Gene identification for the cblD defect of vitamin B12 metabolism

Background Vitamin B12 (cobalamin) is an essential cofactor in several metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. One of these defects, the cblD defect, can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Affected persons present with multisystem clinical abnormalities, including developmental, hematologic, neurologic, and metabolic findings. The gene responsible for the cblD defect has not been identified. Methods We studied seven patients with the cblD defect, and skin fibroblasts from each were investigated in cell culture. Microcell-mediated chromosome transfer and refined genetic mapping were used to localize the responsible gene. This gene was transfected into cblD fibroblasts to test for the rescue of adenosylcobalamin and methylcobalamin synthesis. Results The cblD gene was localized to human chromosome 2q23.2, and a candidate gene, designated MMADHC (methylmalonic aciduria, cblD type, and homocystinuria), was identified in this region. Transfection of wild-type MMADHC rescued the cellular phenotype, and the functional importance of mutant alleles was shown by means of transfection with mutant constructs. The predicted MMADHC protein has sequence homology with a bacterial ATP-binding cassette transporter and contains a putative cobalamin binding motif and a putative mitochondrial targeting sequence. Conclusions Mutations in a gene we designated MMADHC are responsible for the cblD defect in vitamin B12 metabolism. Various mutations are associated with each of the three biochemical phenotypes of the disorder

The Implementation, Interpretation, and Justification of Likelihoods in Cosmology

I discuss the formal implementation, interpretation, and justification of likelihood attributions in cosmology. I show that likelihood arguments in cosmology suffer from significant conceptual and formal problems that undermine their applicability in this context

Antivitamin B12 Inhibition of the Human B12‐Processing Enzyme CblC: Crystal Structure of an Inactive Ternary Complex with Glutathione as the Cosubstrate

B12 antivitamins are important and robust tools for investigating the biological roles of vitamin B12. Here, the potential antivitamin B12 2,4‐difluorophenylethynylcobalamin (F2PhEtyCbl) was prepared, and its 3D structure was studied in solution and in the crystal. Chemically inert F2PhEtyCbl resisted thermolysis of its Co−C bond at 100 °C, was stable in bright daylight, and also remained intact upon prolonged storage in aqueous solution at room temperature. It binds to the human B12‐processing enzyme CblC with high affinity (KD=130 nm) in the presence of the cosubstrate glutathione (GSH). F2PhEtyCbl withstood tailoring by CblC, and it also stabilized the ternary complex with GSH. The crystal structure of this inactivated assembly provides first insight into the binding interactions between an antivitamin B12 and CblC, as well as into the organization of GSH and a base‐off cobalamin in the active site of this enzyme.Antivitamins in action: A new, chemically robust antivitamin B12 was used for biochemical analysis of the inhibition of CblC, the key B12‐processing enzyme of humans. The crystal structure of the inactive enzyme complex provides detailed insight into CblC loaded with a cobalamin and its cosubstrate glutathione.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137373/1/anie201701583.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137373/2/anie201701583_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137373/3/anie201701583-sup-0001-misc_information.pd

Tumor BRCA Testing in High Grade Serous Carcinoma: Mutation Rates and Optimal Tissue Requirements

Background: Approximately 25% of women diagnosed with tubo-ovarian high-grade serous carcinoma have germline deleterious mutations in BRCA1 or BRCA2, characteristic of hereditary breast and ovarian cancer syndrome, while somatic mutations have been detected in 3–7%. We set out to determine the BRCA mutation rates and optimal tissue requirements for tumor BRCA testing in patients diagnosed with tubo-ovarian high-grade serous carcinoma. Methods: Sequencing was performed using a multiplexed polymerase chain reaction-based approach on 291 tissue samples, with a minimum sequencing depth of 500X and an allele frequency of >5%. Results: There were 253 surgical samples (87%), 35 biopsies (12%) and 3 cytology cell blocks (1%). The initial failure rate was 9% (25/291), including 9 cases (3%) with insufficient tumor, and 16 (6%) with non-amplifiable DNA. Sequencing was successful in 78% (228/291) and deemed indeterminate due to failed exons or variants below the limit of detection in 13% (38/291). Repeat testing was successful in 67% (28/42) of retested samples, with an overall success rate of 86% (251/291). Clinically significant (pathogenic, likely pathogenic) variants were identified in 17% (48/276) of complete and indeterminate cases. Successful sequencing was dependent on sample type, tumor cellularity and size (p ≤ 0.001) but not on neoadjuvant chemotherapy or age of blocks (p > 0.05). Conclusions: Our study shows a 17% tumor BRCA mutation rate, with an overall success rate of 86%. Biopsy and cytology samples and post-chemotherapy specimens can be used for tumor BRCA testing, and optimal tumors measure ≥5 mm in size with at least 20% cellularity

Inhibierung des humanen B12‐verarbeitenden Enzyms CblC durch Antivitamine B12 – Kristallstruktur des inaktiven ternären Komplexes mit dem Kosubstrat Glutathion

Antivitamine B12 gewinnen als robuste B12‐Dummys zunehmend biomedizinisches Interesse. Das potenzielle Antivitamin B12 2,4‐Difluorphenylethinylcobalamin (F2PhEtyCbl) wurde hergestellt, und seine 3D‐Struktur in Lösung und im Kristall wurde untersucht. Das chemisch inerte F2PhEtyCbl zeigte sich gegen Thermolyse seiner Co‐C‐Bindung bei 100 °C resistent, war stabil bei Bestrahlung mit (hellem) Tageslicht und blieb auch bei längerer Aufbewahrung in wässriger Lösung bei Raumtemperatur intakt. Es wurde vom humanen B12‐verarbeitenden Enzym CblC in Gegenwart des Kosubstrats Glutathion (GSH) mit hoher Affinität (KD=130 nm) gebunden. F2PhEtyCbl stabilisierte den ternären Komplex von CblC mit GSH und widerstand der Verarbeitung. Die Kristallstruktur dieses Komplexes lieferte erste Einblicke in die Wechselwirkungen eines Antivitamins B12 mit CblC sowie in die Anordnung von GSH und Base‐off‐Cobalamin im aktiven Zentrum dieses blockierten Enzyms.Antivitamin in Aktion: Antivitamine B12 sind vom Vitamin abstammende „B12‐Dummys”. Ein neues, robustes Antivitamin B12 wurde bei der Inhibierung von CblC biochemisch analysiert, dem humanen B12‐verarbeitenden Schlüsselenzym. Die Kristallstruktur des inaktivierten Enzym‐Komplexes ermöglichte detaillierte Einblicke in das mit Cobalamin und dem Kosubstrat Glutathion vollbeladene CblC.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138308/1/ange201701583_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138308/2/ange201701583-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138308/3/ange201701583.pd

Reconstruction of a Nonminimal Coupling Theory with Scale-invariant Power Spectrum

A nonminimal coupling single scalar field theory, when transformed from Jordan frame to Einstein frame, can act like a minimal coupling one. Making use of this property, we investigate how a nonminimal coupling theory with scale-invariant power spectrum could be reconstructed from its minimal coupling counterpart, which can be applied in the early universe. Thanks to the coupling to gravity, the equation of state of our universe for a scale-invariant power spectrum can be relaxed, and the relation between the parameters in the action can be obtained. This approach also provides a means to address the Big-Bang puzzles and anisotropy problem in the nonminimal coupling model within Jordan frame. Due to the equivalence between the two frames, one may be able to find models that are free of the horizon, flatness, singularity as well as anisotropy problems.Comment: 31 pages, 4 figure

Vacuum Stability, Perturbativity, and Scalar Singlet Dark Matter

We analyze the one-loop vacuum stability and perturbativity bounds on a singlet extension of the Standard Model (SM) scalar sector containing a scalar dark matter candidate. We show that the presence of the singlet-doublet quartic interaction relaxes the vacuum stability lower bound on the SM Higgs mass as a function of the cutoff and lowers the corresponding upper bound based on perturbativity considerations. We also find that vacuum stability requirements may place a lower bound on the singlet dark matter mass for given singlet quartic self coupling, leading to restrictions on the parameter space consistent with the observed relic density. We argue that discovery of a light singlet scalar dark matter particle could provide indirect information on the singlet quartic self-coupling.Comment: 25 pages, 10 figures; v2 - fixed minor typos; v3 - added to text discussions of other references, changed coloring of figures for easier black and white viewin

Arcobacter Species in Humans1

During an 8-year study period, Arcobacter butzleri was the fourth most common Campylobacter-like organism isolated from 67,599 stool specimens. Our observations suggest that A. butzleri displays microbiologic and clinical features similar to those of Campylobacter jejuni; however, A. butzleri is more frequently associated with a persistent, watery diarrhea

Access to Organometallic Arylcobaltcorrins through Radical Synthesis: 4‐Ethylphenylcobalamin, a Potential “Antivitamin B 12 ”

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96761/1/anie_201209651_sm_miscellaneous_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96761/2/2606_ftp.pd

The Secret to Successful User Communities: An Analysis of Computer Associates’ User Groups

This paper provides the first large scale study that examines the impact of both individual- and group-specific factors on the benefits users obtain from their user communities. By empirically analysing 924 survey responses from individuals in 161 Computer Associates' user groups, this paper aims to identify the determinants of successful user communities. To measure success, the amount of time individual members save through having access to their user networks is used. As firms can significantly profit from successful user communities, this study proposes four key implications of the empirical results for the management of user communities