Effects of maternal nutrient restriction during gestation on bovine serum microRNA abundance.

The objective was to determine the effects of maternal nutrient restriction during gestation on serum microRNA (miRNA) abundance in cattle. Primiparous Angus-cross cows (n=22) were fed either control (CON; to gain 1 Kg/week) or nutrient restricted (NR; 0.55% NEm) diets based on National Research Council requirements. On day 30 of gestation, cows were blocked by body condition and randomly assigned to one of three diets: CON (n=8) days 30-190; NR (n=7) days 30-110 followed by CON days 110-190 (NR/C); or CON (n=7) days 30-110 followed by NR days 110-190 (C/NR). At 190 days of gestation, maternal serum was collected for RNA isolation and analyzed using a miRNA microarray of known Bos taurus sequences. Data were normalized using LOWESS and analyzed via ANOVA. At 190 days of gestation, 16 miRNAs exhibited differential abundance (P<0.05) between treatments. Cows that underwent NR, irrespective of when the insult occurred, had downregulated bta-miR-126-3p compared to CON cows. Bta-miR-16b was downregulated and three miRNAs upregulated in NR/C compared to C/NR and CON cows. Additionally, seven miRNAs were downregulated and four miRNAs upregulated in C/NR compared to NR/C and CON cows. Comparison of NR/C and C/NR cows revealed three differentially abundant (P<0.04) miRNAs (bta-miR-2487_L-2R-3_1ss15CT, bta-miR-215, and bta-miR-760-5p). Top KEGG pathway enrichment of target genes included: pathways in cancer, PI3K-Akt signaling, focal adhesion, Ras signaling, proteoglycans in cancer, and MAPK signaling. In summary, maternal nutrient restriction altered serum miRNA abundance profiles irrespective of the time at which the nutritional insult was induced.

EGFR mutation subtypes and response to immune checkpoint blockade treatment in non-small-cell lung cancer.

BACKGROUND: Although EGFR mutant tumors exhibit low response rates to immune checkpoint blockade overall, some EGFR mutant tumors do respond to these therapies; however, there is a lack of understanding of the characteristics of EGFR mutant lung tumors responsive to immune checkpoint blockade. PATIENTS AND METHODS: We retrospectively analyzed de-identified clinical and molecular data on 171 cases of EGFR mutant lung tumors treated with immune checkpoint inhibitors from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, University of California Los Angeles, and Dana Farber Cancer Institute. A separate cohort of 383 EGFR mutant lung cancer cases with sequencing data available from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, and The Cancer Genome Atlas was compiled to assess the relationship between tumor mutation burden and specific EGFR alterations. RESULTS: Compared with 212 EGFR wild-type lung cancers, outcomes with programmed cell death 1 or programmed death-ligand 1 (PD-(L)1) blockade were worse in patients with lung tumors harboring alterations in exon 19 of EGFR (EGFRΔ19) but similar for EGFRL858R lung tumors. EGFRT790M status and PD-L1 expression did not impact response or survival outcomes to immune checkpoint blockade. PD-L1 expression was similar across EGFR alleles. Lung tumors with EGFRΔ19 alterations harbored a lower tumor mutation burden compared with EGFRL858R lung tumors despite similar smoking history. CONCLUSIONS: EGFR mutant tumors have generally low response to immune checkpoint inhibitors, but outcomes vary by allele. Understanding the heterogeneity of EGFR mutant tumors may be informative for establishing the benefits and uses of PD-(L)1 therapies for patients with this disease.

Evaluation of dietary betaine in lactating Holstein cows subjected to heat stress.

Betaine (BET), a natural, organic osmolyte, improves cellular efficiency by acting as a chaperone, refolding denatured proteins. To test if dietary BET reduced the effect of heat stress (HS) in lactating dairy cows, multiparous, lactating Holstein cows (n=24) were blocked by days in milk (101.4±8.6 d) and randomly assigned to 1 of 3 daily intakes of dietary BET: the control (CON) group received no BET, mid intake (MID) received 57mg of BET/kg of body weight, and high dose (HI) received 114mg of BET/kg of body weight. Cows were fed twice daily and BET was top-dressed at each feeding. Cows were milked 2 times/d and milk samples were taken daily for analysis. Milk components, yield, feed intake, and water intake records were taken daily. Rectal temperature and respiration rate were taken 3 times/d at 0600, 1400, and 1800h. Cows were housed in environmentally controlled rooms and were allowed acclimation for 7d at thermoneutral (TN) conditions with a mean temperature-humidity index of 56.6. Cows were then exposed to 7d of TN followed by 7d of HS represented by a temperature-humidity index of 71.5 for 14d. This was followed by a recovery period of 3d at TN. Dietary BET increased milk yield during the TN period. No differences were found between BET and CON in total milk production or milk composition during HS. The increase in water intake during HS was not as great for cows fed BET compared with controls. The cows on CON diets had higher p.m. respiration rate than both MID and HI BET during HS, but lower rectal temperature compared with BET. No difference was found in serum glucose during TN, but cows given HI had elevated glucose levels during HS compared with CON. No differences were found in serum insulin levels between CON and BET but an intake by environment interaction was present with insulin increasing in HI-treated lactating dairy cows during HS. The heat shock response [heat shock protein (HSP) 27 and HSP70] was upregulated in bovine mammary epithelial cells in vitro. Blood leukocyte HSP27 was downregulated at the HI dose under TN conditions and HSP70 was upregulated at the HI dose and this effect was increased by HS. No effect was seen with the MID dose with HSP27 or HSP70. The lack of effect of BET at MID may be associated with uptake across the gut. We conclude that BET increased milk production under TN conditions and was associated with reduced feed and water intake and slightly increased body temperatures during HS of cows fed BET. The effect of BET on milk production was lost during HS with HI BET, whereas serum glucose levels increased during HS.

The effect of early to mid-gestational nutrient restriction on female offspring fertility and hypothalamic-pituitary-adrenal axis response to stress.

Primiparous ewes born as singletons to Rambouillet x Columbia crossbred ewes fed either 100% of NRC recommendations (control, Con; n = 7) or 50% of NRC (nutrient restricted, NR; n = 7) from d 28 through 78 postmating were utilized for this study. At 1 yr of age, a subset of ewes born to Con (n = 4) and NR (n = 4) mothers received jugular catheters and were subjected to a corticotrophin-releasing hormone (CRH)/arginine vasopressin (AVP) challenge, an ACTH challenge, and an isolation stress test, in which ACTH and cortisol responses were determined. A week after these challenges, estrus was monitored twice daily in all ewes from Con (n = 7) and NR mothers (n = 7). Once estrus was observed (d 0), daily blood samples were collected from ewes for progesterone through the subsequent estrus. Estrous detection and daily blood sampling were repeated during an estrous cycle in the next year, ewes were hand mated at the second estrus, and pregnancy was determined by delivery of a live lamb(s). Ewes from NR mothers tended (P = 0.10) to have a greater peak ACTH response after an intravenous CRH/AVP injection than ewes from Con mothers. The cortisol response of ewes to a CRH/AVP or ACTH challenge was not influenced by maternal nutrition. In contrast, ewes from Con mothers tended (P = 0.10) to release more ACTH in response to the isolation stress test and showed a greater (P = 0.04) cortisol release than ewes from NR mothers. Ewes from NR mothers exhibited decreased (P < 0.05) plasma progesterone in both yr 1 and 2 of the study compared with ewes from Con mothers. Furthermore, fewer (P < 0.0001) ewes from NR mothers produced a lamb (1 of 7) than ewes from Con mothers (7 of 7) during yr 2 of the study. These findings indicate that maternal undernutrition during early gestation may affect stress responses by the offspring, but has limited impact on hypothalamo-pituitary-adrenal sensitivity. Furthermore, offspring of NR ewes exhibited reduced progesterone secretion during the luteal phase of their estrous cycles and a markedly reduced fertility compared with offspring from Con ewes.