINGS: We utilized a novel in vitro model of transient focal ischemia to analyze the brain distribution of neurosphere-derived cells (NCs) in the early 3 hours that follow transient occlusion of the medial cerebral artery (MCA). NCs obtained from newborn C57/BL6 mice are immature cells with self-renewal properties that could differentiate into neurons, astrocytes and oligodendrocytes. MCA occlusion for 30 minutes in the in vitro isolated guinea pig brain preparation was followed by arterial perfusion with 1x10(6) NCs charged with a green fluorescent dye, either immediately or 60 minutes after reperfusion onset. Changes in extracellular pH and K(+) concentration during and after MCAO were measured through ion-sensitive electrodes. CONCLUSION/SIGNIFICANCE: It is demonstrated that NCs injected through the vascular system do not accumulate in the ischemic core and preferentially distribute in non-ischemic areas, identified by combined electrophysiological and morphological techniques. Direct measurements of extracellular brain ions during and after MCA occlusion suggest that anoxia-induced tissue changes, such as extracellular acidosis, may prevent NCs from entering the ischemic area in our in vitro model of transitory focal ischemia and reperfusion suggesting a role played by the surrounding microenviroment in driving NCs outside the ischemic core. These findings strongly suggest that the potential beneficial effect of NCs in experimental focal brain ischemia is not strictly dependent on their homing into the ischemic region, but rather through a bystander mechanism possibly mediated by the release of neuroprotective factors in the peri-infarct region.     

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.     

Antidepressant-like effect of a Ginkgo biloba extract (EGb761) in the mouse forced swimming test: role of oxidative stress

EGb761 is a well-defined mixture of active compounds extracted from Ginkgo biloba leaves. This extract is used clinically due to its neuroprotective effects, exerted probably via its potent antioxidant or free radical scavenger action. Previous studies suggest that oxidative stress, via free radical production, may play an important role in depression and animal models for depression-like behavior. Preclinical studies have suggested that antioxidants may have antidepressants properties. The aim of this study was to investigate the antidepressant-like of EGb761 due to its antioxidant role against oxidative stress induced in the forced swimming test, the most widely used preclinical model for assessing antidepressant-like behavior. Male BALB/c mice were pretreated with EGb761 (10 mg/kg, ip) daily for 17 days followed by the forced swimming test and spontaneous locomotor activity. Animals were sacrificed to evaluate lipid peroxidation, different antioxidant enzyme activities, serotonin and dopamine content in midbrain, hippocampus and prefrontal cortex. EGb761 significantly decreased the immobility time (39%) in the forced swimming test. This antidepressant-like effect of EGb761 was associated with a reduction in lipid peroxidation and superoxide radical production (indicated by a downregulation of Mn-superoxide dismutase activity), both of which are indicators of oxidative stress. The protective effect of EGb761 is not related to excitatory or inhibitory effects in locomotor activity, and was also associated with the modulation of serotonergic and dopaminergic neurotransmission. It is suggested that EGb761 produces an antidepressant-like effect, and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

Molecular Characterization of Microbial Population Dynamics during Sildenafil Citrate Degradation

Little is known about pharmaceutical and personal care products pollutants (PPCPs), but there is a growing interest in how they might impact the environment and microbial communities. The widespread use of Viagra (sildenafil citrate) has attracted great attention because of the high usage rate, the unpredictable disposal and the unknown potential effects on wildlife and the environment. Until now information regarding the impact of Viagra on microbial community in water environment has not been reported. In this research, for the first time, the genetic profile of the microbial community, developing in a Viagra polluted water environment, was evaluated by means of the 16S and 18S rRNA genes, for bacteria and fungi, respectively, amplified by polymerase chain reaction (PCR) and separated using the denaturing gradient gel electrophoresis (DGGE) technique. The DGGE results revealed a complex microbial community structure with most of the population persisting throughout the experimental period. DNA sequences from bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria and fungi found previously mainly in polluted environmental and treating bioreactors. Biotransformation ability of sildenafil citrate by the microbial pool was studied and the capability of these microorganisms to detoxify a polluted water ecosystem was assessed. The bacterial and fungal population was able to degrade sildenafil citrate entirely. Additionally, assays conducted on Daphnia magna, algal growth inhibition assay and cell viability determination on HepG2 human cells showed that biotransformation products obtained from the bacterial growth was not toxic. The higher removal efficiency for sildenafil citrate and the lack of toxicity by the biotransformation products obtained showed that the microbial community identified here represented a composite population that might have biotechnological relevance to retrieve sildenafil citrate contaminated sites.     

A New Species of Thraulodes (Ephemeroptera: Leptophlebiidae) from Mexico

A new species of Thraulodes from Mexico is described from male and female imagines. The male imagines of this species have very characteristic genitalia that allow its easy separation from the other known species of the genus.     

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.