Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2

The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.     

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.     

Randomized comparison of two non-bismuth-containing second-line rescue therapies for Helicobacter pylori

Background: Classical second‐line anti‐Helicobacter pylori includes proton‐pump inhibitor, tetracycline, metronidazole, and bismuth salts, but alternative therapies are required owing to the restricted availability of the latter. Levofloxacin‐containing triple therapy is recommended but is expensive. Besides, quinolone resistance in an endemic tuberculosis infection area like Taiwan is concerned. The low in vitro antibiotic resistance to amoxicillin and tetracycline in Taiwanese H. pylori strains implies that in vivo esomeprazole/amoxicillin/tetracycline (EAT) second‐line rescue therapy may be effective. This study compared the efficacy of esomeprazole/amoxicillin/levofloxacin (EAL) and EAT second‐line eradication therapies and determines the clinical factors influencing the efficacy of salvage regimens. Materials and methods: One hundred and twenty‐eight patients who failed H. pylori eradication using the standard triple therapy for 7 days are randomly assigned to either EAL group (esomeprazole 40 mg twice daily, amoxicillin 1 g twice daily, and levofloxacin 500 mg once daily) for 7 days or EAT group (esomeprazole 40 mg twice daily, amoxicillin 1 g twice daily, tetracycline 500 mg four times daily) for 14 days. Follow‐up endoscopy or urea breath test was performed 8 weeks later to assess treatment response. Results: The eradication rates of EAL and EAT groups were 78.1 versus 75.0%, p = .676 (in intention‐to‐treat analysis) and 80.3 versus 80%, p = .0964 (per‐protocol analysis). Both groups exhibited similar drug compliance (95.3 vs 96.9%, p = .952) but more adverse events in the EAT group (6.3 vs 12.5%, p = .225). Conclusions: Despite low in vitro drug resistances to amoxicillin and tetracycline, the efficacy of 14‐day EAT regimens attained an unacceptable report card of 75% eradication rates in intention‐to‐treat analysis and was not even superior to the 7‐day EAL regimen. Drug–drug interaction between combined antibiotics should be considered other than in vivo drug resistances.     

Impact of mucosal inflammation on cervical human immunodeficiency virus (HIV-1)-specific CD8 T-cell responses in the female genital tract during chronic HIV infection

The female genital tract is the major route of heterosexual human immunodeficiency virus (HIV) acquisition and transmission. Here, we investigated whether HIV-specific CD8 T-cell-mediated immune responses could be detected in the genital mucosa of chronically HIV-infected women and whether these were associated with either local mucosal HIV shedding or local immune factors. We found that CD8⁺ T-cell gamma interferon responses to Gag were detectable at the cervix of HIV-infected women but that the magnitude of genital responses did not correlate with those similarly detected in blood. This indicates that ex vivo HIV responses in one compartment may not be predictive of those in the other. We found that increased genital tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) levels correlated significantly with levels of Gag-specific CD8⁺ T cells at the cervix. Women who were detectably shedding virus in the genital tract had significantly increased cervical levels of TNF-α, IL-1β, IL-6, and IL-8 compared to women who were not detectably shedding virus. We were, however, unable to detect any association between the magnitude of cervical HIV-specific responses and mucosal HIV shedding. Our results support the hypothesis that proinflammatory cytokines in the female genital tract may promote HIV replication and shedding. In addition, we further show that inflammatory cytokines are associated with increased levels of HIV-specific CD8 effector cells at the genital mucosa but that these were not able to control genital HIV shedding.     

Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.     

Intraperitoneal implantation of life-long telemetry transmitters in otariids

Background

Pinnipeds, including many endangered and declining species, are inaccessible and difficult to monitor for extended periods using externally attached telemetry devices that are shed during the annual molt. Archival satellite transmitters were implanted intraperitoneally into four rehabilitated California sea lions (Zalophus californianus) and 15 wild juvenile Steller sea lions (Eumetopias jubatus) to determine the viability of this surgical technique for the deployment of long-term telemetry devices in otariids. The life history transmitters record information throughout the life of the host and transmit data to orbiting satellites after extrusion following death of the host.

Results

Surgeries were performed under isoflurane anesthesia and single (n = 4) or dual (n = 15) transmitters were inserted into the ventrocaudal abdominal cavity via an 8.5 to 12 cm incision along the ventral midline between the umbilicus and pubic symphysis or preputial opening. Surgeries lasted 90 minutes (SD = 8) for the 19 sea lions. All animals recovered well and were released into the wild after extended monitoring periods from 27 to 69 days at two captive animal facilities. Minimum post-implant survival was determined via post-release tracking using externally attached satellite transmitters or via opportunistic re-sighting for mean durations of 73.7 days (SE = 9.0, Z. californianus) and 223.6 days (SE = 71.5, E. jubatus).

Conclusion

The low morbidity and zero mortality encountered during captive observation and post-release tracking periods confirm the viability of this surgical technique for the implantation of long-term telemetry devices in otariids.

     

Effect of fixation period on HER2/neu gene amplification detected by fluorescence in situ hybridization in invasive breast carcinoma.

This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration.