DNA Methyltransferase 3B-Mediated Intratumoral Heterogeneity and Therapeutic Targeting in Breast Cancer Recurrence and Metastasis.

The mechanisms of how cancer cells are selected and evolve to establish distant metastatic colonies remain unclear. Tumor heterogeneity and lack of biomarkers are some of the most difficult challenges in cancer biology and treatment. Here using mouse models for triple-negative breast cancer (TNBC) metastasis, we report heterogeneous expression of DNA methyltransferase 3B (DNMT3B) in both mouse and human primary tumors. High levels of DNMT3B were correlated with poor clinical outcomes in multiple human breast cancer datasets. Mechanistically, clonal cells with high DNMT3B (DNMT3BH) showed higher vimentin (VIM) expression and displayed enhanced epithelial-to-mesenchymal transition capacity. Deletion of VIM diminished the metastatic phenotype of DNMT3BH cells. Importantly, in preclinical mouse models in which the primary tumors were surgically removed, perioperative targeting of DNMT3B in combination with chemotherapy markedly suppressed tumor recurrence and metastasis. Our studies identify DNMT3B-mediated transcription regulation as an important mediator of tumor heterogeneity and show that DNMT3B is critical for tumor invasion and metastasis, reinforcing its potential as a target for treating metastatic disease. IMPLICATIONS: Our findings of transcriptome changes mediated by DNMT3B provide new mechanistic insight for intratumor heterogeneity and chemoresistance, and therapeutic targeting of DNMT3B in combination with chemotherapy offer additional treatment options for metastatic disease especially for patients with TNBC.

Induction of DNMT3B by PGE2 and IL6 at Distant Metastatic Sites Promotes Epigenetic Modification and Breast Cancer Colonization.

Current cancer treatments are largely based on the genetic characterization of primary tumors and are ineffective for metastatic disease. Here we report that DNA methyltransferase 3B (DNMT3B) is induced at distant metastatic sites and mediates epigenetic reprogramming of metastatic tumor cells. Multiomics analysis and spontaneous metastatic mouse models revealed that DNMT3B alters multiple pathways including STAT3, NFκB, PI3K/Akt, ß-catenin, and Notch signaling, which are critical for cancer cell survival, apoptosis, proliferation, invasion, and colonization. PGE2 and IL6 were identified as critical inflammatory mediators in DNMT3B induction. DNMT3B expression levels positively correlated with human metastatic progression. Targeting IL6 or COX-2 reduced DNMT3B induction and improved chemo or PD1 therapy. We propose a novel mechanism linking the metastatic microenvironment with epigenetic alterations that occur at distant sites. These results caution against the "Achilles heel" in cancer therapies based on primary tumor characterization and suggests targeting DNMT3B induction as new option for treating metastatic disease. SIGNIFICANCE: These findings reveal that DNMT3B epigenetically regulates multiple pro-oncogenic signaling pathways via the inflammatory microenvironment at distant sites, cautioning the clinical approach basing current therapies on genetic characterization of primary tumors.

Nutritional, Microbial, and Allergenic Changes during the Fermentation of Cashew 'Cheese' Product Using a Quinoa-Based Rejuvelac Starter Culture.

Fermentation has been applied to a multitude of food types for preservation and product enhancing characteristics. Interest in the microbiome and healthy foods makes it important to understand the microbial processes involved in fermentation. This is particularly the case for products such as fermented cashew (Anacardium occidentale). We hereby describe the characterisation of cashew samples throughout an entire fermentation production process, starting at the quinoa starter inoculum (rejuvelac). The viable bacterial count was 108 -109 colony forming units/g. The nutritional composition changed marginally with regards to fats, carbohydrates, vitamins, and minerals. The rejuvelac starter culture was predominated by Pediococcus and Weissella genera. The 'brie' and 'blue' cashew products became dominated by Lactococcus, Pediococcus, and Weissella genera as the fermentation progressed. Cashew allergenicity was found to significantly decrease with fermentation of all the end-product types. For consumers concerned about allergic reactions to cashew nuts, these results suggested that a safer option is for products to be made by fermentation.

Muddy conditions reduce hygiene and lying time in dairy cattle and increase time spent on concrete.

Dairy cattle spend less time lying and show signs of increased stress when housed in rainy and windy conditions, but no work has separated the effects of exposure to inclement weather from muddy conditions underfoot. Our objective was to evaluate the effects of muddy conditions alone on lying behavior, hygiene, and physiological responses. We housed pairs of pregnant, nonlactating dairy cattle (n = 12; 6 primigravid heifers, 6 multiparous cows) in enclosed pens with dirt floors and a concrete feed apron. Cattle were exposed to 3 levels of soil moisture: 90 (dry), 74 (muddy), or 67% (very muddy) dry matter for 5 d each in a replicated 3 × 3 Latin square design. Lying time was measured on all days with data loggers, and lying locations and postures were recorded on the final day of each treatment. Before and after each treatment, blood samples were collected, and the percentage of dirty surface area was measured on the udder, hind leg, and side of each animal. Cattle spent less time lying down in muddier conditions, especially in the first 24 h of exposure, when cows and heifers spent only 3.2 and 5.8 h, respectively, lying down in the muddiest treatment compared with 12.5 and 12.7 h on dry soil. When the soil was dry, cattle never chose to lie down on concrete, but in muddier conditions they spent a greater proportion of their lying time on concrete (mean ± SE: 56 ± 14 and 10 ± 8% in the very muddy and muddy treatments, respectively). The shift in lying location was more marked for heifers, and all 6 spent ≥87% of their lying time on concrete in the muddiest treatment. When cattle chose to lie down on wetter soil, they limited the surface area exposed to their surroundings by tucking their legs beneath their bodies (mean ± SE: 30 ± 11, 15 ± 4, and 5 ± 2% of lying observations in the very muddy, muddy, and dry treatments, respectively). Despite cattle spending less time on wetter soil, all 3 measured body parts became dirtier in muddier conditions (1.4-, 1.6-, and 1.8-fold more on the leg, udder, and side, respectively, in the muddiest compared with the driest treatment). In addition, higher soil moisture levels resulted in greater reductions in white blood cell counts relative to baseline levels (-0.95, -0.43, and 0.34 × 109/L relative to baseline levels in the very muddy, muddy, and dry treatments). Muddy conditions, even in the absence of wind or rain, are aversive for cattle and have negative implications for their welfare.

Technical note: Comparison of instantaneous sampling and continuous observation of dairy cattle behavior in freestall housing.

Recording behavior at fixed intervals (instantaneous sampling) can reduce labor relative to observing continuously. However, instantaneous sampling may inaccurately estimate potentially important responses, such as how frequently cows perform a behavior (i.e., the number of bouts). Our objective was to validate the use of instantaneous sampling for capturing how long and how frequently cows in freestall housing lie down or visit the feed bunk and water trough. We predicted that more frequent sampling would be needed to accurately reflect the behaviors that cows spent less time performing. In addition, we predicted that instantaneous sampling would underestimate how often cows engaged in behaviors that they frequently performed in short bouts or with short intervals between bouts, as some of these events may occur between sample intervals. Continuous video observations of 18 lactating Holstein-Friesian dairy cows were conducted for 48-h periods. Instantaneous samples (1 and 30 s, and 1, 3, 5, 10, 15, and 30 min) were generated from continuous data, with the samples recorded at 1-s intervals representing true values. Estimates from each sample interval ≥30 s were compared pairwise to true values with regression analysis. Sample intervals were considered accurate if they met 3 criteria: coefficient of determination ≥0.9 (i.e., strongly related to true values), slope=1, and intercept=0 (i.e., did not over- or underestimate true values). The amount of time cows spent lying (12.1±1.8h/24h, mean ± standard deviation) or visiting the water trough (1.1±0.8h/24h) and feed bunk (5.6±0.8h/24h) were accurately captured using sample intervals ≤30, 10, and 5 min, respectively. In addition, sample intervals ≤3 min accurately estimated the number of lying bouts (10.3±2.4 per 24h), likely because cows were recumbent for long periods (74.0±17.4 min, on average, with <6% of bouts lasting <5 min) and rarely resumed lying soon after standing up (0.4% of intervals between lying bouts were <30 s). However, shorter sample intervals may be needed in situations where cows more frequently transition between lying and standing. In contrast to lying in this study, cows visited the water trough and feed bunk for shorter periods (3.5±1.7 and 25.6±5.8 min, respectively) and frequently returned to these resources soon after leaving (17 and 7% of intervals between visits were <30 s long). As some of these events likely occurred between sample intervals, all sample intervals ≥30 s underestimated the number of times cows visited the water trough and feed bunk (18.5±6.2 and 14.1±4.4 per 24h, respectively). Therefore, continuous observation is needed to determine how often cows visit these resources.

Cooling cows efficiently with water spray: Behavioral, physiological, and production responses to sprinklers at the feed bunk.

Dairies commonly mount nozzles above the feed bunk that intermittently spray cows to dissipate heat. These sprinklers use potable water-an increasingly scarce resource-but there is little experimental evidence for how much is needed to cool cows in loose housing. Sprinkler flow rate may affect the efficacy of heat abatement, cattle avoidance of spray (particularly on the head), and water waste. Our objectives were to determine how sprinkler flow rate affects cattle behavioral, physiological, and production responses when cows are given 24-h access to spray in freestall housing, and to evaluate heat abatement in relation to water use. We compared 3 treatments: sprinklers that delivered 1.3 or 4.9L/min (both 3min on and 9min off, 24h/d) and an unsprayed control. Nine pairs of high-producing lactating Holstein cows received each treatment at a shaded feed bunk for 2d in a replicated 3×3 Latin square design [air temperature (T): 24-h maximum=33±3°C, mean ± SD]. Cows spent 5.8±0.9h/24h (mean ± SD) at the feed bunk overall, regardless of treatment. With few exceptions, cows responded similarly to the 1.3 and 4.9L/min flow rates. Sprinklers resulted in visits to the feed bunk that were on average 23 to 27% longer and 13 to 16% less frequent compared with the control, perhaps because cows avoided walking through spray. Indeed, when the sprinklers were on, cows left the feed bunk half as often as expected by chance, and when cows chose to walk through spray, they lowered their heads on average 1.7- to 3-fold more often than in the control. Despite possible reluctance to expose their heads to spray, cows did not avoid sprinklers overall. In warmer weather, cows spent more time at the feed bunk when it had sprinklers (on average 19 to 21min/24h for each 1°C increase in T), likely for heat abatement benefits. Compared with the control, sprinklers resulted in 0.3 to 0.7°C lower body temperature from 1300 to 1500h and 1700 to 2000h overall and attenuated the rise in this measure on warmer days (for each 10°C increase in T, body temperature increased by on average 0.5 to 0.7°C with sprinklers vs. 1.6°C without). Sprinkler access also resulted in milk yield that was, on average, 3.3 to 3.7kg/24h higher than in the control treatment. In this hot and dry climate, 1.3L/min cooled cows more efficiently than 4.9L/min, as the lower flow rate achieved equivalent reduction in body temperature and increase in milk yield relative to no spray, despite using 73% less water.

Cooling cows efficiently with sprinklers: Physiological responses to water spray.

Dairies in the United States commonly cool cattle with sprinklers mounted over the feed bunk that intermittently spray the cows' backs. These systems use potable water-an increasingly scarce resource--but there is little experimental evidence about how much is needed to cool cows or about droplet size, which is thought to affect hair coat penetration. Our objectives were to determine how sprinkler flow rate and droplet size affect physiological measures of heat load in a hot, dry climate, and to evaluate cooling effectiveness against water use. The treatments were an unsprayed control and 6 soaker nozzles that delivered four 3-min spray applications of 0.4, 1.3, or ≥ 4.5 L/min (with 2 droplet sizes within each flow rate) and resulting in 30 to 47% of spray directly wetting each cow. Data were collected from high-producing lactating Holsteins (n = 19) tested individually in ambient conditions (air temperature = 31.2 ± 3.8°C, mean ± standard deviation). Cows were restrained in headlocks for 1h and received 1 treatment/d for 3d each, with order of exposure balanced in a crossover design. When cows were not sprayed, physiological measures of heat load increased during the 1-h treatment. All measures responded rapidly to spray: skin temperature decreased during the first water application, and respiration rate and body temperature did so before the second. Droplet size had no effect on cooling, but flow rate affected several measures. At the end of 1h, 0.4 L/min resulted in lower respiration rate and skin temperature on directly sprayed body parts relative to the control but not baseline values, and body temperature increased to 0.2°C above baseline. When 1.3 or ≥ 4.5 L/min was applied, respiration rate was lower than the control and decreased relative to baseline, and body temperature stayed below baseline for at least 30 min after treatment ended. The treatment that best balanced cooling effectiveness against water usage was 1.3 L/min: although ≥ 4.5 L/min reduced respiration rate relative to baseline by 4 more breaths/min than 1.3 L/min did (-13 vs. -9 breaths/min, respectively), each additional liter of water decreased this measure by only ≤ 0.1 breaths/min (≤ 1% of the total reduction achieved using 1.3 L/min). We found similar water efficiency patterns for skin temperature and the amount of time that body temperature remained below baseline after treatment ended. Thus, when using this intermittent spray schedule in a hot, dry climate, applying at least 1.3 L/min improved cooling, but above this, additional physiological benefits were relatively minor.

Dairy cows use and prefer feed bunks fitted with sprinklers.

Sprinklers reduce heat load in cattle, but elicit variable behavioral responses: cows readily use water in some studies, but in others either avoid it or show no preference. Nevertheless, on US dairies, a common way to cool cows is with nozzles mounted over the feed bunk that intermittently spray (i.e., 5 min on, 10 min off, as in this study) animals' backs while they feed. The objectives of this study were to determine how this type of sprinkler system affects behavior (single-treatment phase), and to assess preferences when cows were allowed to choose between feed bunks with or without sprinklers (choice phase). Data were collected 24h/d for lactating Holsteins tested in groups of 3 cows (n=8 groups) in warm ambient conditions [air temperature (mean ± standard deviation): 24-h average=24.5±2.5°C, maximum=36.0±3.5°C]. In the single-treatment phase, cows were fed from shaded bunks with or without sprinklers for 2 d/treatment, with order of exposure balanced in a crossover design. When sprinklers were present, cows spent more time at the bunk, both feeding [sprinkler vs. no sprinkler: 3.5 vs. 2.5h/24h, standard error (SE)=0.12h] and standing without feeding (4.3 vs. 2.3h/24h, SE=0.32 h) than when no sprinklers were present. Sprinklers lowered the average 24-h core temperature (38.8 vs. 39.2°C, SE=0.08°C), particularly on warmer days. Water cooling also mitigated the effects of weather on feeding time, which decreased with increasing heat load (air temperature and temperature-humidity index) when cows did not have sprinklers, but was unchanged when sprinklers were provided. In the choice phase, feed was provided ad libitum in both treatments for 5 d and preference was assessed. All groups preferred the feed bunk with sprinklers (78 vs. 22% of time spent near both feed bunks, SE=3.9%), and the magnitude of this preference increased linearly with heat load. In both phases of the study, cows protected their heads from direct spray when head position was elective (i.e., standing without feeding): cows were more likely to put their heads through the head gates when the sprinklers were on than off (single-treatment phase: 78 vs. 59%, respectively, of time spent standing without feeding, SE=2.8%; choice phase: 71 vs. 52%, SE=2.0%). In conclusion, although cows avoided wetting their heads, this is the first study to demonstrate that cattle readily use and clearly prefer sprinklers mounted above the feed bunk, possibly due to the cooling provided by this resource.

Retrieval monitoring and anosognosia in Alzheimer's disease.

This study explored the relationship between episodic memory and anosognosia (a lack of deficit awareness) among patients with mild Alzheimer's disease (AD). Participants studied words and pictures for subsequent memory tests. Healthy older adults made fewer false recognition errors when trying to remember pictures compared with words, suggesting that the perceptual distinctiveness of picture memories enhanced retrieval monitoring (the distinctiveness heuristic). In contrast, although participants with AD could discriminate between studied and nonstudied items, they had difficulty recollecting the specific presentation formats (words or pictures), and they had limited use of the distinctiveness heuristic. Critically, the demands of the memory test modulated the relationship between memory accuracy and anosognosia. Greater anosognosia was associated with impaired memory accuracy when participants with AD tried to remember words but not when they tried to remember pictures. These data further delineate the retrieval monitoring difficulties among individuals with AD and suggest that anosognosia measures are most likely to correlate with memory tests that require the effortful retrieval of nondistinctive information.

Molecular species of acylglycerols incorporating radiolabeled fatty acids from castor (Ricinus communis L.) microsomal incubations.

Sixty-one molecular species of triacylglycerols (TAG) and diacylglycerols produced from castor microsomal incubations incorporating six different (14)C-labeled fatty acids have been identified and quantified. The preference for incorporation into TAG was in the order ricinoleate > oleate > linoleate > linolenate > stearate > palmitate. Ricinoleate was the major fatty acid incorporated, whereas stearate, linolenate, and palmitate were incorporated at low levels. Twenty-one molecular species of acylglycerols (HPLC peaks) in castor oil have also been assigned. The levels of TAG in castor oil are RRR (triricinolein) >> RR-TAG >> R-TAG > no R-TAG. The levels of the molecular species within the groups of RR-TAG, RL-TAG, and LL-TAG individually are ricinoleate > linoleate > oleate > linolenate, stearate, and palmitate. The results of the labeled fatty acid incorporation are consistent with ricinoleate being preferentially driven into TAG and oleate being converted to ricinoleate in castor oil biosynthesis.

Molecular species of PC and PE formed during castor oil biosynthesis.

As part of a program to elucidate castor oil biosynthesis, we have identified 36 molecular species of PC and 35 molecular species of PE isolated from castor microsomes after incubations with [14C]-labeled FA. The six [14C]FA studied were ricinoleate, stearate, oleate, linoleate, linolenate, and palmitate, which were the only FA identified in castor microsomal incubations. The incorporation of each of the six FA into PC was better than that into PE. The [14C]FA were incorporated almost exclusively into the sn-2 position of both PC and PE. The incorporation of [14C]stearate and [14C]palmitate into 2-acyl-PC was slower compared to the other four [14C]FA. The incorporation does not show any selectivity for the various lysoPC molecular species. The level of incorporation of [14C]FA in PC was in the order of: oleate > linolenate > palmitate > linoleate > stearate > ricinoleate, and in PE: linoleate > linolenate > oleate > palmitate > stearate > ricinoleate. In general, at the sn-1 position of both PC and PE, linoleate was the most abundant FA, palmitate was the next, and oleate, linolenate, stearate, and ricinoleate were minor FA. The activities of oleoyl-12-hydroxylase, oleoyl-12-desaturase seem unaffected by the FA at the sn-1 position of 2-oleoyl-PC. The FA in the sn-1 position of PC does not significantly affect the activity of phospholipase A2, whereas ricinoleate is preferentially removed from the sn-2 position of PC. The results show that (i) [14C]oleate is most actively incorporated to form 2-oleoyl-PC, the immediate substrate of oleoyl-12-hydroxylase; (ii) 2-ricinoleoyl-PC is formed mostly by the hydroxylation of 2-oleoyl-PC, not from the incorporation of ricinoleate into 2-ricinoleoyl-PC; and (iii) 2-oleoyl-PE is less actively formed than 2-oleoyl-PC.