Ecology and Evolutionary Biology
Understanding the consequences of environmental change for the microbial regulation of carbon and nutrient cycling is a critical need. We collected data on extracellular enzyme activities from an 18-month experiment where microbial communities were reciprocally transplanted along a Southern California elevation gradient with inverse variation in temperature and precipitation. The microbial communities were from desert, scrubland, grassland, pine-oak, and sub-alpine ecosystems. By simulating different temperature and precipitation changes for ecosystem-specific microbial communities, this study was designed to predict microbial community response and resilience to future environmental changes. Our first hypothesis proposed a “home field advantage” (HFA) of enzyme activity and litter decomposition, where microbial communities in their home ecosystem outperform microbial communities transplanted from other ecosystems. Our alternate hypothesis states that transplanted microbial communities will exhibit functional resilience by producing enzymes and decomposing litter at the same rate as native microbial communities when exposed to temperature and precipitation changes. Microbial community extracellular enzyme activities were evaluated with analysis of variance. Enzyme datasets yielding a significant (p < 0.05) site by microbial community interaction were further analyzed with Tukey post hoc comparisons within each site to compare the extracellular enzyme activities associated with different microbial communities. Our results partially supported the microbial community resilience hypothesis with respect to extracellular enzyme activity. Significant interaction effects did not support our HFA hypothesis and instead indicated a home field disadvantage, where the transplanted microbial community displayed higher extracellular enzyme activity than the native community. Our findings suggest that transplanted microbes are resilient to climate change, which may be due to their previous adaptations to extreme temperatures and drought. These enzymatic patterns are consistent with litter decomposition rates, which also did not exhibit home field advantage.
Molecular Biology and Biochemistry
Toxoplasma gondii is a food-borne obligate intracellular parasite that infects one-third of the global population. T. gondii disseminates through the bloodstream and enters the CNS via the blood-brain barrier (BBB). Recent work has demonstrated that T. gondii infects and lyses microvascular endothelial cells at the BBB. To investigate the effects of T. gondii¬ infection on the integrity and activation of the BBB, we infected C57BL/6J mice via intraperitoneal injection (i.p.) with Type II T. gondii or PBS as a control. During acute infection the mice were perfused, and the brains of control and infected mice were harvested. Parasites were detected in the brain by PCR within 9 days post-infection (dpi), and immunofluorescence microscopy of brain sections revealed elevated levels of ICAM-1, an adhesion molecule and marker of endothelial cell activation, covering greater than 10-fold more of the blood vessel area in the brains of T. gondii-infected mice compared to control mice. Increased platelet and fibrin(ogen) deposition were also observed in infected mice compared to control mice, indicating activation of the clotting cascade. Finally, cranial windows were installed in transgenic mice expressing eGFP-claudin-5 under the control of the Tie2 promoter, which allows for delineation of the tight junctions of the brain endothelial cells. Intravital two-photon imaging of the brain microvasculature in these mice during T. gondii infection demonstrated increased vessel tortuosity. Furthermore, injection of tracer dye during imaging provided evidence of vascular occlusion in T. gondii-infected mice. This research reveals an underappreciated aspect of T. gondii pathogenesis and contributes to our understanding of the effects of the parasite on the brain vasculature during infection.
Ajay Nair Sharma
School of Medicine
Skin cancer, composed of both melanoma and non-melanoma skin cancer (NMSC), is the most common type of malignancy in the United States. A number of different treatment modalities exist, including surgical excision. The primary endpoint for surgical excision of melanoma-in-situs (MIS) and NMSC’s is tumor clearance rate, determined postoperatively by the presence of tumor-free resection margins in the excised sample. This study explores whether the specialty of the treating provider and the location in which they work impacts the success of a MIS or NMSC excision.
We performed a retrospective study of 5,800 standard excisions of MIS and basal cell carcinomas (BCC, a type of NMSC) performed by dermatologists, general surgeons, otolaryngologists, and plastic surgeons from 13 different Kaiser Permanente centers. The primary outcome measure was presence of histological evidence of tumor in the surgical margins of excision specimens (incomplete excision). Statistical analysis included both univariable and multivariable models.
An incomplete excision was found in 23% of all specimens. There was no significant difference between the proportion of incomplete excisions for a BCC versus a MIS. Per specialty, the proportion of incomplete excisions was 24% for dermatology, 26% for plastic surgery, 28% for otolaryngology, and 12% for general surgery. Variables associated with a higher probability of incomplete excision included increasing age, head and neck location, smaller tumor size, and medical center location (all p<0.05). Variables with no statistically significant bearing on incomplete excision rate included sex, ethnicity, provider degree (PA or NP vs physician), and clinical experience.
Given the enormous prevalence of BCC and MIS tumors worldwide, the optimal treatment for a particular skin cancer relies on a number of factors. Factors outside the patient can influence the success of achieving tumor-free resection margins, such as the treating specialty and performing medical center.
Developmental & Cell Biology
Congenital heart defects (CHDs) are structural defects of the heart that impair the heart’s ability to pump blood throughout the body. CHDs affect approximately 1% of all individuals and are the leading cause of birth-associated deaths. Progress towards understanding why and how CHDs develop has been difficult due to the polygenic nature of most CHDs, wherein multiple genes contribute to their development. However, the study of genetic syndromes known as transcriptomopathies, in which a single gene mutation results in changes to the expression of many genes, is paving the way towards understanding the developmental origin of CHDs. Among these is Cornelia de Lange Syndrome (CdLS), the majority of which result from the loss of one functional copy of or haploinsufficiency for the gene NIPBL – the downstream effect of which changes the expression of thousands of genes. These changes are so small that each one on their own would not be expected to produce any physical defect. Yet, collectively, they cause severe physical defects throughout the whole body. Among these are heart defects, which occur at a frequency of 30% in Nipbl-haploinsufficient mice. Because heart defects in the Nipbl-haploinsufficient mouse arise from the collective effects of small gene expression changes, it can be seen as a biological model for studying the polygenic origin of CHDs. Here, I present evidence indicating that CHDs in CdLS originate from an early developmental deficiency in the number of heart progenitor cells involved in forming the heart and introduce a novel paradigm for viewing how CHDs and birth defects develop: failure to correct for early alterations in the progenitor populations responsible for forming the affected tissue/organ.
Ecology and Evolutionary Biology
Botanists have cataloged an incredible diversity of flower shapes, colors, and scents that attract different types of pollinators. Recurring suites of flower characteristics that are associated with a given pollinator group, such as birds, bats, or bees, are stereotyped as pollination “syndromes”. But what happens to these signals when plants evolve a way to transfer pollen without having to attract an animal pollinator? Repeated transitions from moth to wind and self pollination in the Hawaiian genus Schiedea allow us to ask whether floral scent was reduced following these transitions, whether scent followed similar evolutionary trajectories across independent transitions, and if there are scent compounds typical of each mode of pollination. We used dynamic headspace sampling and GC-MS to quantify the scent composition of 24 Schiedea species. By comparing these scent blends across dramatic transitions in pollination and controlling for relatedness, we determined that floral scent was reduced in amount and diversity in transitions to self and bird pollination, but not from moth to wind pollination, where floral scent surprisingly increased. There was no convergence in scent across independent pollination shifts, and highly variable scent compositions in the moth-pollinated species. We identified compounds that were associated after phylogenetic correction with moth vs. wind pollination, and others that occur in wind but not moth pollinated species. These key compounds are being assessed for their moth-attractive abilities in the field. This study shows how a complex signal has rapidly evolved as this plant lineage radiated into new habitats and courted novel pollinators or did without them.
Molecular Biology & Biochemistry
The study of rare cellular sub-types such as cancer stem cells (CSCs) at the single-cell level has skyrocketed in the last decade. Many groups study these cells, but given their low percentage in tumors, in order to do so they must be enriched for in some way. We believe that these culturing methods are likely to alter the gene expression landscape of these cells over time, further complicating their study and characterization in a medical context. A clear understanding of the molecular make-up of these cells is imperative for an accurate and complete model of cancer progression, metastasis, drug resistance and relapse. Here, we aim to characterize these putative cancer stem cell populations (from multiple tissues using an a priori determined phenotype) at the molecular level with minimal perturbation and manipulation.
We employ the Micro-pallet Array for the single-cell isolation of rare cells in a complex, heterogeneous mixture. Briefly, thousands of microscopic polymer towers are arranged in an array. Tumors are dissociated to a single-cell suspension and allowed to adhere to the top of the micro-pallets. The array is introduced to an antibody cocktail tailored to the identification of specific cellular cell-types of interest. Upon discovery of a desired cell, a laser pulse ejects the tower of interest from the rest of the array. A ferro-magnetic Ni core enables mechanical manipulation of the pallet into lysis buffer for subsequent downstream molecular analyses.
Accurate enumeration and characterization of these rare cellular CSC sub-types (and others) from patient tumor samples will serve to better equip physicians in administering therapeutics and designing treatment plans; in understanding the molecular basis behind disease progression & metastasis; and in providing novel therapeutic targets that will help eradicate the CSC pool, while sparing normal cells needed to promote tissue regeneration.
Chemistry and MB&B
As the global population continues to grow, agricultural production must increase as well. Currently, industrially-produced fertilizer is used to provide crops with essential nutrients. One component of fertilizer is a nitrogen source in the form of ammonia, which is derived from atmospheric nitrogen gas. Industrial production of nitrogen-containing fertilizer requires large amounts of energy input and generates pollution as a byproduct. However, a biological alternative can provide plants with the same important nitrogen source without the energy requirement or associated production of pollution. This system relies on a bacterial enzyme called nitrogenase. The process by which nitrogenase creates fertilizer is not well understood, but this research works to better understand nitrogenase and improve its efficiency. By studying nitrogenase, we are developing an environmentally-friendly method of fertilizer production to feed the global population.
Ecology & Evolutionary Biology
The musculoskeletal system is critical to locomotion that allows the body to safely generate and dissipate mechanical energy. Rapid decelerations upon contact with the ground are common across various modes of terrestrial locomotion including running downhill, landing from a jump and hopping. Variation in the mechanical properties of the environment can impact the rate and magnitude of energy that needs to be dissipated by the musculoskeletal system, which may have to alter strategy in order to minimize the risk of injury. Specifically, the stiffness of a substrate may be used to temporarily store energy and reduce the demand on the body to dissipate energy upon impact. Rhinella marina – the cane toad – has become a model system to understand controlled deceleration and using force-plate ergometry and high-speed videography we can compare the landing performance across four compliance treatments relative to an individual’s body weight (BW); 0, 2.5, 5, and 10 mm BW-1. Center of mass analyses are paired with inverse dynamics to extract not only global body dynamics and energy dissipation but also at the level of individual joints within the forelimb. Our results imply that while energetic demand on the forelimbs decreases with increasing compliance of the substrate the overall kinematics remain similar suggesting a conserved control mechanism for landing.