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A synthesis of data including over 9,300 head of cattle and over 980 pen means was conducted to evaluate the effect of corn processing method, distillers grains type, oil removal from distillers, and dietary distillers concentration on cattle performance and total feeding costs under different historical price scenarios. The corn processing method was either steam-flaked corn, high-moisture or dry-rolled corn or a blend of the latter two and their effects on performance with and without distillers grains. Wet, modified, and dry distillers were analyzed as either full fat or de-oiled products in diets at various dietary concentrations with either corn type to determine cattle performance. Performance estimates were used to determine the inclusion level that minimized total finishing costs ($/head) for each diet given various assumptions on corn price, corn-to-distillers price ratio, supplemental protein cost, and steam flake processing cost. Feeding distillers grains in steam-flaked or high-moisture and dry-rolled corn diets resulted in improved feed conversion and reduced cattle feeding costs, including lower supplemental protein costs and lower total feeding costs compared with a diet without distillers. However, feeding diets with steam-flaked corn improved feed conversion regardless of distillers type or level of inclusion. Feeding full fat distillers improved feed conversion relative to de-oiled distillers. Cattle fed de-oiled distillers were more efficient than those fed diets containing no distillers grains. At all corn price and corn-to-distillers price ratios examined, feeding some level of distillers of any type produced a lower total feeding costs relative to a diet without distillers. Different robustness tests were conducted to determine the impact of varying steam flake corn processing costs and the cost of supplemental protein on the optimal level of distillers inclusion level. The break-even cost a producer could pay for steam-flaking corn varied but was significantly higher when corn price was high. Feeding distillers grains in steam-flaked or high-moisture and dry-rolled corn diets resulted in improved feed efficiency, reduced cattle feeding costs, including replacement of other supplemental protein and associated costs compared to a diet without distillers grains.A pooled analysis of research was conducted to evaluate the effect of corn processing method, distillers grains type, oil removal from distillers, and distillers inclusion on cattle performance within studies conducted at the University of Nebraska-Lincoln between 1992-2020. Two corn processing methods were included in the dataset, steam-flaked corn (SFC) or any combination of high-moisture (HMC) and dry-rolled corn (DRC), fed with and without distillers gains, a by-product of the dry milling ethanol industry. Distillers products were either wet, modified, or dry distillers grains plus solubles and were analyzed as both full fat and de-oiled products. Cattle performance resulting from feeding the six by-product combinations with both corn types was analyzed and then used to find the inclusion level that minimized the total feeding cost ($/head) given different corn prices and corn-to-distillers price ratios. Cattle had improved feed conversion when fed SFC compared with HMC:DRC in all diets. Cattle fed full-fat distillers grains had slightly improved feed conversions compared with de-oiled distillers grains. Cattle fed distillers grains diets had improved feed conversion compared with cattle fed control diets without distillers. Overall, feeding distillers grains in combination with either corn type offers benefits including improved feed conversions, lower finishing costs, and reduced reliance on supplemental protein sources compared to a diet without distillers.

Crossbred, yearling beef steers (n = 210: initial BW = 430 ± 22 kg) were used in a randomized blocked design finishing experiment to evaluate the impact of removing modified distillers grains plus solubles (MDGS) on finishing performance and carcass characteristics. Treatments included feeding 20% MDGS (DM basis) during the entire feeding period (124 d); 20% MDGS until day 79 and then 0% MDGS until the end of the feeding period; or 20% MDGS until day 43 and then 0% MDGS until the end of the feeding period. A total of 21 pens (10 steers/pen) were used with 7 pens/treatment. Cattle were fed a 60:40 blend of high-moisture and dry-rolled corn, with 15% corn silage, 20% MDGS and 5% supplement. Distillers were replaced with the corn blend and urea when removed. Cattle were stepped down to 10% MDGS for 4 days before the complete removal of MDGS. Cattle with MDGS removed on either d 79 or d 43 had reduced (P < 0.05) final BW, DMI, and ADG. Cattle with MDGS removed on d 43 were 5% less efficient (P < 0.05) than cattle fed 20% MDGS throughout the entire feeding period. Cattle fed 20% MDGS throughout the whole feeding had the greatest (P < 0.05) HCW and LM area. There were no differences (P ≥ 0.35) in backfat and marbling scores among treatments, and no differences (P = 0.59) in percent of abscessed livers, although 38% of steers fed 20% MDGS continuously had liver abscesses compared with 46% for steers with MDGS removed on d 43. Based on the results of this study, removing MDGS from finishing diets on either day 43 or day 79 of the feeding period had a negative impact on cattle performance compared with feeding 20% MDGS continuously throughout the entire finishing period.

Background Bipolar percutaneous radiofrequency (RF) is an established method for ablation of distal peripheral branches of the facial nerve to produce weakness of the corrugator muscle. Recent developments to the Serene RF system (Serene Medical, Inc., Pleasanton, CA) allowing for safer and more predictable results highlighted a need for prospective clinical data. Objectives The authors sought to assess the ability of percutaneous RF to safely improve the appearance of dynamic glabellar lines for a minimum of 3 months and up to 1 year. Methods This prospective study enrolled 78 patients across 5 centers in the United States. The most distal branches of the medial and lateral facial nerve innervation to the brow depressor/corrugator complex were treated. Follow-up was at 3 days by telephone and in-office at 7 days and 3, 6, 9, and 12 months posttreatment. Patients were evaluated employing the Merz scale for dynamic glabellar lines. Results At 3 months, 93.6% of patients had a ≥1-point Merz scale improvement in dynamic glabellar lines based on independent, blinded review. Sustained ≥2-point improvement was present in 60% and 20% of patients at 3 and 12 months, respectively. Overall satisfaction remained >60% through 6 months and >50% at 12 months. All device- or procedure-related adverse events were mild (90%) or moderate (10%) and resolved without medical intervention. Conclusions Bipolar percutaneous RF may be adopted in conjunction with surgery or as an office-based procedure where it can serve as a minimally invasive alternative to, or in concert with, Botulinum Toxin A therapy. Level of Evidence: 4

Although immunotherapy works well in glioblastoma (GBM) pre-clinical mouse models, the therapy has unfortunately not demonstrated efficacy in humans. In melanoma and other cancers, the composition of the gut microbiome has been shown to determine responsiveness or resistance to immune checkpoint inhibitors (anti-PD-1). Most pre-clinical cancer studies have been done in mouse models using mouse gut microbiomes, but there are significant differences between mouse and human microbial gut compositions. To address this inconsistency, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a pre-clinical mouse model of GBM. We used five healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate five independent humanized mouse lines (HuM1-HuM5). Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. Interestingly, we found that the HuM lines responded differently to anti-PD-1. Specifically, we demonstrate that HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls, while HuM1, HuM4, and HuM5 mice are resistant to anti-PD-1. These mice are genetically identical, and only differ in the composition of the gut microbiome. In a correlative experiment, we found that disrupting the responder HuM2 microbiome with antibiotics abrogated the positive response to anti-PD-1, indicating that HuM2 microbiota must be present in the mice to elicit the positive response to anti-PD-1 in the GBM model. The question remains of whether the “responsive” microbial communities in HuM2 and HuM3 can be therapeutically exploited and applicable in other tumor models, or if the “resistant” microbial communities in HuM1, HuM4, and HuM5 can be depleted and/or replaced. Future studies will assess responder microbial transplants as a method of enhancing immunotherapy.

Background Although immunotherapy works well in glioblastoma (GBM) preclinical mouse models, the therapy has not demonstrated efficacy in humans. To address this anomaly, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a preclinical mouse model of GBM. Methods We used 5 healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate 5 independent humanized mouse lines (HuM1-HuM5). Results Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. All HuM mouse lines were susceptible to GBM transplantation, and exhibited similar median survival ranging from 19 to 26 days. Interestingly, we found that HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM1, HuM4, and HuM5 mice are nonresponders to anti-PD-1, while HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls. Bray-Curtis cluster analysis of the 5 HuM gut microbial communities revealed that responders HuM2 and HuM3 were closely related, and detailed taxonomic comparison analysis revealed that Bacteroides cellulosilyticus was commonly found in HuM2 and HuM3 with high abundances. Conclusions The results of our study establish the utility of humanized microbiome mice as avatars to delineate features of the host interaction with gut microbial communities needed for effective immunotherapy against GBM.

The objective of this experiment was to evaluate the impact of feeding biochar in a growing diet on cattle performance, methane (CH4) and carbon dioxide (CO2) emissions. Biochar was sourced from ponderosa pine wood waste (High Plains Biochar, Laramie, WY) and was 83% C with 426 m2/g surface area. Crossbred steers (n = 160; initial BW = 357 kg ± 44 kg) were utilized in a randomized block design (blocked by BW), steers assigned randomly to pen (n = 16), and pens assigned randomly to treatment. Two treatments were evaluated, a control (CON) without biochar (40% wheat straw, 40% corn silage, 15% modified distillers grains, 5% supplement) and the same diet with biochar included at 0.8% of diet dry matter replacing corn in the supplement (CHAR). Four pen replications per treatment were paired within BW block and rotated randomly through an emissions barn with two chambers (each treatment evaluated simultaneously) to capture average weekly CH4 and CO2 production. Pen was experimental unit and chamber was included as a fixed effect for emissions data. There were no statistical differences in performance outcomes between CHAR and CON steers (P ≥ 0.23). Numerically, average daily gain (ADG) was greater (P = 0.46) and dry matter intake (DMI) was lower (P = 0.23) for CHAR steers, resulting in a 2.8% improvement in feed efficiency for CHAR steers (P = 0.25). Emissions of CO2 and CH4 did not statistically differ between treatments (P ≥ 0.22). Numerically, CO2 and CH4 emissions were lower for CON compared to CHAR steers when reported as g per day (3.6% lower) or g per kg of DMI (2.4% lower). Based on the results from this study, there was no indication that feeding biochar, supplemented at 0.8% of diet, reduces GHG emissions in growing steers when compared to negative control.

The objective of this experiment was to evaluate the effect of feeding biochar in a finishing diet on cattle performance, carcass quality, methane (CH4) and carbon dioxide (CO2) emissions. Biochar was sourced from ponderosa pine wood waste (High Plains Biochar, Laramie, WY) and was 83% C with 426 m2/g surface area. Crossbred steers (n = 128; initial BW = 480 kg ± 82 kg) were utilized in a randomized block design (blocked by BW), steers assigned randomly to pen (n = 16), and pen was assigned randomly to treatment. Two treatments were evaluated, a finisher control (CON) without biochar and the same finisher with biochar included at 1.0% of diet dry matter replacing corn (CHAR). Four pen replications per treatment were paired within BW block and rotated randomly through an emissions barn with two chambers (each treatment evaluated simultaneously) to capture average weekly emissions of CH4 and CO2. Pen was experimental unit and chamber was included as a fixed effect for emissions data. Dry matter intake (DMI; P < 0.01) and average daily gain (ADG; P = 0.02) were 2.4 and 5.9% lower for CHAR steers, respectively. Feed efficiency (P = 0.22) and production of CO2 and CH4 (P ≥ 0.60) did not differ between treatments. Methane production was numerically lower for CHAR steers when reported as g per day (1.8% lower) or g per kg of DMI (4.8% lower). Hot carcass weight tended to be lighter (P = 0.10) and calculated USDA yield grade was decreased (P = 0.02) for CHAR steers. There was no difference between treatments for LM area, USDA quality grade, or 12th rib fat (P ≥ 0.12). In conclusion, biochar supplementation at 1.0% of diet DM reduced DMI and ADG and had no effect on CH4 and CO2 emissions in finishing steers.

Steam-flaked corn (SFC) has been widely used in feedlots in the southern United States to improve feed efficiency (G:F) by increasing starch digestibility. Similarly, feedlots in the Midwest have commonly fed high moisture corn (HMC), both to ensure corn supply for the year and to improve G:F. However, popularity of SFC in the Midwest is on the rise, but producers still recognize the benefits of HMC utilization. A study was conducted at the Eastern Nebraska Research and Extension Center (ENREC) to compare finishing cattle performance when fed HMC, SFC, or a blend. The objective was to determine the optimum inclusion of the grains when modified distillers grains (MDGS) was included at 20% of the diet. This study utilized 90 yearling steers (353 kg; SD=5.8 kg) individually fed using the Calan gate system in a completely randomized design with cattle stratified by body weight (BW). Treatments included: 100% HMC, 75% HMC blended with 25% SFC, a 50% blend of the grains, 25% HMC blended with 75% SFC, or 100% SFC (DM basis; % of the 70% grain in the diet). Cattle were on feed 168 days with 3-day initial BW and 1 day final BW. Animal was the experimental unit with 18 replications per treatment. No differences in initial BW, ribeye area, or marbling score between treatments were detected (P > 0.12). Ending BW, hot carcass weight, average daily gain, and G:F all linearly increased with increasing inclusion of SFC in the diet (P < 0.05). Carcass adjusted G:F improved from 0.158 to 0.170 for 100% HMC compared to 100% SFC diets, respectively. In conclusion, feeding higher inclusions of SFC in finishing diets resulted in improved performance compared to HMC in diets containing 20% MDGS.

Although the immunotherapy anti-PD-1 works well in glioblastoma (GBM) pre-clinical mouse models, the therapy has not demonstrated a similar efficacy in patient clinical trials. Recent studies have linked the gut microbe composition to tumor growth and response to immunotherapy in some cancers. To date, all GBM pre-clinical studies have been done in mouse models using mouse gut microbiomes. There are significant differences between mouse and human microbial gut compositions, with up to 85% of gut bacteria found in laboratory mice not found in humans. Because it is known that the gut microbe composition can impact the immune system, we hypothesize that the non-responsiveness of GBM patients to immunotherapy may be due to the composition of the gut microbiome. Therefore, we have generated a humanized microbiome mouse model in which mice have been colonized by human donor microbes in their GI tract (two different healthy human donors (HuM1 and HuM2)). In preliminary results, we have found that HuM1 mice are resistant to anti-PD-1, while HuM2 mice are responders to anti-PD-1 in the GL261 syngeneic intracranial model. These mice are genetically identical and only differ in gut microbiome composition. Furthermore, we found that HuM2 mice exhibited a significant increase in cytotoxic CD8+T-cells producing IFN-γ and significant increased CD8+/Treg ratio in the spleen following anti-PD-1 treatment, which was not observed in the HuM1 mice. When testing the efficacy of standard of care temozolomide (TMZ) in our humanized mice, we found that TMZ significantly prolonged survival of both HuM1 and HuM2 mice with intracranial tumors. However, HuM2 mice exhibited superior efficacy (p< 0.001; 57% survival), compared to HuM1 mice (p< 0.01; 0% survival). We are extending these studies to analyze additional humanized microbiome lines as well as GBM patient donor lines to more accurately understand individual responses to tumor growth and responsiveness to therapies.

Cancer immunotherapies, including the checkpoint inhibitors, demonstrate remarkable success in patients with melanoma, but have not shown a similar efficacy in patients with glioblastoma (GBM). Recently, the composition of the gut microbiome has been shown to promote resistance to immune checkpoint inhibitors in melanoma and other cancers suggesting a favorable composition of the gut microbiome is needed to produce an optimal response to checkpoint inhibitors and subsequent anti-tumor immune responses. We propose that the gut microbiome of GBM patients promotes resistance to immunotherapies. To investigate this, we have collected and analyzed the gut microbiome from GBM patients (short-term and long-term survivors) and healthy controls by microbial 16S and metagenomic sequencing. These results will determine microbiome differences between GBM patients and controls, as well as differences within each patient as the disease progressed. Furthermore, all GBM pre-clinical studies to date have been performed in mouse models using mouse gut microbiota. We have previously found that human fecal samples can be transplanted into gnotobiotic (germ-free) mice to successfully colonize the mouse GI tract with human microbes. Herein, we have established humanized microbiome mice utilizing human GBM donor fecal samples previously obtained and analyzed. This model is critical because it allows us to study the relationship between the human microbiome among different GBM patients, and the responsiveness to therapies using the syngeneic GL261 intracranial model. We are currently using these humanized microbiome mouse models to analyze pre-clinical testing of anti-PD-1 in GBM, and endpoints to assess efficacy will include survival times, tumor growth, and examining the periphery and tumor environment for phenotype(s) of infiltrating immune cells. These studies will enhance our understanding of the mechanism of GBM patient’s microbiome in the resistance to immunotherapies and lead to new therapeutic strategies to alter the microbiome composition to enhance immunotherapy for GBM.

Like many cancers, glioma cells have an altered metabolic state and utilize glycolysis even in the presence of oxygen, and thus require a large amount of glucose. The ketogenic diet is a high fat, low carbohydrate, low protein diet that limits the amount of glucose, and theoretically starves tumor growth. In addition, recent reports have indicated that the gut microbiome can influence tumor growth as well as the effectiveness of certain therapeutic agents. In this study, we sought to determine if the ketogenic diet is an effective treatment for glioma, and to correlate changes in the healthy gut bacteria in response to the ketogenic diet. In an orthotopic mouse model of glioma, we found that mice fed the ketogenic diet had slightly increased survival compared to mice fed a normal diet. Comparing the composition of gut bacteria from animals on normal versus ketogenic diets, we found significant differences in several key microbes. Interestingly, we observed that there were several long-term ketogenic survivors implanted with intracranial tumors and displayed a significant increase in the percentage of microbes found in the murine gastrointestinal tract (F. rodentium and related microbes). We found that F. rodentium growth does not occur until day 21 on the diet; thus, F. rodentium could be introduced early as a probiotic and enhance anti-tumor effects of the ketogenic diet. Additionally, we found that the ketogenic diet increased the percentage of mature Th17 cells, potentially collaborating with the growth of F. rodentium to decrease tumor growth. No studies have ever evaluated a probiotic for therapeutic efficacy in glioma. As the ketogenic diet is very strict, and can negatively affect quality of life, a ketogenic-diet probiotic (healthy gut bacteria, similar to the probiotics in yogurt) may offer an alternative supplement and patients could possibly eat a modified (less strict) ketogenic diet.

Glioblastoma (GBM), a particularly devastating type of brain tumor, remains a challenging and difficult disease to treat. Despite aggressive therapies, patients diagnosed with GBM have a median survival of 12-16 months. On a cellular level, GBM tumors are extremely heterogeneous, consisting of resident tumor cells, tumor initiating cells, infiltrating immune cells, endothelial cells, and other tumor associated stromal cells, which makes developing targeted therapies a challenge. In GBM, microglia (MG) and macrophages (MP) are the largest population of tumor-infiltrating cells. GBM tumor cells recruit MG/MP via the secretion of chemo-attractants; however, upon arrival at the tumor, the MG/MP adopt an anti-inflammatory phenotype, and in turn accidentally aide in tumor promotion through the secretion of various cytokines. We have previously determined that macrophages with deletion of Suppressor of Cytokine Signaling 3 (SOCS3), the negative regulator of Signal Transducer and Activator of Transcription 3 (STAT3), have an enhanced M1 (pro-inflammatory) response. Herein, we sought to determine the role of SOCS3 in MG/MP functionality in GBM. In this report, we utilized a conditional model in which SOCS3 deletion is restricted to the myeloid cell population. We found that deletion of SOCS3 in the myeloid cell population delays intracranial tumor growth and increases survival of mice bearing orthotopic glioma tumors. Intracranial tumors from mice with SOCS3 deficient myeloid cells have an increase in the number of infiltrating immune cells, including monocytes, neutrophils and macrophages. Lastly, SOCS3 deficient bone marrow derived macrophages display enhanced and prolonged expression of pro-inflammatory M1 cytokines (TNF-α, IL-6) when exposed to tumor cell conditioned medium in vitro. Therefore, loss of SOCS3 in MG/MP in the tumor promotes an inflammatory, immune response early and thus delays tumor growth. These preliminary findings highlight an important role of the STAT3/SOCS3 signaling axis in MG/MP in GBM tumors.