Treetop: A Shiny-based application and R package for extracting forest information from LiDAR data for ecologists and conservationists

Individual tree detection (ITD) and crown delineation are two of the most relevant methods for extracting detailed and reliable forest information from LiDAR (Light Detection and Ranging) datasets. However, advanced computational skills and specialized knowledge have been normally required to extract forest information from LiDAR.The development of accessible tools for 3D forest characterization can facilitate rapid assessment by stakeholders lacking a remote sensing background, thus fostering the practical use of LiDAR datasets in forest ecology and conservation. This paper introduces the treetop application, an open-source web-based and R package LiDAR analysis tool for extracting forest structural information at the tree level, including cutting-edge analyses of properties related to forest ecology and management.We provide case studies of how treetop can be used for different ecological applications, within various forest ecosystems. Specifically, treetop was employed to assess post-hurricane disturbance in natural temperate forests, forest homogeneity in industrial forest plantations and the spatial distribution of individual trees in a tropical forest.treetop simplifies the extraction of relevant forest information for forest ecologists and conservationists who may use the tool to easily visualize tree positions and sizes, conduct complex analyses and download results including individual tree lists and figures summarizing forest structural properties. Through this open-source approach, treetop can foster the practical use of LiDAR data among forest conservation and management stakeholders and help ecological researchers to further understand the relationships between forest structure and function.The authors thank Nicholas L. Crookston for co‐developing the web‐LiDAR treetop tool, and the two anonymous reviewers for their helpful suggestions on the first version of the manuscript. This study is based on the work supported by the Department of Defence Strategic Environmental Research and Development Program (SERDP) under grants No. RC‐2243, RC19‐1064 and RC20‐1346 and USDA Forest Service (grand No. PRO00031122

Prevention and Mitigation of Acute Radiation Syndrome in Mice by Synthetic Lipopeptide Agonists of Toll-Like Receptor 2 (TLR2)

Bacterial lipoproteins (BLP) induce innate immune responses in mammals by activating heterodimeric receptor complexes containing Toll-like receptor 2 (TLR2). TLR2 signaling results in nuclear factor-kappaB (NF-κB)-dependent upregulation of anti-apoptotic factors, anti-oxidants and cytokines, all of which have been implicated in radiation protection. Here we demonstrate that synthetic lipopeptides (sLP) that mimic the structure of naturally occurring mycoplasmal BLP significantly increase mouse survival following lethal total body irradiation (TBI) when administered between 48 hours before and 24 hours after irradiation. The TBI dose ranges against which sLP are effective indicate that sLP primarily impact the hematopoietic (HP) component of acute radiation syndrome. Indeed, sLP treatment accelerated recovery of bone marrow (BM) and spleen cellularity and ameliorated thrombocytopenia of irradiated mice. sLP did not improve survival of irradiated TLR2-knockout mice, confirming that sLP-mediated radioprotection requires TLR2. However, sLP was radioprotective in chimeric mice containing TLR2-null BM on a wild type background, indicating that radioprotection of the HP system by sLP is, at least in part, indirect and initiated in non-BM cells. sLP injection resulted in strong transient induction of multiple cytokines with known roles in hematopoiesis, including granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin-6 (IL-6). sLP-induced cytokines, particularly G-CSF, are likely mediators of the radioprotective/mitigative activity of sLP. This study illustrates the strong potential of LP-based TLR2 agonists for anti-radiation prophylaxis and therapy in defense and medical scenarios

Protein and Cell Surface Engineering with Oxime Conjugates

In the biological and medical fields, there is a heavy reliance on using genetic tools to manipulate proteins and investigate cellular functions. While these methods have proven hugely successful, they are limited in their ability to introduce novel functionality into biomolecules or interrogate the distinct chemical entities that control cellular responses. To address these restrictions, chemists have developed a set of chemoselective reactions with minimal cross-reactivity and toxicity in biological systems; termed the bioorthogonal reaction. The work in this dissertation has centered on the use of one such chemistry, oxime formation, which is obtained by the reaction of an aminooxy functionality with the carbonyl group of a ketone or aldehyde. I have utilized this chemistry to create unique protein assemblies (Chapter 1), site-specifically glycosylate proteins (Chapter 3,4), and introduce homogeneous glycans onto cell surfaces (Chapter 5). An emphasis of this work is the generation of homogeneous glycoconjugates that can be utilized to study the role of certain glycan structures in biology. This includes advancing methodology in cell surface modification for introducing multivalent glycomimics onto cellular membranes as well as utilizing protein modification techniques to create homogeneous glycoproteins. Chapter 1 outlines my work on utilizing the genetically encoded consensus sequence termed the aldehyde tag to introduce a bioorthogonal carbonyl group onto protein backbones. The technique exploits the known function of the formylglycine generating enzyme to oxidize the thiol group of a cysteine residue within a specific peptide sequence to an aldehyde forming the non-natural amino acid, formylglycine. My work has focused on optimizing the conjugation of small molecules and large proteins to the formylglycine residue to create new protein scaffolds for studying protein function and for the creation of improved biotherapeutics. The remaining chapters reside largely in the realm of glycobiology and thus Chapter 2 is devoted to providing a brief overview of the field specifically as it pertains to the role of glycans in modulating and defining medical treatment. Many therapeutics consist of glycoproteins or glycans themselves and though they have been used for decades, only recently have scientists been able to understand and engineer these treatments at the molecular level. As more tools become available to study how the location and chemical makeup of saccharides affect their function, we have begun to see a better appreciation of the significant part that glycans play in medicine. This will presumably lead to a surgence in novel types of treatment that rely solely on the function of specific glycan structures. In the pursuit of creating new methods to define the role of glycan structures on protein function, Chapter 3 presents the use of the aldehyde tag technology to conjugate a wide range of chemically synthesized glycans to proteins. This approach relies on the development of an improved synthetic route of aminooxy glycans that utilizes chemical and enzymatic synthesis to create a small library of aminooxy sugars. I also show that these glycans can be conjugated site-specifically to the human growth hormone to create a novel set of chemically glycosylated therapeutic proteins. This system was taken a step further by placing the aldehyde tag on the capsid of human adenovirus as a conjugation point to attach glycan targeting moieties to the virus for gene therapy. The glycosylated protein should then retarget the virus to infect specific leukocyte lineages based on their unique expression of lectins that have specific glycan binding preferences. More work is needed to demonstrate the ability to direct the virus to infect nonnative cellular types such as leukocytes. The aminooxy glycans generated thus far have been limited to small concise structures that one would normally find on the antennary regions of a full-length glycoconjugate. Chapter 4 outlines new work to create chemical mimics of full-length N-glycans with established dendrimer chemistry. We produced a novel glycodendrimer structure that consists of a piperidine-melamine core decorated with monosaccharides at the antennary and an aminooxy functionality for site-specific conjugation to aldehyde-tagged proteins. We have successfully conjugated a biantennary N-glycan mimic to an hGH construct and have begun work to create a IFN-β "glycodendriprotein" as a biotherapeutic surrogate. The final chapter is the culmination of my thesis work, where we have utilized phospholipid containing glycopolymers to engineer cell surfaces with chemically defined glycan structures to study their role in regulating cellular activities. Chapter 5 focuses on the use of this approach to define the role of Siglec-7 on regulating the activity of Natural Killer (NK) cells to target and kill cancer cells. The overexpression of sialic acid has been well documented for many human cancers but few reports have provided the molecular details for how this provides a selective advantage to cancer cells to survive. We provide evidence that increasing sialosides on cancer cell surfaces can engage the inhibitory receptor Siglec-7 on NK cells and dampen the ability of NK cells to recognize and kill tumorigenic cells

Protein Glycoengineering Enabled by the Versatile Synthesis of Aminooxy Glycans and the Genetically Encoded Aldehyde Tag

Homogeneously glycosylated proteins are important targets for fundamental research and for biopharmaceutical development. The use of unnatural protein–glycan linkages bearing structural similarity to their native counterparts can accelerate the synthesis of glycoengineered proteins. Here we report an approach toward generating homogeneously glycosylated proteins that involves chemical attachment of aminooxy glycans to recombinantly produced proteins via oxime linkages. We employed the recently introduced aldehyde tag method to obtain a recombinant protein with the aldehyde-bearing formylglycine residue at a specific site. Complex aminooxy glycans were synthesized using a new route that features <i>N</i>-pentenoyl hydroxamates as key intermediates that can be readily elaborated chemically and enzymatically. We demonstrated the method by constructing site-specifically glycosylated variants of the human growth hormone

Illuminating Vital Surface Molecules of Symbionts in Health and Disease

The immunomodulatory surface molecules of commensal and pathogenic bacteria are critical to the microbe’s survival and the host’s response.1,2 Recent studies have highlighted the unique and important responses elicited by commensal-derived surface macromolecules;3–5 however, the technology available to track these molecules in host cells and tissues remains primitive. We report here an interdisciplinary approach that uses metabolic labeling combined with bioorthogonal click chemistry (i.e., reactions performed in living organisms)6 to specifically tag up to three prominent surface immunomodulatory macromolecules – peptidoglycan (PGN), lipopolysaccharide (LPS), and capsular polysaccharide (CPS) – either simultaneously or individually in live anaerobic commensal bacteria. Importantly, the PGN labeling enables for the first time the specific labeling of live endogenous, anaerobic bacteria within the mammalian host. This approach has allowed us to image and track the path of labeled surface molecules from live, luminal bacteria into specific intestinal immune cells in the living murine host during health and disease. The chemical labeling of three specific macromolecules within a live organism offers the potential for in-depth visualization of host-pathogen interactions

Scapular spine stress fractures: To fix or not to fix, our experience in a patient with bilateral fractures and review of the literature

We report a case of bilateral scapular spine stress fracture, treated conservatively on one side and operatively on the other side. Besides, we performed a literature review to establish management options. A 61-year-old right-handed gentleman came to our clinic with acute on chronic deterioration of shoulder pain and loss of arm function. Clinical assessment and investigations revealed long-standing bilateral rotator cuff tear and scapular spine stress fractures. The fracture on the right side united with conservative management for 2 months. However, his left side remained symptomatic with pain, abnormal mobility and no radiological evidence of union. The fracture progressed to union after fixation and bone grafting. At the final follow-up at 2 years, the patient was asymptomatic with regards to the fractures with Oxford Shoulder Score (OSS)-30 and Disabilities of the Arm, Shoulder and Hand (DASH)-30.8. Fracture union either by conservative or operative treatment is associated with good functional outcome and is supported by our review