N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor

A series of potent antagonists of the ion channel transient receptor potential A1 (TRPA1) was developed by modifying lead structure 16 that was discovered by high-throughput screening. Based on lead compound 16, a SAR was established, showing a narrow region at the nitro-aromatic R(1) moiety and at the warhead, while the R(2) side had a much wider scope including ureas and carbamates. Compound 16 inhibits Ca(2+)-activated TRPA1 currents reversibly in whole cell patch clamp experiments, indicating that under in vivo conditions, it does not react covalently, despite its potentially electrophilic ketone.     

Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior

Background

Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity.

Methods

MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses.

Results

After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months.

Conclusions

These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.     

The antibody mining toolbox: An open source tool for the rapid analysis of antibody repertoires

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.     

Resource attractiveness of the male beaugregory damselfish and his decision to court or defend

We examined the decisions territorial male beaugregory damselfish (Stegastes leucostictus) make when presented with mating and aggressive situations simultaneously. Specifically, we testedhow males responded to simultaneous and consecutive presentationsof conspecific males and females in bottles. We hypothesizedthat (1) territorial males would show lower levels of courtshiptoward females when a competing male is also present comparedto when only the female is present and (2) territorial maleswould show higher levels of aggression toward intruding maleswhen a female is also present on their territories than whenonly the intruder is present. We predicted that males withhigh-quality breeding sites would court females and attackcompetitors more vigorously than males with low-quality breedingsites. Males on low-quality breeding sites exhibited fewerconspicuous courtship displays toward bottled females and alower bite rate toward bottled males than did the males onhigh-quality breeding sites. We concluded that poor breeding-sitequality was responsible for the decline in the most conspicuous aspects of both courtship and aggression. When presented simultaneouslywith a bottled male and a bottled female, defenders of bothbreeding-site types drastically reduced the number of courtshipdisplays and the amount of time they spent near the female.Thus, all aspects of courtship were reduced in the presenceof a male intruder. Time spent near the bottled male also decreased in these simultaneous presentations; however, this decreasewas less dramatic than the reduction in time spent near bottledfemales. Habitat quality did not affect these trade-off decisions,but the bite rate exhibited toward the bottled male increasedin the simultaneous presentations compared to the single presentations.Due to this increase in a territorial male's defensive behaviorwhen a female is present, we conclude that the presence of afemale temporarily increased the value of a territory. We suggestthat the possible loss of a territory to an intruding maletakes priority over the potential benefits of successfullycourting a single female and that it is a male's future reproduction,as well as his current reproduction, by which he assesses hisbenefits.     

Shuttle vector expression in Thermococcus kodakaraensis: contributions of cis elements to protein synthesis in a hyperthermophilic archaeon

Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli have been constructed. Plasmid pTN1 from Thermococcus nautilis was ligated to the commercial vector pCR2.1-TOPO, and selectable markers were added so that T. kodakaraensis transformants could be selected by DeltatrpE complementation and/or mevinolin resistance. Based on Western blot measurements, shuttle vector expression of RpoL-HA, a hemagglutinin (HA) epitope-tagged subunit of T. kodakaraensis RNA polymerase (RNAP), was approximately 8-fold higher than chromosome expression. An idealized ribosome binding sequence (5'-AGGTGG) was incorporated for RpoL-HA expression, and changes to this sequence reduced expression. Changing the translation initiation codon from AUG to GUG did not reduce RpoL-HA expression, but replacing AUG with UUG dramatically reduced RpoL-HA synthesis. When functioning as translation initiation codons, AUG, GUG, and UUG all directed the incorporation of methionine as the N-terminal residue of RpoL-HA synthesized in T. kodakaraensis. Affinity purification confirmed that an HA- plus six-histidine-tagged RpoL subunit (RpoL-HA-his(6)) synthesized ectopically from a shuttle vector was assembled in vivo into RNAP holoenzymes that were active and could be purified directly from T. kodakaraensis cell lysates by Ni(2+) binding and imidazole elution.     

How does a parent respond when its mate emphasizes the wrong role? A test using a monogamous fish

The convict cichlid fish, Archocentrus nigrofasciatus, is biparental: the male spends the majority of his time defending the territory and the female spends much of her time close to the offspring. Under natural conditions, this separation into sex-typical roles is somewhat blurred as males do spend some time with the offspring and females do attack intruders. Here we tested whether an individual selects a parental role based on the location (i.e. parental role) of its mate. For example, do females emphasize offspring care because the male is away from the offspring? Will males be more likely to care for the offspring when the female is away from the offspring? We manipulated the location of one parent by placing it in a transparent plastic box, either near the offspring or at the far end of the tank near a clear plastic compartment that held a conspecific male intruder. We tested both male and female parent under the following four conditions: boxed mate near offspring with no intruder present, boxed mate near offspring with intruder present, boxed mate near intruder compartment with no intruder present, and boxed mate near intruder compartment with intruder present. We found that both parents spent more time with the offspring and less time attacking the intruder when the mate was positioned near the offspring and more time away from the offspring and more time attacking the intruder when the mate was near the intruder. Males were more affected by the location of their mates than were females and we concluded that males were attracted both to their mates' location and their offspring while females were mostly attracted to their offspring. Overall, the location of the mate had little effect on the types of aggressive activities used against the intruder. We did find that males increased their aggression towards boxed females when they were positioned far from the offspring, whereas the aggressive behaviour of females towards boxed males when they were positioned near the offspring was ambiguous. We suggest that males in particular enforce the separation of sex-specific parental roles via this aggression.     

Role of the JAK/STAT signal transduction pathway in the regulation of gene expression in CNS

Over the last 20 years the JAK/STAT signal transduction pathway has been extensively studied. An enormous amount of data on different cell signal transduction pathways is now available. The JAK/STAT signal transduction pathway is one of the intracellular signaling pathways activated by cytokines and growth factors that was first studied in the hematopoietic system, but recent data demonstrate that this signal transduction is also greatly utilized by other systems. The JAK/STAT pathway is a signaling cascade that links the activation of specific cell membrane receptors to nuclear gene expression. This review is focused on the role of JAK/STAT signal transduction pathway activation in the central nervous system (CNS).