A splice site mutation in the TSEN2 causes a new syndrome with craniofacial and central nervous system malformations, and atypical hemolytic uremic syndrome.

Recessive mutations in the genes encoding the four subunits of the tRNA splicing endonuclease complex (TSEN54, TSEN34, TSEN15, and TSEN2) cause various forms of pontocerebellar hypoplasia, a disorder characterized by hypoplasia of the cerebellum and the pons, microcephaly, dysmorphisms, and other variable clinical features. Here, we report an intronic recessive founder variant in the gene TSEN2 that results in abnormal splicing of the mRNA of this gene, in six individuals from four consanguineous families affected with microcephaly, multiple craniofacial malformations, radiological abnormalities of the central nervous system, and cognitive retardation of variable severity. Remarkably, unlike patients with previously described mutations in the components of the TSEN complex, all the individuals that we report developed atypical hemolytic uremic syndrome (aHUS) with thrombotic microangiopathy, microangiopathic hemolytic anemia, thrombocytopenia, proteinuria, severe hypertension, and end-stage kidney disease (ESKD) early in life. Bulk RNA sequencing of peripheral blood cells of four affected individuals revealed abnormal tRNA transcripts, indicating an alteration of the tRNA biogenesis. Morpholino-mediated skipping of exon 10 of tsen2 in zebrafish produced phenotypes similar to human patients. Thus, we have identified a novel syndrome accompanied by aHUS suggesting the existence of a link between tRNA biology and vascular endothelium homeostasis, which we propose to name with the acronym TRACK syndrome (TSEN2 Related Atypical hemolytic uremic syndrome, Craniofacial malformations, Kidney failure).

A toxic epidermal necrolysis-like presentation of linear IgA bullous dermatosis treated with dapsone.

Linear IgA bullous dermatosis is a rare autoimmune vesiculobullous disease characterized by linear deposition of IgA along the basement membrane zone. It is classically idiopathic, but may also arise secondary to drug exposure. A heterogeneous spectrum of clinical features has been described, including a rare, morbid variant mimicking toxic epidermal necrolysis. Herein, we present a case of vancomycin-induced linear IgA bullous dermatosis that manifested clinically as toxic epidermal necrolysis and resolved with dapsone therapy.

Glutamine and hyperammonemic crises in patients with urea cycle disorders.

UNLABELLED: Blood ammonia and glutamine levels are used as biomarkers of control in patients with urea cycle disorders (UCDs). This study was undertaken to evaluate glutamine variability and utility as a predictor of hyperammonemic crises (HACs) in UCD patients. METHODS: The relationships between glutamine and ammonia levels and the incidence and timing of HACs were evaluated in over 100 adult and pediatric UCD patients who participated in clinical trials of glycerol phenylbutyrate. RESULTS: The median (range) intra-subject 24-hour coefficient of variation for glutamine was 15% (8-29%) as compared with 56% (28%-154%) for ammonia, and the correlation coefficient between glutamine and concurrent ammonia levels varied from 0.17 to 0.29. Patients with baseline (fasting) glutamine values >900 µmol/L had higher baseline ammonia levels (mean [SD]: 39.6 [26.2]µmol/L) than patients with baseline glutamine ≤ 900 µmol/L (26.6 [18.0]µmol/L). Glutamine values >900 µmol/L during the study were associated with an approximately 2-fold higher HAC risk (odds ratio [OR]=1.98; p=0.173). However, glutamine lost predictive significance (OR=1.47; p=0.439) when concomitant ammonia was taken into account, whereas the predictive value of baseline ammonia ≥ 1.0 upper limit of normal (ULN) was highly statistically significant (OR=4.96; p=0.013). There was no significant effect of glutamine >900 µmol/L on time to first HAC crisis (hazard ratio [HR]=1.14; p=0.813), but there was a significant effect of baseline ammonia ≥ 1.0 ULN (HR=4.62; p=0.0011). CONCLUSIONS: The findings in this UCD population suggest that glutamine is a weaker predictor of HACs than ammonia and that the utility of the predictive value of glutamine will need to take into account concurrent ammonia levels.

Epithelial to mesenchymal transition (EMT) seems to be regulated differently in endometriosis and the endometrium.

PURPOSE: Epithelial-mesenchymal transition (EMT) endows cells with migratory and invasive properties, a prerequisite for the establishment of endometriotic lesions. However, the role EMT might play in the pathophysiology of endometriosis is still unknown. Therefore, we examined five recognized markers for EMT in endometrium and endometriosis: E-cadherin, N-cadherin, Twist, Snail and Slug. METHODS: Immunohistochemistry was used for peritoneal, ovarian and rectovaginal endometriotic lesions (n = 27) and endometrium (n = 13). Reverse transcription polymerase chain reaction was applied to tissue samples and primary cell cultures of endometriotic lesions (n = 9) and endometrium (n = 8). RESULTS: In endometriosis and endometrium E-cadherin, N-cadherin, Twist, Snail and Slug were expressed on protein and mRNA level. E-cadherin expression was strong in epithelial cells, but single E-cadherin-negative cells were frequently present in endometriosis. In endometriosis N-cadherin, Twist and Snail expression were upregulated in comparison with endometrium. The expression of E- and N-cadherin was inversely correlated, while that of N-cadherin and Twist was positively correlated. CONCLUSION: This study strongly suggests that EMT may be regulated differently in endometriosis and the endometrium. Future research should further elucidate the regulation of EMT in the endometrium and endometriosis.

Verruciform xanthoma.

A 50-year-old man presented with a several month history of a polypoid papule on the scrotum. A dense accumulation of macrophages with foamy cytoplasm was exhibited in the biopsy specimen leading to a diagnosis of verruciform xanthoma.

Elevated phenylacetic acid levels do not correlate with adverse events in patients with urea cycle disorders or hepatic encephalopathy and can be predicted based on the plasma PAA to PAGN ratio.

BACKGROUND: Phenylacetic acid (PAA) is the active moiety in sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB, HPN-100). Both are approved for treatment of urea cycle disorders (UCDs) - rare genetic disorders characterized by hyperammonemia. PAA is conjugated with glutamine in the liver to form phenylacetyleglutamine (PAGN), which is excreted in urine. PAA plasma levels ≥ 500 µg/dL have been reported to be associated with reversible neurological adverse events (AEs) in cancer patients receiving PAA intravenously. Therefore, we have investigated the relationship between PAA levels and neurological AEs in patients treated with these PAA pro-drugs as well as approaches to identifying patients most likely to experience high PAA levels. METHODS: The relationship between nervous system AEs, PAA levels and the ratio of plasma PAA to PAGN were examined in 4683 blood samples taken serially from: [1] healthy adults [2], UCD patients of ≥ 2 months of age, and [3] patients with cirrhosis and hepatic encephalopathy (HE). The plasma ratio of PAA to PAGN was analyzed with respect to its utility in identifying patients at risk of high PAA values. RESULTS: Only 0.2% (11) of 4683 samples exceeded 500 µg/ml. There was no relationship between neurological AEs and PAA levels in UCD or HE patients, but transient AEs including headache and nausea that correlated with PAA levels were observed in healthy adults. Irrespective of population, a curvilinear relationship was observed between PAA levels and the plasma PAA:PAGN ratio, and a ratio>2.5 (both in µg/mL) in a random blood draw identified patients at risk for PAA levels>500 µg/ml. CONCLUSIONS: The presence of a relationship between PAA levels and reversible AEs in healthy adults but not in UCD or HE patients may reflect intrinsic differences among the populations and/or metabolic adaptation with continued dosing. The plasma PAA:PAGN ratio is a functional measure of the rate of PAA metabolism and represents a useful dosing biomarker.

Urinary phenylacetylglutamine as dosing biomarker for patients with urea cycle disorders.

UNLABELLED: We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). STUDY DESIGN: These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6-17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. RESULTS: Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8 g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r = 0.730, p < 0.001) or as total 24-hour excretion (r = 0.791 p<0.001), followed by plasma phenylacetylglutamine AUC(24-hour), plasma phenylacetic acid AUC(24-hour) and phenylbutyric acid AUC(24-hour). Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. CONCLUSION: The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring.

A TFAP2C Gene Signature Is Predictive of Outcome in HER2-Positive Breast Cancer.

The AP-2γ transcription factor, encoded by the TFAP2C gene, regulates the expression of estrogen receptor-alpha (ERα) and other genes associated with hormone response in luminal breast cancer. Little is known about the role of AP-2γ in other breast cancer subtypes. A subset of HER2+ breast cancers with amplification of the TFAP2C gene locus becomes addicted to AP-2γ. Herein, we sought to define AP-2γ gene targets in HER2+ breast cancer and identify genes accounting for physiologic effects of growth and invasiveness regulated by AP-2γ. Comparing HER2+ cell lines that demonstrated differential response to growth and invasiveness with knockdown of TFAP2C, we identified a set of 68 differentially expressed target genes. CDH5 and CDKN1A were among the genes differentially regulated by AP-2γ and that contributed to growth and invasiveness. Pathway analysis implicated the MAPK13/p38δ and retinoic acid regulatory nodes, which were confirmed to display divergent responses in different HER2+ cancer lines. To confirm the clinical relevance of the genes identified, the AP-2γ gene signature was found to be highly predictive of outcome in patients with HER2+ breast cancer. We conclude that AP-2γ regulates a set of genes in HER2+ breast cancer that drive cancer growth and invasiveness. The AP-2γ gene signature predicts outcome of patients with HER2+ breast cancer and pathway analysis predicts that subsets of patients will respond to drugs that target the MAPK or retinoic acid pathways. IMPLICATIONS: A set of genes regulated by AP-2γ in HER2+ breast cancer that drive proliferation and invasion were identified and provided a gene signature that is predictive of outcome in HER2+ breast cancer.

North-South contraction of the mojave block and strike-slip tectonics in southern california.

The Mojave block of southern California has undergone significant late Cenozoic north-south contraction. This previously unappreciated deformation may account for part of the discrepancy between neotectonic and plate-tectonic estimates of Pacific-North American plate motion, and for part of the Big Bend in the San Andreas fault. In the eastern Mojave block, contraction is superimposed on early Miocene crustal extension. In the western Mojave block, contractional folds and reverse faults have been mistaken for extensional structures. The three-dimensional complexity of the contractional structures may mean that rigid-block tectonic models of the region based primarily on paleomagnetic data are unreliable.

A dual track system to give more-rapid access to new drugs: applying a systems mindset to the US food and drug administration (FDA).

A widely applicable lesson learned from systems analysis is that a proposed change should always be studied in terms of value to the customer and not a gain in efficiency of any particular component of the system. A systems mindset reveals invalid assumptions that have caused the FDA to substitute its own needs for the needs of its customers (patients). Further, the key constraint to overall system improvement is the lack of consumer choice and competition due to FDA's monopoly over access to drugs. Therefore, we need legislation to implement a proposed dual track system for access to drugs that have successfully passed Phase I safety trials. On one track, an experimental drug would continue with conventional FDA clinical trials. On a new, free-to-choose track, patients, advised by their doctors, would make informed decisions about immediate access to not-yet-approved drugs. Internet access to a government-operated tradeoff evaluation database would provide patients and doctors with up-to-date information on all drug treatment outcomes for both tracks. Dual tracking is a dynamic process that overcomes the limitations of a static FDA regulatory process that ignores individual risk preferences.

Estrogen and progestogen receptor positive endometriotic lesions and disseminated cells in pelvic sentinel lymph nodes of patients with deep infiltrating rectovaginal endometriosis: a pilot study.

BACKGROUND: Deep infiltrating endometriosis (DIE) shows similarities to malignant diseases. A recent study involving DIE patients found endometriosis in mesorectal lymph nodes (LNs) after segmental bowel resection. However, it is unclear whether this observation is a local phenomenon or a sign of systemic disease. Therefore, we conducted a prospective study to investigate the occurrence of endometriosis in pelvic sentinel lymph nodes (SLNs) in patients with DIE. METHODS: Fourteen patients underwent primary surgery for symptomatic DIE. Combined vaginal laparoscopic-assisted resection of the rectovaginal septum was performed. Dye was injected into the visible/palpable nodule. SLNs were removed from the iliac region. In order to identify endometriotic cells, immunohistochemical analysis of estrogen and progestogen receptors, CD10 and cytokeratin was performed. RESULTS: In 12 out of 14 patients with DIE, SLNs were detected. The localization of the SLN followed the typical LN spread of the upper vagina. In three patients, we could detect typical endometriotic lesions in the LNs. Ten out of 12 (83.3%) SLNs showed disseminated estrogen and/or progestogen positive cells. CONCLUSIONS: By using immunohistochemistry, we could demonstrate endometriotic lesions and endometriotic-like cells in pelvic SLNs of patients with DIE suggesting the potential for lymphatic spread of the disease.

A neocentromere derived from a supernumerary marker deleted from the long arm of chromosome 6.

Parental chromosome studies were referred to us after initial finding of a balanced translocation involving chromosomes 4 and 15 in their phenotypically abnormal male child (cytogenetic analysis was done at another laboratory). In addition to the same 4;15 translocation, the father also had an interstitial deletion of the long arm of one chromosome 6 and a marker chromosome. In this article, we report a neocentromere on this marker, which was determined to be composed of chromosome 6 material by FISH. The child's karyotype was re-interpreted to be unbalanced due to the presence of the abnormal chromosome 6, but without the marker. The clinical phenotype associated with the interstitial deletion of chromosome 6 is also reported.

Multiple molecular forms of glucose-6-phosphate dehydrogenase in normal, preneoplastic, and neoplastic mammary tissues of mice.

Multiple molecular forms of glucose-6-phosphate dehydrogenase (G6PD) in normal, preneoplastic, and neoplastic mammary tissues were separated by polyacrylamide gel electrophoresis and identified by specific straining for enzyme activity. Mammary tissue from lactating BALB/c mice showed considerable amounts (up to 50%) of a slower-migrating G6PD species, G6PD-III, which was essentially absent from glands of pregnant mice, preneoplastic nodules, and mammary carcinomas. All tissues possessed a faster-migrating species, G6PD-II, which accounted for up to 85% of the total G6PD in the glands of pregnant mice. A third species, G6PD-I, migrating more rapidly than G6PD-II, was found in both abnormal tissues (preneoplastic and neoplastic) and accounted for up to 35% of the total enzymatic activity. G6PD-I was present in moderate amounts (less than 15%) in glands from pregnant mice and was essentially absent from the lactating gland (approximately 5%). The addition of dithiothreitol did not alter the measurable G6PD activity but did increase the relative activity of G6PD-II or G6PD-I, as judged by the intensity of the bands on the gels. Mild oxidation (stirring overnight at 4 degrees in air) resulted in a loss of G6PD activity, but preparations had greater amounts of G6PD-III; presence of dithiothreitol during aeration partially prevented loss of G6PD activity and largely prvented the appearance of G6PD-III. Molecular-weight estimations with preparations from lactating mice yielded a value of 118,000 for G6PD-II and 260,000 for G6PD-III, suggesting a monomer and dimer, respectively. The addition of nicotinamide adenine dinucleotide phosphate stabilized G6PD activity by preventing heat inactivation at 47 degrees; nicotinamide adenine dinucleotide phosphate did not alter the pattern of species present. The data from heat inactivation studies suggest that G6PD-III (dimer) was the more stable species. The addition of nicotinamide adenine dinucleotide phosphate to samples after oxidation in the absence of dithiothreitol (about 70% loss of activity) resulted in no change in patterns and in recovery of full G6PD activity during heating at 47 degrees. A potential relationship between glutathione reductase activity and the pattern of G6PD species observed in the various tissues is noted.

Identity, release, and binding of mitochondrial-bound hexokinases in mammary glands and adenocarcinomas of lactating mice.

The mitochondrial-bound hexokinases (adenosine triphosphate:D-hexose 6-phosphotransferase) of mammary adenocarcinoma and of normal gland were compared in lactating C3H mice. Treatment of mitochondria isolated from both the normal and neoplastic tissue with 0.5 m NaCl or 0.1 mM glucose 0-phosphate effected the release of about 50% of the bound hexokinase. In the presence of magnesium ion, enzyme from either source attached to mitochondria from either tissue and in all combinations to the same extent. Identification of the isoenzyme complement in the mitochondrial extract by diethylaminoethylcellulose chromatography revealed only types I and II. In the tumor, the hexokinase activity in both the cytosol and the fraction solubilized from mitochondria was predominantly in the form of type I ( 60%). In contrast, the activity released from mitochondria isolated from normal gland was predominately type II, while the cytosol contained almost equivalent amounts of types I and II. While this difference does not explain differences in glucose utilization between the normal and neoplastic tissue, it may provide a means of distinguishing between the two.

In vitro transformation of mouse mammary epithelial cells grown serum-free inside collagen gels.

Mammary epithelial cells from 4-month-old virgin BALB/c mice were cultured inside collagen gels in the following serum-free media: Dulbecco's modified Eagle's medium:Hams's F-12 (1:1) supplemented with: insulin (10 micrograms/ml), bovine serum albumin (5 mg/ml), and epidermal growth factor (5 ng/ml); insulin, bovine serum albumin, progesterone (0.05 microgram/ml), and prolactin (1 microgram/ml); insulin, bovine serum albumin, progesterone, prolactin, and linoleic acid (10 micrograms/ml). Cells proliferated in all these media. The cells were treated with 0.01 micrograms/ml of 7,12-dimethylbenz(a)anthracene or 100 micrograms/ml of N-nitroso-N-methylurea on day 3 of culture and, subsequently, at 1-week intervals for 3-6 weeks. Tetradecanoylphorbol acetate (0.1 micrograms/ml) was added to selected cultures. The cultures were maintained for up to 9 weeks; the cells were then removed from the collagen gels, placed in monolayer culture for 2 days, and removed from monolayer culture, and 5 X 10(5) cells were transplanted to each of the gland-free mammary fat pads of 3-week-old female mice. Approximately 10 weeks after transplantation, the transplanted mammary fat pads were examined for outgrowths. Cells that were not treated with carcinogen and cultured for up to 9 weeks in different serum-free media and transplanted to the gland-free mammary fat pad produced only ductal outgrowths similar in morphology to the ducts of the virgin host's mammary glands. Six treatments with 7,12-dimethylbenz(a)anthracene, of cells grown in the presence of epidermal growth factor, induced 31% spindle cell tumors, 17% ductal hyperplasias, and 5% lobuloalveolar hyperplasias. Cells that were grown in epidermal growth factor and treated three times with N-nitroso-N-methylurea produced 23% ductal hyperplasias and 17% lobuloalveolar hyperplasias. Cells grown in the presence of progesterone and prolactin and treated three times with 7,12-dimethylbenz(a)anthracene produced up to 23% lobuloalveolar hyperplasias and 12% ductal hyperplasias. Three treatments with N-nitroso-N-methylurea of cells grown in progesterone- and prolactin-containing media produced a maximum of 50% lobuloalveolar hyperplasias and 33% ductal hyperplasias. The lobuloalveolar hyperplasias have the characteristics of the precancerous hyperplastic alveolar nodules found in mouse mammary tumorigenesis. The in vitro carcinogen-induced lobuloalveolar hyperplasias were transplantable, maintained their lobuloalveolar morphology in virgin hosts, and produced carcinomas.

A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior.

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. In vitro models that mimic in vivo tumor neovascularization facilitate exploration of this role. Here we investigated lung adenocarcinoma cancer cells (344SQ) and endothelial and pericyte vascular cells encapsulated in cell-adhesive, proteolytically-degradable poly(ethylene) glycol-based hydrogels. 344SQ in hydrogels formed spheroids and secreted proangiogenic growth factors that significantly increased with exposure to transforming growth factor beta 1 (TGF-ß1), a potent tumor progression-promoting factor. Vascular cells in hydrogels formed tubule networks with localized activated TGF-ß1. To study cancer cell-vascular cell interactions, we engineered a 2-layer hydrogel with 344SQ and vascular cell layers. Large, invasive 344SQ clusters (area > 5,000 µm(2), circularity < 0.25) developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed cluster morphological changes required close proximity to vascular cells. Additionally, TGF-ß1 inhibition blocked endothelial cell-driven 344SQ migration. Our findings suggest vascular cells contribute to tumor progression and establish this culture system as a platform for studying tumor vascularization.

Cancer-Associated Fibroblasts Induce a Collagen Cross-link Switch in Tumor Stroma.

UNLABELLED: Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used Kras(LA1) mice, which develop lung adenocarcinomas from somatic activation of a Kras(G12D) allele. The lung tumors in Kras(LA1) mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCC) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. IMPLICATIONS: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells.

Serine isotopmer analysis by 13C-NMR defines glycine-serine interconversion in situ in the renal proximal tubule.

[2-(13)C]glycine metabolism was studied in freshly isolated rat renal proximal tubules. Mitochondrial coupling of the glycine cleavage complex (GC) and serine hydroxymethyltransferase (SHMT) was confirmed by the formation of three serine isotopomers, [2-(13)C]-, [3-(13)C]- and [2,3-(13)C]serine, detected by 13C-NMR. Incubation with different fractions of 13C-labelled glycine altered the labelling pattern of the serine isotopomers predictably and allowed calculation of the 13C-labelled fractions of total glycine and methylene in N5,N10-methylenetetrahydrofolate (m-THF) available for serine metabolism. Within 20 min there was a fall in labelled glycine (to 42 +/- 3, 68 +/- 3 and 93 +/- 2%, (n = 4, mean +/- S.D.) from 50%, 75% and 100% 13C-labelled added glycine respectively), followed by a slow rate of endogenous glycine formation for up to 80 min incubation. The C2 of glycine was the source of more than 90% of the methylene group of m-THF formed. Gas chromatography-mass spectroscopy (GC-MS) showed that greater than 50% of serine formed was unlabelled. GC and SHMT proceeded in the direction of serine formation. Serine isotopomer analysis by NMR and GC-MS allowed the actions of GC and SHMT and de novo contributions to glycine, serine and m-THF to be monitored in situ in fresh renal proximal tubules.

A clinical trial for the Food and Drug Administration's clinical trial process.

The U.S. Food and Drug Administration (FDA) is tasked with opposing goals which demand tradeoffs--the time and cost of clinical trials to assure safety and efficacy while also encouraging speedy drug advances and affordability. On top of that, there is no feedback mechanism to inform the FDA of the effectiveness of their tradeoff decisions. Dual Tracking is a proposed public policy field experiment that would provide needed feedback information. The proposal is rooted in the right of informed patients to choose among FDA-approved or new, experimental drugs still in clinical trials. All new drugs would continue along the FDA's clinical testing track. On a new track independent of the FDA (but only after successful FDA Phase I safety evaluations), drug development firms would have the option to legally contract with individual patients and their doctors to sell them a not-yet-FDA-approved drug. The contract would require ongoing Internet reporting in a specified format of all drug-related experiences. The results of the Dual Tracking process compared to the FDA's process would constitute the critical information now missing.