👤 Cory Greer

Individuals born very low birthweight (VLBW) are at increased risk of impaired cardiovascular and respiratory function in adulthood. To identify markers to predict future risk for VLBW individuals, we analyzed DNA methylation at birth and at 28 years in the New Zealand (NZ) VLBW cohort (all infants born < 1500 g in NZ in 1986) compared with age-matched, normal birthweight controls. Associations between neonatal methylation and cardiac structure and function (echocardiography), vascular function and respiratory outcomes at age 28 years were documented. Genomic DNA from archived newborn heel-prick blood (n = 109 VLBW, 51 controls) and from peripheral blood at ~ 28 years (n = 215 VLBW, 96 controls) was analyzed on Illumina Infinium MethylationEPIC 850 K arrays. Following quality assurance and normalization, methylation levels were compared between VLBW cases and controls at both ages by linear regression, with genome-wide significance set to p < 0.05 adjusted for false discovery rate (FDR, Benjamini-Hochberg). In neonates, methylation at over 16,400 CpG methylation sites differed between VLBW cases and controls and the canonical pathway most enriched for these CpGs was Cardiac Hypertrophy Signaling (p = 3.44E These findings suggest that methylation patterns in VLBW neonates may be informative about future adult cardiovascular and respiratory outcomes and have value in guiding early preventative care to improve adult health. Show less

The protocol used to induce cell death for generating vaccines from whole tumor cells is a critical consideration that impacts vaccine efficacy. Here we compared how different protocols used to induce cell death impacted protection provided by a prophylactic whole tumor cell vaccine in a mouse melanoma model. We found that melanoma cells exposed to γ-irradiation or lysis combined with UV-irradiation (LyUV) provided better protection against tumor challenge than lysis only or cells exposed to UV-irradiation. Furthermore, we found that the immunoregulatory cytokine, IL-27 enhanced protection against tumor growth in a dose-dependent manner when combined with either LyUV or γ-irradiated whole tumor cell vaccine preparations. Taken together, this data supports the use of LyUV as a potential protocol for developing whole tumor cell prophylactic cancer vaccines. We also showed that IL-27 can be used at low doses as a potent adjuvant in combination with LyUV or γ-irradiation treated cancer cells to improve the protection provided by a prophylactic cancer vaccine in a mouse melanoma model. Show less

Were Neanderthals and Denisovans (referred here also as extinct hominidae) carrying deleterious variants in genes regulating reproduction? The majority of extinct hominidae analyzed here, presented a considerable number of deleterious variants per individual in proteins regulating different aspects of reproduction, including gonad and uterine function, and gametogenesis. Neanderthals, Denisovans and extant humans were interfertile and hybridized while occupying geographically overlapping areas in Europe and Asia. This is evidenced by the small archaic genome component (average ∼2%) present in non-African extant humans. The genome of eight extinct hominidae, together with five human genome databases, plus 44 mothers and 48 fathers (fertile controls), were screened to look for deleterious variants in 1734 protein-coding genes regulating reproduction. Ancient DNA from six Neanderthals and two Denisovans dated between ∼82 000 and 43 000 calibrated years was retrieved from the public European Nucleotide Archive. The hominins analyzed include Altai, Vindija 33.15, 33.19, 33.25 and 33.26, El Sidron 1253, Denisova 3 and 11. Their DNA was analyzed using the CLC Genomics Workbench 12, by mapping overlapping paired-end reads (Illumina, FASTQ files) to the human genome assembly GRCh37 (hg19) (Vindija 33.19, 33.25, 33.26, Denisova 3 and Denisova 11) or by analyzing BAM files (Altai, El Sidron 1253 and Vindija 33.15) (human genome reference, GRCh37 (hg19)). Non-synonymous reproductive variants were classified as deleterious or tolerated (PolyPhen-2 and SIFT analyses) and were compared to deleterious variants obtained from extant human genome databases (Genome Aggregation Database (GnomAD), 1000 Genomes, the Haplotype Map (HapMap), Single Nucleotide Polymorphism Database (dbSNPs)) across different populations. A genetic intersection between extant or extinct DNA variants and other genetic disorders was evaluated by annotating the obtained variants with the Clinical Variant (ClinVar) database. Among the eight extinct hominidae analyzed, a total of 9650 non-synonymous variants (only coverage ≥20 reads included; frameshift mutations were excluded) in 1734 reproductive protein-coding genes were found, 24% of which were classified as deleterious. The majority (73%) of the deleterious alleles present in extant humans that are shared between extant humans and extinct hominidae were found to be rare (<1%) in extant human populations. A set of 8044 variants were found uniquely in extinct hominidae. At the single-gene level, no extinct individual was found to be homozygous for deleterious variants in genes necessary for gamete recognition and fusion, and no higher chance of embryo-lethality (calculated by Mendelian Genetics) was found upon simulated mating between extant human and extinct hominidae compared to extant human-extant human. However, three of the eight extinct hominidae were found to be homozygous for 48-69 deleterious variants in 55 genes controlling ovarian and uterine functions, or oogenesis (AKAP1, BUB1B, CCDC141, CDC73, DUSP6, ESR1, ESR2, PATL2, PSMC3IP, SEMA3A, WT1 and WNT4). Moreover, we report the distribution of nine Neanderthal variants in genes associated with a human fertility phenotype found in extant human populations, one of which has been associated with polycystic ovarian syndrome and primary congenital glaucoma. While analyzing archaic DNA, stringent filtering criteria were adopted to screen for deleterious variants in Neanderthals and Denisovans, which could result in missing a number of variants. Such restraints preserve the potential for detection of additional deleterious variants in reproductive proteins in extinct hominidae. This study provides a comprehensive overview of putatively deleterious variants in extant human populations and extinct individuals occurring in 1734 protein-coding genes controlling reproduction and provides the fundaments for future functional studies of extinct variants in human reproduction. This study was supported by the Department of Biological Science and by the Office of Research and Sponsored Programs at the University of Tulsa (Faculty Research Grant and Faculty Research Summer Fellowship) to M.A. and the University of Tulsa, Tulsa Undergraduate Research Challenge (TURC) program to E.L.; no conflict of interest to declare. N/A. Show less

It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases - polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) - localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (G(olf), AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia. Show less

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P < 0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P < 0.05) and insulin (1.5-fold; P < 0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P < 0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P < 0.05) and partially corrected the obesity-related islet hypertrophy and beta-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes. Show less

The calculated rate of urea production [U(p); mmol urea/(h. kg(0. 75))], based on urinary urea-N (UUN) excretion and changes in total body urea-N, was compared with the calculated total body V(max) of carbamoyl phosphate synthetase (CPS-1) of 24 neonatal piglets from four treatments as follows: 6 h baseline control (n = 8), 18 h of alanine intravenously (IV) at 50% of resting energy expenditure (REE; n = 4), 36 h of alanine IV at 50% of REE (n = 6), or 36 h of glucose IV at 50% of REE (n = 6). The following significant increases from baseline were seen in piglets infused with alanine for 36 h: 1) UUN excretion [10.6 +/- 5.9 mg N/(h. kg(0.75)) to 53.2 +/- 11.1]; 2) BUN concentrations (9.1 +/- 3.0 mmol urea N/L to 51.2 +/- 7.0); 3) calculated urea production [0.34 +/- 0.21 mmol urea/(h. kg(0.75)) to 2.39 +/- 0.53]; and 4) CPS-1 V(max) [2.0 +/- 0.81 mmol citrulline/(h. kg (0.75)) to 4.4 +/- 1.5], (P < 0.05). With the exception of CPS-1 activity, significant decreases from baseline were seen in these values in piglets infused with glucose for 36 h (P < 0.05). Comparison of calculated urea production with calculated total body CPS-1 V(max) at baseline, 18 or 36 h after the start of infusion of alanine or glucose revealed a positive relationship (slope = 0.263; P < 0.002). At all enzyme activities, infusion of alanine resulted in a significant increase in the rate of urea production compared with controls (P < 0.001). Total body CPS-1 activity varied from 1.8 to 5.8 times that of urea production, suggesting that CPS-1 did not limit urea production. Show less