mTOR signaling is required for phagocyte free radical production, GLUT1 expression, and control of Staphylococcus aureus infection.

Mammalian target of rapamycin (mTOR) is a key regulator of metabolism in the mammalian cell. Here, we show the essential role for mTOR signaling in the immune response to bacterial infection. Inhibition of mTOR during infection with Staphylococcus aureus revealed that mTOR signaling is required for bactericidal free radical production by phagocytes. Mechanistically, mTOR supported glucose transporter GLUT1 expression, potentially through hypoxia-inducible factor 1α, upon phagocyte activation. Cytokine and chemokine signaling, inducible nitric oxide synthase, and p65 nuclear translocation were present at similar levels during mTOR suppression, suggesting an NF-κB-independent role for mTOR signaling in the immune response during bacterial infection. We propose that mTOR signaling primarily mediates the metabolic requirements necessary for phagocyte bactericidal free radical production. This study has important implications for the metabolic requirements of innate immune cells during bacterial infection as well as the clinical use of mTOR inhibitors.IMPORTANCESirolimus, everolimus, temsirolimus, and similar are a class of pharmaceutics commonly used in the clinical treatment of cancer and the anti-rejection of transplanted organs. Each of these agents suppresses the activity of the mammalian target of rapamycin (mTOR), a master regulator of metabolism in human cells. Activation of mTOR is also involved in the immune response to bacterial infection, and treatments that inhibit mTOR are associated with increased susceptibility to bacterial infections in the skin and soft tissue. Infections caused by Staphylococcus aureus are among the most common and severe. Our study shows that this susceptibility to S. aureus infection during mTOR suppression is due to an impaired function of phagocytic immune cells responsible for controlling bacterial infections. Specifically, we observed that mTOR activity is required for phagocytes to produce antimicrobial free radicals. These results have important implications for immune responses during clinical treatments and in disease states where mTOR is suppressed.

Influence of BMI percentile on craniofacial morphology and development in adolescents,Part II: elevated BMI is associated with larger final facial dimensions.

BACKGROUND: Prevalence of adolescent obesity has markedly increased from 5.2% in 1974 to 19.7% in 2021. Understanding the impacts of obesity is important to orthodontists, as growth acceleration and greater pre-pubertal facial dimensions are seen in children with elevated body mass index (BMI). METHODS: To identify whether adolescent obesity shifts the timing and rate of craniofacial growth resulting in larger post-treatment dimensions, we evaluated cephalometric outcomes in overweight/obese (BMI > 85%, n = 168) and normal weight (n = 158) adolescents (N = 326 total). Cephalometric measurements were obtained from pre- and post-treatment records to measure growth rates and final dimensions and were statistically evaluated with repeated measures analysis of variance and linear regression models. RESULTS: Overweight and obese adolescents began and finished treatment with significantly larger, bimaxillary prognathic craniofacial dimensions, with elevated mandibular length [articulare-gnathion (Ar-Gn)], maxillary length [condylion-anterior nasal spine (Co-ANS), posterior nasal spine-ANS (PNS-ANS)], and anterior lower face height (ANS-Me), suggesting overweight children grow more overall. However, there was no difference between weight cohorts in the amount of cephalometric change during treatment, and regression analyses demonstrated no correlation between change in growth during treatment and BMI. BMI percentile was a significant linear predictor (P < 0.05) for cephalometric post-treatment outcomes, including Ar-Gn, Co-ANS, ANS-Me, upper face height percentage (UFH:total FH, inverse relationship), lower face height percentage (LFH:total FH), sella-nasion-A-point (SNA), and SN-B-point (SNB). LIMITATIONS: The study is retrospective. CONCLUSIONS: Growth begins earlier in overweight and obese adolescents and continues at a rate similar to normal-weight children during orthodontic treatment, resulting in larger final skeletal dimensions. Orthodontics could begin earlier in overweight patients to time care with growth, and clinicians can anticipate that overweight/obese patients will finish treatment with proportionally larger, bimaxillary-prognathic craniofacial dimensions.

Chronic early life stress alters the neuroimmune profile and functioning of the developing zebrafish gut.

Chronic early life stress (ELS) potently impacts the developing central nervous and immune systems and is associated with the onset of gastrointestinal disease in humans. Though the gut-brain axis is appreciated to be a major target of the stress response, the underlying mechanisms linking ELS to gut dysfunction later in life is incompletely understood. Zebrafish are a powerful model validated for stress research and have emerged as an important tool in delineating neuroimmune mechanisms in the developing gut. Here, we developed a novel model of ELS and utilized a comparative transcriptomics approach to assess how chronic ELS modulated expression of neuroimmune genes in the developing gut and brain. Zebrafish exposed to ELS throughout larval development exhibited anxiety-like behavior and altered expression of neuroimmune genes in a time- and tissue-dependent manner. Further, the altered gut neuroimmune profile, which included increased expression of genes associated with neuronal modulation, correlated with a reduction in enteric neuronal density and delayed gut transit. Together, these findings provide insights into the mechanisms linking ELS with gastrointestinal dysfunction and highlight the zebrafish model organism as a valuable tool in uncovering how "the body keeps the score."

Patient and Caregiver Perceptions of Animal Assisted Activity in Orthodontics.

Dental anxiety affects up to 21% of children and 80% of adults and is associated with lifelong dental avoidance. Animal assisted activity (AAA) is widely used to reduce anxiety and pain in medical settings and has promise in dentistry. The primary objective of this study was to evaluate caregiver and patient perceptions of canine AAA in orthodontics. A cross-sectional survey consisting of pre-tested and validated questions was conducted (n = 800) including orthodontic patients (n = 352 minors, n = 204 adults) and parents/caregivers (n = 244) attending university orthodontic clinics. In this study, AAA and dog therapy were not used or tested for dental anxiety management. More than a third of orthodontic patients (37%) had moderate or greater anxiety related to care. Participants believed that therapy animals would make dental experiences more enjoyable (75%) and reduce anxiety (82%). There was little to no concern expressed regarding cleanliness (83%), allergies (81%), and safety (89%) with a therapy animal in dental settings. Almost half of the participants would preferentially select an orthodontic office offering AAA. In light of the COVID-19 pandemic, we assessed whether perceptions of AAA changed before and after the shutdown of dental offices, with no significant differences. Across patients and caregivers, the responses support the use of AAA in orthodontic settings with minimal concerns.

Orthodontic loading activates cell-specific autophagy in a force-dependent manner.

INTRODUCTION: Orthodontic tooth movement (OTM) relies on bone remodeling and controlled aseptic inflammation. Autophagy, a conserved homeostatic pathway, has been shown to play a role in bone turnover. We hypothesize that autophagy participates in regulating bone remodeling during OTM in a force-dependent and cell type-specific manner. METHODS: A split-mouth design was used to load molars with 1 of 3 force levels (15, 30, or 45 g of force) in mice carrying a green fluorescent protein-LC3 transgene to detect cellular autophagy. Fluorescent microscopy and quantitative polymerase chain reaction analyses were used to evaluate autophagy activation and its correlation with force level. Cell type-specific antibodies were used to identify cells with green fluorescent protein-positive puncta (autophagosomes) in periodontal tissues. RESULTS: Autophagic activity increased shortly after loading with moderate force and was associated with the expression of bone turnover, inflammatory, and autophagy markers. Different load levels resulted in altered degrees of autophagic activation, gene expression, and osteoclast recruitment. Autophagy was specifically induced by loading in macrophages and osteoclasts found in the periodontal ligament and alveolar bone. Data suggest autophagy participates in regulating bone turnover during OTM. CONCLUSIONS: Autophagy is induced in macrophage lineage cells by orthodontic loading in a force-dependent manner and plays a role during OTM, possibly through modulation of osteoclast bone resorption. Exploring the roles of autophagy in OTM is medically relevant, given that autophagy is associated with oral and systemic inflammatory conditions.

Zebrafish harbor diverse intestinal macrophage populations including a subset intimately associated with enteric neural processes.

Intestinal macrophages are essential for gut health but remain understudied outside of human and mouse systems. Here, we establish zebrafish as a powerful model that provides superior imaging capabilities for whole-gut analysis along all dimensions (anterior-posterior and center-outer axes) for dissecting macrophage biology in gastrointestinal health and disease. We utilized high-resolution imaging to show that the zebrafish gut contains bona fide muscularis and mucosal macrophages, as well as surprisingly large subsets intimately associated with enteric neural processes. Interestingly, most muscularis macrophages span multiple gut layers in stark contrast to their mammalian counterparts typically restricted to a single layer. Using macrophage-deficient irf8 zebrafish, we found a depletion of muscularis but not mucosal macrophages, and that they may be dispensable for gross intestinal transit in adults but not during development. These characterizations provide first insights into intestinal macrophages and their association with the enteric nervous system from development to adulthood in teleosts.

Lipid mediators and biomarkers associated with type 1 diabetes development.

Type 1 diabetes (T1D) is a consequence of autoimmune ß cell destruction, but the role of lipids in this process is unknown. We previously reported that activation of Ca2+-independent phospholipase A2ß (iPLA2ß) modulates polarization of macrophages (MΦ). Hydrolysis of the sn-2 substituent of glycerophospholipids by iPLA2ß can lead to the generation of oxidized lipids (eicosanoids), pro- and antiinflammatory, which can initiate and amplify immune responses triggering ß cell death. As MΦ are early triggers of immune responses in islets, we examined the impact of iPLA2ß-derived lipids (iDLs) in spontaneous-T1D prone nonobese diabetic mice (NOD), in the context of MΦ production and plasma abundances of eicosanoids and sphingolipids. We find that (a) MΦNOD exhibit a proinflammatory lipid landscape during the prediabetic phase; (b) early inhibition or genetic reduction of iPLA2ß reduces production of select proinflammatory lipids, promotes antiinflammatory MΦ phenotype, and reduces T1D incidence; (c) such lipid changes are reflected in NOD plasma during the prediabetic phase and at T1D onset; and (d) importantly, similar lipid signatures are evidenced in plasma of human subjects at high risk for developing T1D. These findings suggest that iDLs contribute to T1D onset and identify select lipids that could be targeted for therapeutics and, in conjunction with autoantibodies, serve as early biomarkers of pre-T1D.

Adrenergic Signaling in Muscularis Macrophages Limits Infection-Induced Neuronal Loss.

Enteric-associated neurons (EANs) are closely associated with immune cells and continuously monitor and modulate homeostatic intestinal functions, including motility and nutrient sensing. Bidirectional interactions between neuronal and immune cells are altered during disease processes such as neurodegeneration or irritable bowel syndrome. We investigated the effects of infection-induced inflammation on intrinsic EANs (iEANs) and the role of intestinal muscularis macrophages (MMs) in this context. Using murine models of enteric infections, we observed long-term gastrointestinal symptoms, including reduced motility and loss of excitatory iEANs, which was mediated by a Nlrp6- and Casp11-dependent mechanism, depended on infection history, and could be reversed by manipulation of the microbiota. MMs responded to luminal infection by upregulating a neuroprotective program via ß2-adrenergic receptor (ß2-AR) signaling and mediated neuronal protection through an arginase 1-polyamine axis. Our results identify a mechanism of neuronal death post-infection and point to a role for tissue-resident MMs in limiting neuronal damage.

Critical Role for a Subset of Intestinal Macrophages in Shaping Gut Microbiota in Adult Zebrafish.

The gut microbiota is strongly influenced by environmental factors, although host contribution is far less understood. We leveraged macrophage-deficient interferon regulatory factor irf8 zebrafish mutants to investigate the role of macrophages in this process. In conventionally raised adult irf8-deficient mutants, we found a significant loss of intestinal macrophages associated with a strikingly altered gut microbiota when compared to co-housed siblings. The destabilization of the gut commensal microbiota was associated with a severe reduction in complement C1q genes and outgrowth of a rare bacterial species. Consistent with a critical function of irf8 in adult intestinal macrophages, irf8 is abundantly expressed in these cells normally, and restoring macrophage irf8 expression in irf8 mutants was sufficient to recover commensal microbes and C1q genes expression. This study reports an important subpopulation of intestinal macrophages that requires irf8 to establish in the gut, ensure normal colonization of gut microbes, and prevent immune dysregulation.

Targeting resolution of neuroinflammation after ischemic stroke with a lipoxin A4 analog: Protective mechanisms and long-term effects on neurological recovery.

BACKGROUND: Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A4 (LXA 4) is an anti-inflammatory, pro-resolution lipid mediator that reduces neuroinflammation in stroke. Since LXA 4 is rapidly inactivated, potent analogs have been synthesized, including BML-111. We hypothesized that post-ischemic, intravenous treatment with BML-111 for 1 week would provide neuroprotection and reduce neurobehavioral deficits at 4 weeks after ischemic stroke in rats. Additionally, we investigated the potential protective mechanisms of BML-111 on the post-stroke molecular and cellular profile. METHODS: A total of 133 male Sprague-Dawley rats were subjected to 90 min of transient middle cerebral artery occlusion (MCAO) and BML-111 administration was started at the time of reperfusion. Two methods of week-long BML-111 intravenous administration were tested: continuous infusion via ALZET ® osmotic pumps (1.25 and 3.75 µg µl-1 hr-1), or freshly prepared daily single injections (0.3, 1, and 3 mg/kg). We report for the first time on the stability of BML-111 and characterized an optimal dose and a dosing schedule for the administration of BML-111. RESULTS: One week of BML-111 intravenous injections did not reduce infarct size or improve behavioral deficits 4 weeks after ischemic stroke. However, post-ischemic treatment with BML-111 did elicit early protective effects as demonstrated by a significant reduction in infarct volume and improved sensorimotor function at 1 week after stroke. This protection was associated with reduced pro-inflammatory cytokine and chemokine levels, decreased M1 CD40+ macrophages, and increased alternatively activated, anti-inflammatory M2 microglia/macrophage cell populations in the post-ischemic brain. CONCLUSION: These data suggest that targeting the endogenous LXA 4 pathway could be a promising therapeutic strategy for the treatment of ischemic stroke. More work is necessary to determine whether a different dosing regimen or more stable LXA 4 analogs could confer long-term protection.

Aging, inflammation, immunity and periodontal disease.

The increased prevalence and severity of periodontal disease have long been associated with aging, such that this oral condition affects the majority of the adult population over 50 years of age. Although the immune system is a critical component for maintaining health, aging can be characterized by quantitative and qualitative modifications of the immune system. This process, termed 'immunosenescence', is a progressive modification of the immune system that leads to greater susceptibility to infections, neoplasia and autoimmunity, presumably reflecting the prolonged antigenic stimulation and/or stress responses that occur across the lifespan. Interestingly, the global reduction in the host capability to respond effectively to these challenges is coupled with a progressive increase in the general proinflammatory status, termed 'inflammaging'. Consistent with the definition of immunosenescence, it has been suggested that the cumulative effect of prolonged exposure of the periodontium to microbial challenge is, at least in part, a contributor to the effects of aging on these tissues. Thus, it has also been hypothesized that alterations in the function of resident immune and nonimmune cells of the periodontium contribute to the expression of inflammaging in periodontal disease. Although the majority of aging research has focused on the adaptive immune response, it is becoming increasingly clear that the innate immune compartment is also highly affected by aging. Thus, the phenomenon of immunosenescence and inflammaging, expressed as age-associated changes within the periodontium, needs to be more fully understood in this era of precision and personalized medicine and dentistry.

Intestinal Epithelial Cell Regulation of Adaptive Immune Dysfunction in Human Type 1 Diabetes.

Environmental factors contribute to the initiation, progression, and maintenance of type 1 diabetes (T1D), although a single environmental trigger for disease has not been identified. Studies have documented the contribution of immunity within the gastrointestinal tract (GI) to the expression of autoimmunity at distal sites. Intestinal epithelial cells (IECs) regulate local and systemic immunologic homeostasis through physical and biochemical interactions with innate and adaptive immune populations. We hypothesize that a loss in the tolerance-inducing nature of the GI tract occurs within T1D and is due to altered IECs' innate immune function. As a first step in addressing this hypothesis, we contrasted the global immune microenvironment within the GI tract of individuals with T1D as well as evaluated the IEC-specific effects on adaptive immune cell phenotypes. The soluble and cellular immune microenvironment within the duodenum, the soluble mediator profile of primary IECs derived from the same duodenal tissues, and the effect of the primary IECs' soluble mediator profile on T-cell expansion and polarization were evaluated. Higher levels of IL-17C and beta-defensin 2 (BD-2) mRNA in the T1D-duodenum were observed. Higher frequencies of type 1 innate lymphoid cells (ILC1) and CD8+CXCR3+ T-cells (Tc1) were also observed in T1D-duodenal tissues, concomitant with lower frequencies of type 3 ILC (ILC3) and CD8+CCR6+ T-cells (Tc17). Higher levels of proinflammatory mediators (IL-17C and BD-2) in the absence of similar changes in mediators associated with homeostasis (interleukin 10 and thymic stromal lymphopoietin) were also observed in T1D-derived primary IEC cultures. T1D-derived IEC culture supernatants induced more robust CD8+ T-cell proliferation along with enhanced polarization of Tc1 populations, at the expense of Tc17 polarization, as well as the expansion of CXCR3+CCR6+/- Tregs, indicative of a Th1-like and less regulatory phenotype. These data demonstrate a proinflammatory microenvironment of the T1D-duodenum, whereby IECs have the potential to contribute to the expansion and polarization of innate and adaptive immune cells. Although these data do not discern whether these observations are not simply a consequence of T1D, the data indicate that the T1D-GI tract has the capacity to foster a permissive environment under which autoreactive T-cells could be expanded and polarized.

Human norovirus culture in B cells.

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h.

Downstream mediators of the intratumoral interferon response suppress antitumor immunity, induce gemcitabine resistance and associate with poor survival in human pancreatic cancer.

The cancer microenvironment allows tumor cells to evade immune surveillance through a variety of mechanisms. While interferon-γ (IFNγ) is central to effective antitumor immunity, its effects on the microenvironment are not as clear and have in some cancers been shown to induce immune checkpoint ligands. The heterogeneity of these responses to IFNγ remains poorly characterized in desmoplastic malignancies with minimal inflammatory cell infiltration, such as pancreatic cancer (PC). Thus, the IFNγ response within and on key cells of the PC microenvironment was evaluated. IFNγ induced expression of human leukocyte antigen (HLA) class I and II on PC cell lines, primary pancreatic cancer epithelial cells (PPCE) and patient-derived tumor-associated stroma, concomitant with an upregulation of PDL1 in the absence of CD80 and CD86 expression. As expected, IFNγ also induced high levels of CXCL10 from all cell types. In addition, significantly higher levels of CXCL10 were observed in PC specimens compared to those from chronic pancreatitis, whereby intratumoral CXCL10 concentration was an independent predictor of poor survival. Immunohistochemical analysis revealed a subset of CXCR3-positive cancer cells in over 90 % of PC specimens, as well as on a subset of cultured PC cell lines and PPCE, whereby exposure to CXCL10 induced resistance to the chemotherapeutic gemcitabine. These findings suggest that IFNγ has multiple effects on many cell types within the PC microenvironment that may lead to immune evasion, chemoresistance and shortened survival.

H2O2 production rate in Lactobacillus johnsonii is modulated via the interplay of a heterodimeric flavin oxidoreductase with a soluble 28 Kd PAS domain containing protein.

Host and commensals crosstalk, mediated by reactive oxygen species (ROS), has triggered a growing scientific interest to understand the mechanisms governing such interaction. However, the majority of the scientific studies published do not evaluate the ROS production by commensals bacteria. In this context we recently showed that Lactobacillus johnsonii N6.2, a strain of probiotic value, modulates the activity of the critical enzymes 2,3-indoleamine dioxygenase via H2O2 production. L. johnsonii N6.2 by decreasing IDO activity, is able to modify the tryptophan/kynurenine ratio in the host blood with further systemic consequences. Understanding the mechanisms of H2O2 production is critical to predict the probiotic value of these strains and to optimize bacterial biomass production in industrial processes. We performed a transcriptome analysis to identify genes differentially expressed in L. johnsonii N6.2 cells collected from cultures grown under different aeration conditions. Herein we described the biochemical characteristics of a heterodimeric FMN reductase (FRedA/B) whose in vitro activity is controlled by LjPAS protein with a typical Per-Arnst-Sim (PAS) sensor domain. Interestingly, LjPAS is fused to the FMN reductase domains in other lactobacillaceae. In L. johnsonii, LjPAS is encoded by an independent gene which expression is repressed under anaerobic conditions (>3 fold). Purified LjPAS was able to slow down the FRedA/B initial activity rate when the holoenzyme precursors (FredA, FredB, and FMN) were mixed in vitro. Altogether the results obtained suggest that LjPAS module regulates the H2O2 production helping the cells to minimize oxidative stress in response to environmental conditions.

Enteric bacteria promote human and mouse norovirus infection of B cells.

The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual's histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.

A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors.

Evolutionary history and metabolic insights of ancient mammalian uricases.

Uricase is an enzyme involved in purine catabolism and is found in all three domains of life. Curiously, uricase is not functional in some organisms despite its role in converting highly insoluble uric acid into 5-hydroxyisourate. Of particular interest is the observation that apes, including humans, cannot oxidize uric acid, and it appears that multiple, independent evolutionary events led to the silencing or pseudogenization of the uricase gene in ancestral apes. Various arguments have been made to suggest why natural selection would allow the accumulation of uric acid despite the physiological consequences of crystallized monosodium urate acutely causing liver/kidney damage or chronically causing gout. We have applied evolutionary models to understand the history of primate uricases by resurrecting ancestral mammalian intermediates before the pseudogenization events of this gene family. Resurrected proteins reveal that ancestral uricases have steadily decreased in activity since the last common ancestor of mammals gave rise to descendent primate lineages. We were also able to determine the 3D distribution of amino acid replacements as they accumulated during evolutionary history by crystallizing a mammalian uricase protein. Further, ancient and modern uricases were stably transfected into HepG2 liver cells to test one hypothesis that uricase pseudogenization allowed ancient frugivorous apes to rapidly convert fructose into fat. Finally, pharmacokinetics of an ancient uricase injected in rodents suggest that our integrated approach provides the foundation for an evolutionarily-engineered enzyme capable of treating gout and preventing tumor lysis syndrome in human patients.

Adenine Synthesis in a Model Prebiotic Reaction: Connecting Origin of Life Chemistry with Biology.

Many high school laboratory experiments demonstrate concepts related to biological evolution, but few exist that allow students to investigate life's chemical origins. This series of laboratory experiments has been developed to allow students to explore and appreciate the deep connection that exists between prebiotic chemistry, chemical evolution, and contemporary biological systems. In the first experiment of the series, students synthesize adenine, one of the purine nucleobases of DNA and RNA, from plausibly prebiotic precursor molecules. Students compare their product to authentic standards using thin-layer chromatography. The second and third experiments of the series allow students to extract DNA from a familiar organism, the strawberry, and hydrolyze it, releasing adenine, which they can then compare to the previously chemically-synthesized adenine. A fourth, optional experiment is included where the technique of thin-layer chromatography is introduced and chromatographic skills are developed for use in the other three experiments that comprise this series. Concepts relating to organic and analytical chemistry, as well as biochemistry and DNA structure, are incorporated throughout, allowing this series of laboratory experiments to be easily inserted into existing laboratory courses and to reinforce concepts already included in any high school chemistry or biology curriculum.