Genetically Engineered Alginate Lyase-PEG Conjugates Exhibit Enhanced Catalytic Function and Reduced Immunoreactivity

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.Cystic Fibrosis Foundation (Research Development Program)National Center for Research Resources (U.S.) (P20RR018787-06

Harmful Elements in Estuarine and Coastal Systems

Estuaries and coastal zones are dynamic transitional systems which provide many economic and ecological benefits to humans, but also are an ideal habitat for other organisms as well. These areas are becoming contaminated by various anthropogenic activities due to a quick economic growth and urbanization. This chapter explores the sources, chemical speciation, sediment accumulation and removal mechanisms of the harmful elements in estuarine and coastal seawaters. It also describes the effects of toxic elements on aquatic flora and fauna. Finally, the toxic element pollution of the Venice Lagoon, a transitional water body located in the northeastern part of Italy, is discussed as a case study, by presenting the procedures adopted to measure the extent of the pollution, the impacts on organisms and the restoration activities

Perinatal induction of Cre recombination with tamoxifen

Temporal control of site-specific recombination is commonly achieved by using a tamoxifen-inducible form of Cre or Flp recombinases. Although powerful protocols of induction have been developed for gene inactivation at adult stages or during embryonic development, induction of recombination at late gestational or early postnatal stages is still difficult to achieve. In this context, using the ubiquitous CMV-CreERT2 transgenic mice, we have tested and validated two procedures to achieve recombination just before and just after birth. The efficiency of recombination was evaluated in the brain, which is known to be more problematic to target. For the late gestation treatment with tamoxifen, different protocols of complementary administration of progesterone and estrogen were tested. However, delayed delivery and/or mortality of pups due to difficult delivery were always observed. To circumvent this problem, pups were collected from tamoxifen-treated pregnant dams by caesarian section at E18.5 and given to foster mothers. For postnatal treatment, different dosages of tamoxifen were administered by intragastric injection to the pups during 3 or 4 days after birth. The efficiency of these treatments was analyzed at P7 using a transgenic reporter line. They were also validated with the Hoxa5 conditional allele. In conclusion, we have developed efficient procedures that allow achieving efficient recombination of floxed alleles at perinatal stages. These protocols will allow investigating the late/adult functions of many developmental genes, whose characterization has been so far restricted to embryonic development

Impact of sedimentation history for As distribution in Late Pleistocene-Holocene sediments in the Hetao Basin, China

PURPOSE: To understand the impact of geochemical sedimentation history for arsenic (As) distribution in the sediment profiles of the Hetao Basin, we (1) evaluated sediments provenance and variations of weathering intensities, (2) attempted to reconstruct the depositional environments, and (3) explored the As and Fe speciation in the sediments. Combining the information above, different sedimentation facies were distinguished in the vertical profiles. METHODS: Two sediments cores were drilled up to 80 m depth. Major and trace element compositions, including rare earth elements (REE), were analyzed. Carbon isotope ratios (δ¹³Cₒᵣg) of embedded organic matter in the sediments were analyzed by isotope ratio mass spectrometry (IR-MS). Arsenic and Fe speciation of the sediments were determined by sequential extractions. RESULTS AND DISCUSSION: The similar REE geochemistry of rocks from the Lang Mountains and sediments in the Hetao Basin indicated that the sediments originated from the Lang Mountains. The C/N ratio (~ 4 to ~ 10) in combination with δ¹³Cₒᵣg (− 27‰ to −2 4‰) suggested that sediments were mainly deposited in aquatic environments. The unconfined aquifer equaled the lacustrine deposit with less intensive weathering during last glacial maximum (LGM). Here, the As content (average, 5.4 mg kg⁻¹) was higher than in the aquifer sediments below (average, 3.6 mg kg⁻¹). CONCLUSION: Higher content of releasable As in combination with paleolake-derived organic matter aquifer sediments probably contributes to higher groundwater As concentration in the unconfined aquifer. This study provides the first insight into the impact of sedimentation history on As distributions in sediment profiles in the Hetao Basin