Suppression of Bile Acid Synthesis in a Preterm Infant Receiving Prolonged Parenteral Nutrition.

Bile acid metabolism is altered in neonates on parenteral nutrition (PN), predisposing them to parenteral nutrition-associated liver disease. Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the bile acid synthesis pathway, is repressed by fibroblast growth factor 19 (FGF19) and phytosterols (PS). We describe a case of a preterm infant who developed necrotizing enterocolitis (NEC) and received exclusive PN for over 2 months. Our objective was to serially assess CYP7A1 activity and plasma FGF19 and PS concentrations in this infant case compared to five healthy preterm infants. We found that CYP7A1 activity increased during the first 2 weeks of life in control infants but was undetectable in the infant case. FGF19 concentrations were high at birth in all infants and subsequently declined and did not differ between the case and control infants. As expected, PS concentrations were elevated in the infant case and continued to increase despite lipid minimization. In conclusion, CYP7A1 activity was gradually upregulated in healthy preterm infants but remained suppressed in the infant requiring prolonged PN. Preterm infants also had elevated FGF19 concentrations at birth, which decreased with advancing postnatal age.

Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection and vaccination.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and COVID-19 vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Social and legal implications of urine drug screen analysis in the neonate: A case of suspected specimen mishandling.

BACKGROUND: The legal implications associated with illicit drug use during pregnancy are significant, as providers are required to notify child protective services when a drug-exposed infant is identified. CASE REPORT: The case presented involves possible specimen mishandling in two infants at risk for in utero drug exposure and describes alternative methodologies available to confirm specimen identity. CONCLUSIONS: It is critical that institutions establish and adhere to stringent procedures when screening newborns.

Developmental regulation of the gut-liver (FGF19-CYP7A1) axis in neonates.

Introduction: Fibroblast growth factor 19 (FGF19) is a gut-derived hormone that regulates the expression of CYP7A1, the rate-limiting enzyme in bile acid (BA) synthesis pathway. Dysregulation of the FGF19-CYP7A1 (gut-liver) axis is associated with cholestatic liver disease. Infants, especially preterm infants and those with intestinal failure are at high risk for developing cholestatic liver disease. The activity of the gut-liver axis has not been characterized in this population. Our objective was to assess relationships between circulating FGF19 concentrations and CYP7A1 activity in neonates.Materials and methods: Plasma samples were obtained longitudinally from term and preterm infants (22-41-week gestation) hospitalized in a neonatal intensive care unit. Infants with congenital and acquired gastrointestinal disorders were excluded. Plasma levels of 7α-hydroxy-4-cholesten-3-one (C4), a marker of CYP7A1 activity, were quantified using HPLC-MS/MS. Plasma FGF19 concentrations were quantified by ELISA. Data were analyzed using linear regression models and structural equation modeling.Results: One hundred eighty-one plasma samples were analyzed from 62 infants. C4 concentrations were undetectable prior to 30 weeks' gestation and, thereafter, increased with advancing gestational age and with volume of enteral feeds. They did not correlate with serum FGF19 concentrations, which decreased with advancing gestational age and volume of enteral feeds.Discussion: The activity of CYP7A1, the rate-limiting BA synthetic enzyme in adults, is developmentally regulated and undetectable in newborns less than 30 weeks' gestation. FGF19 concentrations do not correlate with CYP7A1 activity, suggesting that the gut-liver axis is not functional in infants. High FGF19 concentrations at birth in infants less than 37 weeks' gestation is a novel finding, and its source and role in preterm infants warrants further investigation.Rationale: The intestinal hormone, fibroblast growth factor 19 (FGF19), is a major regulator of CYP7A1, the rate limiting enzyme in bile acid (BA) synthesis. Recently, dysregulation of the gut-liver (FGF19-CYP7A1) axis has been implicated in adult cholestatic liver disease, and animal studies have shown that exogenous FGF19 protects against liver injury. Given the therapeutic potential related to this signaling pathway, we sought to characterize the association between CYP7A1 and FGF19 in term and preterm infants. We conducted a prospective, observational study that measured in vivo CYP7A1 activity and FGF19 concentrations in 62 term and preterm infants (n = 181 samples). We found that CYP7A1 activity is developmentally regulated; its activity is undetectable prior to 30 weeks' gestation and increases with advancing gestational age and volume of enteral feeds. Contrary to expectation, we demonstrated that FGF19 is expressed at birth in preterm infants and decreases over time, even as enteral feeds increase. Using structural equation modeling, we were able to show that CYP7A1 activity does not correlate with FGF19 concentrations. Our results suggest that the gut-liver axis is not upregulated in preterm and term infants and that neonates with cholestatic liver disease will unlikely benefit from supplemental FGF19. We also report novel findings of elevated FGF19 concentrations in preterm infants at birth and speculate that there may be an extra-intestinal source of FGF19 that is developmentally expressed in these infants.

Heterophile antibody interference affecting multiple Roche immunoassays: A case study.

BACKGROUND: Analysis of many clinically important analytes is dependent on antibody-based assays. However, depending on the design, these assays are vulnerable to interference from endogenous molecules including circulating antibodies and free biotin. In this case report, we describe a patient whose laboratory findings from immunoassay based methodologies, are inconsistent with the clinical presentation. CASE PRESENTATION: A 14-year-old male was referred to Pediatric Endocrinology for suspected hyperthyroidism based on critically elevated free thyroxine (fT4) levels although clinical assessment was inconsistent with hyperthyroidism. Because repeat testing was discrepant, Endocrinology questioned the validity of the results prompting consultation with the laboratory to investigate the source of the inconsistent findings. Review of discordant results revealed that fT4 levels measured in laboratories utilizing Roche instrumentation were critically high, while results from laboratories using alternative platforms (i.e. Siemens Centaur) were within normal limits. CONCLUSION: After a comprehensive evaluation which included testing of paired specimens on multiple platforms, measurement of serially diluted specimens and a formal evaluation for the presence of heterophile antibodies, it was determined that a heterophile antibody interference was the most likely cause of the aberrant results in this patient.

GLUT3 gene expression is critical for embryonic growth, brain development and survival.

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly.

Slc2a8 deficiency in mice results in reproductive and growth impairments.

SLC2A8, also known as GLUT8, is a facilitative glucose transporter expressed in the testis, brain, liver, heart, uterus, ovary, and fat. In this study we examined the effect of Slc2a8 deficiency on mouse gamete, preimplantation embryo, and implantation phenotype, as well as postnatal growth and physiology. For this model, the transcriptional start site and exons 1-4 were targeted and a lack of protein expression was confirmed by Western immunoblot. Oocytes obtained from Slc2a8(-/-) mice demonstrated abnormal metabolism and ATP production. In addition, deletion of Slc2a8 resulted in impaired decidualization, a critical step in the differentiation of endometrial stromal cells (ESCs), necessary for implantation. This indicates a role for SLC2A8 in decidualization, which is supported by Slc2a8 mRNA expression in both mouse and human ESCs, which increases dramatically in response to hormonal changes occurring during the process of implantation. Ovarian transplantation studies confirm that lack of SLC2A8 affects both the embryo and the implantation processes. This phenotype leads to decreased litter size, and smaller pups at weaning that continue to display an abnormally small growth phenotype into adulthood. The Slc2a8 null mice display decreased body fat by magnetic resonance imaging, and, interestingly, they are resistant to a diet high in fat and carbohydrates.

Akt2 modulates glucose availability and downstream apoptotic pathways during development.

Glucose is the primary energy substrate for eukaryotic cells and the predominant substrate for the brain. Studies suggest that glucose serves an additional role in the regulation of cellular functions, including viability. Zebrafish is a tractable system for defining the cellular and molecular mechanisms perturbed by impaired glucose transport and metabolism. Previously, we demonstrated a critical role for the facilitative glucose transporter, Glut1, in the regulation of embryonic brain development. In this study, we aim to identify mediators in this Glut1-sensitive process by investigating the role of the antiapoptotic kinase, Akt2. Results show that abrogating expression of akt2 causes a phenotype strikingly similar to that observed when glut1 expression is inhibited. akt2-deficient embryos exhibit increased neuronal apoptosis, impaired glucose uptake, and death by 72 h postfertilization. Similar to what was observed in the glut1 morphants, inhibiting the expression of the proapoptotic protein, bad, in the context of impaired akt2 expression results in the inhibition of apoptosis and rescue of the morphant embryos. Intriguingly, overexpression of glut1 in the akt2 morphants was also able to rescue these embryos. Quantitative reverse transcription-PCR analysis revealed decreased glut1 transcript expression in akt2 morphant embryos. Taken together, these data suggest that Akt2 modulates glucose availability by regulating Glut1 expression at the transcript level. These data support a role for akt2 in an integrative pathway directly linking glucose, Glut1 expression, and activation of apoptosis and demonstrate the dependence of akt2 on glucose availability for the maintenance of cellular viability, particularly in the central nervous system.

Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection.

Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20 min. The dynamic range of each amino acid was generally 1-1000 micromol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88-125%. Deming regression with the Beckman 7300 yielded slopes from 0.4-1.2, intercepts from -21 to 65 micromol/L, correlation coefficients from 0.84-0.99 and Syx from 2-125 micromol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, beta-alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors.

GLUT1 deficiency links nutrient availability and apoptosis during embryonic development.

GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.

Phosphatidylinositol 3-kinase activity is critical for glucose metabolism and embryo survival in murine blastocysts.

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is a well known mediator of cell growth, proliferation, and survival signals. Whereas the expression and function of this pathway has been documented during mammalian development, evidence demonstrating the physiologic importance of this pathway in murine preimplantation embryos is beginning to emerge. This study demonstrates that inhibition of the PI3K pathway leads to the induction of apoptosis in both murine blastocysts and trophoblast stem cells. The apoptosis induced in both model systems correlates with a decrease in the expression of the glucose transporter GLUT1 at the plasma membrane. In addition, blastocysts cultured in the presence of the PI3K inhibitor LY-294002 display a decrease in both 2-deoxyglucose uptake and hexokinase activity as compared with control blastocysts. To determine the impact of PI3K inhibition on pregnancy outcome, embryo transfer experiments were performed. Blastocysts cultured in the presence of LY-294002 demonstrate a dramatic increase in fetal resorptions as compared with control embryos. Finally, we demonstrate that impairment of glucose metabolism via iodoacetate, a glyceraldehyde-3-phosphate dehydrogenase inhibitor, is sufficient to induce apoptosis in both blastocysts and trophoblast stem cells. Moreover, blastocysts treated with iodoacetate result in poor pregnancy outcome as determined by embryo transfer experiments. Taken together these data demonstrate the critical importance of the PI3K pathway in preimplantation embryo survival and pregnancy outcome and further emphasize the importance of glucose utilization and metabolism in cell survival pathways.

Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes.

A novel glucose transporter (GLUT), mouse GLUT9 (mGLUT9), was recently cloned from mouse 7-d embryonic cDNA. Several splice variants of mGLUT9 were described, two of which were cloned (mGLUT9a and mGLUT9a Delta 209-316). This study describes the cloning and characterization of another splice variant, mGLUT9b. Cloned from adult liver, mGLUT9b is identical to mGLUT9a except at the amino terminus. Based on analysis of the genomic structure, the different amino termini result from alternative transcriptional/translational start sites. Expression and localization of these two mGLUT9 splice variants were examined in control and diabetic adult mouse tissues and in cell lines. RT-PCR analysis demonstrated expression of mGLUT9a in several tissues whereas mGLUT9b was observed primarily in liver and kidney. Using a mGLUT9-specific antibody, Western blot analysis of total membrane fractions from liver and kidney detected a single, wide band, migrating at approximately 55 kDa. This band shifted to a lower molecular mass when deglycosylated with peptide-N-glycosidase F. Both forms were present in liver and kidney. Immunohistochemical localization demonstrated basolateral distribution of mGLUT9 in liver hepatocytes and the expression of mGLUT9 in specific tubules in the outer cortex of the kidney. To investigate the alternative amino termini, mGLUT9a and mGLUT9b were overexpressed in kidney epithelium cell lines. Subcellular fractions localized both forms to the plasma membrane. Immunofluorescent staining of polarized Madin Darby canine kidney cells overexpressing mGLUT9 depicted a basolateral distribution for both splice variants. Finally, mGLUT9 protein expression was significantly increased in the kidney and liver from streptozotocin-induced diabetic mice compared with nondiabetic animals.

The PI3K/Akt pathway is present and functional in the preimplantation mouse embryo.

The PI3K/Akt signal transduction pathway is a well-known mediator of growth promoting and cell survival signals. While the expression and function of this pathway have been documented during early and late stages of the reproductive process, currently, there is no evidence demonstrating either the presence or function of the PI3K/Akt pathway in murine preimplantation embryos. We found, using confocal immunofluorescent microscopy and Western blot analysis, that the p 85 and p110 subunits of PI3K and Akt are expressed from the 1-cell through the blastocyst stage of murine preimplantation embryo development. These proteins were localized predominantly at the cell surface from the 1-cell through the morula stage. At a blastocyst stage, both PI3K and Akt exhibited an apical staining pattern in the trophectoderm cells. Interestingly, phosphorylated Akt was detected throughout murine preimplantation development, and its presence at the plasma membrane is a reflection of its activation status. Inhibition of Akt activity had significant effects on the normal physiology of the blastocyst. Specifically, inhibition of this pathway resulted in a reduction in insulin-stimulated glucose uptake. In addition, inhibiting Akt activity resulted in a significant delay in blastocyst hatching, a developmental step facilitating implantation. Finally, we established the presence of this pathway in trophoblast stem (TS) cells, a potentially useful in vitro model to study this signaling cascade. Taken together, these data are the first to demonstrate the presence and function of the PI3K/Akt pathway in mammalian preimplantation embryos.

Identification and characterization of human glucose transporter-like protein-9 (GLUT9): alternative splicing alters trafficking.

The recently cloned human GLUT9 gene, which maps to chromosome 4p15.3-p16, consists of 12 exons coding for a 540-amino acid protein. Based on a sequence entry (NCBI accession number BC018897) and screening of expressed sequence tags, we have cloned an alternative splice variant of GLUT9 from human kidney cDNA. The RNA of this splice variant consists of 13 exons and codes for a putative protein of 512 amino acids (GLUT9DeltaN). The predicted proteins differ only in their N terminus, suggesting a different subcellular localization and possible physiological role. Screening human tissue RNA by reverse transcription-PCR showed that GLUT9 is expressed mainly in kidney, liver, placenta, and leukocytes, whereas GLUT9DeltaN was detected only in kidney and placenta. The GLUT9 protein localized by immunohistochemistry to human kidney proximal tubules, and subcellular fractionation of human kidney revealed the GLUT9 protein in plasma membranes and high density microsomal membranes. Treatment of kidney membrane proteins with peptide N-glycosidase F showed that GLUT9 and GLUT9DeltaN are expressed in vivo. Localization of GLUT9 and GLUT9DeltaN in three kidney-derived cell lines revealed a plasma membrane distribution for GLUT9 in COS-7 and HEK293 cells, whereas GLUT9DeltaN showed a perinuclear pattern and plasma membrane staining in COS-7 and HEK293 cells, respectively. In polarized Madin-Darby canine kidney cells, GLUT9 trafficked to the basolateral membrane, whereas GLUT9DeltaN localized to the apical membrane. Using heterologous expression of GLUT9 in Xenopus oocytes, GLUT9 appears to be a functional isoform with low affinity for deoxyglucose. Deoxyglucose transport mediated by GLUT9 was not inhibited by cytochalasin B. GLUT9 did not bind cytochalasin B as shown by a cytochalasin B binding assay, indicating a similar behavior of GLUT9 compared with GLUT5.

GLUT9 is differentially expressed and targeted in the preimplantation embryo.

During preimplantation development in the mouse, it is crucial that glucose metabolism not be compromised. Any decrease in glucose uptake at this stage in development can compromise the developing embryo. We have cloned another member of the glucose transporter family, GLUT9, which is expressed embryonically. Three different isoforms were identified. We have shown that two of the mouse GLUT9 isoforms transport glucose at a rate significantly greater than controls. Expression analysis of the preimplantation blastocyst identifies only the presence of the shorter GLUT9 isoform, RT-PCR and Western immunoblot confirmed this finding. A differential pattern of expression was seen with GLUT9 present at the plasma membrane in one- and two-cell zygotes and in an intracellular compartment in trophectoderm cells at a blastocyst stage. Although blocking GLUT9 expression during preimplantation development had no effect on glucose transport or apoptosis, transfer of these embryos into pseudopregnant mice resulted in increased pregnancy loss, suggesting that GLUT9 is critical for early preimplantation development.

Syntaxin 4 expression affects glucose transporter 8 translocation and embryo survival.

Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.