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Ascorbic acid functions as an antioxidant and facilitates other biochemical processes such as collagen triple helix formation, and iron uptake by cells. Animals which endogenously produce ascorbic acid have a functional gulonolactone oxidase gene (GULO); however, humans have a GULO pseudogene (GULOP) and depend on dietary ascorbic acid. In this study, the conservation of GULOP sequences in the primate haplorhini suborder were investigated and compared to the GULO sequences belonging to the primates strepsirrhini suborder. Phylogenetic analysis suggested that the conserved GULOP exons in the haplorhini primates experienced a high rate of mutations following the haplorhini/strepsirrhini divergence. This high mutation rate has decreased during the evolution of the haplorhini primates. Additionally, indels of the haplorhini GULOP sequences were conserved across the suborder. A separate analysis for GULO sequences and well-conserved GULOP sequences focusing on placental mammals identified an in-frame GULO sequence in the Brazilian guinea pig, and a potential GULOP sequence in the pika. Similar to haplorhini primates, the guinea pig and lagomorph species have experienced a high substitution rate when compared to the mammals used in this study. A shared synteny to examine the conservation of local genes near GULO/GULOP identified a conserved inversion around the GULO/GULOP locus between the haplorhini and strepsirrhini primates. Fischer's exact test did not support an association between GULOP and the chromosomal inversion. Mauve alignment showed that the inversion of the length of the syntenic block that the GULO/GULOP genes belonged to was variable. However, there were frequent rearrangements around ~ 2 million base pairs adjacent to GULOP involving the KIF13B and MSRA genes. These data may suggest that genes acquiring deleterious mutations in the coding sequence may respond to these deleterious mutations with rapid substitution rates. [ABSTRACT FROM AUTHOR]

Non-coding RNAs (ncRNAs) are, arguably, the enigma of the RNA transcriptome. Even though there are more annotated ncRNAs (25,967) compared to mRNAs (19,827), we know far less about each of the genes that produce ncRNA, especially in terms of their regulation, molecular functions, and interactions. Further, we are only beginning to understand the role of differential regulation or function of ncRNAs caused by genetic and epigenetic perturbations, such as single nucleotide variants (SNV), deletions, insertions, and histone/DNA modifications. The 22 papers in this Special Issue describe the emerging roles of ncRNAs in neurological, cardiovascular, immune, and hepatic systems, to name a few, as well as in diseases such as cancer, Prader–Willi Syndrome, cardiac arrhythmias, and diabetes. As we begin to understand the function and regulation of this class of RNAs, strategies targeting ncRNAs could lead to improved therapeutic interventions for some conditions. [ABSTRACT FROM AUTHOR]

Continual advances in our understanding of the human genome have led to exponential increases in known single nucleotide variants. The characterization of each of the variants lags behind. For researchers needing to study a single gene, or multiple genes in a pathway, there must be ways to narrow down pathogenic variants from those that are silent or pose less pathogenicity. In this study, we use the NHLH2 gene which encodes the nescient helix-loop-helix 2 (Nhlh2) transcription factor in a systematic analysis of all missense mutations to date in the gene. The NHLH2 gene was first described in 1992. Knockout mice created in 1997 indicated a role for this protein in body weight control, puberty, and fertility, as well as the motivation for sex and exercise. Only recently have human carriers of NHLH2 missense variants been characterized. Over 300 missense variants for the NHLH2 gene are listed in the NCBI single nucleotide polymorphism database (dbSNP). Using in silico tools, predicted pathogenicity of the variants narrowed the missense variants to 37 which were predicted to affect NHLH2 function. These 37 variants cluster around the basic-helix-loop-helix and DNA binding domains of the transcription factor, and further analysis using in silico tools provided 21 SNV resulting in 22 amino acid changes for future wet lab analysis. The tools used, findings, and predictions for the variants are discussed considering the known function of the NHLH2 transcription factor. Overall use of these in silico tools and analysis of these data contribute to our knowledge of a protein which is both involved in the human genetic syndrome, Prader–Willi syndrome, and in controlling genes involved in body weight control, fertility, puberty, and behavior in the general population, and may provide a systematic methodology for others to characterize variants for their gene of interest. [ABSTRACT FROM AUTHOR]

The Small Nucleolar Host Gene 14 (SNHG14) is a host gene for small non-coding RNAs, including the SNORD116 small nucleolar C/D box RNA encoding locus. Large deletions of the SNHG14 locus, as well as microdeletions of the SNORD116 locus, lead to the neurodevelopmental genetic disorder Prader–Willi syndrome. This review will focus on the SNHG14 gene, its expression patterns, its role in human cancer, and the possibility that single nucleotide variants within the locus contribute to human phenotypes in the general population. This review will also include new in silico data analyses of the SNHG14 locus and new in situ RNA expression patterns of the Snhg14 RNA in mouse midbrain and hindbrain regions. [ABSTRACT FROM AUTHOR]

Objective: To assess the role of body mass index (BMI) and exercise levels in self-perception of critical thinking skills. Participants: Three hundred forty-seven students from an upper-division nutrition class over two consecutive years. Methods: A pre/post survey with a 15-week intervention assessed perceived critical thinking skills in a blended classroom. Results: Students gained in perceived critical thinking skills in six areas over the semester. A higher BMI was associated with decreased perception of one's ability to think logically, along with increased perception that memorization was the key to success. Those that exercised reported that they had strong critical thinking skills compared to those that exercised less frequently. Conclusions: A blended classroom approach was effective in increasing multiple areas of perceptions of critical thinking. However, some perceptions of critical thinking are viewed differently for those of different BMIs and exercise frequency. Consequently, designing interventions specifically targeting those with higher BMIs, could work to erase these inequities. [ABSTRACT FROM AUTHOR]

Metabolism has a role in determining the time of pubertal development and fertility. Nonetheless, molecular/cellular pathways linking metabolism/body weight to puberty/reproduction are unknown. The KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons in the arcuate nucleus of the hypothalamus constitute the GnRH (gonadotropin-releasing hormone) pulse generator. We previously created a mouse model with a whole-body targeted deletion of nescient helix-loop-helix 2 (Nhlh2; N2KO), a class II member of the basic helix-loop-helix family of transcription factors. As this mouse model features pubertal failure and late-onset obesity, we wanted to study whether NHLH2 represents a candidate molecule to link metabolism and puberty in the hypothalamus. Exome sequencing of a large Idiopathic Hypogonadotropic Hypogonadism cohort revealed obese patients with rare sequence variants in NHLH2, which were characterized by in-silico protein analysis, chromatin immunoprecipitation, and luciferase reporter assays. In vitro heterologous expression studies demonstrated that the variant p.R79C impairs Nhlh2 binding to the Mc4r promoter. Furthermore, p.R79C and other variants show impaired transactivation of the human KISS1 promoter. These are the first inactivating human variants that support NHLH2's critical role in human puberty and body weight control. Failure to carry out this function results in the absence of pubertal development and late-onset obesity in humans. [ABSTRACT FROM AUTHOR]

This article describes the development and assessment of a Nutritional Genomics course, designed to be held in a regular classroom during normal class periods, with few extra costs to the students or the department. The course was run as an upper‐level undergraduate and lower‐level graduate student course. Student taking the course spent 11 weeks learning and then 4 weeks using various in silico methods to independently characterize genes of interest in the field. During the last 4 weeks of the course, students combined their methods to test a hypothesis they generated about a gene they have not yet studied and completed a final report in the form of a journal article. Two students have published or are in the process of publishing work from their final project. Validated surveys of genetic knowledge given at least 6 months following the course indicated a very high level of genetic knowledge retainment, and favorable attitudes toward genetics testing and medical use of genetics. Finally, self‐perceived critical thinking skills were high, and students indicated that they perceived these skills to be gained by their participation in the course. Materials and syllabus provided in the manuscript makes this CURE easily transferrable to other disciplines. [ABSTRACT FROM AUTHOR]

Hereditary hemochromatosis (HH) is a disease that causes excess iron absorption from the diet. This excess iron can be stored in the liver, skin, heart, pancreas, and joints, and then can lead to other health conditions, as the human body has no way of actively excreting iron. The human hemochromatosis protein (HFE protein) is encoded by the HFE gene, and mutations in this gene can lead to a dysfunction of the protein resulting in HH or iron overload later in adulthood. The objective of this study was to analyze the mutant allele frequency and the penetrance of the H63D mutation (SNP rs1799945) of the HFE gene in a cohort of Virginia Tech students. This study had a total of 69 participants. Fifty-two participants provided saliva samples, genomic data from 23andMe®, and surveys with phenotypic information. Of these, 6 were genotyped using the RFLP technique and served as controls for genotype confirmation. An additional 17 participants provided saliva samples, but did not provide 23andMe® data; genomic DNA from these participants were genotyped using the RFLP technique. Our results showed that although none of the participants had been diagnosed with HH, the mutant allele frequency of this population was 13.04%. In conclusion, as HH is usually diagnosed in older adults, we could not identify any students with a phenotype of HH, even though we could detect the mutant allele. This data suggests that affordable and accessible genetic ancestry and health kits such as the 23andMe® kit, could provide an efficient way to identify, prevent, and manage HH and other genetic diseases before symptoms arise. [ABSTRACT FROM AUTHOR]

The SNORD116 small nucleolar RNA locus (SNORD116@) is contained within the long noncoding RNA host gene SNHG14 on human chromosome 15q11-q13. The SNORD116 locus is a cluster of 28 or more small nucleolar (sno) RNAs; C/D box (SNORDs). Individual RNAs within the cluster are tandem, highly similar sequences, referred to as SNORD116-1, SNORD116-2, etc., with the entire set referred to as SNORD116@. There are also related SNORD116 loci on other chromosomes, and these additional loci are conserved among primates. Inherited chromosomal 15q11-q13 deletions, encompassing the SNORD116@ locus, are causative for the paternally-inherited/maternally-imprinted genetic condition, Prader-Willi syndrome (PWS). Using in silico tools, along with molecular-based and sequenced-based confirmation, phylogenetic analysis of the SNORD116@ locus was performed. The consensus sequence for the SNORD116@ snoRNAs from various species was determined both for all the SNORD116 snoRNAs, as well as those grouped using sequence and location according to a human grouping convention. The implications of these findings are put in perspective for studying SNORD116 in patients with inherited Prader-Willi syndrome, as well as model organisms. [ABSTRACT FROM AUTHOR]

Recently, conjugated linoleic acid (CLA) has been reported to prevent body weight gain and fat accumulation in part by improving physical activity in mice. The current study was conducted to determine the role of CLA on skeletal muscle metabolism in nescient basic helix-loop-helix 2 knock-out (N2KO) mice, an adult-onset inactivity induced obese model. Five-week-old female N2KO and wild type mice were fed either control or CLA containing diet (0.5%) for 10 wk. Voluntary activity was determined biweekly and markers for muscle metabolisms were determined from the gastrocnemius muscle. CLA fed N2KO animals showed significant increased voluntary movement and gastrocnemius muscle mass compared to control group, whereas in wild type animals, no differences were observed. CLA treatment up-regulated AMP-activated protein kinase (AMPK), mitochondria biogenic markers, peroxisome proliferator-activated receptor-δ (PPARδ), and mitochondrial transcription factor A (Tfam) compared to control animals. These observations indicate that CLA supplementation activates AMPKα-PPARδ and/or -Tfam signaling cascade for stimulating mitochondria biogenesis. Taken together, these results suggest that CLA may in part activate the underlying biomarkers involved in muscle metabolism via stimulation of mitochondrial biogenesis, resulting in increased voluntary activity and muscle mass, potentially contributing to regulating weight gain. Practical applications: Lack of physical activity is a global public health problem, which induces obesity and its associated pathologies. Approximately, 3.2 million deaths per year are attributable to lack of physical activity. CLA has previously been reported to increase voluntary and endurance activities in mice. However, the exact mode of action is not completely understood. Thus, the purpose of the study was to shed light on positive effects of CLA on physical activity through modulation of molecular targets in skeletal muscle of Nhlh2 knockout mice, which are an adult-onset inactivity induced obesity model. The current results suggest that CLA acts as a potential exercise-mimetic, resulting in increased voluntary activity and muscle mass, potentially contributing to regulating weight gain. Conjugated linoleic acid partially improves muscle metabolism via stimulation of mitochondrial biogenesis, resulting in increased voluntary activity. [ABSTRACT FROM AUTHOR]

The hypothalamus integrates energy balance information from the periphery using different neuronal subtypes within each of the hypothalamic areas. However, the effects of prandial state on global mRNA, microRNA and long noncoding (lnc) RNA expression within the whole hypothalamus are largely unknown. In this study, mice were given either a 24-h fast, or ad libitum access to food. RNA samples were analyzed by microarray, and then a subset was confirmed using quantitative real-time PCR (QPCR). A total of 540 mRNAs were either up- or downregulated with food deprivation. Since gene ontology enrichment analyses identified several categories of mRNAs related to cell cycle processes, ten cell-cycle-related genes were further analyzed using QPCR with six confirmed to be significantly up-regulated and one downregulated in response to 24-h fasting. While 22 independent microRNAs were differentially expressed by microarray, secondary analysis by QPCR failed to confirm significant changes with fasting. There were 622 lncRNAs identified as differentially expressed, and of three tested by QPCR, two were confirmed. Overall, this is the first time that expression of hypothalamic lncRNAs has been shown to be responsive to food deprivation. In addition, this study is the first to identify a list of lncRNAs with high expression in RNA extracted from hypothalamus. Individual contributions from specific miRNA, lncRNA and mRNAs to the food deprivation response can now be further studied at the physiological and biochemical levels. [ABSTRACT FROM AUTHOR]

Prader–Willi Syndrome (PWS) is a human genetic condition that affects up to 1 in 10,000 live births. Affected infants present with hypotonia and developmental delay. Hyperphagia and increasing body weight follow unless drastic calorie restriction is initiated. Recently, our laboratory showed that one of the genes in the deleted locus causative for PWS, Snord116, maintains increased expression of hypothalamic Nhlh2, a basic helix–loop–helix transcription factor. We have previously also shown that obese mice with a deletion of Nhlh2 respond to a conjugated linoleic acid (CLA) diet with weight and fat loss. In this study, we investigated whether mice with a paternal deletion of Snord116 (Snord116m+/p−) would respond similarly. We found that while Snord116m+/p− mice and mice with a deletion of both Snord116 alleles were not significantly obese on a high-fat diet, they did lose body weight and fat on a high-fat/CLA diet, suggesting that the genotype did not interfere with CLA actions. There were no changes in food intake or metabolic rate, and only moderate differences in exercise performance. RNA-seq and microbiome analyses identified hypothalamic mRNAs, and differentially populated gut bacteria, that support future mechanistic analyses. CLA may be useful as a food additive to reduce obesity in humans with PWS. [ABSTRACT FROM AUTHOR]

Background: Mice with a targeted deletion of the basic helix-loop-helix transcription factor, Nescient Helix-Loop-Helix 2 (Nhlh2), display adult-onset obesity with significant increases in their fat depots, abnormal responses to cold exposure, and reduced spontaneous physical activity levels. These phenotypes, accompanied by the hypothalamic expression of Nhlh2, make the Nhlh2 knockout (N2KO) mouse a useful model to study the role of central nervous system (CNS) control on peripheral tissue such as adipose tissue. Methodology: Differences in body temperature and serum analysis of leptin were performed in fasted and ad lib fed wildtype (WT) and N2KO mice. Histological analysis of white (WAT) and brown adipose tissue (BAT) was performed. Gene and protein level expression of inflammatory and metabolic markers were compared between the two genotypes. Principal Findings: We report significant differences in serum leptin levels and body temperature in N2KO mice compared with WT mice exposed to a 24-hour fast, suggestive of a defect in both white (WAT) and brown adipose tissue (BAT) function. As compared to WT mice, N2KO mice showed increased serum IL-6 protein and WAT IL-6 mRNA levels. This was accompanied by slight elevations of mRNA for several macrophage markers, including expression of macrophage specific protein F4/80 in adipose, suggestive of macrophage infiltration of WAT in the mutant animals. The mRNAs for b3-adrenergic receptors (b3-AR), b2-AR and uncoupling proteins were significantly reduced in WAT and BAT from N2KO mice compared with WT mice. Conclusions: These studies implicate Nhlh2 in the central control of WAT and BAT function, with lack of Nhlh2 leading to adipose inflammation and altered gene expression, impaired leptin response to fasting, all suggestive of a deficient torpor response in mutant animals. [ABSTRACT FROM AUTHOR]

Biotechnology 385 is a team-taught upper level undergraduate course where students acquire hands-on experience with advanced laboratory techniques. Although student evaluations are usually very high for this course, students feel a "disconnection" among the four professors and four different modules, and indicate that they feel unprepared to go into lab and "do the experiment" even after having read the laboratory manual. To address these issues, a WebCT site for each of the four modules and Flash animations for the Molecular Genetics module were developed. The data indicate that communication between students and teachers in this course was improved by use of WebCT and student understanding of the laboratory procedures was enhanced by the Flash animations. The usage statistics show that students viewed the animations immediately prior to the laboratories where they used the procedures, and the assessments indicate that this was an effective use of web-enhancement for a laboratory-based course. [ABSTRACT FROM AUTHOR]

Demonstrates that mouse embryos lacking the early-response gene SIL have axial midline defects, a block in midline Sonic hedgehog (Shh) signaling and randomized cardiac looping. Comparison with Shh mutant embryos, which have axial defects but normal cardiac looping; Indications.

MicroRNAs (miRNAs) are small non-coding RNA molecules that control the post-transcriptional gene expression. They play a pivotal role in the regulation of important physiological processes. Variations in miRNA genes coding for mature miRNA sequences have been implicated in several diseases. However, the association of variants in miRNAs genes with Type 2 Diabetes Mellitus (T2DM) in the Pakistani population is rarely reported. Therefore, the current study was designed to investigate the association of rs11614913 T/C (MIR196A2), rs2910164 G/C (MIR146A), and rs6505162 C/A (MIR423) in clinicopathological proven T2DM patients and gender-matched healthy controls. The tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) reaction method was used to determine the genotypes and to establish the association of each variant with T2DM through inherited models. In conclusion, the present study showed that variants rs11614913 T/C and rs2910164 G/C were linked with the risk of T2DM. The data suggested that rs11614913 T/C and rs2910164 G/C could be considered as novel risk factors in the pathogenesis of T2DM in the Pakistani population. [ABSTRACT FROM AUTHOR]