Dietary microRNAs Research Articles

Differential Digestive Stability of Food-Derived microRNAs: The Case of miR-30c-5p and miR-92a-3p in Polyfloral Honey.

Dietary microRNAs (miRs) represent a new area in food science. Although they have been found in many foods, including honey, more research is needed about their stability and fate during digestion. Hence, this study aimed to analyze the digestive stability of two selected miRs in honey. We extracted miR-92a-3p and miR-30c-5p from pasteurized and unpasteurized forms of polyfloral honey using two different methods and kits: a column-based manual method and a phenol-free semi-automated magnetic-bead-based method. The latter option was used for the subsequent analysis of samples according to the INFOGEST static in vitro digestion protocol. Also, the honey samples were examined for exosome-like particles using dynamic light scattering. Although the expression levels of both miRs were significantly lower following intestinal digestion, we found a difference in the resilience of the miRs to gastrointestinal conditions, with miR-30c-5p being relatively stable compared to miR-92a-3p following digestion, regardless of the honey's pasteurization treatment. Moreover, there was marked heterogeneity in the extracellular vesicle profile of the pasteurized sample. We identified the presence of two broadly conserved miRs in honey: miR-92a-3p and miR-30c-5p. Despite honey exhibiting high digestibility, miR-92a-3p was less resilient than miR-30c-5p, demonstrating considerable resistance under gastrointestinal conditions. Although further research is needed, the results obtained from this study may represent a starting point for utilizing honey as a source of exogenous miRNAs for preventive strategies and more "natural" treatments.

A decade later: future perspectives on dietary microRNAs

A decade later: future perspectives on dietary microRNAs

Regulation of microRNA with food. Part 1. Food of plant origin

Тhe scientific review presents the mechanisms of microRNA regulation of biological processes in the human body with the help of food products of plant origin. To write the article, information was searched using Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library databases. The obtained information on dietary microRNAs is organized into the Dietary MicroRNA Databases (http://sbbi.unl.edu/dmd/), which is a repository for archiving and analyzing structural and functional microRNAs that enter the human body with food. It is stated that exogenous plant miRNAs entering the body with food are present in blood serum, tissues of humans and various animals, and regulate the expression of target genes. More than 50 miRNAs of plant origin have been identified in human blood serum using next-generation sequencing. The authors list some representatives from the multitude of dietary plant miRNAs. It is emphasized that several miRNAs show their functional activity both in plants and in mammals and easily overcome physiological barriers (miR-155, miR-168 and miR-854). Scientists believe that the ambiguous effect of plant products on the expression of human microRNA was shown by the results of a study conducted in vegetarians. Thus, with a comprehensive review using the latest information search databases, it was found that in modern scientific literature, the authors determine the horizontal transfer of numerous microRNA molecules from plants to the human body. Exogenous xenogenic xenomiRs are identified in most samples of human tissues and biological fluids. Entering the body together with food, they can significantly change the structure of the human transcriptome. Food products not only bring xenogeneic miRNAs, but also act as modulators of the endogenous miRNA generation by cells of the human body.

Dietary microRNAs: Could They Be Considered Nutraceuticals?

ADVERTISEMENT RETURN TO ARTICLES ASAPPREVViewpointNEXTDietary microRNAs: Could They Be Considered Nutraceuticals?Daniel A. Jacobo-Velázquez*Daniel A. Jacobo-VelázquezTecnologico de Monterrey, Institute for Obesity Research, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, NL MexicoTecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Campus Guadalajara, Av. General Ramon Corona 2514, C.P. 45201, Zapopan, Jalisco Mexico*Email: [email protected]More by Daniel A. Jacobo-Velázquezhttps://orcid.org/0000-0002-9478-2570, Griselda Rabadán-ChávezGriselda Rabadán-ChávezTecnologico de Monterrey, Institute for Obesity Research, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, NL MexicoMore by Griselda Rabadán-Chávez, and Edwin E. Reza-ZaldívarEdwin E. Reza-ZaldívarTecnologico de Monterrey, Institute for Obesity Research, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, NL MexicoMore by Edwin E. Reza-ZaldívarCite this: ACS Food Sci. Technol. 2023, XXXX, XXX, XXX-XXXPublication Date (Web):June 15, 2023Publication History Received31 May 2023Accepted6 June 2023Published online15 June 2023https://doi.org/10.1021/acsfoodscitech.3c00210© 2023 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views-Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (2 MB) Get e-AlertscloseSUBJECTS:Bacteria,Food,Genetics,Plant derived food,Vesicles Get e-Alerts

Dietary microRNAs (miRNAs) and Their Cutting-Edge Use in Food Science

Food quality and dietary habits have a significant impact on human health. Growing evidence suggests that miRNAs have been extensively detected in various dietary sources, including plants and animals. miRNAs are a class of small non-coding RNA that regulates gene expression in a sequence-specific manner and modulates various biological processes. These dietary miRNAs are transported through the circulatory system and affect the expression of the gene in the recipient cells through a process called cross-kingdom regulation of the gene. This review will provide insight into the role of dietary miRNAs, their stability and transport mechanisms, as well as their impact on human health. Likewise, we update and discuss the future consequences of dietary miRNAs and their possible use in the treatment of various human diseases.

Dietary microRNAs: Current status and perspective in food science.

Food safety and health conditions are global issues that are primarily caused by insufficient nutrition or contaminated food, which have enormous social, economic, and public health implications. Foodomics study mainly involves nutrigenomics, nutrigenetics, and nutritional epigenetics, which help in finding the correlation between genetic variation and nutrient-driven epigenetic alterations that are suggested as a primary challenge to nutritional needs. Micro-ribonucleic acids (miRNAs) are non-coding RNA molecules that have recently gained in popularity as a result of their critical roles in gene regulation and nutritional studies. In this commentary, the relevance of miRNAs to human nutrition is discussed. This study emphasizes the importance of dietary miRNAs based on numerous independent studies, which have explained the unique characteristics of dietary miRNAs in terms of gene expression regulation mechanism. There is an urgent requirement of measurement techniques to detect miRNA in food and evolutionary samples. These techniques may be helpful to find even low levels of miRNAs because of their high susceptibility and selectivity. There is a desperate requisite to evaluate the impact of dietary supplement compounds on circulating and tissue miRNAs. Food science studies in humans may aid in the identification of novel biomarkers and other necessary mode of action of certain dietary compounds, with the goal of facilitating how nutrients and bioactive components can influence miRNAs and enforce biological effects. PRACTICAL APPLICATIONS: Dietary microRNAs may be helpful to find even the low levels of miRNAs due to their high susceptibility and selectivity. There is also desperate need to evaluate the impact of dietary supplement compounds effect on circulating and tissue miRNAs. There is also requirement for new analytical tools to study the role of gene regulations and nutrition in different diseases. The quality of dietary miRNAs should be investigated, particularly the exogenous types which are used as high doses in food supplements. It would also be of great interest to use antagomir technology in food science research, in addition to omics technology. Dietary miRNAs may also aid in the identification of novel biomarkers. These are some of the practical applications of dietary miRNAs.

Dietary microRNAs and cancer: A new therapeutic approach?

Cancer is one of the leading causes of premature death and constitutes a challenge for both low- and high-income societies. Previous evidence supports a close association between modifiable risk factors, including dietary habits, and cancer risk. Investigation of molecular mechanisms that mediate the pro-oncogenic and anti-oncogenic effects of diet is therefore fundamental. MicroRNAs (miRNAs) have received much attention in the past few decades as crucial molecular elements of human physiology and disease. Aberrant expression patterns of these small noncoding transcripts have been observed in a wide array of cancers. Interestingly, human miRNAs not only can be modulated by bioactive dietary components, but it has also been proposed that diet-derived miRNAs may contribute to the pool of human miRNAs. Results from independent groups have suggested that these exogenous miRNAs may be functional in organisms. These findings open the door to novel and innovative approaches to cancer therapy. Here, we provide an overview of the biology of miRNAs, with a special focus on plant-derived dietary miRNAs, summarize recent findings in the field of cancer, address the possible applications to clinical practice and discuss obstacles and challenges in the field.

SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs

Dietary microRNAs have been shown to be absorbed by mammals and regulate host gene expression, but the absorption mechanism remains unknown. Here, we show that SIDT1 expressed on gastric pit cells in the stomach is required for the absorption of dietary microRNAs. SIDT1-deficient mice show reduced basal levels and impaired dynamic absorption of dietary microRNAs. Notably, we identified the stomach as the primary site for dietary microRNA absorption, which is dramatically attenuated in the stomachs of SIDT1-deficient mice. Mechanistic analyses revealed that the uptake of exogenous microRNAs by gastric pit cells is SIDT1 and low-pH dependent. Furthermore, oral administration of plant-derived miR2911 retards liver fibrosis, and this protective effect was abolished in SIDT1-deficient mice. Our findings reveal a major mechanism underlying the absorption of dietary microRNAs, uncover an unexpected role of the stomach and shed light on developing small RNA therapeutics by oral delivery.

Delineating effect of corn microRNAs and matrix, ingested as whole food, on gut microbiota in a rodent model.

Dietary microRNAs (miRNAs) are thought to regulate a wide range of biological processes, including the gut microbiota. However, it is difficult to separate specific effect(s) of miRNA from that of the food matrix. This study aims to elucidate the specific effect(s) of dietary corn miRNAs, ingested as a whole food, on the gut microbiota. We developed an autoclave procedure to remove 98% of miRNA from corn. A mouse feeding study was conducted comparing autoclaved corn to nonautoclaved corn and purified corn miRNA. Compared to nonspecific nucleotides and corn devoid of miRNAs, feeding purified corn miRNAs or corn to C57BL/6 mice via gavage or diet supplementation for two weeks lead to a decrease in total bacteria in the cecum. The effect appeared to be due to changes in Firmicutes. Additionally, corn matrix minus miRNA and processing also affected gut bacteria. In silico analysis identified corn miRNAs that aligned to Firmicutes genome sequences lending further support to the interaction between corn miRNAs and this bacterium. These data support interactions between plant food miRNA, as well as matrix, and the gut microbiota exist but complex. However, it provides additional support for mechanism by which bioactive dietary components interact with the gut microbiota.

Dietary microRNA—A Novel Functional Component of Food

MicroRNAs are a class of small RNAs that play essential roles in various biological processes by silencing genes. Evidence emerging in recent years suggests that microRNAs in food can be absorbed into the circulatory system and organs of humans and other animals, where they regulate gene expression and biological processes. These food-derived dietary microRNAs may serve as a novel functional component of food, a role that has been neglected to date. However, a significant amount of evidence challenges this new concept. The absorption, stability, and physiological effects of dietary microRNA in recipients, especially in mammals, are currently under heavy debate. In this review, we summarize our current understanding of the unique characteristics of dietary microRNAs and concerns about both the mechanistic and methodological basis for studying the biological significance of dietary microRNAs. Such efforts will benefit continuing investigations and offer new perspectives for the interpretation of the roles of dietary microRNA with respect to the health and disease of humans and animals.

Dietary non-coding RNAs from plants: Fairy tale or treasure?

The past two decades have witnessed soaring interest in the field of non-coding RNAs, largely attributed by its regulatory role in controlling two third of human transcriptional output. Though, there are several classes of non-coding RNAs found in nature, microRNAs takes the central stage because of their pleiotropic roles. In particular, extracellular microRNAs are gaining traction due to their relative stability and bio availability. Extracellular microRNAs has been shown to occur in all living organisms, including dietary plants. Some of the recent reports suggest that these dietary microRNAs pass through the gut, enter systemic circulation and exert biological effects on animal physiology. However, evidences against this hypothesis are also presented in literature and hence this area has been strongly debated. In this review, I will briefly summarise the evidences accumulated for and against this hypothesis and discuss potential implications of such findings in human health.

Nutritive implications of dietary microRNAs: facts, controversies, and perspectives.

As a group of non-coding RNA molecules, microRNAs have recently become more well-known due to their pivotal role in gene regulation. A large number of endogenous microRNAs naturally occur in the human body, and some of them act as regulatory targets of diet and its components. The wide presence of microRNAs in various food materials has inspired food scientists and nutritionists to explore their nutritive and bioactive significance. This article comprehensively reports updated insights into the accessibility, stability, absorbability, and bioactivity of dietary microRNAs by combining the current knowledge into figures and tables for reader's convenience. As one frontier in food science and nutrition, the research platform on dietary microRNAs is imperfect and even defective as indicated by the inconsistent and even contradictory results concluded by different investigations. The pros and cons as well as the limitations of current investigations have been critically discussed with attention chiefly paid to experimental designs and protocols. Moreover, future research directions have been recommended. Thus, this paper may not only provide a quick glance at the state-of-the-art of dietary microRNAs but also guide further research to clarify the present controversies and make the results more credible and persuasive.

RNase H2-Dependent Polymerase Chain Reaction and Elimination of Confounders in Sample Collection, Storage, and Analysis Strengthen Evidence That microRNAs in Bovine Milk Are Bioavailable in Humans

Evidence suggests that dietary microRNAs (miRs) are bioavailable and regulate gene expression across species boundaries. Concerns were raised that the detection of dietary miRs in plasma might have been due to sample contamination or lack of assay specificity. Objectives: The objectives of this study were to assess potential confounders of plasma miR analysis and to detect miRs from bovine milk in human plasma. Potential confounders of plasma miR analysis (circadian rhythm, sample collection and storage, calibration, and erythrocyte hemolysis) were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR) by using blood from healthy adults (7 men, 6 women; aged 23-57 y). Bovine miRs were analyzed by RNase H2-dependent PCR (rhPCR) in plasma collected from a subcohort of 11 participants before and 6 h after consumption of 1.0 L of 1%-fat bovine milk. The use of heparin tubes for blood collection resulted in a complete loss of miRs. Circadian variations did not affect the concentrations of 8 select miRs. Erythrocyte hemolysis caused artifacts for some miRs if plasma absorbance at 414 nm was >0.300. The stability of plasma miRs depended greatly on the matrix in which the miRs were stored and whether the plasma was frozen before analysis. Purified miR-16, miR-200c, and cel-miR-39 were stable for ≤24 h at room temperature, whereas losses equaled ≤80% if plasma was frozen, thawed, and stored at room temperature for as little as 4 h. rhPCR distinguished between bovine and human miRs with small variations in the nucleotide sequence; plasma concentrations of Bos taurus (bta)-miR-21-5p and bta-miR-30a-5p were >100% higher 6 h after milk consumption than before milk consumption. Confounders in plasma miR analysis include the use of heparin tubes, erythrocyte hemolysis, and storage of thawed plasma at room temperature. rhPCR is a useful tool to detect dietary miRs.

Diet and cancer prevention: Dietary compounds, dietary MicroRNAs, and dietary exosomes.

Cancer is one of main health public problems worldwide. Several factors are involved in beginning and development of cancer. Genetic and internal/external environmental factors can be as important agents that effect on emerging and development of several cancers. Diet and nutrition may be as one of important factors in prevention or treatment of various cancers. A large number studies indicated that suitable dietary patterns may help to cancer prevention or could inhibit development of tumor in cancer patients. Moreover, a large numbers studies indicated that a variety of dietary compounds such as curcumin, green tea, folat, selenium, and soy isoflavones show a wide range anti-cancer properties. It has been showed that these compounds via targeting a sequence of cellular and molecular pathways could be used as suitable options for cancer chemoprevention and cancer therapy. Recently, dietary microRNAs and exosomes have been emerged as attractive players in cancer prevention and cancer therapy. These molecules could change behavior of cancer cells via targeting various cellular and molecular pathways involved in cancer pathogenesis. Hence, the utilization of dietary compounds which are associated with powerful molecules such as microRNAs and exosomes and put them in dietary patterns could contribute to prevention or treatment of various cancers. Here, we summarized various studies that assessed effect of dietary patterns on cancer prevention shortly. Moreover, we highlighted the utilization of dietary compounds, dietary microRNAs, and dietary exosomes and their cellular and molecular pathways in cancer chemoprevention.

Milk exosomes: beyond dietary microRNAs

Extracellular vesicles deliver a variety of cargos to recipient cells, including the delivery of cargos in dietary vesicles from bovine milk to non-bovine species. The rate of discovery in this important line of research is slowed by a controversy whether the delivery and bioactivity of a single class of vesicle cargos, microRNAs, are real or not. This opinion paper argues that the evidence in support of the bioavailability of microRNAs encapsulated in dietary exosomes outweighs the evidence produced by scholars doubting that phenomenon is real. Importantly, this paper posits that the time is ripe to look beyond microRNA cargos and pursue innovative pathways through which dietary exosomes alter metabolism. Here, we highlight potentially fruitful lines of exploration.

Do microRNAs play a role in the activity of plant-based vaccines?

ABSTRACT Introduction: An important trend in vaccinology is the development of oral vaccines based on genetically modified plants. Areas covered: Several studies have suggested that dietary microRNAs from plants and other organisms are bioavailable upon oral ingestion exerting biological events in the host such as the modulation of gene expression in several cell types. Since oral plant-based vaccines rely on whole cells as vaccine delivery vehicles, miRNAs could play a role in the immunogenic activity of this type of vaccine. In the present report, this hypothesis is discussed under the light of recent evidence on the immunomodulatory activity exerted by miRNAs using in vitro and in vivo evaluations. Expert commentary: The ways to generate new knowledge and exploit the potential of miRNAs in the development of oral vaccines are discussed.

Biological Activities of Extracellular Vesicles and Their Cargos from Bovine and Human Milk in Humans and Implications for Infants

Extracellular vesicles (EVs) in milk harbor a variety of compounds, including lipids, proteins, noncoding RNAs, and mRNAs. Among the various classes of EVs, exosomes are of particular interest, because cargo sorting in exosomes is a regulated, nonrandom process and exosomes play essential roles in cell-to-cell communication. Encapsulation in exosomes confers protection against enzymatic and nonenzymatic degradation of cargos and provides a pathway for cellular uptake of cargos by endocytosis of exosomes. Compelling evidence suggests that exosomes in bovine milk are transported by intestinal cells, vascular endothelial cells, and macrophages in human and rodent cell cultures, and bovine-milk exosomes are delivered to peripheral tissues in mice. Evidence also suggests that cargos in bovine-milk exosomes, in particular RNAs, are delivered to circulating immune cells in humans. Some microRNAs and mRNAs in bovine-milk exosomes may regulate the expression of human genes and be translated into protein, respectively. Some exosome cargos are quantitatively minor in the diet compared with endogenous synthesis. However, noncanonical pathways have been identified through which low concentrations of dietary microRNAs may alter gene expression, such as the accumulation of exosomes in the immune cell microenvironment and the binding of microRNAs to Toll-like receptors. Phenotypes observed in infant-feeding studies include higher Mental Developmental Index, Psychomotor Development Index, and Preschool Language Scale-3 scores in breastfed infants than in those fed various formulas. In mice, supplementation with plant-derived MIR-2911 improved the antiviral response compared with controls. Porcine-milk exosomes promote the proliferation of intestinal cells in mice. This article discusses the above-mentioned advances in research concerning milk exosomes and their cargos in human nutrition. Implications for infant nutrition are emphasized, where permitted, but data in infants are limited.

Potential relevance of microRNAs in inter-species epigenetic communication, and implications for disease pathogenesis

ABSTRACTMicroRNAs are short non-protein coding RNA molecules involved in the epigenetic regulation of gene expression. Recently, extracellular microRNAs have been described in body fluids that might enable epigenetic communication between distant tissues. Being highly conserved molecules, exogenous xeno-microRNAs from different species could affect gene expression in the host even in a cross-kingdom fashion. Several data underline the relevance of microRNA-mediated communication between virus and host, and there are some experimental data showing that plant- or animal-derived dietary microRNAs might have gene expression modulating activity in humans. Milk-derived microRNAs might be involved in the “epigenetic priming” of the baby. Exogenous microRNAs might be hypothesized to be implicated in disease pathogenesis, e.g. in tumors. Major questions remain to be addressed including the amount of xeno-microRNAs needed for biological action or routes for microRNA delivery. In this brief review, experimental data and hypotheses on the potential pathogenic inter-species relevance of microRNA are presented.

Hydroxytyrosol supplementation modulates the expression of miRNAs in rodents and in humans

Dietary microRNAs (miRNAs) modulation could be important for health and wellbeing. Part of the healthful activities of polyphenols might be due to a modulation of miRNAs' expression. Among the most biologically active polyphenols, hydroxytyrosol (HT) has never been studied for its actions on miRNAs. We investigated whether HT could modulate the expression of miRNAs in vivo. We performed an unbiased intestinal miRNA screening in mice supplemented (for 8 weeks) with nutritionally relevant amounts of HT. HT modulated the expression of several miRNAs. Analysis of other tissues revealed consistent HT-induced modulation of only few miRNAs. Also, HT administration increased triglycerides levels. Acute treatment with HT and in vitro experiments provided mechanistic insights. The HT-induced expression of one miRNA was confirmed in healthy volunteers supplemented with HT in a randomized, double-blind and placebo-controlled trial. HT consumption affects specific miRNAs' expression in rodents and humans. Our findings suggest that the modulation of miRNAs' action through HT consumption might partially explain its healthful activities and might be pharmanutritionally exploited in current therapies targeting endogenous miRNAs. However, the effects of HT on triglycerides warrant further investigations.

Depletion of Dietary microRNAs from Cow's Milk Causes an Increase of Purine Metabolites in Human Body Fluids and Mouse Livers

BackgroundMicroRNAs (miRNAs) play essential roles in gene regulation. We have recently reported the ground‐breaking discovery that miRNAs are not exclusively derived from endogenous synthesis but can also be obtained from dietary sources such as cow's milk. Evidence suggests that milk consumption is inversely correlated with serum uric acid (UA) concentrations, and 19 miRNAs regulate 11 pathways in purine metabolism.HypothesisConsumption of a diet depleted of milk miRNAs causes changes in purine metabolism.MethodsIn a first screen, C57BL/6 mice were fed a diet depleted of miRNAs (encapsulated in exosomes) from age 3 to 7 weeks [denoted Exosome (E) Minus, E−], whereas controls were fed a miRNA‐sufficient diet (E+). Mouse liver metabolites were analyzed using non‐targeted LC/MS‐MS, and targeted enzymatic assays for purine metabolites. Subsequently, we assessed purine metabolites in urine and plasma from human dairy avoiders and dairy consumers. The statistical significance of differences was assessed by t‐test.ResultsPrincipal Components Analysis (PCA), a dendrogram performed with hierarchical clustering, Variable Important in Projection (VIP) scores, and a heat map analysis consistently suggested that concentrations of purine metabolites were higher in E− livers than in E+ livers, and that purine metabolites were affected by milk miRNA intake to a greater extent than any of the other metabolites that were identified (p<0.01; n=5). As for effect size, the concentrations of xanthine were 16.6 ± 3.3 μM and 10.1 ± 1.0 μM in E − and E+ livers, respectively (p<0.05). Likewise, the concentrations of plasma xanthine and urinary uric acid were 81.6% and 19.3% higher in human dairy avoiders compared with dairy consumers (n=6).ConclusionDietary depletion of milk miRNAs causes an increase in purine metabolites in mouse tissues and human body fluids.Future plansA reversed‐phased HPLC was developed to quantify eight purine metabolites in a comprehensive analysis of purine metabolism; gene expression analysis and purine feeding studies will be used to identify the step(s) in purine metabolism targeted by milk miRNAs.Support or Funding InformationNIFA/USDA 2015‐67017‐23181, NIH 1P20GM104320, Gerber Foundation, Egg Nutrition Center, USDA W‐3002, Hatch and NIH P30GM103335