A clinically validated human saliva metatranscriptomic test for global systems biology studies.

The authors report here the development of a high-throughput, automated, inexpensive and clinically validated saliva metatranscriptome test that requires less than 100 µl of saliva. RNA is preserved at the time of sample collection, allowing for ambient-temperature transportation and storage for up to 28 days. Critically, the RNA preservative is also able to inactivate pathogenic microorganisms, rendering the samples noninfectious and allowing for safe and easy shipping. Given the unique set of convenience, low cost, safety and technical performance, this saliva metatranscriptomic test can be integrated into longitudinal, global-scale systems biology studies that will lead to an accelerated development of precision medicine, diagnostic and therapeutic tools.

Gut Microbiome Activity Contributes to Prediction of Individual Variation in Glycemic Response in Adults.

Limiting postprandial glycemic response (PPGR) is an important intervention in reducing the risk of chronic metabolic diseases and has been shown to impart significant health benefits in people with elevated levels of blood sugar. In this study, we collected gut microbiome activity data by assessing the metatranscriptome, and we measured the glycemic responses of 550 adults who consumed more than 30,000 meals, collectively, from omnivore or vegetarian/gluten-free diets. We demonstrate that gut microbiome activity, anthropometric factors, and food macronutrients modulate individual variation in glycemic response. We employ two predictive models, including a mixed-effects linear regression model (R = 0.77) and a gradient boosting machine model (Rtrain = 0.80/R2train = 0.64; Rtest = 0.64/R2test = 0.40), which demonstrate variation in PPGR between individuals when ingesting the same foods. All features in the final mixed-effects linear regression model were significant (p < 0.05) except for two features which were retained as suggestive: glutamine production pathways (p = 0.08) and the interaction between tyrosine metabolizers and carbs (p = 0.06). We introduce molecular functions as features in these two models, aggregated from microbial activity data, and show their statistically significant contributions to glycemic control. In summary, we demonstrate for the first time that metatranscriptomic activity of the gut microbiome is correlated with PPGR among adults.

Blood sugar dysregulation is caused by various underlying conditions, including type 2 diabetes, and this may lead to extended periods of hypoglycemia or hyperglycemia, which can be harmful or deadly. Clinically, glycemic control is a primary therapeutic target for dysglycemia, and food and nutrition are frequent interventions used to reduce postprandial blood glucose excursions. Primary determinants of postprandial glycemic response (PPGR) include dietary carbohydrates, individual phenotypes, and individual molecular characteristics which include the gut microbiome. Typical investigations of gut microbiomes depend on analysis methods which have poor taxonomic resolution, cannot identify certain microorganisms, and are prone to errors. In this study, each RNA molecule was identified and counted, allowing quantitative strain-level taxonomic classification and molecular pathway analysis. The primary goal of the study was to assess the impact of microbial functional activity on PPGR. The study was conducted in the USA and involved a multiethnic population of healthy adults with HbA1c levels below 6.5. All participants received 14-day omnivore diets or vegetarian/gluten-free diets, depending on nutritional requirements (omnivore diets include meat while vegetarian/gluten-free diets exclude both gluten and meat). Over this timeframe, blood glucose levels were measured in 15-min intervals, 24 h per day, capturing postprandial responses for more than 27,000 meals, including more than 18,000 provided meals which spanned a wide range of foods and macronutrient characteristics. Computational modeling demonstrated the statistical significance of all features and identified new features which may be relevant to glycemic control. These results show, for the first time, that a person's glycemic response depends on individual traits, including both their anthropometrics and their gut metatranscriptome, representing the activity of gut microbiomes.

The salivary metatranscriptome as an accurate diagnostic indicator of oral cancer.

Despite advances in cancer treatment, the 5-year mortality rate for oral cancers (OC) is 40%, mainly due to the lack of early diagnostics. To advance early diagnostics for high-risk and average-risk populations, we developed and evaluated machine-learning (ML) classifiers using metatranscriptomic data from saliva samples (n = 433) collected from oral premalignant disorders (OPMD), OC patients (n = 71) and normal controls (n = 171). Our diagnostic classifiers yielded a receiver operating characteristics (ROC) area under the curve (AUC) up to 0.9, sensitivity up to 83% (92.3% for stage 1 cancer) and specificity up to 97.9%. Our metatranscriptomic signature incorporates both taxonomic and functional microbiome features, and reveals a number of taxa and functional pathways associated with OC. We demonstrate the potential clinical utility of an AI/ML model for diagnosing OC early, opening a new era of non-invasive diagnostics, enabling early intervention and improved patient outcomes.

Discoidin-domain receptor coordinates cell-matrix adhesion and collective polarity in migratory cardiopharyngeal progenitors.

Integrated analyses of regulated effector genes, cellular processes, and extrinsic signals are required to understand how transcriptional networks coordinate fate specification and cell behavior during embryogenesis. Ciona cardiopharyngeal progenitors, the trunk ventral cells (TVCs), polarize as leader and trailer cells that migrate between the ventral epidermis and trunk endoderm. We show that the TVC-specific collagen-binding Discoidin-domain receptor (Ddr) cooperates with Integrin-ß1 to promote cell-matrix adhesion. We find that endodermal cells secrete a collagen, Col9-a1, that is deposited in the basal epidermal matrix and promotes Ddr activation at the ventral membrane of migrating TVCs. A functional antagonism between Ddr/Intß1-mediated cell-matrix adhesion and Vegfr signaling appears to modulate the position of cardiopharyngeal progenitors between the endoderm and epidermis. We show that Ddr promotes leader-trailer-polarized BMP-Smad signaling independently of its role in cell-matrix adhesion. We propose that dual functions of Ddr integrate transcriptional inputs to coordinate subcellular processes underlying collective polarity and migration.

FGF and TGFß signaling link form and function during jaw development and evolution.

How does form arise during development and change during evolution? How does form relate to function, and what enables embryonic structures to presage their later use in adults? To address these questions, we leverage the distinct functional morphology of the jaw in duck, chick, and quail. In connection with their specialized mode of feeding, duck develop a secondary cartilage at the tendon insertion of their jaw adductor muscle on the mandible. An equivalent cartilage is absent in chick and quail. We hypothesize that species-specific jaw architecture and mechanical forces promote secondary cartilage in duck through the differential regulation of FGF and TGFß signaling. First, we perform transplants between chick and duck embryos and demonstrate that the ability of neural crest mesenchyme (NCM) to direct the species-specific insertion of muscle and the formation of secondary cartilage depends upon the amount and spatial distribution of NCM-derived connective tissues. Second, we quantify motility and build finite element models of the jaw complex in duck and quail, which reveals a link between species-specific jaw architecture and the predicted mechanical force environment. Third, we investigate the extent to which mechanical load mediates FGF and TGFß signaling in the duck jaw adductor insertion, and discover that both pathways are mechano-responsive and required for secondary cartilage formation. Additionally, we find that FGF and TGFß signaling can also induce secondary cartilage in the absence of mechanical force or in the adductor insertion of quail embryos. Thus, our results provide novel insights on molecular, cellular, and biomechanical mechanisms that couple musculoskeletal form and function during development and evolution.

Surrounding tissues canalize motile cardiopharyngeal progenitors towards collective polarity and directed migration.

Collectively migrating cells maintain group polarity and interpret external cues to reach their destination. The cardiogenic progenitors (also known as trunk ventral cells, TVCs) of the ascidian Ciona intestinalis provide a simple chordate model with which to study collective migration. Bilateral pairs of associated TVCs undergo a stereotyped polarized migration away from the tail towards the ventral trunk, arguably constituting the simplest possible example of directed collective migration. To identify tissues contributing to TVC polarity and migration, we quantified the contact between TVCs and surrounding tissues, and blocked the secretory pathway in a tissue-specific manner. Even though TVCs normally migrate as an invariably determined leader-trailer polarized pair of adherent cells, they are capable of migrating individually, albeit a shorter distance and with altered morphology. The mesenchyme contacts newborn TVCs and contributes to robust specification of the trailer but appears to have only minor effects on directed migration. The notochord does not contact the TVCs but contributes to the onset of migration. The trunk endoderm first contacts the leader TVC, then 'encases' both migrating cells and provides the inputs maintaining leader-trailer polarity. Migrating TVCs adhere to the epidermis and need this contact for their cohesion. These phenomenological studies reveal that inherently motile cardiopharyngeal progenitors are channeled into stereotyped behaviors by interactions with surrounding tissues.

Interleukin-1beta induces increased transcriptional activation of the transforming growth factor-beta-activating integrin subunit beta8 through altering chromatin architecture.

The integrin αvß8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-ß. Through its association with LAP, TGF-ß is maintained in a latent form that must be activated to function. Binding to the integrin αvß8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-ß activation in vivo. Altered expression of the integrin ß8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1ß (IL-1ß) increases ITGB8 expression on lung fibroblasts, which increases αvß8-mediated TGF-ß activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1ß. Our data support a model where the chromatin architecture of the ITGB8 core promoter is altered by nucleosomal repositioning that enhances the interaction of an AP1 complex (containing c-Jun and ATF2). This repositioning is caused by the dissociation of HDAC2 with the ITGB8 core promoter, leading to increased histone H4 acetylation and a loosening of nucleosomal-DNA interactions allowing "opening" of the chromatin structure and increased association of c-Jun and ATF-2. These changes are mediated through NFκB- and p38-dependent pathways. Ultimately, these events culminate in increasing ITGB8 transcription, αvß8 surface expression, and αvß8-mediated TGFß activation.

Lineage analysis of micromere 4d, a super-phylotypic cell for Lophotrochozoa, in the leech Helobdella and the sludgeworm Tubifex.

The super-phylum Lophotrochozoa contains the plurality of extant animal phyla and exhibits a corresponding diversity of adult body plans. Moreover, in contrast to Ecdysozoa and Deuterostomia, most lophotrochozoans exhibit a conserved pattern of stereotyped early divisions called spiral cleavage. In particular, bilateral mesoderm in most lophotrochozoan species arises from the progeny of micromere 4d, which is assumed to be homologous with a similar cell in the embryo of the ancestral lophotrochozoan, more than 650 million years ago. Thus, distinguishing the conserved and diversified features of cell fates in the 4d lineage among modern spiralians is required to understand how lophotrochozoan diversity has evolved by changes in developmental processes. Here we analyze cell fates for the early progeny of the bilateral daughters (M teloblasts) of micromere 4d in the leech Helobdella sp. Austin, a clitellate annelid. We show that the first six progeny of the M teloblasts (em1-em6) contribute five different sets of progeny to non-segmental mesoderm, mainly in the head and in the lining of the digestive tract. The latter feature, associated with cells em1 and em2 in Helobdella, is seen with the M teloblast lineage in a second clitellate species, the sludgeworm Tubifex tubifex and, on the basis of previously published work, in the initial progeny of the M teloblast homologs in molluscan species, suggesting that it may be an ancestral feature of lophotrochozoan development.

High resolution cell lineage tracing reveals developmental variability in leech.

Knowing the normal patterns of embryonic cell proliferation, migration, and differentiation is a cornerstone for understanding development. Yet for most species, the precision with which embryonic cell lineages can be determined is limited by technical considerations (the large numbers of cells, extended developmental times, opacity of the embryos), and these are exacerbated by the inherent variability of the lineages themselves. Here, we present an improved method of cell lineage tracing in the leech Helobdella, driving the expression of a nuclearly localized histone H2B:GFP (green fluorescent protein) fusion protein in selected lineages by microinjection of a plasmid vector. This construct generates a long lasting and minimally mosaic signal with single cell resolution, and does not disrupt the development of most lineages tested. We have validated this technique by elucidating details of cell lineages contributing to segmental and prostomial tissues that could not be observed with standard dextran lineage tracers.

Integrin alpha(v)beta8-mediated activation of transforming growth factor-beta by perivascular astrocytes: an angiogenic control switch.

Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the alpha(v)beta8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the alpha(v)beta8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-beta by astrocytes influences endothelial cell function. Thus, we identified the integrin alpha(v)beta8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic alpha(v)beta8 was a major cell surface receptor for latent TGF-beta, and alpha(v)beta8-dependent activation of TGF-beta was the major mechanism of TGF-beta activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-beta was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic alpha(v)beta8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-beta activation and expression of TGF-beta-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.

A 50-A separation of the integrin alpha v beta 3 extracellular domain C termini reveals an intermediate activation state.

The integrin alpha(v)beta(3) has been shown to exist in low and high affinity conformations. Activation to the high affinity state is thought to depend on the "switchblade-like" opening, from a low affinity bent conformation with a closed headpiece to an extended form of the integrin with an open headpiece. Activation has been shown to depend on separation of the cytoplasmic domains. How cytoplasmic domain separation is related to separation of the transmembrane domains is unknown, and the distance of separation of the transmembrane domains required for activation has not been defined. A constrained secreted form of alpha(v)beta(3) was engineered that introduced a 50-A separation of the integrin C-terminal tails of the extracellular domains of the alpha(v) and beta(3) subunits. Receptor binding and recognition by ligand-induced binding state (LIBS) monoclonal antibodies demonstrated that the mutant receptor was locked into a low affinity state that was likely in a partially extended conformation but with a closed headpiece. In the presence of RGD peptide, the constrained receptor was able to fully extend, as determined by full exposure of LIBS epitopes. In the presence of the appropriate LIBS antibody, high affinity ligand binding of the constrained receptor was achieved. The results support the existence of transient intermediate activation states of secreted alpha(v)beta(3). Furthermore, these results with the secreted alpha(v)beta(3) receptor support a model for the full-length membrane-bound form of alpha(v)beta(3), whereby a 50-A lateral separation of the integrin alpha(v) and beta(3) transmembrane domains would be sufficient to enforce the switchblade-like opening to the extended conformation but insufficient for full receptor activation.