Managing Culturally Significant Land: The Badger-Two Medicine Area as a Case Study

Located in Helena-Lewis and Clark National Forest, Badger-Two Medicine area (B2M) spans 130,000 acres and is situated on the Rocky Mountain Front. The area borders the Blackfeet Reservation, Glacier National Park, and Great Bear and Bob Marshall Wilderness areas. B2M possesses immense cultural and spiritual importance for the Blackfeet tribe, who have occupied the area since time immemorial. The area is also one of the last remaining refuges for vulnerable fish and wildlife species such as westslope cutthroat, grizzly bears, wolverines, and mountain goats. Altogether, B2M possesses vast spiritual, cultural, and ecological importance throughout. However, extractive development, disagreements over protection status, and varying tribal and federal interests have created management complications within its boundaries. Through an education-based project that focuses on communication and understanding, we explored current and future policy actions, opinions, and considerations for managing B2M’s culturally significant land. Our research involves interviewing tribal, federal, state, private, and non-profit stakeholders in the area to better understand where each group stands on current and future management actions in the area, and culturally significant land more broadly. Further, we will also host a Zoom panel in April consisting of individuals representing tribal, federal, conservation, and academic areas of expertise regarding B2M. In doing so, we hope to facilitate an informal yet informative discussion regarding management actions that inform the public and decision makers. We hope that our project will convey the complexities of managing culturally significant land, yet also inform others of the B2M’s management landscape. Communication between relevant groups and accessible information appears to be lacking, and this panel would help communicate the nuanced and broad distinctions between policy approaches to managing B2M. Through our case study, we also aim to provoke a wider discussion on policies pertaining to culturally significant land in other areas of the U.S. and globally

A Stiff Injectable Biodegradable Elastomer

Injectable materials often have shortcomings in mechanical and drug-eluting properties that are attributable to their high water contents. A water-free, liquid four-armed PEG modified with dopamine end groups is described which changes from liquid to elastic solid by reaction with a small volume of Fe3+ solution. The elastic modulus and degradation times increase with increasing Fe3+ concentrations. Both the free base and the water-soluble form of lidocaine can be dissolved in the PEG4-dopamine and released in a sustained manner from the cross-linked matrix. PEG4-dopamine is retained in the subcutaneous space in vivo for up to 3 weeks with minimal inflammation. This material's tailorable mechanical properties, biocompatibility, ability to incorporate hydrophilic and hydrophobic drugs and release them slowly are desirable traits for drug delivery and other biomedical applications.National Institute on Deafness and Other Communication Disorders (U.S.) (NIDCD R21 DC 009986)National Institutes of Health (U.S.) (NIH Ruth L. Kirschstein National Research Service Award (no. F32GM096546))National Institutes of Health (U.S.) (NIH R01 EB00244

Ribonucleotide reductases of Salmonella Typhimurium : transcriptional regulation and differential role in pathogenesis

Ribonucleotide reductases (RNRs) are essential enzymes that carry out the de novo synthesis of deoxyribonucleotides by reducing ribonucleotides. There are three different classes of RNRs (I, II and III), all having different oxygen dependency and biochemical characteristics. Salmonella enterica serovar Typhimurium (S. Typhimurium) harbors class Ia, class Ib and class III RNRs in its genome. We have studied the transcriptional regulation of these three RNR classes in S. Typhimurium as well as their differential function during infection of macrophage and epithelial cells. Deletion of both NrdR and Fur, two main transcriptional regulators, indicates that Fur specifically represses the class Ib enzyme and that NrdR acts as a global repressor of all three classes. A Fur recognition sequence within the nrdHIEF promoter has also been described and confirmed by electrophoretic mobility shift assays (EMSA). In order to elucidate the role of each RNR class during infection, S. Typhimurium single and double RNR mutants (as well as Fur and NrdR mutants) were used in infection assays with macrophage and epithelial cell lines. Our results indicate class Ia to be mainly responsible for deoxyribonucleotide production during invasion and proliferation inside macrophages and epithelial cells. Neither class Ib nor class III seem to be essential for growth under these conditions. However, class Ib is able to maintain certain growth in an nrdAB mutant during the first hours of macrophage infection. Our results suggest that, during the early stages of macrophage infection, class Ib may contribute to deoxyribonucleotide synthesis by means of both an NrdR and a Fur-dependent derepression of nrdHIEF due to hydrogen peroxide production and DNA damage associated with the oxidative burst, thus helping to overcome the host defenses

High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells

Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (VH) and light (VL) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of VH and VL domains, further validating the here-reported expression system

DnaC Inactivation in Escherichia coli K-12 Induces the SOS Response and Expression of Nucleotide Biosynthesis Genes

Background: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations. Methodology/Principal Findings: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38uC and 42uC. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation. Conclusion/Significance: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart

In situ conjugation of dithiophenol maleimide polymers and oxytocin for stable and reversible polymer–peptide conjugates

The in situ one-pot synthesis of peptide–polymer bioconjugates is reported. Conjugation occurs efficiently without the need for purification of dithiophenol maleimide functionalized polymer as a disulfide bridging agent for the therapeutic oxytocin. Conjugation of polymers was demonstrated to be reversible and to significantly improve the solution stability of oxytocin

Promoter Complexity and Tissue-Specific Expression of Stress Response Components in Mytilus galloprovincialis, a Sessile Marine Invertebrate Species

The mechanisms of stress tolerance in sessile animals, such as molluscs, can offer fundamental insights into the adaptation of organisms for a wide range of environmental challenges. One of the best studied processes at the molecular level relevant to stress tolerance is the heat shock response in the genus Mytilus. We focus on the upstream region of Mytilus galloprovincialis Hsp90 genes and their structural and functional associations, using comparative genomics and network inference. Sequence comparison of this region provides novel evidence that the transcription of Hsp90 is regulated via a dense region of transcription factor binding sites, also containing a region with similarity to the Gamera family of LINE-like repetitive sequences and a genus-specific element of unknown function. Furthermore, we infer a set of gene networks from tissue-specific expression data, and specifically extract an Hsp class-associated network, with 174 genes and 2,226 associations, exhibiting a complex pattern of expression across multiple tissue types. Our results (i) suggest that the heat shock response in the genus Mytilus is regulated by an unexpectedly complex upstream region, and (ii) provide new directions for the use of the heat shock process as a biosensor system for environmental monitoring

Understanding Marine Mussel Adhesion

In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion

Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein

Marine mussels secret protein-based adhesives, which enable them to anchor to various surfaces in a saline, intertidal zone. Mussel foot proteins (Mfps) contain a large abundance of a unique, catecholic amino acid, Dopa, in their protein sequences. Catechol offers robust and durable adhe-sion to various substrate surfaces and contributes to the curing of the adhesive plaques. In this article, we review the unique features and the key functionalities of Mfps, catechol chemistry, and strategies for preparing catechol-functionalized poly- mers. Specifically, we reviewed recent findings on the contributions of various features of Mfps on interfacial binding, which include coacervate formation, surface drying properties, control of the oxidation state of catechol, among other features. We also summarized recent developments in designing advanced biomimetic materials including coacervate-forming adhesives, mechanically improved nano- and micro-composite adhesive hydrogels, as well as smart and self-healing materials. Finally, we review the applications of catechol-functionalized materials for the use as biomedical adhesives, therapeutic applications, and antifouling coatings

Toxin-Based Therapeutic Approaches

Protein toxins confer a defense against predation/grazing or a superior pathogenic competence upon the producing organism. Such toxins have been perfected through evolution in poisonous animals/plants and pathogenic bacteria. Over the past five decades, a lot of effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes that neutralize their action. In parallel, many research groups turned to explore the pharmaceutical potential of such toxins when they are used to efficiently impair essential cellular processes and/or damage the integrity of their target cells. The following review summarizes major advances in the field of toxin based therapeutics and offers a comprehensive description of the mode of action of each applied toxin