Background: Photobiomodulation therapy (PBMT) is widely used in clinical settings, particularly for treating oral mucositis. Although PBMT has shown promise in aiding tissue healing, its safety and potential effects on tumorigenesis remain controversial. Objective: Xenopus laevis (X. laevis) tadpoles have proven to be an effective in vivo model system to study how different therapies affect epithelial biology. Because of this, the goal of this study was to investigate how PBMT influences epithelial tissue in X. laevis tadpoles. Methods: X. laevis tadpoles were treated with PBMT three times, every 24 h, using a 660 nm low-level laser at a fluence of 2.08 J/cm2 and an irradiance of 0.208 W/cm2, resulting in a cumulative dose of 2.25 J. Tadpole tails were then fixed, and the abnormal tissue area was analyzed using fluorescent microscopy. Results: PBMT induced the formation of abnormal tissue structures along the epithelial edges. The altered tissue increased from 0.03% in control tadpoles to 0.37% in PBMT-treated tadpoles (p < 0.0001; Mann-Whitney nonparametric test), and the number of tadpoles displaying this abnormal phenotype increased from 3.8% of control tadpoles to 30.6% of PBMT-treated tadpoles (p < 0.001; chi-squared test). Conclusion: This study demonstrates that PBMT can have significant effects on the epithelial tissue of X. laevis. The PBMT-induced abnormal tissue structures represent a loss of tissue polarization and cellular organization along the tail edge, both of which are essential to maintaining tissue homeostasis. For the first time, we show that X. laevis may provide an in vivo model system for examining the effects and mechanisms of PBMT.

This review explores the regenerative capacity of Xenopus laevis, focusing on tail regeneration, as a model to uncover cellular, molecular, and developmental mechanisms underlying tissue repair. X. laevis tadpoles provide unique insights into regenerative biology due to their regeneration-competent and -incompetent stages and ability to regrow complex structures in the tail, including the spinal cord, muscle, and skin, after amputation. The review delves into the roles of key signaling pathways, such as those involving reactive oxygen species (ROS) and signaling molecules like BMPs and FGFs, in orchestrating cellular responses during regeneration. It also examines how mechanotransduction, epigenetic regulation, and metabolic shifts influence tissue restoration. Comparisons of regenerative capacity with other species shed light on the evolutionary loss of regenerative abilities and underscore X. laevis as an invaluable model for understanding the constraints of tissue repair in higher organisms. This comprehensive review synthesizes recent findings, suggesting future directions for exploring regeneration mechanisms, with potential implications for advancing regenerative medicine.

Lung epithelial development relies on the proper balance of cell proliferation and differentiation to maintain homeostasis. When this balance is disturbed, it can lead to diseases like cancer, where cells undergo hyperproliferation and then can undergo migration and metastasis. Lung cancer is one of the deadliest cancers, and even though there are a variety of therapeutic approaches, there are cases where treatment remains elusive. The rho-associated protein kinase (ROCK) has been thought to be an ideal molecular target due to its role in activating oncogenic signaling pathways. However, in a variety of cases, inhibition of ROCK has been shown to have the opposite outcome. Here, we show that ROCK inhibition with y-27632 causes abnormal epithelial tissue development in Xenopus laevis embryonic skin, which is an ideal model for studying lung cancer development. We found that treatment with y-27632 caused an increase in proliferation and the formation of ciliated epithelial outgrowths along the tail edge. Our results suggest that, in certain cases, ROCK inhibition can disturb tissue homeostasis. We anticipate that these findings could provide insight into possible mechanisms to overcome instances when ROCK inhibition results in heightened proliferation. Also, these findings are significant because y-27632 is a common pharmacological inhibitor used to study ROCK signaling, so it is important to know that in certain in vivo developmental models and conditions, this treatment can enhance proliferation rather than lead to cell cycle suppression.

OBJECTIVES To investigate the effects of dietary folate and sex on histopathology of oral squamous cell carcinoma in mice. MATERIALS AND METHODS Mice (C57Bl/6, 30/sex) were fed either a deficient folate or sufficient folate diet. Vehicle or 4-nitroquinoline1-oxide (50 μg/mL) in vehicle were administered in drinking water for 20 weeks, followed by 6 weeks of regular drinking water. Oral lesions were observed weekly. Tongues were studied for histopathologic changes. Immunohistochemical techniques were used to measure cell proliferation (Ki67+), and to quantify expression of folate receptor, reduced folate carrier, and proton-coupled folate transporter. T cells, macrophages, and neutrophils were counted and normalized to area. RESULTS All 4NQO-treated mice developed oral tumors. Dietary folate level did not affect tumor burden. More tumors were observed on the ventral aspect of the tongue than in other locations within the oral cavity. 4-nitroquinoline-1-oxide-treated mice displayed 27%-46% significantly lower expression of all three folate transport proteins; diet and sex had no effect on folate transporter expression. T-cell and neutrophil infiltration in tongues were 9.1-fold and 18.1-fold increased in the 4-nitroquinoline-1-oxide-treated mouse tongues than in controls. CONCLUSION Treatment with 4NQO was the primary factor in determining cancer development, decreased folate transport expression, and lymphoid cell infiltration.

Risk factors for liver cancer include tobacco use, alcohol consumption, obesity, and male sex. Administration of 4-nitroquinonline-1-oxide (4NQO) in drinking water mimics the effects of tobacco and leads to oral carcinoma in mice. This study compared the effects of diets high and low in saturated fat (HF and LF, respectively), and sex, on liver histopathology in 4NQO-treated mice and controls. We hypothesized that 4NQO would cause histopathological changes in liver, and that a HF diet would increase hepatic pathology when compared to the LF diet. Mice (C57Bl/6, 36/sex), were divided into a low fat (10 kcal% fat; LF) or high fat (60 kcal% fat, HF) diet. Mice were further subdivided into one of 3 water treatment groups for 17 weeks: water (control), vehicle (1.25% propylene glycol in water [PG]), or 4NQO in (50 μg/ml; 4NQO). All mice were subsequently given water alone for 6 more weeks. Upon euthanasia, livers were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E). H&E slides were graded for histopathology; frozen liver samples were analyzed for triglyceride content. Trichrome stained sections were graded for fibrosis. CD3+ T cells, CD68+ macrophages, and Ly6+ neutrophils were detected by immunohistochemistry. Compared to water controls, 4NQO-treatment caused mouse liver histopathological changes such as fibrosis, and increases in hepatic neutrophils, T cells, and macrophages. HF diet exacerbated pathological changes compared to LF diet. Male controls, but not females, demonstrated severe steatosis and increased triglyceride content. 4NQO treatment decreased hepatic fat accumulation, even in animals on a HF diet. In conclusion, this murine model of oral cancer may serve as a model to study the effects of tobacco and diet on liver.

Objective One-carbon metabolism is a metabolic network that integrates nutritional signals with biosynthesis, redox homeostasis, and epigenetics. There are sex differences in hepatic one-carbon metabolism. However, it is unclear whether there are sex differences in dietary deficiencies of one-carbon metabolism in the brain. The aim of this study was to investigate if sex modulates the effects of dietary folic acid deficiency in brain tissue using a mouse model. Methods Male and female C57Bl/6J mice were placed on a folic acid deficient (FD) or control diet (CD) at six weeks of age. Mice were maintained on these diets for six months, after which animals were euthanized and brain tissue and serum were collected for analysis. Serum folate levels were measured. In brain tissue, hippocampal volume and morphology including Cornu Ammonis 1 and 3 (CA1; CA3), and dentate gyrus thickness were measured. Apoptosis within the hippocampus was assessed using active caspase-3 immunofluorescence staining. Additionally, cortical acetylcholine metabolism was measured in brain tissue using immunofluorescence staining of acetylcholinesterase (AChE), or choline acetyltransferase (ChAT), and neuronal nuclei (NeuN). Results Male and female FD mice had reduced serum levels of folate. Both males and females maintained on a FD showed a decrease in the thickness of the hippocampal CA1-CA3 region. Interestingly, there was a sex difference in the levels of active caspase-3 within the CA3 region of the hippocampus. In cortical tissue, there were increased levels of neuronal ChAT and reduced levels of AChE in FD females and male mice. Conclusions The results indicated that FD impacts hippocampal morphology and cortical neuronal acetylcholine metabolism. The data from our study indicate that there was only one sex difference and that was in hippocampal apoptosis. Our study provides little evidence that sex modulates the effects of dietary folate deficiency on hippocampal morphology and cortical neuronal acetylcholine metabolism.

IntroductionWorldwide, liver cancer remains among the top ten most common cancers. Risk factors for liver cancer include tobacco usage, alcohol consumption, obesity, and sex. Administration of 4‐nitroquinoline 1‐oxide (4NQO) in drinking water mimics the effects of tobacco and leads to oral carcinoma in mice. The purpose of this study was to compare the effects of high and low in saturated fat diets (HF and LF, respectively), and sex, on liver histopathology in 4NQO‐treated mice and controls. We hypothesized that 4NQO’s carcinogenic effects would cause histopathological changes in the liver. We also hypothesized that a HF diet would increase inflammation, steatosis, and fibrosis when compared to the LF diet.Materials and methodsMale and female C57Bl/6 mice (Jackson Labs, 36 each gender), 5 weeks old, were divided into a low fat (10 kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H2O); or 4NQO in PG‐H2O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 weeks prior to euthanasia. Livers were harvested, portions were frozen or formalin fixed, sectioned, and stained with hematoxylin and eosin (H&E). Histopathology criteria used for tissue assessment included steatosis, fibrosis, and lymphoid cell infiltration. Frozen liver samples were hydrolyzed and a colorimetric test was used to measure triglyceride content. Trichrome staining of collagen was performed, and slides were graded visually for fibrosis using an adapted Metavir scale. T‐cells (CD3+ cells), macrophages (CD68+ cells), and neutrophils (Ly6+ cells) were detected by immunohistochemistry, counted, and normalized to tissue area. Statistical analysis was done using GraphPad Prism 7.0. All animal work was in compliance with the regulations of IACUC at Midwestern University, Downers Grove, IL.ResultsCompared to water controls, 4NQO‐treatment significantly increased hepatic inflammatory cell infiltration by neutrophils (11± 1 vs. 31± 3 cells/mm2), T cells (76 ± 7 vs. 99 ± 6 cells/mm2), and macrophages (468 ± 40 vs. 562 ± 25 cells/mm2). Infiltration with inflammatory cells was accompanied by liver fibrosis. Interestingly, HF diet caused hepatic histopathologic changes with severe fatty changes in males, and only moderate fatty changes in female control mice. Thus, triglyceride content was significantly higher in HF males when compared to all other control groups (HF male 14.0 ± 3.2 %, LF male 2.9 ± 0.8 %, HF female 3.2 ± 0.4 %, and LF female 2.3 ± 0.5 %). Fatty change was significantly reduced by 4NQO treatment, possibly as a result of cachexia.ConclusionThis mouse model of oral cancer, initially established to mimic the effects of smoking, may be extended to serve as a model for the effects of tobacco and diet on liver as well.Support or Funding InformationWe recognize the Midwestern University Deans of the College of Dental Medicine and the College of Graduate studies for their support of this research. Student research was supported by the Office of Research and Sponsored Programs.

Objective To compare the effects of dietary fat and sex on murine oral squamous cell carcinoma pathology. Materials and methods Male and female C57Bl/6 mice (36/sex) received a low-fat (10 kcal%) or high-fat (60 kcal%) diet. Water (control), vehicle, or 4-nitroquinoline-1-oxide in vehicle (50 mu g/ml) was provided for 17 weeks followed by six additional weeks of water. Oral lesion development was recorded weekly. Histopathologic changes in tongues were examined, and T cells (CD3+), macrophages (CD68+), and neutrophils (Ly6+) were quantified. Results All 4-nitroquinoline-1-oxide-treated mice developed oral tumors. High-fat diet exacerbated pathology, demonstrated by an increased final tumor burden (10.9 +/- 4.5 vs. 7.9 +/- 2.5, mm/mouse,p < .05; high-fat diet vs. low-fat diet, respectively), and a greater histopathology score. When dietary groups were combined, 4-nitroquinoline-1-oxide-treated males displayed higher histopathology scores than females (4.2 +/- 0.3 vs. 3.6 +/- 0.2, respectively,p < .05). Lymphoid cell infiltration was greater in the 4-nitroquinoline-1-oxide mouse tongues than controls: T cells (14.0 vs. 0.96 cells/mm(2)), macrophages (3.6 vs. 1.8 cells/mm(2)), and neutrophils (12.0 vs. 0.38 cells/mm(2)). Conclusion High-fat diet and male sex increased the pathology of 4-nitroquinoline-1-oxide-induced oral cancer. Elevated lymphoid cell infiltration contributed to disease pathology.

SPECC1L mutations have been identified in patients with rare atypical orofacial clefts and with syndromic cleft lip and/or palate (CL/P). These mutations cluster in the second coiled-coil and calponin homology domains of SPECC1L and severely affect the ability of SPECC1L to associate with microtubules. We previously showed that gene-trap knockout of Specc1l in mouse results in early embryonic lethality. We now present a truncation mutant mouse allele, Specc1lΔC510, that results in perinatal lethality. Specc1lΔC510/ΔC510 homozygotes showed abnormal palate rugae but did not show cleft palate. However, when crossed with a gene-trap allele, Specc1lcGT/ΔC510 compound heterozygotes showed a palate elevation delay with incompletely penetrant cleft palate. Specc1lcGT/ΔC510 embryos exhibit transient oral epithelial adhesions at E13.5, which may delay shelf elevation. Consistent with oral adhesions, we show periderm layer abnormalities, including ectopic apical expression of adherens junction markers, similar to Irf6 hypomorphic mutants and Arhgap29 heterozygotes. Indeed, SPECC1L expression is drastically reduced in Irf6 mutant palatal shelves. Finally, we wanted to determine if SPECC1L deficiency also contributed to non-syndromic (ns) CL/P. We sequenced 62 Caucasian, 89 Filipino, 90 Ethiopian, 90 Nigerian and 95 Japanese patients with nsCL/P and identified three rare coding variants (p.Ala86Thr, p.Met91Iso and p.Arg546Gln) in six individuals. These variants reside outside of SPECC1L coiled-coil domains and result in milder functional defects than variants associated with syndromic clefting. Together, our data indicate that palate elevation is sensitive to deficiency of SPECC1L dosage and function and that SPECC1L cytoskeletal protein functions downstream of IRF6 in palatogenesis.

IntroductionHead and neck cancer is the sixth most common cancer worldwide. Risk factors include tobacco exposure, alcohol use, male sex, and a poor diet. Administration of 4‐nitroquinoline‐1‐oxide (4NQO) in drinking water causes oral cancer in mice, and mimics the effects of tobacco use. Folate is essential for nucleotide biosynthesis as well as epigenetic regulation. Folate is imported by folate receptors (FR), proton‐coupled folate transporters (PCFT), and the reduced folate carrier (RFC). This study aimed to investigate the effects of a high saturated fat (HF) or low fat (LF) diet, sex, and 4NQO treatment on expression of folate transport proteins in mice tongues. We hypothesized that there would be a decreased expression of these proteins in the HNSCC tongue samples compared to the control tongue samples.Materials and MethodsFive week old mice (C57BI/6, 36 per sex) were provided either a low saturated fat diet (10 kcal% fat; LF) or a high fat diet (60 kcal% fat, HF). After one week, mice were then randomly assigned to 17 weeks on one of three water treatment groups: water alone (control); propylene glycol in water (1.25%, PG‐water); or 4NQO in PG‐water (50 mg/ml; 4NQO). After 17 weeks, the mice were provided water alone for six more weeks but maintained on their diet. Tongues from euthanized animals were fixed in formalin and processed for histological examination. Immunohistochemistry was performed to detect the expression of FR, PCFT, and RFC in these samples. Slides were visually graded, and the total score for the expression of each protein was acquired by adding the intensity grade (0–3) and distribution grade (0–3). Statistical analysis was performed using Graphpad Prism 7.0. All animal work was in compliance with the Institutional Animal Care and Use Committee at Midwestern University, Downers Grove, IL.ResultsCompared to the control tongues, there was decreased expression of all three folate transport proteins in tumors (p<0.001). The total score (intensity + distribution) in oral cancer versus control tissue for FR, RFC, and PCFT, respectively were: 3.21 ± 0.10 vs 4.48 + 0.14; 4.38 ± 0.11 vs 5.13 ± 0.10; and 4.22 ± 0.11 vs 5.65 ± 0.69. All slides displayed a 100% distribution rate of FR, PCFT, and RFC in the epithelium. There were no significant differences in folate transport protein expression in males versus females. In the 4NQO tongues, a statistically significant decrease in FR expression was observed between the HF diet group and the LF group (3.97 ± 0.20 vs 3.05 ± 0.10, p <0.0002). No differences were observed in the expression of RFC and PCFT proteins with regards to diet or sex.ConclusionWe measured decreased expression in all three folate transport proteins in oral cancers when compared to normal tongue epithelium. We speculate that mutations leading to decreased folate import can promote carcinogenesis through epigenetic mechanisms.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Introduction Risk factors for oral cancer include tobacco use, male sex, and possibly dietary fat. Exposure to 4‐nitroquinoline 1‐oxide (4NQO) in the drinking water causes oral cancer in mice and models the effects of tobacco use. This study was undertaken to investigate the effects of a high saturated fat (HF) or low fat (LF) diet and sex on presence of lymphoid cells in cancerous tongues. We hypothesized that inflammation, characterized by the increased presence of lymphoid cells, would accompany development of tumor lesions and that sex and diet would affect the levels of inflammation. Methods Male and female C57Bl/6 mice (36 each gender), 5 weeks old, were divided into a low fat (10kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H 2 O); or 4NQO in PG‐H 2 O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 more weeks. Tongues harvested from the euthanized animals were fixed in formalin and processed for histological examination. T cells (CD3 + cells), macrophages (CD68 + cells), and neutrophils (Ly6 + cells) were detected by immunohistochemistry, counted, and normalized to tissue area. Analysis was done using GraphPad Prism 7.0. All animal work was in compliance with the regulations of IACUC at Midwestern University, Downers Grove, IL. Results Oral cancer developed in all mice exposed to 4NQO and was absent in all controls. All three lymphoid cell types were significantly higher (p<0.05) in the 4NQO mice versus the control mice: CD3 + (11.2 vs 0.9 cells/mm 2 ), CD68 + (3.6 vs 1.5 cells/mm 2 ), and Ly6 + (9.4 vs 0.38 cells/mm 2 ). Among the 4NQO treated mice, there were no significant differences between males and females, or between HF and LF mice for T cells or macrophages. 4NQO treated males had a significantly higher numbers of neutrophils than females (12 vs 6.6 cells/mm 2 ). LF diet increased the levels of neutrophils in both males and females (15 and 7.7 cells/mm 2 respectively) when compared to the HF diet in both males and females (9.3 and 5.3 cells/mm 2 respectively); however, this trend was not statistically significant. We speculate that the higher carbohydrate content of the LF diet may support increased anaerobic glycolytic metabolism via the Warburg effect. Conclusion Development of 4NQO‐induced oral cancer was accompanied by inflammatory responses as manifested by the presence of T cells, macrophages, and neutrophils. Neutrophils were the only cell type affected by sex and possibly by the content of dietary fat. Support or Funding Information This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

IntroductionHMGB1 is a nuclear protein that binds to DNA and regulates gene transcription. HMGB1 can be released from the nucleus to function as a stress signal and/or a chemokine. HMGB1 overexpression has been reported in various cancers. The purpose of this study was to characterize the patterns of cytoplasmic HMGB1 expression in normal and cancerous mouse tongue tissues. Oral squamous cell carcinoma was induced by administration of 4‐nitroquinonline 1‐oxide (4NQO) in drinking water. 4NQO mimics the effects of tobacco and is a well‐established model of oral cancer in mice. In addition, we examined whether gender and high and low in saturated fat diets (HF and LF, respectively) could modify cytoplasmic levels of HMGB1. We hypothesized that higher levels of cytoplasmic HMGB1 will be present in tumor lesions and that gender and diet will affect the levels of expression.Materials and methodsMale and female C57Bl/6 mice (36 each gender), 5 weeks old, were divided into a low fat (10kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H2O); or 4NQO in PG‐H2O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 weeks prior to euthanasia. The presence of oral lesions, their size and location were monitored and recorded weekly. Upon sacrifice, tongues were harvested, formalin fixed, and processed for histological evaluation. Tongue tissue sections were stained with anti‐HMGB1 antibody (AB18256, Abcam Inc.) and examined microscopically. To evaluate localization and intensity of HMGB1 staining ImageJ program IHC profiler was used. The scale 0–3 was employed to measure the intensity of HMGB1 cytoplasmic staining.ResultsEpithelial cancer lesions were characterized by significantly higher levels of cytoplasmic HMGB1 than control epithelium (1.4–1.8 versus 0.8, p<0.01). Expression of cytoplasmic HMGB1 was limited to cancer lesions since unaffected areas of tongue epithelium from 4NQO animals showed low levels of HMGB1 (0.74–1.0), similar to the control animals (0.8). HMGB1 levels were higher in cancer lesions from HF diet than from LF diet animals (1.8 in HF, 1.4 in LF, p=0.0003). In addition, cancer lesions in females (LF and HF diets combined) showed higher levels of HMGB1 than in males (1.7 versus 1.3, p=0.008).ConclusionCytoplasmic expression of HMGB1 is limited to oral squamous carcinoma cells. HMGB1 may contribute to the tumor progression. Higher levels of HMGB1 in tumors from HF diet animals provide more evidence that HF diet may exacerbate cancer development. HMGB1 could serve as a cancer biomarker and as a therapeutic target.Support or Funding InformationWe recognize the Midwestern University Deans of CDMI and the Basic Science Division for their support of this research. Student research was supported by the Office of Research and Sponsored Programs and the Biomedical Sciences Division.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IntroductionA variety of factors, including use of tobacco, diet, gender, and genetics impact cancer risk. It is well established that administration of 4‐nitroquinonline 1‐oxide (4NQO) in drinking water mimics the effects of tobacco and leads to development of oral carcinoma in mice. The purpose of this study was to determine whether administration of 4NQO induced liver pathology and to examine the effects of gender, and high and low saturated fat diets (HF and LF, respectively) on the liver histopathology. We hypothesized that 4NQO's carcinogenic effects would cause pathological changes in liver and a HF diet would exacerbate this process.Materials and methodsMale and female C57Bl/6 mice (36 each gender), 5 weeks old, were divided into a low fat (10 kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H2O); or 4NQO in PG‐H2O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 more weeks. Livers harvested from the euthanized animals were weighed, then fixed in formalin and processed for histological examination. In addition, neutrophils and macrophages were detected by immunocytochemistry (Ly6 and CD68 positive cells, respectively) and counted in a standard area in liver sections. T‐tests were used to confirm no difference between water and PG controls, which were then combined for further statistical analysis using GraphPad Prism 7.0.ResultsAdministration of 4NQO resulted in histopathological changes in liver in all treatment groups. Morphological changes in hepatocytes, the presence of lobular and portal lymphoid cell infiltrates, and periportal fibrosis were observed in hematoxylin and eosin‐stained tissues. In addition, HF diet resulted in severe steatosis and liver enlargement (3× higher liver index in HF males versus all other treatment groups) of male but not female controls. Treatment with 4NQO significantly increased the number of neutrophils in liver, when compared to non‐4NQO‐treated control livers in HF males (208 versus 24.7), HF females (100 versus 29.2), and LF females (80 versus 26.7; for all groups, p<0.05). Also, treatment with 4NQO increased the number of macrophages in HF males, when compared to non‐4NQO‐treated males (121 versus 55, p<0.05). In addition, HF diet exacerbated the effect of 4NQO in males, with 121 neutrophils in HF 4NQO‐treated males versus 94.1 neutrophils in LF 4NQO‐treated males (p = 0.05).ConclusionOral administration of 4NQO, which mimics the effects of tobacco, induces histopathological changes in the liver, and those changes are exacerbated by a HF diet, especially in males.Support or Funding InformationWe recognize the Midwestern University Deans of the College of Dental Medicine and the Basic Science Division for their support of this research. Student research was supported by the Office of Research and Sponsored Programs and the Biomedical Sciences Division.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

OBJECTIVES:The purpose of this study was to assess the expression of the 3 major folate transporters-folate receptors (FRs), reduced folate carrier (RFC), and proton-coupled folate transporter (PCFT)-in oral squamous cell carcinoma (OSCC). We hypothesized that patterns of expression of folate transporters would be different in OSCC compared with normal oral epithelium.STUDY DESIGN:We used immunohistochemistry to examine the expression of FR, RFC, and PCFT in 15 primary specimens collected from patients with OSCC, 2 human cadaveric samples of OSCC, and 12 normal human cadaveric oral tissues from a medical gross anatomy laboratory. Possible correlations between the expression of each folate transporter and patients' clinical data were determined.RESULTS:All 3 folate transporters were highly expressed in normal oral epithelium. In contrast, OSCC samples generally demonstrated low expression of FR, RFC, and PCFT, with wide distribution in the invading cancer cells. There were no differences in folate transporter expression between OSCC samples collected from patients and from human cadavers. The lowest expression of FR and PCFT characterized less-differentiated tumors, and the lowest expression of RFC correlated with higher lymph node involvement.CONCLUSIONS:Human oral cancer samples expressed decreased amounts of all 3 major folate transport proteins compared with controls from normal cadaveric oral tissues.

Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer worldwide, and is more common in men than women. Since the diagnosis of HNSCC is typically late in disease progression, the prognosis is often poor, with significant co‐morbid effects. Common risk factors include tobacco exposure, alcohol use, and human papilloma virus infection. Effects of a high fat diet (HF) on oral cancer development and severity have not been clearly defined. The purpose of this study was to compare the effects of a high saturated fat diet and a low saturated fat diet on the induction and severity of oral SCC using a mouse model of 4‐nitroquinoline 1‐oxide (4NQO) induced SCC. We hypothesized that HF would augment 4NQO‐induced SCC when compared to the LF diet. Materials and methods Male (n=36) and female (n=36) C57Bl/6 mice (Jackson Labs), 5 weeks old, were equally distributed into a low fat (10kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H 2 O); or 4NQO in PG‐H 2 O (50 μg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 weeks prior to euthanasia. Initial and weekly body weights were measured. Weekly assessments of the oral cavity began after 5 weeks and continued for the remaining 18 weeks. The presence of oral lesions, their size, and location were monitored and recorded weekly. Statistically significant differences in body weight, tumor size and severity were determined by ANOVA, p<0.05. Results All 4‐NQO treated mice developed tumors in the oral cavity; no control mice developed oral tumors. Overall tumor burden at 23 weeks for both sexes combined was statistically higher in the HF mice compared to the LF group (10.3±1.1 vs. 7.9±0.6, p=0.055). There was no significant difference in tumor burden as a result of gender. Significant changes in weekly body weights were determined, with 4NQO mice demonstrating lower final body weights than the H 2 O control group and the PG‐H 2 O control mice on the respective diets (p<0.05). Twenty‐four hour food intake was determined at week 19, demonstrating a significant reduction in calorie intake in the 4‐NQO male mice (p<0.05). Conclusion This mouse model of SCC, initially established to mimic the effects of smoking, demonstrated significantly greater tumor burden as a consequence of elevated fat intake. The contributory effects of HF associated metabolic and inflammatory changes in SCC development can be discerned using this model. The clinical implication of these findings may be helpful in modifying lifestyle to improve outcomes. Support or Funding Information Acknowledgements: Andrew Nguyen was supported by the Midwestern University Biomedical Sciences Program. We also recognize the Midwestern University Deans of CDMI and the Basic Science Division for their support of this research.

Introduction Folic acid is a water‐soluble vitamin that is essential for many cellular processes such as biosynthesis of DNA, RNA, and amino acids. In E. coli , the abg region encodes proteins that enable the uptake and cleavage of p ‐aminobenzoyl‐glutamate (PABA‐GLU), a breakdown product of folic acid. These genes comprise an apparent operon oriented divergently from abgR, encoding a putative transcriptional regulator. Since this operon is only found in bacterial chromosomes, understanding its cellular role may provide new drug targets. Additionally, it has been noted in some studies that changes in the abgR region are characteristic of some pathogenic strains of E. coli . The goal of this study was to characterize expression and regulation of the abg operon. Our hypothesis was that AbgR regulated expression of the divergently transcribed abgABT genes. Methods E coli strains studied included wild type Keio strain BW25113, JW1333 ( abgR ::kan) and a strain with a point mutation in the intergenic region between abgR and abgA (BN1140). Cells were grown in Luria Broth (LB) and in Vogel Bonner (VB) glucose minimal medium with and without supplemental PABA‐GLU (100 μM). Cell growth was monitored by measuring absorbance at 600 nm. Cells were harvested at several points in the growth curve: ~0.2 OD 600 , ~0.5 OD 600 , and in early stationary phase (~1.0 OD 600 ). RNA was isolated using a RiboPure‐Bacteria kit (Ambion, Austin, TX). Primers were designed for abgR, abgA , abgB, and abgT . Primers were tested for dynamic range and efficiency. Quantitative real time PCR was performed using Power SYBR Green from Applied Biosystems (Carlsbad, CA) and the 7300 Real Time PCR Applied Biosystems instrument. Standard curves were used to determine the absolute transcript level for each gene. Data were analyzed using GraphPad Prism 5. Results In JW1333 cells grown in LB to stationary phase, mRNA levels of abgA, abgB , and abgT were higher (7.0‐fold, 2.3‐fold, and 2.3‐fold, respectively) when compared to expression in BW25113; this is consistent with AbgR functioning as a transcriptional repressor. When comparing expression of these four genes in BW25113 cells grown in LB or minimal medium, harvested at 0.2 OD, 0.5 OD, and 1.0 OD, results varied depending on the gene. AbgR was expressed more highly at every condition than the abgABT genes. At 0.2 OD, abgA, abgB and abgT were more highly expressed in minimal medium (8.6‐, 5.3‐ and 6.2 fold respectively) than in LB. In cells harvested at 0.5 OD and 1.0 OD, expression varied much less for each growth condition, between 0.8 and 2.5‐fold. BN1140 expressed 9–30‐fold more copies of AbgABT than wildtype (BW25113) cells; little change in AbgR expression was observed. Thus, the point mutation in the intergenic region affected abgABT expression alone. Wild type cells expressing a high‐copy plasmid encoding abgT , enabling import of PABA‐GLU, and grown in the absence and presence of 100 μM PABA‐GLU, demonstrated no differences in expression. This showed that expression of this region is independent of the cellular concentration of PABA‐GLU. Conclusion AbgR acts as a transcriptional repressor of divergently transcribed abgA, abgB and abgT. Expression of the abg region is dependent on nutrient availability. PABA‐GLU does not control the expression of the abg operon. Support or Funding Information Acknowledgements: This work was supported by Midwestern University.

Gene trap mutagenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays to enable directional gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional gene trap cassettes, we developed a generic strategy for generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the Prdm16 transcription factor gene. We demonstrate that the gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other Prdm16 mutant mice. Consecutive breeding to Flpe and Emx1IREScre deleter mice spatially restricted Prdm16 loss to regions of the forebrain expressing the homeobox gene Emx1, demonstrating the utility of the technology for the analysis of tissue-specific gene functions.

Introduction A variety of factors, including use of tobacco, diet, gender, and genetics impact cancer risk. Smoking is a risk factor for about 1/3 of all human cancers. It is well established that administration of 4‐nitroquinonline 1‐oxide (4NQO) in drinking water mimics the effects of tobacco and leads to development of oral carcinoma in mice. The purpose of this study was to compare the effects of high and low in saturated fat diets (HF and LF, respectively) on the incidence and severity of 4NQO‐induced cancers. We hypothesized that 4NQO's carcinogenic effects extend beyond oral tissues and will result in pathological changes in organs beyond the oral cavity. We also hypothesized that a HF diet would increase inflammation and tissue changes when compared to the LF diet. Materials and methods Male and female C57Bl/6 mice (Jackson Labs, 36 each gender), 5 weeks old, were divided into a low fat (10kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H 2 O); or 4NQO in PG‐H 2 O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 weeks prior to euthanasia. Animals were weighed weekly and upon sacrifice. Salivary glands, lungs, liver, kidney, spleen, and portions of the gastrointestinal tract were harvested, formalin fixed, microtome sectioned, and stained with hematoxylin and eosin (H&E) for histological evaluation. Histopathology criteria used for tissue assessment were based on the presence and degree of cellular atypia, tissue hyperplasia and dysplasia, and lymphoid cells infiltration. Results Histopathological changes in 4NQO‐treated animals were identified in esophagus (epithelial hyperplasia, dysplasia, inflammation, and development of carcinoma), lungs (significant peribronchial lymphoid cell infiltrates and – characteristic of emphysema – increased in size air spaces), and liver (lymphoid cell infiltrates in portal tracts and liver parenchyma and periportal fibrosis). Kidney, spleen, salivary glands, and pancreas demonstrated little changes based on H&E‐stained tissue analysis. Gender had no significant effect on a degree of 4NQO‐induced tissue pathology. However, HF diet exacerbated pathological changes compared to LF diet. This was particularly evident in liver, with males but not females demonstrating severe steatosis and liver enlargement (3× higher liver index in HF males versus all other treatment groups). Conclusion This mouse model of oral cancer, initially established to mimic the effects of smoking, may be extended to serve as a model for the effects of tobacco and diet on extra‐oral tissues as well. Support or Funding Information We recognize the Midwestern University Deans of the College of Dental Medicine and the Basic Science Division for their support of this research. Student research was supported by the Office of Research and Sponsored Programs and the Biomedical Sciences Division.