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Responses of Bos taurus (BT) and B. indicus (BI) beef cows were compared according to their body condition score (BCS) at initiation of breeding season. Cows were assigned to an estrus synchronization + AI protocol and classified as BCS < 5.0 (LBCS, BT, n = 158; BI, n = 248) or BCS ≥ 5.0 (HBCS; BT, n = 173; BI, n = 122). Pregnancy status was verified via transrectal ultrasonography at 30 and 40 d after AI for BI and BT cows, respectively. All BT cows were exposed to natural service for 50 d (beginning 18 d after AI), and all non-pregnant BI cows were immediately exposed to natural service for 60 d. Pregnancy rates to AI did not differ (P ≥ 0.16) between HBCS or LBCS cows (60.9 vs. 53.5% for BT; 37.0 vs. 31.4% for BI; respectively). Pregnancy rates to natural service were greater (P = 0.03) in HBCS BT cows compared with LBCS (82.7 vs. 67.9%; respectively) but did not differ in BI cows (78.9 vs. 71.5%; respectively). Calving rate was greater (P < 0.03) in HBCS cows, which also calved earlier (P < 0.01) compared with LBCS (93.7 vs. 85.1% in BT, 83.6 vs. 73.3% in BI; respectively). HBCS Cows weaned more (P ≤ 0.05) calves compared with LBCS, which were older (P ≤ 0.04) (215 vs. 211 d in BT, 219 vs. 209 d in BI; respectively) and heavier (P ≤ 0.09) at weaning (258 vs. 252 kg in BT, 213 vs. 203 d in BI; respectively). Lastly, kg of calf weaned/cow was greater (P ≤ 0.03) in HBCS cows compared with LBCS (221 vs. 200 d in BT, 159 vs. 129 d in BI; respectively). Therefore, HBCS cows had greater reproductive efficiency and overall productivity, and this was observed in both BT and BI.

Genomic selection has been adopted nationally and internationally in different livestock and plant species. However, understanding whether genomic selection has been effective or not is an essential question for both industry and academia. Once genomic evaluation started being used, estimation of breeding values with pedigree BLUP became biased because this method does not consider selection using genomic information. Hence, the point of divergence of genetic trends obtained with BLUP and Single-step genomic BLUP (ssGBLUP) may indicate the start date of genomic selection. This study aimed to find the start date of genomic selection for a set of economically important traits in pigs and Angus cattle by comparing genetic and Realized Mendelian sampling (RMS) trends obtained using BLUP and ssGBLUP. In pigs, the genetic trends from ssGBLUP and BLUP diverged for all traits with different intensity. In beef cattle, the trends started diverging in 2009 for weaning weight and in 2016 for postweaning gain, with little divergence for birth weight. In pigs, the RMS trends from ssGBLUP and BLUP diverged strongly for one trait and weakly for another trait. In beef cattle, the RMS trends started diverging in 2009 for weaning weight and in 2016 for postweaning gain, with little trend for birth weight. Divergence of the genetic trends from ssGBLUP and BLUP indicate onset of the genomic selection. Presence of trends for RMS indicates selective genotyping, with or without the genomic selection. The onset of genomic selection and genotyping strategies agree with industry practices across the two species. In summary, the effective start of genomic selection can be detected by the divergence between genetic and RMS trends from BLUP and ssGBLUP.

The objective of this study was to evaluate the effects of a synthetic analogue of the bovine appeasing pheromone (i.e. bovine appeasing substance; BAS) on growth and temperament of heifers. At weaning (d 0), 30 heifers (Aberdeen Angus ′ Nelore; 8 ± 1 mo) were stratified by body weight (199.8 ± 16 kg) and randomly assigned to receive a single dose of BAS (n = 15; SecureCattle; Nutricorp, Araras, SP, Brazil) or saline (CON; n = 15; saline 0.9% NaCl). Treatments (5 ml) were topically applied to the nuchal skin area of each animal on d0. Body weight was collected on d 0, 6, 15, 45, and 150. Chute score (1 to 5; 1 = calm, no movement; 5 = violent and continuous struggling) and chute entrance and exit score (1 to 3; 1 = slow; 3 = fast) were collected on d 0, 2, 6, 15, 45 and 150. Scores were averaged across 4 trained technicians. Data were analyzed using the MIXED procedure of SAS. Heifers assigned to BAS had greater (P < 0.01) average daily gain (ADG) than heifers assigned to CON treatment from d 6 to 15 (2.35 and 1.88 kg/d ± 0.16) and from d 15 to 45 (1.79 vs. 1.56 kg/d ± 0.08). Heifers assigned to BAS had lower (P < 0.01) chute entrance score on d 6, 15, and 45, and chute exit score on d 2 to 45 (P = 0.05) when compared to heifers assigned to CON. Additionally, heifers assigned to BAS tended (P = 0.08) to have lower chute score, from d 1 to 150 than heifers assigned to CON. The application of BAS at weaning improved heifers ADG, likely due to an improvement in temperament, suggesting that BAS application has calming effects in the beef cattle herd.

This study evaluated the effect of heat stress abatement strategies on the faecal microbiome of finishing beef steers in the Southeastern United States. The study was designed as a completely randomized block using 32 steers (BW 453k 4 kg) stratified by weight and randomly assigned to one of four treatment groups: covered with fans (CWF), covered no fans (CNF), outside with shade (SHADE), and outside without shade (OUT). All steers were housed outside from June to July and were brought up on a common feedlot ration. After-which steers were placed into treatments on d -10 and covered steers were given 10 days to acclimate Calan-gate system. During the acclimation phase, both covered and outside steers were brought up at a similar rate. Steers were weighed and faecal samples were collected on d 0, 50, and 85. Microbial DNA was extracted from the samples using a combination of mechanical and enzymatic processes, and the DNA sequences were analyzed using QIIME v2.0. There was a treatment x day interaction for Actinobacteriota, Bacteroidota, and Patescibacteria phyla. Additionally, there was a day effect for all three phyla (P < 0.001), but there was only a treatment effect (P < 0.001) for Patescibacteria. There was not an interaction for Fibrobacterota, Firmicutes, or Proteobacteria (P > 0.226), but there was a day effect (P < 0.019) for all three phyla. There was a treatment effect (P = 0.036) for Fibrobacterota, however there was not a treatment effect for Firmicutes and Proteobacteria (P > 0.394). Moreover, there was no interaction, treatment, or day effect (P > 0.39) for the Firmicutes to Bacteroidota ratio. These results suggest that stress abatement strategies may influence the fecal microbiome over time, and they might help better understand how stress affects the microbiome and nutrient digestibility in the lower gut.

The effects of live yeast (Saccharomyces cerevisiae) and ruminal environment on in vitro true digestibility of dry matter, organic matter, and fiber components were evaluated. Ruminally-cannulated beef steers (n = 4; BW = 520 ± 30 kg) were used in a cross-over design to serve as donors of ruminal content. In vitro substrates (6 grower and 6 finisher diets) and individual ingredients (steam-flaked corn, wet corn gluten feed, and old-world bluestem hay) were dehydrated (55o C), ground (1 mm), and incubated using a 2 × 2 factorial arrangement of treatments: a) the presence or not of yeast (8×109 CFU/animal-daily adjusted to a 70 L ruminal content volume), and b) two ruminal inoculums collected from donors fed a grower (50:50) or a finisher (92:08) diet (concentrate:forage). Four incubation batches (experimental unit) were performed, in which the initial two batches were performed followed by another two after animals switched diets and were adapted again during 21d. A Daisy in vitro incubation system was used, and samples incubated for 48h (39°C). The GLIMMIX procedure of SAS was used for the statistical analysis. No 3-way interactions (P ≥ 0.71) or 2-way interactions with substrate were observed (P ≥ 0.25). The in vitro true digestibility of DM, OM, NDF, and hemicellulose increased when yeast was added to the incubator containing a 50:50 ruminal content (Rumen × Yeast; P ≤ 0.02). The ADF digestibility increased (P = 0.02) with yeast addition regardless of ruminal content type (50:50 or 92:08). As expected, finisher substrate diets were more (P < 0.01) digestible than grower diets. The combination of live yeast and a specific ruminal environment seemed to be more important than substrate type, in which live yeast improved in vitro digestion within a 50:50 ruminal inoculum more evidently.

A farmlet study was established to evaluate the effect of four sward types on the performance of heifers and lambs co-grazing: a perennial ryegrass only sward (PRG) receiving 170 kg N/ha each year; a permanent pasture (PP) receiving 135 kg N/ha each year; a six species mix (2 grasses, 2 legumes, 2 herbs) at 70 kg N/ha each year (6SP); and a 12 species mix (3 grasses, 4 legumes and 5 herbs) at 70 kg N/ha each year (12SP). Each farmlet (9 ha) was rotationally grazed with 20 dairy cross heifers (13 months old) and 22 ewes plus lambs from April to November 2020. Heifers and lambs were finished for slaughter from herbage only diets. Heifers were weighed monthly and drafted for slaughter on three occasions when their estimated fat class on the EUROP grid scale reached 3-. Lambs were weighed fortnightly and drafted for slaughter at 42 kg [lambing to weaning (July 7th)], 44 kg (weaning to September) and 46 kg (after September 1st) to obtain a target carcass weight of 21 kg. Average daily gain (ADG) was higher for heifers grazing the 6SP sward (1.13 kg/day) than the PRG (0.99 kg/day) or PP swards (1.03 kg/day; P < 0.05). Kill out percentage (KO%) was higher for heifers grazing the 12SP sward (48.9%) compared to the PP sward (47.2%; P < 0.01). Lamb ADG was higher for lambs grazing the 6SP sward (0.38kg/day) compared to lambs grazing either the PP (0.32kg/day) or the PRG sward (0.29kg/day) (P < 0.01). Lamb KO% was higher for lambs grazing the 12SP sward (49.4%) compared to those grazing the PRG (45.7%) and the PP swards (44.5%; P < 0.01). Lambs grazing the 6SP sward had reduced days to slaughter (109) compared to lambs grazing the PP (137) and PRG (149) swards (P < 0.01). In conclusion, co-grazing multispecies swards improved overall heifer and lamb performance.

The role of the reproductive tract microbiota is an emerging field receiving considerable attention in human research, whereby the vaginal microbiota has been characterized in relation to fertility, conception, pregnancy, and parturition. However, the acidic, Lactobaccillus dominated vaginal microbiota in humans contrasts the neutral, dynamic microbial environment characterized in cattle. Over the past few years, many postpartum studies in dairy cattle described a mechanism recently proposed in humans, the ascension of the vaginal microbiota into the uterine body at the onset of labor. Although bovine fetal sterility prior to parturition is under debate, the vaginal microbiota is an early contact for the neonate with microorganisms and inoculates the uterus during parturition. Thus, any changes occurring in the vaginal microbiota could have implications in dam and calf health; this, combined with ease of access, led to the current research focusing on the bovine vaginal microbiota. Characterization studies during the estrous cycle, gestation, and postpartum period have been performed, concluding the bovine vaginal microbiota is dynamic. However, due to the low abundance in the vaginal microbiota, the seemingly small taxonomic changes reported in these studies could be of great importance. Secondly, researchers have begun to evaluate the effect of exogenous and endogenous hormone concentrations on the vaginal microbiota. Little to no differences have been found except with exogenous melatonin supplementation. In pregnant heifers, exogenous melatonin altered the beta diversity of the vaginal tract by increasing aerobic bacteria present. Moreover, a study evaluating the effects of nutrient restriction on the vaginal microbiota found no taxonomic or structure differences. These findings have allowed researchers to target paucities, improve methodology, and realize the importance of the reproductive tract microbiota. In short, based on these studies, future research examining the role of the reproductive tract microbiota could uncover mechanisms vital to increasing reproductive performance.

Publication in scientific journals remains the primary method to disseminate research findings; however, the landscape of scientific publication is rapidly changing. For instance, although open access publication has led to unprecedented opportunities to share information with the global scientific community, it has also contributed to the rise of “predatory journals.” These journals accept fees to publish articles without promised quality checks (e.g. peer review). In order to better understand current publication practices and the threat predatory journals pose, this session will: 1) Briefly summarize the history of scientific publication and how the current model of peer-reviewed publication developed. 2) Define predatory journals and review components of the international consensus definition (false or misleading information, deviation from best editorial and publication practices, lack of transparency, aggressive/indiscriminate solicitation; Nature doi.org/10.1038/d41586-019-03759-y). 3) Summarize empirical studies that have assessed the current burden of predatory journals. A broad group of stakeholders are affected by these journals, including researchers and the public. 4) Provide a practical approach for audience members to distinguish between predatory and legitimate journals. 5) Highlight some key developments that will lead to new publication models in the future.

Pregnant sheep have been used extensively for investigating pregnancy physiology, providing valuable information about the progression of ruminant pregnancy. The ability to place indwelling catheters, within both maternal and fetal vessels, allows for steady-state investigation of blood flow, nutrient uptakes and utilization, and hormone secretion, under non-stressed and non-anesthetized conditions. As such, our understanding of the in vivo physiology of pregnancy in sheep is unrivalled by any other species. However, until recently, a significant deficit existed in determining the specific function or significance of individual genes expressed by the placenta in livestock. To that end, we developed and have been using in vivo RNA interference (RNAi) within the sheep placenta to examine the function and relative importance of genes involved in conceptus development (PRR15 and LIN28), placental nutrient transport (SLC2A1 and SLC2A3), and placenta derived hormones (CSH). The lentiviral vector LL3.7 is used to generate virus that is stably integrated into the infected cell’s genome, thereby expressing a short-hairpin RNA (shRNA), that when processed within the cell, combines with the RNA Induced Silencing Complex (RISC) resulting in specific mRNA degradation or translational blockage. To accomplish in vivo RNAi, day 9 hatched and fully expanded blastocysts are infected with the lentivirus for 4–5 hours, and then surgically transferred to synchronized recipient uteri. Only the trophectoderm cells are infected by the replication deficient virus, leaving the inner cell mass unaltered, and we typically obtain 70–80% pregnancy rates following transfer of a single blastocyst. Data will be presented from two projects. One is focused on generating a deficiency in placental glucose transporters at mid-gestation, and the other on the impact of CSH RNAi during late gestation, demonstrating the utility of this experimental approach for examining gene function within the placenta of livestock. Supported by NIH-NICHD grants HD093701 and HD094952.

The objective of this study was to determine the effects of in ovo injection of high yield broilers with nicotinamide riboside (NR) on broiler pectoralis major muscle (PMM) development, growth, and mRNA expression. Fertilized Cobb 700 broiler eggs were randomly assigned to 1 of 4 treatments within a 2×2 factorial design with NR dose (DOS; 0 or 2.5 mM) and injection location (LOC; albumen or yolk) as main effects. Eggs were injected with their assigned treatment at d 10 of incubation. Day-15 and -18 embryos and hatched chicks were euthanized, right PMM and muscle fiber morphometric measurements were collected, and left PMM were utilized for mRNA expression analysis of sirtuin-1 and cyclin D1, 2 and 3. There were no LOC×DOS interactions or main effects for d-15 and -18 PMM measurements (P > 0.23). There were LOC×DOS interactions for hatched chick PMM weight and length, and muscle fiber density (P < 0.04). When NR was injected into the albumen, PMM weight decreased (P = 0.02) and length and fiber density were unaffected (P > 0.33), while PMM weight and fiber density increased when NR was injected into the yolk (P < 0.05). There were no LOC×DOS interactions for all genes analyzed for d-15 and 18-embryos (P > 0.23), but NR embryos had greater expression of all genes (P < 0.03) except d-18 cyclin-D2 (P = 0.08). There were LOC×DOS interactions for hatched chick expression of all genes (P < 0.04). There was no difference in sirturin-1 expression when chicks were injected with 0 mM NR, but 2.5-mM yolk chicks had greater expression than 2.5-mM albumen chicks. When NR was injected into the albumen, expression of all cyclins was unaffected (P > 0.08), while expression increased when NR was injected into the yolk (P < 0.01). Increased expression of sirturin-1 and the cyclins may be responsible for NR catalyzed increase in PMM weight and fiber density.

Prenatal and early postnatal life represents critical periods of development across species for many organ systems including immune, nervous, reproductive, and gastrointestinal systems. A high level of plasticity exists during these periods, and thus maternal and environmental cues including stress, immune stimulation, and nutrition, can alter the normal developmental programming of the fetus and neonate and impact the trajectory for disease risk and productivity across the lifespan. This presentation will focus on the impact of and biological mechanisms by which prenatal and early postnatal stressors, including psychological immune and nutritional stressors, alter the normal developmental programming of the immune, gastrointestinal, and neuroendocrine stress axes in the offspring and how this may link to increased disease risk and reduced productivity across the lifespan in animals and humans. Further, specifically how host factors such as biological sex interact with early life stress to shape gut and systemic neuroimmune development and later life disease risk will be discussed.

Lameness is a significant health issue in Canadian feedlots resulting in substantial economic losses. However, the high frequency of misdiagnosis of lameness using traditional methods leads to ineffective treatment, suggesting a new diagnostic method is needed. Growing evidence indicates that microRNAs (miRNAs) can be used as biomarkers for identifying the animals’ physiological status and the diagnosis of certain diseases, but this approach has not been utilized in beef cattle. The objective of this study was to compare blood miRNA profiles between lame and healthy cattle to investigate the relationship between miRNA expression patterns and specific lameness phenotypes. Blood samples were collected from 156 feedlot cattle at 0, 1, 2 and 3 weeks after being diagnosed with either digital dermatitis (DD; n=62), toe tip necrosis syndrome (TTNS; n = 40), or footrot (FR; n = 40) and healthy controls (HC; n = 12) for miRNA libraries construction and sequencing. A total of 314 expressed miRNAs were identified in 89 blood samples collected at week 0 across all groups, with TTN having the largest number of expressed miRNAs (291, P < 0.01) compared to all other groups (HC=276, DD=281, FR=278). Although miRNA profiles did not differ among the lameness types, type-specific miRNAs were identified; 6 in DD, 10 in TTN, 5 in FR and 7 in HC cattle. In addition, 3, 6 and 7 DE miRNAs were detected in DD, TTNS and FR when compared with HC cattle. Most of the DE and group-specific miRNAs are related to inflammation and skin diseases. The DE miRNAs were different between week 0 and all other weeks, indicating miRNA profiles may differ over time and with disease progression and recovery. These findings provide an initial understanding of the relationship between the cattle blood miRNAome and lameness and suggest that miRNA expression holds promise in the discovery of novel biomarkers for identifying lameness.

Domestic ruminants utilize extensive pasture and rangelands as a primary source of nutrition. These forage sources vary greatly in nutritional content and availability due to season, plant species and climatic factors. Therefore, ruminants are exposed to nutrient excesses and deficiencies at varying points during the reproductive cycle. The objectives of this presentation are 1) to review our understanding of the impact of nutritional changes experienced by the dam during gestation on subsequent reproductive performance of female offspring, 2) examine the timing and duration of nutrient restriction on indicators of fertility in domestic ruminants and 3) compare responses under production conditions to mechanisms determined in controlled experiments. Depending on the time and severity of the nutrient deprivation, physiological effects include reduced gonadal weight, reduction in 2° and 3° follicles, alterations in the hypothalamic-pituitary axis, fetal weight and attainment of puberty. Anti-Müllerian hormone (AMH) and antral follicle count (AFC) are indicators of fertility in ruminants. Severe nutrient deprivation of heifers during the first trimester of gestation reduced AMH and AFC in female offspring. In contrast, a more moderate nutritional challenge to mature cows during early gestation did not result in changes in AMH and AFC in female offspring. Heifers from dams that grazed protein deficient range during the third trimester had delayed puberty onset and reduced pregnancy rates compared to heifers from supplemented dams. Recently, we compared indictors of fertility in heifers from dams that grazed range or irrigated pastures during the first two trimesters. Based on preliminary data, cows that grazed range were nutritionally challenged; however, heifers from range dams did not appear to differ in indicators of fertility from heifers from dams grazing irrigated pastures. Continued investigations on impacts of maternal nutrition on reproduction in offspring under production conditions are warranted.

How do you teach a plant-based course focused on forage physiology, production and management to an animal science student? How do you engage them and keep them engaged? Empathy. Put yourself in the shoes of the animal science student who is sitting in a plant-science course. Make it applied and relatable to the animal scientist. In this talk I will take a case-study approach to sharing my experience as an animal science student tasked with knowing something about plants and how I use that experience to guide my teaching methods in a Forages course at the University of Missouri.

Assessment of embryo quality and viability has traditionally been based on morphological evaluation. However, morphological evaluation, though somewhat reliable, is subjective in nature and varies greatly depending upon the skill and experience of the evaluator. Although research has been conducted with the goal of identifying more accurate and objective methods of evaluation, the issue of repeatability and consistency in predicting the likelihood of a successful pregnancy remains. Previous research has proposed the ability to utilize specific gravity to predict developmental energy reserves based on embryonic weight, theoretically identifying those embryos with an increased chance of success following standard embryonic transfer. The objective of this study was to determine if specific gravity could accurately identify those embryos that were most likely to succeed in generating positive pregnancies. Bovine embryos (n = 20) were dropped through media in a specific gravity chamber. Utilizing an embryo tracking software program, researchers recorded the time elapsed as the embryos descended 1 cm through the media. Embryos were then transferred into recipient cattle, and pregnancy was detected via ultrasound approximately 40 days post transfer. Of the 20 embryos transferred, 12 resulted in positive pregnancies. Descent times of these positive pregnancies ranged from 25.96 to 90.27 seconds, with an average descent time of 59.72 seconds. An analysis of the relationship between descent time and pregnancy outcome showed no significant difference (P > 0.05). Although there was no significance in the findings of this study, additional trials will be performed to thoroughly evaluate the potential of this technology as an objective measurement of embryonic viability.

Animal welfare often engages diverse values and ethics including those of citizens, consumers, scientists, and farmers. Consequently, AW is one of the seven grand challenges (GCs) identified by the ASAS Public Policy Committee (PPC). Societal concerns can lead to public policy decisions at the state or federal level that impact animal agriculture. The ASAS PPC GCs on animal welfare will be examined, progress evaluated, and recommendations made for setting research priorities. Five key questions and their corresponding expected outcomes were identified to advance AW in the GC areas of sustainable management practices, genetic markers, pain mitigation, transportation, and humane euthanasia/depopulation. The diversity of disciplines required to address these key questions is astounding. A five-year window (2015-present) was used to evaluate each key question and outcome. The published peer-reviewed literature and the North Central 1029 and Western 3173 multi-state project station reports served as the foundation for our evaluation. In brief, research opportunities exist to identify new methods and technologies to improve livestock transportation, humane endpoints for emergency depopulation, and management practices enhancing the sustainability footprint of livestock; and there is an urgent need to identify effective preventative measures against heat stress. The investigation of effective methods for species-specific pain assessment and mitigation strategies should continue to be a priority and include the development of alternative practices that avoid pain. Research into genetic markers associated with various aspects of animal wellbeing is growing and offers ample opportunity to improve animal welfare. Animal welfare research also requires scientists to engage with stakeholders to maintain perspective. One example is demonstrated by members of NC 1029 who report engagement in over thirty advisory boards across the supply chain. Through this type of engagement, animal scientists can influence the food supply system and serve as a conduit between social concerns, policy, and science.

The aim of the present study was to evaluate the effects of reducing dietary crude protein (CP) levels and supplementing rumen-protected methionine (RPM) on production, apparent digestibility of nutrients and economic effectiveness of lactating Holstein cows. A total of 96 (60 ± 7 d in milk; 34.39 ± 5.74 kg/d of milk production; mean ± SD) lactating Holstein dairy cows were randomly assigned to 1 of 2 treatments: diet containing 17.3% CP without RPM (control group; CON); diet containing 16.4% CP with supplementing 15.0 g/d of RPM (treatment group; RPM). All the data were analyzed using one-way analysis of variance (ANOVA) of SAS (SAS version 9.2, SAS Institute Inc., Cary, NC.). No effects were observed of reducing dietary CP on somatic cell counts, milk yield and milk composition including fat, protein, lactose, total solid, solids-not-fat and milk urea nitrogen. The total apparent digestibility of nutrients was similar between treatments, while RPM group had higher calcium digestion (44.24% vs. 42.75% ± 10.13%) and phosphorus digestion (48.50% vs. 45.96% ± 12.90%) numerically. In addition, supplying the diet of 16.4% CP with RPM supplementation to cows could reduce feeding cost by 0.5 $/d per cow and boost net profits. In conclusion, lower dietary CP with RPM supplementation did not limit milk yield, milk composition and apparent digestibility of nutrients, and also could improve the economic benefits of the dairy farms.

Interest in preparing pet foods at home has continued to build momentum over the last decade. Websites, blogs, and social media groups praise the benefits of feeding raw and home-prepared diets. Consistent research has demonstrated the beneficial effects of raw diets on macronutrient digestibility (primarily fat and protein); however, research is lacking on overall health impacts. While it is important to acknowledge the lack of substantial research, owner choice and compliance also should be considered. With well-established nutrient requirements for dogs and cats available through AAFCO and the National Research Council (NRC), it is relatively uncomplicated to meet requirements when typical processing methods are not required (extrusion or canning). Although it is not complicated to meet the requirements with home-based fresh or lightly cooked ingredients, care must be taken to properly ensure that vitamin and mineral requirements are met. In review of over 100 homemade diets submitted for evaluation, 0% met all vitamin and mineral requirements for dogs or cats. All diets were supplemented with some form of calcium. Approximately 25% of submitted diets utilized bones as the source of calcium. Bones can be problematic depending on type, particularly for digestion, broken pieces that can cause intestinal blockages, and broken teeth. None of the diets were properly balanced for copper, zinc and iron. It is likely, this triad of minerals has been overlooked in diet formulations. It is critical to note that improper balance of these minerals could play a significant role in oxidative stress and aging. Iodine is typically met through additions of kelp; however, variations in quality can result in over or under-supplementation having detrimental effects on the thyroid. Vitamins E and D also are often under-supplemented in these diets. Educating owners on nutrient requirements and ratios, and providing proper supplementation, can assure a quality and balanced diet.

Designing research for beef cattle production in rangeland environments is an ongoing challenge for researchers worldwide. Specifically, creating study designs that mirror actual production environments yet have enough observations for statistical inference is a challenge that often hinders researchers in efforts to publish their observations. Numerous journals will accept “case study” or observational results that lack valid statistical inference. However, these journals are limited in number and often lack impact. Approaches are available to gain statistical inference by creating multiple observations within a common group of animals. Approaches to increasing statistical observations will be discussed in this presentation. Modeling animal behavior and performance on extensive rangeland landscapes is commonly practiced in wildlife ecology and, more recently, has been published in Animal Science journals. Additionally, new technology has made it possible to apply treatments (e.g., supplementation studies) to individual animals on extensive environments where large, diverse herds/flocks of cattle/sheep are managed as a single group. Use of individual animal identification (EID) and feed intake technology has opened a wide range of research possibilities for beef cattle production systems research in rangeland environments. Likewise, global positioning system (GPS) collars and activity monitors have created the opportunity to evaluate animal grazing behavior in remote and extensive landscapes. The use of multiple regression models to evaluate resource use in extensive environments will, in turn, help managers optimize beef cattle production and the sustainable use of forage/rangeland resources. Embracing new technologies such as GPS, activity monitors, EID tags, and feed intake monitors combined with multiple regression modeling tools will aid in designing and publishing beef cattle production research in extensive rangeland environments.

Distilling the alcoholic beverage category down, sugar + yeast = ethanol. However, the manufacturing processes for these beverages are complex and unique. Utilizing a myriad of raw material inputs to produce numerous outputs is no small feat. This presentation will provide an overview of the distilled beverage landscape and will discuss some of the key product types, substrates, ancillary ingredients, and their manufacturing processes. On a fundamental level, we will also consider what by-products are created during the process of making these beloved liquids and how that might relate back to the animal industry.

Enterotoxigenic Escherichia coli (ETEC) F4 and F18 are the two most dominant pathogenic strains in weaned pigs. The objective of this experiment was to test the effects of dietary monobutyrin and monovalerin on performance and systemic immunity of weanling piglets coinfected with F4/F18 ETEC. Twenty weaned pigs (8.21 ± 1.23 kg) were individually housed and were randomly allotted to one of three diets: control (n = 6), 0.1% monobutyrin (n = 7), or 0.1% monovalerin (n = 7). The experiment was conducted 14 days, including 7 days’ adaption and 7 days post-inoculation (PI). On d 0, d 1, and d 2 PI, pigs were inoculated with 0.5 × 109 CFU/1.5 mL each of F4 and F18 ETEC for three consecutive days. Diarrhea score was recorded daily to determine frequency of diarrhea. Piglets and feeders were weighed throughout the trial to analyze growth performance. Fecal cultures from pigs on d 0, 2, and 4 PI were inspected to identify the absence or presence of hemolytic coliforms. Blood was collected on d 0, 4, and 7 PI for complete blood cells count. All data were analyzed by the Proc Mixed of SAS with randomized complete block design. Pigs supplemented with monovalerin and monobutyrin had numerically higher ADG (249 and 282 g/day) from d 0 to d 7 PI than pigs in control (198 g/day). Supplementation of monovalerin reduced (P < 0.05) frequency of diarrhea throughout the experiment. Pigs fed monovalerin had lower (P < 0.05) neutrophil counts on d 4 PI compared with control. Hemolytic coliforms were observed in all fecal cultures from d 2 and d 4 PI, confirming fecal shedding of ETEC. Results of this study indicate the potential benefits of monovalerin supplementation on performance and disease resistance of weaned pigs coinfected with F4 and F8 ETEC.

To test the hypothesis that average daily gain (ADG) and clinical parameters of steers grazing novel non-toxic (NTE) or toxic KY-31 (TE) endophyte-infected tall fescue would be improved by ad libitum intake of vitamin-mineral mixes (V-M) that contain 27 ppm Se as a 1:1 blend of SELPLEX:sodium selenite (MIX) vs sodium selenite (ISe), 32 TE-naïve beef steers depleted of Se were randomly assigned to ad libitum consumption ISe vs MIX for 35 d and fed enough of a NTE/alfalfa/grain diet to achieve 0.57 kg BW gain/d. Within Se-form treatments, 2 steers were randomly assigned to each of 4, 2-acre NTE (ISe = 316 ± 31 kg, MIX = 315 ± 22 kg) or TE (ISe = 316 ± 37 kg, MIX = 314 ± 39 kg) paddocks for 84 d and had ad libitum access to their respective V-M. The MIXED procedure of SAS was used to assess effects of day, Se-form (ISe, MIX) and endophyte (NTE, TE) treatments, and their interactions. Whole blood Se decreased (P < 0.01) 31% from d 0 to 84 and was 6.2% greater (P < 0.01) for MIX steers. Serum prolactin decreased (P < 0.01) 18% for NTE and 48% for TE steers from d 0 to 84 and was 17% greater (P = 0.01) for MIX vs. ISe TE steers. Alkaline phosphatase activity (AP) decreased (P < 0.02) 27% from d 0 to 84 and was 15% greater (P < 0.02) for MIX steers. Serum urea nitrogen increased (P < 0.02) 8.2% from d 0 to 84 for TE but not NTE steers. Average daily gain (kg/d) was less (P < 0.01) in TE (-0.18) vs NTE (0.09) steers. We conclude that the ad libitum intake of MIX ameliorated the negative effects of consuming TE on serum prolactin and AP but not ADG.

Conflicting anecdotal accounts from producers about their cattle performance and fly-repellence outcomes while receiving garlic-infused supplements have limited feeding decisions. This study evaluated differences in animal performance and insectifugal outcomes among four groups (26 steers per group) over two years (total = 208 steers). The steers either received non-garlic mineral supplement (MS), MS+0.3% garlic oil-based premix (GOil), MS+2.5% garlic powder (LGP), or MS+5% garlic powder (HGP). A completely randomized design was implemented where each steer was assigned to each treatment group while balancing for bodyweight and breed composition. Steers received a feedlot ration and free-choice mineral supplement for 86 and 108 d in the first and second year, respectively. Individual feed and supplement consumption were measured with automatic feeding units. Fly abundance on each animal was determined using digital images collected during weekly fly surveys. The ADG, feed efficiency, ultrasound body composition, and intake-related activities were also evaluated during the study. Data were analyzed using linear- or generalized linear mixed models in SAS. A significant year and Group×year interactions were observed for daily supplement intake (P < 0.02). Similar interactions (P < 0.04) were observed for feeding behaviors associated with supplement intake. Fly abundance during the peak months was below the economic threshold and was not different among the groups (P = 0.25), but the year and Group×year interactions were significant (P < 0.02). There were no differences among the groups for DMI (P = 0.81), ADG (P = 0.54) and gain to feed ratio (P = 0.34). The Group×year interactions were present for the change in ultrasound rib-fat-thickness. The reasons behind the differences in supplement intake are not clear but may be related to the garlic products’ phytochemical profiles. These results suggest that further studies are required to identify the specific bioactive compounds in garlic that affect supplement intake.

Use of cool-season annual cover crops through grazing has been shown to be a potential tool in extending the grazing season, while still mitigating environmental risks associated with warm-season row crop production. Although data describing the effects of grazing on soil health are not novel, effects of grazing length on animal performance and cover crop production are limited. The objective was to determine cattle performance and forage production when grazing a cool-season annual cover-crop. Twelve, 1.2-ha pastures were established in a four species forage mix and randomly allocated to be grazed through either mid-February (FEB), mid-March (MAR), or mid-April (APR) with a non-grazed control (CON). Three tester steers were randomly placed in each paddock and a 1:1 forage allowance was maintained in each paddock using put-and-take steers. Animals were weighed every 30 d for determination of average daily gain (ADG). Forage was harvested bi-weekly and analyzed for forage production, neutral detergent fiber (NDF), and acid detergent fiber (ADF). Fiber fractions were measured using an ANKOM fiber analyzer (ANKOM Tech, Macedon, NY). All data were analyzed using MIXED procedure of SAS version 9.4 (SAS Inst., Cary, NC). Differences in forage mass were detected between CON and FEB (3,694.75 vs. 2,539.68 kg/ha; P < 0.003), CON and MAR (3,694.75 vs. 1,823.45 kg/ha; P < 0.001), and CON and APR (3,694.75 vs. 1,976.23 kg/ha; P < 0.001). Differences in total gain/acre were detected between APR and MAR (212.24 vs. 101.74 kg/ha; P < 0.0001), APR and FEB (212.24 vs 52.65 kg/ha; P < 0.0001), and FEB and MAR (101.74 vs. 52.65 kg/ha; P < 0.003). No differences were detected for tester ADG (1.23 kg/day, P = 0.56), NDF (44.9%, P = 0.99), or ADF (27.2%, P = 0.92) among treatments. These results indicate that cattle removal date effected forage yield and total gain/hectare.

Genetic improvement of health, welfare, efficiency, and fertility traits is challenging due to expensive and fuzzy phenotypes, the polygenic nature of traits, antagonistic genetic correlations to production traits and low heritabilities. Nevertheless, many organizations have introduced large-scale genetic evaluations for such traits in routine selection indexes. Medium and high-density arrays can be applied in genomic selection strategies to improve breeding value accuracy, and also in genome-wide association studies (GWAS) to identify causative mutations responsible for economically important traits. Genomic information is particularly helpful when traits have low heritability. The objective here is to provide a framework for including health, welfare, efficiency, and fertility traits taken from large-scale genetic and genomic analyses and identifying areas of potential improvement in terms of trait definition and performance testing. General tendencies between trait groups confirmed that a number of moderate unfavourable correlations (+/-0.20 or higher) exist between economically important trait complexes and health, welfare, and fertility traits. A number of trait complexes were identified in which “closer-to-biology” phenotypes could provide clear improvements to routine genetic and genomic selection programs. Here we outline development of these phenotypes and describe their collection. While conventional variance component estimation methods have underpinned the genomic component of some traits of economic interest, performance testing for health, welfare, efficiency, and fertility traits remains an elusive goal for breeding programs. Although our results are encouraging, there is much to be done in terms of trait definition and obtaining better measures of physiological parameters for wide-scale application in breeding programs. Close collaboration between veterinarians, physiologists, and geneticists is necessary to attain meaningful advancement in such areas. We would like to acknowledge the support and funding from all national and international partners involved in the RDGP project through the Large Scale Applied Research Project program from Genome Canada

The objectives of this study were to evaluate physiological responses to heat stress of Holstein heifers carrying the SLICK1 allele of the PRLR, i.e., the slick mutation. Slick and non-slick females were produced in central CA (n = 3 farms) and south FL (n = 3 farms) by inseminating cows with semen from two heterozygous slick Holstein sires. In July 2020, a subgroup of heifers of both genotypes (slick=93; non-slick=110) and two age groups (Group 1: 6–8 months old; n = 131; Group 2: 9–51 days old; n = 72) in both states were evaluated for rectal temperature (RT), surface temperature (ST) and sweating rate (SR). Dry bulb temperature, relative humidity and dew point were recorded every 15 min during testing for temperature humidity index (THI) calculation. Statistical models included the effects of genotype, state, farm within state, group, sire, THI, and interactions. Significant effects were considered as those with P < 0.05. Although the THI in CA was lower than FL (80 ± 0.1 vs 84 ± 0.2; P < 0.01), both were above 77 (estimated heat stress threshold for young animals). Slick heifers raised in FL had lower RT compared to non-slick siblings (39.6 ± 0.1 vs 40.1 ±0.1; genotype P < 0.01); this difference was not observed in CA (39.1 ± 0.04 vs 39.1 ± 0.05; genotype P = 0.5; state x genotype P < 0.01). In both states, slick heifers tended to have lower ST (34.7 ± 0.2 vs 35.1 ± 0.2; P = 0.07); CA heifers had lower RT and ST and higher SR (P < 0.01) regardless of genotype. Current results show that presence of the SLICK1 allele can improve the ability of Holstein heifers to regulate body temperature, particularly in high humidity conditions. Next steps will include analysis of performance as study animals enter the reproductive phase. Funding sources: Holstein Association USA and L.E. “Red” Larson Endowment.

Average daily gain is one of the most important measures producers utilize to measure productivity of beef cattle. The objective was to examine how weather variables influence ADG. Data from 790 beef steers collected using an Insentec feeding system were utilized for this study. Intake data were condensed from daily data into weekly averages (n = 13,739 steer-weeks). The variables considered were ADG (-3.0 to 4.86 kg/d), DMI (2.50 to 21.77kg/d), BW (197.3 to 796.1 kg), dietary energy density (NEm; 1.2 to 2.5 Mcal/kg), average ambient temperature (-23.7 to 21.4 °C), range of temperature (2.8 to 19.4 °C), dew point (-27.8 to 14.3 °C), wind speed (2.08 to 6.49 km/h), solar radiation (30.8 to 297.1 W/m2), and two weeks lag (average of previous two weeks values) and monthly lag (average of previous four weeks values) of each weather variable. Relationship between weather variables were considered while developing the model, including controlling for confounding variables. Residuals of ADG generated after fitting week of year as a fixed effect were used in scatter plots with explanatory variables to identify if non-linear relationships existed. The MIXED procedure of SAS was used to assess the fit and regression parameters of explanatory variables as fixed covariates. Repeated measures were handled using the REPEATED statement and within-individual relationship was accounted for using the Toeplitz covariance structure. Body weight and energy density had linear and quadratic relationships with ADG (Table 1). Two weeks lag of wind speed and monthly lag of solar radiation had positive and negative associations with ADG, respectively, and there were several interactions between weather variables. These data indicate that weather variables influence ADG and should be considered in ADG prediction equations.

Thirty-five crossbred Angus heifers (body weight = 359.5 >± 7.1 kg) were randomly assigned to a 2 × 2 factorial design to evaluate the effects of vitamin and mineral supplementation [VMSUP; supplemented (VTM) vs. unsupplemented (NoVTM)] and rate of gain [GAIN; low gain (LG), 0.28 kg/d vs. moderate gain (MG), 0.79 kg/d] during the first 83 d of gestation on trace mineral concentrations in fetal liver, muscle, and allantoic (ALF) and amniotic (AMF) fluids. The VTM treatment (113 g supplement•heifer-1•d-1) was initiated a minimum 71 d before breeding. At breeding, heifers were either maintained on the basal diet (LG) or received the MG diet by adding a protein/energy supplement to the basal diet. On d 83 of gestation, samples of fetal liver, muscle, ALF, and AMF were collected and analyzed for trace mineral concentrations. In fetal liver, Se, Cu, Mn, and Co concentrations were greater (P ≤ 0.04) for VTM than NoVTM, while Mo and Co greater (P ≤ 0.04) for LG than MG. In fetal muscle, VTM increased (P ≤ 0.02) concentrations of Se and Zn, whereas LG increased (P < 0.01) Zn. In ALF, Mo concentrations were affected (P = 0.03) by a VMSUP × GAIN interaction, with VTM-MG greater than NoVTM-MG; while VTM increased (P < 0.01) concentrations of Se and Co. Trace mineral concentrations were not affected (P ≥ 0.13) in AMF. In conclusion, VTM increased fetal liver Se, Cu, Mn, and Co concentrations; fetal muscle Se and Zn; and ALF Se and Co; while LG increased fetal liver Mo and Co concentrations and fetal muscle Zn. Our results confirm that managerial decisions associated with vitamin and mineral supplementation and rate of gain can alter fetal reserves of trace elements during early pregnancy.

The objective was to investigate the effects of a high canola meal-containing diet and multi-carbohydrase supplementation on growth performance and intestinal pH, short-chain fatty acid (SCFA) profile, and microbiome in weaned piglets. Twenty-four piglets (7.46 ± 0.23 kg BW) were individually assigned to 1 of 3 diets in a completely randomized design. Diets were fed for 21 d: SB, corn and soybean meal-based diet; CM, 25% canola meal substituting soybean meal and adjusted to meet energy and nutrient requirements of piglets (NRC, 2012); and CM+E, CM supplemented with multi-carbohydrase (0.01%). Feed disappearance and BW were recorded weekly. On d 21, piglets were euthanized to assess digesta pH, SCFA profile, and gut microbiome. Data were analyzed using the MIXED procedure of SAS using the piglet as the experimental unit. There were no differences (P > 0.10) in growth performance and digesta pH. Piglets fed CM+E tended to (P = 0.051) or had significantly lower (P < 0.05) butyric acid in the cecum than piglets fed CM and SB, respectively. Piglets fed SB had higher (P < 0.05) isobutyric acid in the colon than piglets fed CM+E. Piglets fed SB and CM tended to have higher (P < 0.05) isovaleric acid in the colon than piglets fed CM+E. Piglets fed SB tended to have lower (P < 0.10) proportion of Bifidobacteria, and had lower (P < 0.05) Enterobacteriaceae, Enterococcus, and Lactobacillus than piglets fed CM. Piglets fed CM+E tended to have lower (P < 0.10) Bifidobacteria and higher Clostridium XIVa than piglets fed CM, and had lower (P < 0.05) Enterococcus, but higher (P < 0.05) Lactobacillus than piglets fed CM. In conclusion, feeding 25% of canola meal in substitution to soybean meal with or without multi-carbohydrase supplementation changed the intestinal environment by modifying SCFA profile and relative proportion of microbiome, but without affecting digesta pH or growth performance in nursery piglets.

BACKGROUND In the present study, we characterized the long-term metabolic changes of brain metastases irradiated with stereotactic radiosurgery (SRS) by sequential dynamic PET imaging using the radiolabeled amino acid O-(2-[18F]-fluoroethyl)-L-tyrosine (FET). We hypothesized that this approach is of considerable clinical value to diagnose delayed radiation-induced changes. MATERIAL AND METHODS From 2010–2021, we retrospectively identified patients with brain metastases from solid extracranial primary tumors who (i) were treated with SRS with or without concurrent immunotherapy using checkpoint inhibitors, (ii) had equivocal or progressive MRI findings after SRS, and (iii) subsequently underwent at least two additional dynamic FET PET scans during follow-up for long-term evaluation. Mean tumor-to-brain ratios (TBR) and the dynamic FET PET parameter time-to-peak were obtained. Diagnostic performances were calculated using receiver operating characteristic curve analyses. Diagnoses were confirmed histologically or clinicoradiologically. RESULTS We identified 36 patients with 98 FET PET scans (median number, 3; range, 2–6). Concurrent to SRS, 8 patients (22%) were treated with checkpoint inhibitors. Following SRS, suspicious MRI findings occurred after a median time of 11 months (range, 2–64 months). Subsequently, FET PET scans were acquired over a median period of 13 months (range, 5–60 months). The overall median follow-up time was 26 months (range, 8–101 months). Twenty-one patients (58%) had delayed radiation-induced changes. TBRs calculated from the last available FET PET scan showed the highest accuracy (92%) to identify delayed radiation-induced changes (threshold, 1.95; P CONCLUSION FET PET has a high diagnostic accuracy for characterizing the long-term changes of irradiated brain metastases.

BACKGROUND Although prognosis is poor in high-grade glioma (HGG) patients, some become a long-term survivor (LTS). Health-related quality of life (HRQOL) and cognition are seen as indicators for quality of survival in HGG but data in LTS and their caregivers are scarce. We aim to investigate cognition and HRQOL in LTS with evaluation by their proxies. MATERIAL AND METHODS We included LTS with initial HGG-diagnosis between 1999–2014. Cognition was assessed with: Boston Naming Test, shortened Token Test, verbal (Category and Letter) Fluency, Diagnostic Instrument of Mild Aphasia, Hopkins Verbal Learning Test and Trail Making Test (A, B, B/A) and HRQOL with: EORTC Quality of Life Questionnaire (C30) and Brain Cancer Module (BN20). 15 Proxies evaluated patients’ HRQOL with: C30, BN20 and they filled in a burden scale: Caregiver Strain Index (CSI). RESULTS 21 patients with grade III or IV glioma and 7 years (range 5–20 years) median survival after diagnosis participated. Compared to normative data patients deviated significantly on HVLT (direct recall, delayed recall, recognition true positives), TMT (A, B and B/A), BNT and CF (p12 years survival compared to patients with CONCLUSION Most LTS suffer from cognitive impairments. Despite worse prognosis and cognitive impairments, at group level patients report relatively intact QoL, which is supported by their proxies. However, at the individual level severity of impairments is variable and high caregiver strain is reported in one-third of the proxies. Objective cognitive functioning was only modestly associated with subjective experience in patients. A patient-tailored approach is recommended to investigate individual impairments and needs for rehabilitation and support for patients and also for their carers.

BACKGROUND Mutation of isocitrate dehydrogenase (IDH) is not only an important landmark in the development of low-grade gliomas, but also has prognostic significance and is a potential therapeutic target. There is a high need to determinate IDH mutation status at diagnosis and during the course of therapy in a non-invasive and reliable manner. We established a machine learning approach based on a support vector machine to detect IDH mutation status in in vivo standard 1H-magnetic resonance spectroscopy (1H-MRS) at 3T with an accuracy of 88.2%, a sensitivity of 95.5% (95% CI, 77.2–99.9%), and a specificity of 75% (95% CI, 42.85–94.5%) in a prospective monocentric clinical trial. Here, the same method is applied in a retrospective cohort at 1.5T and tested for transferability. MATERIAL AND METHODS Validation cohort. The validation cohort comprised 100 patients with glioma for which standard in vivo 1H-MRS spectra had been acquired between 2002 and 2007. Standard single voxel spectroscopy had been measured at 1.5T using a PRESS sequence with a TR of 1500ms and a TE of 30ms. One sample had to be excluded due to non-malignant histology and for 15 samples the IDH mutation status was not available. Therefore, the validation cohort comprised 84 samples, of which 35 were bearing an IDH mutation in immunohistochemistry (sequencing for confirmation is outstanding). Machine learning. To transfer our method to an independent validation cohort our previously established machine learning approach was first trained on all samples of the 3T group. The trained algorithm was then applied to the data of the validation cohort. Here, among other factors the different field strengths, with which the spectra were acquired (3T vs. 1.5T) had to be considered. RESULTS 27 samples of the validation cohort had to be excluded due to poor spectra quality. Our approach correctly detected IDH mutation status in 47 of 62 patients (75.8%), although the technical conditions were significantly different from our published prospective cohort. 17 of 30 patients bearing an IDH mutation were correctly identified, while 30 of 32 wild type patients were determined successfully. CONCLUSION Our approach to detect IDH mutation status has promising application in an unselected retrospective cohort, demonstrating transferability across different technical conditions. Further investigations to improve our technique and an advanced neuropathological processing of the samples are planned.

BACKGROUND Neurocognitive deficits are common among brain tumour patients, and may impact on patient awareness of deficits in instrumental activities in daily life (IADL). This study aimed to examine differences between patient-reported and proxy-reported assessments of the patient’s performance of IADL, and whether the level of (dis)agreement is associated with neurocognitive deficits. MATERIAL AND METHODS A phase III EORTC questionnaire measuring IADL in brain tumour patients (EORTC IADL-BN32) and six neurocognitive test measures were administered as part of a larger multicentre international study designed to develop a brain tumour specific IADL questionnaire. Bland-Altman plots and Mann-Whitney U tests were used to evaluated patient- and proxy-reported IADL on a group level. Subsequently, Mann-Whitney U tests were performed to compare patient-proxy difference scores (patient IADL score - their proxy IADL score) between patients who were considered clearly neurocognitively impaired (≥2 neurocognitive test measures; ≤2.0 SD below healthy controls) and patients who were not. Furthermore, multinomial logistic regression analyses were performed to examined which sociodemgraphic, clinical, and particularly neurocognitive variables were independently associated with patients and proxies differing in their evaluation of patient’s IADL. RESULTS Patients (N=81) and proxies (N=81), on group level, did not significantly differ on either the IADL individual item or scale scores. However, significant differences were found on patient-proxy difference scores between patients who were (N=37) and were not (N=44) considered clearly neurocognitively impaired for 10/32 individual items and one of the scales (i.e. Scale 4: Administrative tasks), all showing that the proxies of clearly neurocognitively impaired patients reported more problems relative to the patients themselves, compared to proxies of patients not clearly neurocognitively impaired. Furthermore, for each scale, a neurocognitive variable, either impaired information processing speed, cognitive flexibility, verbal fluency or the number of neurocognitive test measures impaired, was found to be independently associated with proxies reporting more problems. For 4/5 scales, a clinical variable was additionally independently associated with proxies reporting more problems. Only one variable was independently associated with patient reporting more problems, namely being in active treatment was found to be associated with patients reporting more problems on Scale 4: Administrative tasks. CONCLUSION Results imply a consistent trend of clearly neurocognitively impaired patients underreporting problems with IADL compared to their proxies. It would therefore be advised to administer both the patient- and proxy-version of the EORTC IADL-BN32, particularly if neurocognitive deficits are presumed.

BACKGROUND Glioblastoma (GBM) are the most common primary brain tumors. If untreated the average survival is around 12–18 months. Unfortunately, despite extensive research efforts, the therapeutic options remain limited. One major aspect complicating therapeutic development is an immunosuppressive tumor microenvironment. Isocitrate dehydrogenase (IDH)-mutant, WHO grade 4 astrocytomas appear histologically indistinguishable from GBM, but show significantly longer survival times. IDH mutations lead to changes in the tumor microenvironment with accrual of the neometabolite R-2-hydroxyglutarate. Previous studies on bulk transcriptomes have shown differences in the immune compartment of both tumor entities that were linked to the differences in clinical behavior. MATERIAL AND METHODS We have conducted high-dimensional comparative analyses of the myeloid compartment in surgically resected human GBM and IDH-mutant WHO grade 4 astrocytomas using single-cell RNA-Sequencing and immunohistochemistry. Histologically normal brain regions from epilepsy patients were used as controls. For analysis, whole-cell suspensions were prepared from freshly resected tumors or controls. Fluorescence activated cell sorting was used for myeloid cell enrichment. Samples were processed using the high-sensitivity single-cell RNA sequencing protocol CEL-Seq2. Seurat and StemID2 algorithms were used for clustering, differential gene expression and pseudotime analysis. Protein validation was achieved using immunohistochemistry. RESULTS We identified profound transcriptional changes of glioma-associated microglia in GBM with respect to control brain samples. Namely, we observed a global upregulation of major histocompatibility complex associated genes in GBM across all clusters. Additionally, we identified distinct myeloid subsets with phagocytic, hypoxia-associated and chemotactic transcriptomic signatures. Pseudotime analysis finely resolved transitional cell states. These changes were dramatically attenuated in IDH-mutant WHO grade 4 astrocytomas. The myeloid cells in these tumors resembled homeostatic microglia and showed an increased expression of cytokine and chemokine genes. CONCLUSION Here, we present a high-dimensional transcriptomic atlas of the myeloid compartment in human GBM and IDH-mutant WHO grade 4 astrocytomas. The identified differences point towards targeted therapeutic options via the modulation of the tumor microenvironment.

BACKGROUND Paediatric brain tumour survivors may have treatment toxicity associated with signal change on follow-up MRI. Quantitative MRI texture features can potentially be used as surrogates of the underlying tissue changes following radiation therapy. MATERIAL AND METHODS Longitudinal retrospective study in 51 paediatric primary brain tumours treated with photon (N=30) and proton (N=21) radiotherapy (RT). T2 MRI scans at baseline and multiple time point from the date of surgery to 2 years following radiotherapy were selected for the textural analysis. Scans were bias corrected, registered with the CT dose maps and with baseline scan for each patient using 3Dslicer. Regions of interest (ROI) of fixed diameter were drawn in 11 predetermined non-tumoral regions of brain including in peri-tumoural region (PTV). ROIs were placed in homogenous white/grey matter. Radiation dose was calculated in each of these 11 ROIs and texture features were extracted using pyradiomics. Data were analysed using machine learning and statistical analysis. General linear multivariate model was used to corelate primary texture features over period of 24 months and radiation dose, time, effect of dose*time together at each ROI separately. RESULTS There were Brainstem 4, Cerebellar19, Hemispheric cerebral 7 and Supratentorial midline 10 tumours. Median age at diagnosis was 8.26 years (range: 0–20). Median RT treatment dose was 28.52Gy (0-60Gy). Multivariate analysis shows significant corelation (p < 0.001) between radiation dose and longitudinal primary texture features in all 11ROIs. Time showed corelation with feature values only in 3 ROIs and dose* time showed corelation in 5ROIs. Primary (statistical) feature values showing consistent correlation with dose in all 11 ROIs over 24 months are total energy, 10%, 90%, energy, entropy, mean, median, and minimum. CONCLUSION Radiomic texture analysis is a promising modality to understand dose related textural changes in the normal part of brain in paediatric brain tumour patients treated with radiation therapy. Radiomic changes need to be related to neurological outcomes in future research.

BACKGROUND Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, therapy resistance and poor blood-brain-barrier penetration hinder the amelioration of the standard-of-care. Included in the GBM mass are the tumor initiating cells (TICs), representing the driver of GBM growth and relapse in virtue of their stem-like traits and therapy-resistance. Being constantly exposed to environmental stress, including nutrients deficiency, hypoxia and therapeutic insults, all GBM cells -and TICs in particular- have to be highly adaptive in order to survive. Thus, their ability to cope with stress could be targeted to curtail TICs maintenance and the whole GBM aggressiveness. The key of TICs adaptation relies, among the others, on their epigenetic plasticity, hence encouraging epigenetic drugs testing. MATERIAL AND METHODS By exploiting patient-derived GBM TICs and orthotopic xenograft models, we tested the antitumorigenic features of a novel, selective, orally bioavailable and brain-penetrant Lysine-specific histone demethylase 1 inhibitor (LSD1i). We confirmed the specificity of its effects by LSD1 genetic targeting. A combination of RNA-seq, Chromatin Immunoprecipitation(ChIP)-seq, Mass Spectrometry and reverse genetic experiments unraveled LSD1 molecular players in GBM TICs. RESULTS We identified LSD1 as a druggable target in human GBM: LSD1i treatment, mirrored by LSD1 genetic targeting, impairs growth, viability, stem-like traits and in vivo tumorigenicity of GBM TICs. Mechanistically, LSD1 is crucial for the expression of the activating transcription factor 4 (ATF4), which coordinates the integrated stress response (ISR) to manage stressful stimuli as nutrient deprivation and endoplasmic reticulum stress. By mimicking these stress cues in vitro, we found that LSD1i triggers a delayed but unabated ATF4 translation which provokes an over-lasting ISR, eventually culminating in GBM TICs apoptosis. Lastly, LSD1 demethylase activity is dispensable for ATF4 induction. Rather, LSD1i exerts its anti-tumorigenic potential by interfering with LSD1 scaffolding function in GBM TICs. CONCLUSION LSD1-directed therapy is likely a promising strategy to hinder GBM. By sensitizing GBM TICs to stress, LSD1i endangers the GBM TICs pool. The effectiveness of LSD1i administration in different patient-derived GBM TICs and xenografts, regardless of their molecular profile, places a strong rationale toward the clinical translation of this approach for GBM management. FUNDING Italian association for Cancer Research (AIRC) and Italian Ministry of Health

BACKGROUND Numerous epidemiological studies have highlighted the protective role of immunoglobulin E-mediated allergic diseases on glioblastoma (GBM) susceptibility and prognosis. However, the mechanistic explanations behind these phenomena remain unexplored. Our objective was to set up a preclinical model and investigate the mechanisms underlying such protection to improve our understanding of the crosstalk between immune system and brain tumor development. MATERIAL AND METHODS A mouse model of allergic airway inflammation (AAI) induced by repeated nasal instillation of House Dust Mite extract was initiated before intracranial implantation of GL261 glioma cells, in both immunocompetent (C57BL/6) and immunodeficient (RAG-KO) mice. Tumor take and tumor growth were monitored by MRI. Central (microglia) and peripheral (spleen, bone marrow) immune cells were characterized by flow cytometry. The response of microglia was further assessed by RNA sequencing. Impact of candidate genes on patient survival was characterized by Cox regression analysis using data from TCGA and CGGA. RESULTS Following AAI induction in C57BL/6 mice, engraftment of GL261 cells in the brain was delayed and tumor growth rate was reduced. This correlated with an increase in survival of the mice and was accompanied by increased effector memory T-cells in the circulation. Of note, the survival benefit was lost in RAG-KO mice devoid of adaptive immunity. At the level of the brain, we observed enhanced secretion of TNFα and IL6 in microglia ex vivo. AAI induced a transcriptional reprogramming of microglia towards a pro-inflammatory-like state. We identified an allergy-related microglia gene signature that is associated with improved prognosis of glioma patients. CONCLUSION Our results demonstrate that AAI limits both tumor take and GBM progression in mice, providing a preclinical model to study the role of allergic inflammation in GBM susceptibility and prognosis, respectively. At the functional level, we identify a potentiation of microglial and adaptive anti-tumoral immunity. Further investigations are warranted to shed light on the reciprocal crosstalk between microglial reprogramming and peripheral immunity in the context of allergies and brain tumors.

BACKGROUND Glioblastoma has an infiltrative growth pattern that makes complete resection of the tumor virtually impossible. Sooner or later the tumor progresses, even after aggressive treatment with maximal safe resection, radiotherapy and/or chemotherapy. Hematological toxicity is an important cause of treatment delays during 1st line treatment. How often hematological toxicity occurs during 2nd line treatment is unclear. We explored rates of hematological toxicities in patients treated with temozolomide or lomustine at progression and investigated the association between severe toxicity during 1st and 2nd line treatment. METHODS We studied a retrospective cohort study of adult patients (n=247) with a glioblastoma treated with 2nd line alkylating chemotherapy at the Brain Tumor Center Amsterdam between 2000 and 2020. First line treatment of these patients consisted of a combination of radiotherapy combined with different treatments (80% received temozolomide, 4% PCV, 6% other chemotherapy and 10% radiotherapy only). Second line treatment consisted of temozolomide or lomustine. Mild and severe hematological toxicity were defined according to the CTCAE (version 5.0) criteria as a grade 1&2 and grade ≥3, respectively. We used descriptive statistics to analyze frequencies of hematological toxicity in patients with glioblastoma treated with 2nd line chemotherapy. RESULTS Sixty percent (147/247) of patients treated with 2nd line chemotherapy experienced hematological toxicity (grade 1–4). Considering subtypes of hematological toxicities, more patients experienced hematological toxicity during 2nd line treatment; severe thrombocytopenia occurred most frequently observed (6,1 during 1st line vs. 10,5% during 2nd line treatment), followed by neutropenia (3,6 vs. 6,9%), leukocytopenia (4,0 vs. 5,3%) and anemia (0 vs. 0,8%). Fewer patients treated with 2nd line temozolomide (n=113) experienced mild and severe hematological toxicity than patients treated with 2nd line lomustine (n=134; 46% versus 71% (for mild) and 12% vs 21% (severe toxicity), respectively). A subset of 107 patients was initially treated with radiotherapy and concurrent and adjuvant temozolomide; within this subset, patients with none or only mild toxicity during 1st line treatment had only a small risk of severe hematological toxicity during 2nd line treatment (4%). In contrast, the 34,5 % of patients with severe hematological toxicity during 1st line treatment also experienced severe hematological toxicity during 2nd line alkylating chemotherapy. CONCLUSION Hematological toxicity occurs more frequently during 2nd line treatment. Treatment with 2nd line temozolomide results in less hematological toxicity than lomustine. Absence of severe toxicity during 1st line treatment is predictive for the risk of toxicity during 2nd line treatment.

BACKGROUND The evolutionary processes that drive progression in patients with IDH-mutant astrocytoma remain unclear. The GLASS-NL consortium, as part of the larger worldwide GLASS consortium, was initiated to gain insight into the molecular mechanisms underlying glioma evolution and to identify markers of progression in IDH-mutant astrocytomas. Such markers can ultimately assist clinical decision making. Here, we present the first results of genome wide methylation profiling of samples included in the GLASS-NL study. MATERIAL AND METHODS 110 adult patients were identified with an IDH-mutant astrocytoma at first diagnosis. All patients underwent a surgical resection of the tumor at least twice, separated by at least 6 months with a median of 40.9 months (IQR: 24.0, 64.7), in 37% and 18% of the cases, patients were treated with radiotherapy or chemotherapy respectively, before surgical resection of the recurrent tumor. Methylation profiling was done on (macro dissected) DNA isolated of 235 samples from 103 patients (102 1st, 101 2nd, 29 3rd, and 3 4th resection), using the Infinium MethylationEPIC BeadChip array. Copy number variations were also derived from these data. Methylation classes were determined according to Capper et al. (2018). Overall survival (OS) was measured from date of first surgery. RESULTS Of all primary tumors, the methylation-classifier assigned 85 (87%) to the A_IDH (‘low grade’) subclass and 10 (10%) to the A_IDH_HG (‘high grade’) subclass. The relative proportion of high grade tumors increased ~three-fold at tumor recurrence (32/101, 32%) and even further in the second recurrence (15/29, 52%). The overall DNA-methylation levels of recurrent samples was lower than that of primary samples. This difference is explained by the increased number of high grade samples at recurrence, since near identical DNA-methylation levels were observed in samples that remained low grade. In an unsupervised analysis, methylation data derived from first and second resections of individual patients mostly (79%) cluster together. This indicates that variability between tumors is larger than temporal heterogeneity within tumors. Recurrent samples that do not cluster with their primary tumor, form a separate cluster and have relatively low genome-wide DNA-methylation. CONCLUSION Our data demonstrate that methylation profiling identifies a shift towards a higher grade at tumor progression coinciding with reduced genome-wide DNA-methylation levels.

BACKGROUND Fusion genes are chromosomal aberrations in malignancies that can be used as prognostic markers as well as therapeutic targets. The FGFR3-TACC3 (F3-T3) was initially discovered as an oncogenic molecule in glioblastoma and bladder cancer and subsequently found in many other cancer types. Based on clinical evidence, F3-T3 was found in glioblastoma patients before and after TMZ and radiotherapy treatment, suggesting that targeting F3-T3 is a valid strategy for glioblastoma treatment. MATERIAL AND METHODS We profiled the proteins that interacted with F3-T3 fusion protein in U-251 MG cells with F3-T3 through 2-D liquid chromatography-tandem mass spectrometry. To validate the result of proteomic analysis, we performed reverse immunoprecipitation by pulling down Hsp90 or Cdc37 in U-251 MG cells stably expressing F3-T3. To inhibit the association between F3-T3 and the Hsp90-Cdc37 complex, we treated U-251 MG and LN-229 cells stably expressing F3-T3 with Hsp90 inhibitors or siRNA of Cdc37. We applied the CCK8 assay to evaluate the sensitivity of glioblastoma cells stably expressing F3-T3, wild-type FGFR3, kinase-dead F3-T3 (K508R), and empty vectors to TMZ. Immunoblot and immunofluorescence staining were used to detect DNA damage marker pH2AX. The drug combination effect index was analyzed using software CalcuSyn. U-251 MG cells stably expressing F3-T3 infected with luciferase virus were intracranially injected in nude mice. The experimental group was administered with temozolomide (5mg/kg/day) by oral gavage, Hsp90 inhibitor Onalespib (30mg/kg/day) by tail vein injection or the combination of the two for indicated days. RESULTS We identified the proteins that showed increased binding ratios to F3-T3 over full-length FGFR3, the molecular chaperone proteins encoded by the genes HSP90AB1, HSP90AA1, and CDC37 emerged as 5th, 6th, and 7th on the top ten list, showing an approximately 4-fold increase in normalized spectral counts. Using Hsp90 inhibitors or Cdc37 siRNA disrupted the formation of the F3-T3/Hsp90/Cdc37 complex. Disruption of Hsp90-Cdc37 chaperoning caused a ubiquitination-mediated degradation of the glycosylated form of F3-T3 and abrogated the maturation of nascent F3-T3, resulting in suppression of F3-T3 signaling pathways. Additionally, our results provide evidence that the F3-T3 signaling pathway confers drug resistance to TMZ induced DNA damage. However, the resistance of TMZ was disrupted in glioblastoma cells harboring kinase-dead F3-T3 (K508R). We also demonstrated Hsp90 inhibitor significantly sensitized glioblastoma cells harboring the F3-T3 fusion gene to TMZ treatment and improved survival of xenograft model bearing F3-T3 tumor in vivo. CONCLUSION F3-T3 is a strong Hsp90 client that shows strong addiction to the Hsp90-Cdc37 chaperone system. Combination therapy with Hsp90 inhibitor overcomes the TMZ resistance conferred by F3-T3.

BACKGROUND The oncogenic gene fusion between ZFTA (formerly C11orf95) and RELA has in recent years been highlighted as oncogenic driver event ultimately leading to malignant transformation and progression of ependymoma. Representing a genetic hallmark in 17.6% of ependymomas, ZFTA-RELA fusion-positive tumors qualified as separate diagnostic entity in the 2016 revision of the WHO classification of CNS tumors. This tumor entity mainly occurs in pediatric patients and is characterized by poor prognosis and the lack of biology-driven therapy. RESULTS De novo prediction of putative gene fusion-derived neoepitopes in malignant CNS diseases yielded several potential candidates suitable for screening. Of these, vaccination of A2.DR1-transgenic major histocompatibility complex (MHC)-humanized mice with in silico compiled peptide vaccines encompassing the ZFTA-RELA fusion sequence elicited robust antigen-specific CD4+-restricted immune responses. We identified ZFTA-RELA-reactive T cell clones by multiplexed Interferon-γ / Interleukin-2 recall response. Single-cell VDJ-sequencing of reactive T cells demonstrated a highly clonal T cell receptor (TCR) repertoire and shared clonotypes among all vaccinated animals. Matching TCRα/β chains have been assembled from single-cell sequencing data and cloned for functional testing of neoepitope-reactive TCR-transgenic T cells in vitro as well as in vivo using a transposon system-based immunocompetent, MHC-humanized ZFTA-RELA fusion-positive ependymoma model. CONCLUSION Here we identify the oncogenic ZFTA-RELA gene fusion product as a novel tumor-specific neoepitope that is recognized by MHC class II-restricted TCRs. Therapeutic efficacy of TCR-transgenic cell therapy can be further investigated using an immunocompetent MHC-humanized mouse model of ZFTA-RELA fusion-positive ependymoma. Our findings provide initial evidence for neoepitope-specific immunotherapy in pediatric CNS tumors.

BACKGROUND the clinical significance of TERT promoter mutations, telomere length and their interactions with MGMT promoter methylation status in patients with IDH-wildtype GBM patients remain unclear. We performed a large monoinstitutional study to better investigate their impact and their interaction on clinical outcomes MATERIAL AND METHODS TERT promoter mutations (C228T and C250T), relative telomere length (RTL) and MGMT methylation status were assessed in 278 newly diagnosed and in 65 recurrent IDH-wildtype GBM PTS which were treated at Veneto Institute of Oncology (Padua, Italy) from Dec 2016 to Jan 2020. We have retrospectively explored association between gene characteristics and neuroradiological response (RANO criteria), progression-free survival (PFS), overall survival (OS). Telomere length was measured by monochrome multiplex PCR and RTL values were calculated as a telomere/single-copy gene ratio RESULTS characteristics of newly diagnosed GBM PTS were: median age 63 ys, ECOG PS 0–1 in 71% of PTS, radical surgery in 38%, 78% received radiation therapy plus TMZ, MGMT was methylated in 53%, TERT promoter was mutated in 80% (75% C228T, 25% C250T), median RTL was 1.57 (range 0.4–11.37). Objective response rate was reported in 15% of PTS, median OS was 15ms (95% CI 13-18ms), median PFS was 8ms (95% CI 7-9ms). At multivariable analysis, TERT promoter mutations and RTL were not associated with clinical outcomes; about OS, TERT promoter mutations and RTL reported a HR of 1.05 (95% CI 0.64–1.64) and 0.99 (95% CI 0.89–1.10), respectively; MGMT methylated tumors showed significant improved PFS and OS with a HR of 0.54 (95% CI 0.40–0.71) and 0.47 (95% CI 0.34–0.64), respectively. All interactions among MGMT status, TERT mutation status and RTL were not statistically significant. Characteristics of recurrent GBM PTS were: median age 55 ys, ECOG PS 0–1 in 60% of PTS, MGMTmet in 37%, TERT promoter mutations in 75% (75% C228T, 25% C250T), RTL was 1.67 (range 0.68–8.87). At multivariable analysis, only MGMT methylated tumors resulted significantly associated to prolonged OS (HR 0.16; 95% CI 0.07–0.40). No gene interaction was significant CONCLUSION for the first time worldwide, we analyzed the impact of TERT promoter mutations, RTL and MGMT methylation status in both newly diagnosed and recurrent IDH-wildtype GBM PTS. TERT promoter status and RTL were not associated with clinical outcomes at both diagnosis and relapse. MGMT promoter methylation status was the only prognostic factor in both cases. No significant interaction was demonstrated between TERT promoter mutations, RTL and MGMT methylation status

BACKGROUND The diagnosis of brain metastasis involves high morbidity and mortality and remains an unmet clinical need in spite of being the most common tumor in the brain. Exclusion of these cancer patients from clinical trials is a major cause of their limited therapeutic options. MATERIAL AND METHODS We report a novel drug-screening platform (METPlatform) based on organotypic cultures which allows identifying effective anti-metastasis agents in the presence of the organ microenvironment. We have applied this approach to clinically relevant stages of brain metastasis using both experimental models and human tumor tissue (by performing patient-derived organotypic cultures - PDOCs -). We have also used METPlatform to perform unbiased proteomics of brain metastases in situ to identify potential novel mediators of this disease and explore resistance mechanisms to targeted therapy. Finally, we have exploited METPlatform as “avatars” to predict response to therapy in patients with primary brain tumors. RESULTS We identified heat shock protein 90 (HSP90) as a promising therapeutic target for brain metastasis. DEBIO-0932, a blood-brain barrier permeable HSP90 inhibitor, shows high potency against mouse and human brain metastases from different primary origin and oncogenomic profile at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis of brain metastases treated with the chaperone inhibitor revealed non-canonical clients of HSP90 as potential novel mediators of brain metastasis and actionable mechanisms of resistance driven by autophagy. Combined therapy using HSP90 and autophagy inhibitors showed synergistic effects compared to sublethal concentrations of each monotherapy, demonstrating the potential of METPlatform to design and test rationale combination therapies to target metastasis more effectively. Finally, we show that brain tumor PDOCs predict the response of the corresponding patient to standard of care, thus proving the potential of METPlatform for improving personalized care in cancer. CONCLUSION Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is fully compatible with human samples and questions the rationale of excluding patients with brain metastasis from clinical trials. We envision that METPlatform will be established as a clinically relevant strategy to personalize the management of metastatic disease in the brain and elsewhere.

BACKGROUND Patients with primary malignant brain tumors have high symptom burden and commonly rely on family caregivers for practical and emotional support. This can lead to negative mental and physical consequences for caregivers. We investigated effectiveness of an 8-week nurse-led online needs-based support program (SmartCare©) with and without online self-guided cognitive behavioral therapy (CBT) for depression compared to enhanced care as usual (ECAU) on depressive symptoms, caregiving-specific distress, anxiety, mastery, and burden. MATERIAL AND METHODS Family caregivers with depressive symptoms were randomized to three groups: SmartCare© plus/minus self-guided CBT, or ECAU. Primary outcomes (depressive symptoms (CES-D); caregiving-specific distress (Caregiver Needs Screen)) and secondary outcomes (anxiety (POMS-A), caregiver mastery (Caregiver Mastery Scale), and caregiver burden (Caregiver Reactions Assessment)) were assessed online. Intention to treat analyses of covariance corrected for baseline scores were performed for outcomes at four months. RESULTS In total, 120 family caregivers participated. Accrual and CBT engagement were lower than expected, therefore intervention groups were combined (n=80) and compared to ECAU (n=40). For depressive symptoms, no statistically significant group differences were found. Caregiving-specific distress decreased in the intervention group compared with ECAU (p=0.01, partial ɳ 2=0.08). Among secondary outcomes, there was a trend towards improvement in mastery for the intervention group compared with ECAU (p=0.08, partial ɳ 2=0.04). CONCLUSION SmartCare©, with or without self-guided CBT, reduced caregiving-specific distress with a trend towards improving mastery. SmartCare© has the potential to improve the lives of families coping with a brain tumor diagnosis.

BACKGROUND Glioblastomas are among the most heterogeneous tumors, which hampers patient stratification and development of effective therapies. Glioblastomas create a dynamic ecosystem, where heterogeneous tumor cells interact with the tumor microenvironment to establish different niches. Upon tumor growth, Glioblastoma cells manifest remarkable plasticity and respond flexibly to selective pressures by transiting towards states favorable to the new tumor microenvitonment. How this phenotypic plasticity contributes to treatment resistance is currently less clear. The exact nature of treatment resistant, tolerant and sensitive Glioblastoma cells remains unresolved. Further studies at the single cell level are needed to reveal transient and long-term signatures of the resistant states. MATERIAL AND METHODS To investigate long-term phenotypic changes upon treatment at the single cell level we performed single cell RNA-seq (scRNA-seq) on the longitudinal patient-derived xenograft (PDOX) models derived from Glioblastoma patient tumors prior and after the standard-of-care treatment. In addition, direct treatment of PDOXs with temozolomide combined with scRNA-seq allowed revealing short-term transcriptomic changes both in tumor cells and in the mouse-derived cells forming tumor microenvironment. Advanced computational algorithms, including reference-free deconvolution methods, were applied to reveal treatment resistance signatures and master regulators of the identified treatment-resistant subpopulations. RESULTS We show that PDOX models recapitulate all the major cell types and transcriptional programs reported in Glioblastoma patient samples, providing clinically relevant models for investigating treatment resistance signatures of tumor cells and associated tumor microenvironment. Analysis of treatment naïve and treated Glioblastomas at the single cell level revealed presence of pre-existing treatment resistant states as well as newly established resistant subpopulatons. Certain transcriptomic changes are preserved long term, regardless of the lack of genetic evolution of the tumor cells. CONCLUSION Phenotypic plasticity is an important factor contributing to resistance mechanisms in Glioblastoma. Key molecular regulators of tumor cell plasticity towards treatment resistance states represent novel targets for future combinatory treatments.