Daniel Yuan

We report the identification and characterization of CTR1, a gene in the yeast S. cerevisiae that encodes a multispanning plasma membrane protein specifically required for high affinity copper transport into the cell. The predicted protein contains a methionine- and serine-rich domain that includes 11 examples of the sequence Met-X2-Met, a motif noted in proteins involved in bacterial copper metabolism. CTR1 mutants and deletion strains have profound deficiency in ferrous iron uptake, thus revealing a requirement for copper in mediating ferrous transport into the cell. Genetic evidence suggests that the target for this requirement is the FET3 gene (detailed in a companion study), predicted to encode a copper-containing protein that acts as a cytosolic ferro-oxidase. These findings provide an unexpected mechanistic link between the uptake of copper and iron.

Iron must cross biological membranes to reach essential intracellular enzymes. Two proteins in the plasma membrane of yeast--a multicopper oxidase, encoded by the FET3 gene, and a permease, encoded by the FTR1 gene--were shown to mediate high-affinity iron uptake. FET3 expression was required for FTR1 protein to be transported to the plasma membrane. FTR1 expression was required for apo-FET3 protein to be loaded with copper and thus acquire oxidase activity. FTR1 protein also played a direct role in iron transport. Mutations in a conserved sequence motif of FTR1 specifically blocked iron transport.

A network governing DNA integrity was identified in yeast by a global genetic analysis of synthetic fitness or lethality defect (SFL) interactions. Within this network, 16 functional modules or minipathways were defined based on patterns of global SFL interactions. Modules or genes involved in DNA replication, DNA-replication checkpoint (DRC) signaling, and oxidative stress response were identified as the major guardians against lethal spontaneous DNA damage, efficient repair of which requires the functions of the DNA-damage checkpoint signaling and multiple DNA-repair pathways. This genome-wide genetic interaction network also identified novel components (DIA2, NPT1, HST3, HST4, and the CSM1 module) that potentially contribute to mitotic DNA replication and genomic stability and revealed novel functions of well-studied genes (the CTF18 module) in DRC signaling. This network will guide more detailed characterization of mechanisms governing DNA integrity in yeast and other organisms.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.

The CTR1 gene of Saccharomyces cerevisiae encodes a protein required for high affinity copper uptake. The protein is expressed on the plasma membrane, is heavily glycosylated with O-linkages, and exists as an oligomer in vivo. The transcript abundance is strongly regulated by copper availability, being induced by copper deprivation and repressed by copper excess. Regulation occurs at very low, nontoxic levels of available copper and is independent of ACE1, the trans-inducer of yeast metallothionein. Expression of Ctr1p is limiting for copper uptake, since overexpression from a 2 mu high copy number plasmid increases copper uptake. Mutations in CTR1 result in altered cellular responses to extracellular copper, demonstrating a physiologic role for CTR1 in the delivery of copper to the cytosol. A copper-dependent reporter gene construct, CUP1-lacZ, is not expressed in CTR1 mutants to the same level as in wild-type strains, and Cu,Zn superoxide dismutase activity is deficient in these mutants. The growth arrest that occurs in CTR1 mutants grown aerobically in copper-deficient media is attributable to the defect in Cu,Zn superoxide dismutase activity.

To search for a mammalian homologue of <i>ATX1</i>, a human liver cDNA library was screened and a cDNA clone was isolated, which encodes a protein with 47% amino acid identity to Atx1p including conservation of the MTC<i>X</i>GC copper-binding domain. RNA blot analysis using this cDNA identified an abundant 0.5-kilobase mRNA in all human tissues and cell lines examined. Southern blot analysis using this same clone indicated that the corresponding gene exists as a single copy in the haploid genome, and chromosomal localization by fluorescence <i>in situ</i> hybridization detected this locus at the interface between bands 5q32 and 5q33. Yeast strains lacking copper/zinc superoxide dismutase (<i>SOD1</i>) are sensitive to redox cycling agents and dioxygen and are auxotrophic for lysine when grown in air, and expression of this human <i>ATX1</i> homologue (<i>HAH1</i>) in these strains restored growth on lysine-deficient media. Yeast strains lacking <i>ATX1</i> are deficient in high affinity iron uptake and expression of <i>HAH1</i> in these strains permits growth on iron-depleted media and results in restoration of copper incorporation into newly synthesized Fet3p. These results identify HAH1 as a novel ubiquitously expressed protein, which may play an essential role in antioxidant defense and copper homeostasis in humans.

Study of mutant phenotypes is a fundamental method for understanding gene function. The construction of a near-complete collection of yeast knockouts (YKO) and the unique molecular barcodes (or TAGs) that identify each strain has enabled quantitative functional profiling of Saccharomyces cerevisiae. By using these TAGs and the SGA reporter, MFA1pr-HIS3, which facilitates conversion of heterozygous diploid YKO strains into haploid mutants, we have developed a set of highly efficient microarray-based techniques, collectively referred as dSLAM (diploid-based synthetic lethality analysis on microarrays), to probe genome-wide gene-chemical and gene-gene interactions. Direct comparison revealed that these techniques are more robust than existing methods in functional profiling of the yeast genome. Widespread application of these tools will elucidate a comprehensive yeast genetic network.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders. Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

Wilson disease is a disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration due to inherited mutations in a gene encoding a putative copper-transporting P-type ATPase. Polyclonal antisera generated against the amino terminus of the Wilson protein detected a specific 165-kDa protein in HepG2 and CaCo cell lysates. Further analysis revealed that this protein is synthesized as a single-chain polypeptide and localized to the<i>trans</i>-Golgi network under steady state conditions. An increase in the copper concentration resulted in the rapid movement of this protein to a cytoplasmic vesicular compartment. This copper-specific cellular redistribution of the Wilson protein is a reversible process that occurs independent of a new protein synthesis. Expression of the wild-type but not mutant Wilson protein in the<i>ccc2Δ</i> strain of <i>Saccharomyces cerevisiae</i>restored copper incorporation into the multicopper oxidase Fet3p, providing direct evidence of copper transport by the Wilson protein. Taken together these data reveal a remarkable evolutionary conservation in the cellular mechanisms of copper metabolism and provide a unique model for the regulation of copper transport into the secretory pathway of eucaryotic cells.

<h2>Summary</h2> Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants that probes the contribution of each residue to nucleosome function was generated in <i>Saccharomyces cerevisiae</i>. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.

A defect in the yeast GEF1 gene, a CLC chloride channel homolog leads to an iron requirement and cation sensitivity. The iron requirement is due to a failure to load Cu2+ onto a component of the iron uptake system, Fet3. This process, which requires both Gef1 and the Menkes disease Cu2+-ATPase yeast homolog Ccc2, occurs in late- or post-Golgi vesicles, where Gef1 and Ccc2 are localized. The defects of gef1 mutants can be suppressed by the introduction of Torpedo marmorata CLC-0 or Arabidopsis thaliana CLC-c and -d chloride channel genes. The functions of Gef1 in cation homeostasis provide clues to the understanding of diseases caused by chloride channel mutations in humans and cation toxicity in plants.

The Saccharomyces genome-deletion project created >5900 'molecularly barcoded' yeast knockout mutants (YKO mutants). The YKO mutant collections have facilitated large-scale analyses of a multitude of mutant phenotypes. For example, both synthetic genetic array (SGA) and synthetic-lethality analysis by microarray (SLAM) methods have been used for synthetic-lethality screens. Global analysis of synthetic lethality promises to identify cellular pathways that 'buffer' each other biologically. The combination of global synthetic-lethality analysis, together with global protein-protein interaction analyses, mRNA expression profiling and functional profiling will, in principle, enable construction of a cellular 'wiring diagram' that will help frame a deeper understanding of human biology and disease.

Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the histone variant Htz1p at Lys 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic interactions may help illuminate the mechanisms of intricate cellular processes.

We previously discovered a germ cell-specific spermatogenesis and oogenesis basic helix-loop-helix transcription factor, Sohlh2. We generated Sohlh2-deficient mice to understand physiologic consequences of Sohlh2 deletion. We discovered that Sohlh2-knockout adult female mice are infertile due to lack of ovarian follicles. Sohlh2-deficient ovaries can form primordial follicles and, despite limited oocyte growth, do not differentiate surrounding granulosa cells into cuboidal and multilayered structures. Oocytes are rapidly lost in Sohlh2-deficient ovaries, and few are present by 14 days of postnatal life. However, the primordial oocytes are abnormal at the molecular level because they misexpress numerous germ cell- and oocyte-specific genes, including Sohlh1, Nobox, Figla, Gdf9, Pou5f1, Zp1, Zp3, Kit, Oosp1, Nlrp14, H1foo, and Stra8. Our findings show that Sohlh2 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

The CCC2 gene in the yeastSaccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants,sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway. The CCC2 gene in the yeastSaccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants,sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway. Copper enzymes serve vital functions that involve molecular oxygen (1Linder M.C. Biochemistry of Copper. 4. Plenum Publishing Corp., New York1991: 100Google Scholar). In eukaryotic cells, the localization of these enzymes within specific cellular compartments varies with different enzymes and is a crucial determinant of their physiological roles. In both yeast and humans, for example, cytochrome c oxidase plays an essential role in oxygen utilization in mitochondria, while Cu,Zn-dependent superoxide dismutase functions in the detoxification of oxygen-derived free radicals in the cytosol. Most of the copper in the human body is in fact found in ceruloplasmin, a multicopper oxidase and glycoprotein secreted from liver cells into the circulation. This copper enzyme appears to function in iron metabolism (2Roeser H.P. Lee G.R. Nacht S. Cartwright G.E. J. Clin. Invest. 1970; 49: 2408-2417Crossref PubMed Scopus (267) Google Scholar, 3Yoshida K. Furihata K. Takeda S. Nakamura A. Yamamoto K. Morita H. Hiyamuta S. Ikeda S. Shimizu N. Yanagisawa N. Nat. Genet. 1995; 9: 267-272Crossref PubMed Scopus (421) Google Scholar, 4Harris Z.L. Takahashi Y. Miyajima H. Serizawa M. MacGillivray R.T.A. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2539-2543Crossref PubMed Scopus (508) Google Scholar). In the yeast Saccharomyces cerevisiae, a ceruloplasmin homolog, Fet3p, has been identified that participates in high affinity iron uptake at the cell surface (5Askwith C. Eide D. Ho A.V. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (582) Google Scholar). The fact that copper enzymes are localized to various cellular compartments suggests that mechanisms exist for the transport of copper across membranes into these compartments, since it is otherwise difficult to envision how fully folded, copper-loaded proteins might enter membrane-bound compartments from the cytosol. Of particular interest is how copper is transported into the secretory pathway for delivery to secreted or extracellular copper enzymes. In humans, such enzymes carry out a variety of functions, including the biosynthesis of neurotransmitters and the maturation of connective tissue. Specific mechanisms for intracellular copper transport can be expected, given that foodstuffs usually contain only trace amounts of copper, but until recently these mechanisms have been obscure.In 1993, the genes associated with two inherited disorders of human copper metabolism were identified. One of these disorders, Menkes disease (6Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 7Chelly J. Tumer Z. Tonnesen T. Petterson A. Ishikawa-Brush Y. Tommerup N. Horn N. Monaco A.P. Nat. Genet. 1993; 3: 14-19Crossref PubMed Scopus (623) Google Scholar, 8Mercer J.F. Livingston J. Hall B. Paynter J.A. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemieniak D. Glover T.W. Nat. Genet. 1993; 3: 20-25Crossref PubMed Scopus (624) Google Scholar), is characterized by a deficiency of copper in most tissues. The other, Wilson disease (9Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar, 10Tanzi R.E. Petrukhin K. Chernov I. Pellequer J.L. Wasco W. Ross B. Romano D.M. Parano E. Pavone L. Brzustowicz L.M. Devoto M. Peppercorn J. Bush A.I. Sternlieb I. Pirastu M. Gusella J.F. Evgrafov O. Penchaszadeh G.K. Honig Bi Edelman I.S. Soares M.B. Scheinberg I.H. Gilliam T.C. Nat. Genet. 1993; 5: 344-350Crossref PubMed Scopus (1174) Google Scholar, 11Yamaguchi Y. Heiny M.E. Gitlin J.D. Biochem. Biophys. Res. Commun. 1993; 197: 271-277Crossref PubMed Scopus (473) Google Scholar), is marked by failure of the liver to excrete surplus copper into the biliary tract and to deliver copper to ceruloplasmin. Despite different tissue specificities, both disorders appear to involve a defect in the export of copper from the cytosol (12Danks D.M. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York1995: 2211-2236Google Scholar). The genes associated with both of these disorders encode homologous P-type ATPases, suggesting that these ATPases mediate the export of copper from the cytosol (13Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Although functional characterization of these ATPases has proven difficult, some progress has been made in the characterization of a yeast homolog. This homolog, encoded by the CCC2 gene (14Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-292Crossref PubMed Scopus (142) Google Scholar), also appears to export copper from the cytosol, since ccc2 mutant cells fail to deliver copper to the ceruloplasmin homolog, Fet3p, a membrane-bound glycoprotein whose copper-binding domains lie outside the cytosol (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar). Despite a failure to synthesize the holoprotein form of Fet3p,ccc2 mutant cells still produce wild-type amounts of the apoprotein form of Fet3p, and copper uptake remains unimpaired (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar). Thus, ccc2 mutant cells exhibit signs of defective copper export into the secretory pathway that aptly model those observed in Menkes disease and Wilson disease.The aim of the present work was to characterize where copper enters the secretory pathway in yeast, using the genetic tools that are especially advantageous in this organism. Using a variety of yeast mutants with known defects in protein trafficking, the copper export activity of Ccc2p was assessed using biochemical and physiological assays of the copper content of Fet3p, and Ccc2p itself was visualized by immunofluorescence microscopy. It might be conjectured that copper export would occur within the endoplasmic reticulum, given the common requirement for metal cofactors in protein folding (16Lippard S.J. Berg J.M. Principles of Bioinorganic Chemistry. University Science Books, Mill Valley, CA1994: 381-384Google Scholar), or within the Golgi complex, in view of evidence that manganese is exported from the cytosol in yeast by a Golgi-resident P-type ATPase, Pmr1p (17Lapinskas P.J. Cunningham K.W. Liu X.F. Fink G.R. Culotta V.C. Mol. Cell. Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). The studies reported here suggest instead that Ccc2p-dependent copper export is restricted to a late- or post-Golgi compartment in the secretory pathway.EXPERIMENTAL PROCEDURESYeast StrainsThe ccc2::URA3 and Δfet3::TRP1 cells were in the parental background YPH252 as described (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar). TheΔftr1::TRP1 cells were in the same background but opposite mating type (strain 42C) (18Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar). TheΔccc2::LEU2 cells in the parental background 2908 were as described (14Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-292Crossref PubMed Scopus (142) Google Scholar); these cells were mated with strain 3AΔctr1 (MATa, ino1–13 leu2–3,-112 Δctr1::URA3) 1A. Dancis, unpublished observation. and sporulated for isolation of a ccc2 ctr1 strain. Thesec mutant strains (sec1–1, sec4–2,sec7–1, sec18–1, sec21–1,sec23–1, sec53–6, and the congenic wild-type strain X2180–1A) (19Novick P. Field C. Schekman R. Cell. 1980; 21: 205-215Abstract Full Text PDF PubMed Scopus (1249) Google Scholar) were from the Yeast Genetics Stock Center (Berkeley, CA). The vps mutant strains were part of a large collection of yeast strains generously provided by T. Stevens (20Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (671) Google Scholar). When vps strains were available as either vpl orvpt alleles, as listed (20Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (671) Google Scholar), the vpl strains were used. The FET3 gene was disrupted in a vps33(vpl25) strain (MATa, ade6 his4 leu2 ura3 pep4 vpl25–6) by insertion of the URA3 gene at its unique BamHI site, using aNheI-ClaI fragment from the plasmid pΔfet3.1 Insertions were verified by polymerase chain reaction across the insertion junction adjacent to the 3′-end of FET3.67Cu Autoradiography of Fet3pCells were starved for copper and iron by growth for 12–15 h at the indicated temperature to A 600 0.2–0.4 in 100 ml of a defined medium lacking added iron and copper (Bio101, La Jolla, CA). The medium contained glucose (2%, w/v) and MES-Na 2The abbreviations used are: MES, 4-morpholineethanesulfonic acid; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; HA, hemagglutinin; WT, wild type. (50 mm pH 6.1) (each treated with Chelex-100 resin; Bio-Rad), a complete supplement mixture (CSM; Bio101), and ferrozine (100 μm) (an iron chelator for inducing expression of several iron-regulated genes, including FET3 and CCC2) (21Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (288) Google Scholar). Cells were then labeled with 67Cu (Los Alamos National Laboratory, Los Alamos, NM) (1 μCi/ml, 3 nm total copper) for 2 h at the indicated temperature. Membrane extracts were prepared and separated without prior denaturation on 4–20% polyacrylamide gels as described (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar). Loading of gels for autoradiograms was adjusted to compensate for variations in total cellular copper uptake, as measured after cell homogenization and pelleting of cell walls (22Lin C.M. Crawford B.F. Kosman D.J. J. Gen. Microbiol. 1993; 139: 1617-1626Crossref PubMed Scopus (12) Google Scholar). Each lane contained roughly 0.1 μCi of radioactivity. Gels were rinsed briefly with water and exposed to Kodak XAR-2 film with one intensifying screen at −70 °C for 1–5 days.Determination of Protease-accessible Fet3p in SpheroplastsSpheroplasts were prepared using 100 μg of Zymolyase-100T (ICN) from approximately 8 × 108 cells in a buffer (1.2 m sorbitol, 40 mm sodium phosphate (pH 7.0), and 0.5 mm magnesium chloride) supplemented with 10 mm sodium azide and 10 mm sodium fluoride. Cells were then incubated with 2000 units proteinase K (Life Technologies, Inc.) (23Nothwehr S.F Conibear E. Stevens T.H. J. Cell Biol. 1995; 129: 35-46Crossref PubMed Scopus (147) Google Scholar) for 1 h, incubated with 1 mmAEBSF (ICN) for 15 min, and chilled on ice. Mock digestions involved separate incubations of the cells and enzymes/AEBSF in buffer; these were combined after chilling. All cells were washed four times in ice-cold buffer containing 1 mm AEBSF, 10 mmsodium azide, and 10 mm sodium fluoride and homogenized with glass beads in buffer containing 1 mm AEBSF, 30 μm leupeptin (ICN), and 10 μm pepstatin A (ICN). Membrane extracts were then prepared as described (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar). Copper sulfate (50 μm) was added to solubilized membranes to reconstitute oxidase activity for detection of total Fet3p (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar).Oxidase and Immunochemical Assays of Fet3pFet3p was detected in undenatured samples (in oxidase assays) or in denatured samples (in Western blots), as described (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar), using 7.5% polyacrylamide gels (8 μg of protein/lane) and 0.5 mg/mlp-phenylenediamine hydrochloride (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar) or 0.5 mg/mlo-dianisidine hydrochloride (24Schosinsky K.H. Lehmann H.P. Beeler M.F. Clin. Chem. 1974; 20: 1556-1563Crossref PubMed Scopus (519) Google Scholar) as substrates. Gels for oxidase assays were developed in a humid atmosphere at 30 °C for 3 days. Sodium azide (10 mm) was included in the oxidase buffer to diminish background signal. Antiserum to native Fet3p (1971.5) was obtained by immunizing rabbits with a multiple antigenic peptide conjugate (25Tam J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5409-5413Crossref PubMed Scopus (1192) Google Scholar) of the peptide RDLHVDPEVLLNEVD (from within the carboxyl-terminal tail of Fet3p) and was used at a 1:1000 dilution. The antiserum showed a marked preference for high mobility (immaturely glycosylated) forms of Fet3p, even in heat-denatured samples. 3D. S. Yuan unpublished observations. Physiological Assays of Copper-loaded Fet3pIron UptakeHigh affinity iron uptake capacity was measured using 55Fe as described (26Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (561) Google Scholar). Cells were grown for six generations in YPD medium supplemented with 25 mm each of sodium succinate and succinic acid (27Yamashiro C.T. Kane P.M. Wolczyk D.F. Preston R.A. Stevens T.H. Mol. Cell. Biol. 1990; 10: 3737-3749Crossref PubMed Scopus (145) Google Scholar) and 0 or 50 μmcopper sulfate and then grown for another 4 h after 40-fold dilution. Results shown are the differences between means of triplicate determinations at 30 and 0 °C.Respiratory CompetenceCells were first grown for 3 days at 30 °C on standard defined (SD) medium containing a complete supplement. Cells were then washed twice with water and dispensed in 5-μl samples (about 1000 cells) onto ethanol-based growth medium (15Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar) supplemented as indicated. Cells were grown at 30 °C for 5 days.Expression and Visualization of Epitope-tagged Ccc2pThe CCC2 coding region together with 395 bp of 5′-DNA (and no 3′-DNA) was amplified by polymerase chain reaction (28Barnes W.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2216-2220Crossref PubMed Scopus (977) Google Scholar) using genomic DNA derived from the yeast strain YPH274 (29Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) and the oligonucleotides 5′-CTCGCTCGCCCAAATACGCAGATGTTGGTACAGTCTCTTGCAGA-3′ and 5′-CTGGTTCGGCCCAACCAAGACTTCTTCGTTTGACATTAGTCCGGCCTGTGA-3′. Purified polymerase chain reaction products were introduced by ligation-independent cloning (30Aslanidis C. DeJong P.J. Nucleic Acids Res. 1990; 18: 6069-6074Crossref PubMed Scopus (923) Google Scholar) into a low copy number yeast expression vector based on the CEN- and HIS3-containing vector, pRS413 (Stratagene) (29Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). This vector (pDY207) was designed to fuse the 3′-ends of cloned coding sequences with sequences encoding three tandem copies of the influenza hemagglutinin (HA) epitope tag (31Wilson I.A. Niman H.L. Houghten R.A. Cherenson A.R. Connolly M.L. Lerner R.A. Cell. 1984; 37: 767-778Abstract Full Text PDF PubMed Scopus (654) Google Scholar) followed by two stop codons and the CYC1 terminator. The construction of this vector will be described elsewhere. 4D. S. Yuan, manuscript in preparation. ACCC2 fusion construct or pDY207 DNA lacking CCC2sequences was then transferred to the URA3 plasmid pRS416 (Stratagene) (29Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) via EcoRI and XbaI sites in the multiple cloning site. Bacterial cultures (Escherichia colistrain DH5α) of CCC2 plasmids were susceptible to lysis in stationary phase; DNA was prepared from bacteria grown in Terrific Broth (60Tartof K.D. Hobbs C.A. Bethesda Res. Lab. Focus. 1987; 9: 12Google Scholar) to a relatively low culture density (0.4A 600). Expression of aCCC2-containing plasmid (pDY228) or an empty expression vector (pDY225) in yeast cells was accomplished by growth at 30 °C in the defined medium used for copper autoradiography except with CuSO4 added to 0.1 μm (for ccc2cells with epitope-tagged Ccc2p) or 5 μm (forvps cells) and with uracil omitted. Immunofluorescence techniques were essentially as described (32Burns N. Grimwade B. Ross-Macdonald P.B. Choi E.Y. Finberg K. Roeder G.S. Snyder M. Genes Dev. 1994; 8: 1087-1105Crossref PubMed Scopus (461) Google Scholar), except that the primary antibody (1:2000) was a purified mouse monoclonal antibody directed against the HA epitope tag (HA.11, Babco, Berkeley, CA) (33Bosshart H. Humphrey J. Deignan E. Davidson J. Drazba J. Yuan L. Oorschot V. Peters P.J. Bonifacino J.S. J. Cell Biol. 1994; 126: 1157-1172Crossref PubMed Scopus (150) Google Scholar); the secondary antibody (1:200) was a Cy3 conjugate (Jackson ImmunoResearch, West Grove, PA). (Little specific signal was obtained with the anti-HA monoclonal antibody 12CA5 or a Texas Red conjugate.) 4′,6-diamidino-2-phenylindole (Sigma) was added to the mounting medium (1 μg/ml) to stain nuclei. Confocal images were obtained using a laser scanning microscope equipped with Nomarski optics (Zeiss).DISCUSSIONIn the present work, yeast mutants with genetically determined defects in protein trafficking were used to localize Ccc2p-dependent copper export. Temperature-sensitivesec mutants were used to show that copper export requires integrity of the early secretory pathway. In particular, copper export required function of the intra-Golgi protein encoded by the SEC7 gene, while the SEC4 and SEC1gene products involved in transport and fusion of secretory vesicles were dispensable. These findings served to localize copper export to a late Golgi or post-Golgi compartment and led to the finding that Ccc2p is aberrantly distributed in a subset of vps mutants with defects in post-Golgi sorting, thereby suggesting that Ccc2p functions in a post-Golgi compartment diverted from the secretory pathway. A model encompassing these and previous (18Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar) findings is presented in Fig. 6, in which Fet3p reaches this post-Golgi compartment as a complex with Ftr1p (18Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar), receives copper from Ccc2p, and then completes its transit to the plasma membrane by mechanisms involving the SEC4 and SEC1 gene products (Fig. 2 B).It remains to be established whether Ccc2p is in fact localized to a sorted post-Golgi compartment or whether it is actually localized to an unsorted late-Golgi compartment within the constitutive secretory pathway. The effects of vps mutations on copper export are most simply interpreted as showing that Ccc2p functions in a sorted post-Golgi compartment, since by definition vacuolar protein sorting involves the sorting of vacuolar proteins from the constitutive secretory pathway. In this view, Fet3p would travel to this compartment for copper loading and then proceed to the plasma membrane. This maturation pathway for Fet3p apparently occurs independently of any protein sorting motifs that may exist in the membrane-spanning or cytosolic domains of Fet3p, since truncation of those domains does not affect copper loading and secretion.3 Although novel in yeast, such pathways are well described in mammalian cells in connection with major histocompatibility complex class II antigen presentation (e.g. Refs. 43Peters P.J. Neefjes J.J. Oorschot V. Ploegh H.L. Geuze H.J. Nature. 1991; 349: 669-676Crossref PubMed Scopus (554) Google Scholar and 44Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. EMBO J. 1995; 14: 37-49Crossref PubMed Scopus (152) Google Scholar) and with the biogenesis of synaptic vesicles (e.g. Ref. 45Calakos N. Scheller R.H. Physiol. Rev. 1996; 76: 1-29Crossref PubMed Scopus (306) Google Scholar). Interestingly, two copper enzymes involved in mammalian neurotransmitter biosynthesis, dopamine-β-hydroxylase and peptidyl glycine α-amidating enzyme, are sorted away from the constitutive secretory pathway into neurosecretory vesicles through mechanisms that may involve acidic sorting compartments, independently of membrane-spanning or cytosolic domains in these enzymes (46Colomer V. Kicska G.A. Rindler M.J. J. Biol. Chem. 1996; 271: 48-55Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).The effects of vps mutations on Ccc2p localization and function may also be accounted for by the fact that many of these mutations affect the localization of late Golgi proteins as well as that of post-Golgi proteins (23Nothwehr S.F Conibear E. Stevens T.H. J. Cell Biol. 1995; 129: 35-46Crossref PubMed Scopus (147) Google Scholar, 47Robinson J.S. Graham T.R. Emr S.D. Mol. Cell. Biol. 1991; 11: 5813-5824Crossref PubMed Google Scholar, 48Wilsbach K. Payne G.S. EMBO J. 1993; 12: 3049-3059Crossref PubMed Scopus (111) Google Scholar, 49Cereghino J.L. Marcusson E.G. Emr S.D. Mol. Biol. Cell. 1995; 6: 1089-1102Crossref PubMed Scopus (135) Google Scholar). It is therefore also possible that Ccc2p is localized to a late Golgi compartment. An HA-tagged derivative of the late Golgi protein Kex2p was also redistributed in vps1, vps15, and vps33 mutants in a manner indistinguishable from that observed for the HA-tagged Ccc2p.3 Nevertheless, because of the ambiguities inherent in immunofluorescence studies of the late secretory pathway in yeast, double labeling experiments with differently tagged Ccc2p and Kex2p constructs will be necessary to determine whether these two proteins are in fact co-localized. Other phenotypes of certain vpsmutations include disruption of endocytic pathways (40Dulic V. Riezman H. EMBO J. 1989; 8: 1349-1359Crossref PubMed Scopus (39) Google Scholar, 50Davis N.G. Horecka J.L. Sprague Jr., G.F. J. Cell Biol. 1993; 122: 53-65Crossref PubMed Scopus (190) Google Scholar, 51Munn A.L. Riezman H. J. Cell Biol. 1994; 127: 373-386Crossref PubMed Scopus (229) Google Scholar) and the mislocalization of subunits of the vacuolar proton-translocating ATPase (35$$Google Scholar). Notably, vma1 (tfp1) disruption mutants defective in vacuolar acidification (52Shih C.K. Wagner R. Feinstein S. Kanik-Ennulat C. Neff N. Mol. Cell. Biol. 1988; 8: 3094-3103Crossref PubMed Scopus (51) Google Scholar) have severe copper phenotypes similar to ccc2 mutants,3 and another acidification mutant, vma3, has been isolated in a genetic screen for mutants with iron-correctable respiratory deficiency (53Eide D.J. Bridgham J.T. Zhao Z. Mattoon J.R. Mol. Gen. Genet. 1993; 241: 447-456Crossref PubMed Scopus (81) Google Scholar). The acidification of post-Golgi organelles could be required for some aspect of the delivery of copper to Fet3p, e.g. in transporter-metal dissociation kinetics (54Rao K. van Renswoude J. Kempf C. Klausner R.D. FEBS Lett. 1983; 160: 213-216Crossref PubMed Scopus (33) Google Scholar, 55Yamashiro D.J. Maxfield F.R. J. Cell. Biochem. 1984; 26: 231-246Crossref PubMed Scopus (95) Google Scholar) and even in vacuolar protein sorting (Refs. 27Yamashiro C.T. Kane P.M. Wolczyk D.F. Preston R.A. Stevens T.H. Mol. Cell. Biol. 1990; 10: 3737-3749Crossref PubMed Scopus (145) Google Scholar and 56Yaver D.S. Nelson H. Nelson N. Klionsky D.J. J. Biol. Chem. 1993; 268: 10564-10572Abstract Full Text PDF PubMed Google Scholar; see also Ref. 46Colomer V. Kicska G.A. Rindler M.J. J. Biol. Chem. 1996; 271: 48-55Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). It seems clear, however, that metallation of Fet3p by copper in vitro(e.g. in homogenization buffer) does not require an acidic pH and that the localization of copper export to the late secretory pathway is attributable at least in part to the localization of Ccc2p there (Fig. 5).Recent evidence in mammalian cells indicates that the Menkes disease and Wilson disease gene products also function in the late secretory pathway, within trans-Golgi or endosomal compartments (57Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (190) Google Scholar,58Petris M.J. Mercer J.F.B. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). 5J. Gitlin, personal communication. Thus, the exclusion of copper from the early secretory pathway may be a general feature of copper metabolism in eukaryotic cells. A rationale for this may be found in the fact that the lumen of the endoplasmic reticulum has a relatively high oxidation potential (59Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1571) Google Scholar). Copper delivered into this environment would be oxidized to the Cu(II) oxidation state. As this copper species avidly binds imidazole groups in histidine residues and generates hydroxyl radicals in Fenton-type reactions (1Linder M.C. Biochemistry of Copper. 4. Plenum Publishing Corp., New York1991: 100Google Scholar), it would probably disrupt essential functions of the early secretory pathway. Significantly, Fet3p apoprotein cannot be loaded with copper in vitro unless the copper is in the Cu(II) oxidation state.3 Therefore, at least for yeast, the ability of cells to withstand the biosynthesis of a secreted copper enzyme may hinge on their ability to segregate copper loading from other processes within the secretory pathway. Copper enzymes serve vital functions that involve molecular oxygen (1Linder M.C. Biochemistry of Copper. 4. Plenum Publishing Corp., New York1991: 100Google Scholar). In eukaryotic cells, the localization of these enzymes within specific cellular compartments varies with different enzymes and is a crucial determinant of their physiological roles. In both yeast and humans, for example, cytochrome c oxidase plays an essential role in oxygen utilization in mitochondria, while Cu,Zn-dependent superoxide dismutase functions in the detoxification of oxygen-derived free radicals in the cytosol. Most of the copper in the human body is in fact found in ceruloplasmin, a multicopper oxidase and glycoprotein secreted from liver cells into the circulation. This copper enzyme appears to function in iron metabolism (2Roeser H.P. Lee G.R. Nacht S. Cartwright G.E. J. Clin. Invest. 1970; 49: 2408-2417Crossref PubMed Scopus (267) Google Scholar, 3Yoshida K. Furihata K. Takeda S. Nakamura A. Yamamoto K. Morita H. Hiyamuta S. Ikeda S. Shimizu N. Yanagisawa N. Nat. Genet. 1995; 9: 267-272Crossref PubMed Scopus (421) Google Scholar, 4Harris Z.L. Takahashi Y. Miyajima H. Serizawa M. MacGillivray R.T.A. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2539-2543Crossref PubMed Scopus (50

Diabetes foot ulcer (DFU) is a complication of diabetes mellitus. Accurate diagnosis of DFU severity through inflammatory markers will assist in reducing impact on quality of life. We aimed to ascertain the diagnostic test accuracy of commonly used inflammatory markers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), procalcitonin (PCT), and white cell count (WCC) for the diagnosis and differentiation between DFU grades based on the International Working Group on the Diabetic Foot classification system.This systematic review explored studies that investigated one or more of the above-listed index tests aiding in diagnosing infected DFU. This review was registered on PROSPERO database (ID = CRD42021255618) and searched 5 databases including an assessment of the references of included studies. Records were manually screened as per Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. A total of 16 studies were included which were assessed for quality using QUADAS-2 tool and meta-analysed using Meta-Disc v1.4.CRP had the greatest area under the curve (AUC) of 0.893 for diagnosing grade 2 DFU. This returned a pooled sensitivity and specificity of 77.4% (95% CI: 72% to 82%) and 84.3% (95% CI: 79% to 89%) respectively. In terms of diagnosing grade 3 DFU, procalcitonin had the highest AUC value of 0.844 when compared with other markers. The pooled sensitivity of PCT was calculated as 85.5% (95% CI: 79% to 90%) and specificity as 68.9% (95% CI: 63% to 75%).CRP and PCT are the best markers for diagnosing grade 2 and grade 3 DFU respectively. Other markers are also valuable when used in conjunction with clinical judgement. The findings accentuate the necessity of further research to establish standardised cut-off values for these inflammatory markers in diagnosing diabetic foot ulcers.

Linalool, a plant-derived high-value monoterpene, is widely used in the perfume, cosmetic, and pharmaceutical industries. Recently, engineering microbes to produce linalool has become an attractive alternative to plant extraction or chemical synthesis approaches. However, the low catalytic activity of linalool synthase and the shortage of precursor pools have been considered as two key factors for low yields of linalool. In this study, we rationally engineered the entrance of the substrate-binding pocket of linalool synthase (t67OMcLISM) and successfully increased the catalytic efficiency of this enzyme toward geranyl pyrophosphate. Specifically, F447E and F447A, with decreased entrance hydrophobicity and steric hindrance, increased linalool production by 2.2 and 1.9 folds, respectively. Subsequently, cytoplasm and peroxisomes were harnessed to boost linalool synthesis in Saccharomyces cerevisiae, achieving a high titer of linalool (219.1 mg/L) in shake-flask cultivation. Finally, the engineered diploid strain produced 2.6 g/L of linalool by 5 L fed-batch fermentation, which was the highest production in yeast to date. The protein engineering and biosynthetic pathway compartmentalization in the peroxisome provide references for the microbial production of other monoterpenes.

High affinity iron uptake in <i>Saccharomyces cerevisiae</i> requires Fet3p. Fet3p is proposed to facilitate iron uptake by catalyzing the oxidation of Fe(II) to Fe(III) by O₂; in this model, Fe(III) is the substrate for the iron permease, encoded by <i>FTR1</i>. Here, a recombinant Fet3p has been produced in yeast that, lacking the C-terminal membrane-spanning domain, is secreted directly into the growth medium. Solutions of this Fet3p at >1 mg/ml have the characteristic blue color of a type 1 Cu(II)-containing protein, consistent with the sequence homology that placed this protein in the class of multinuclear copper oxidases that includes ceruloplasmin. Fet3p has an intense absorption at 607 nm (ε = 5500 m⁻¹ cm⁻¹) due to this type 1 Cu(II) and a shoulder in the near UV at 330 nm (ε = 5000m⁻¹ cm⁻¹) characteristic of a type 3 binuclear Cu(II) cluster. The EPR spectrum of this Fet3p showed the presence of one type 1 Cu(II) and one type 2 Cu(II) (A_∥ = 91 and 190 × 10⁻⁴cm⁻¹, respectively). Copper analysis showed this protein to have 3.85 g atom copper/mol, consistent with the presence of one each of the three types of Cu(II) sites found in multinuclear copper oxidases. N-terminal analysis demonstrated that cleavage of a signal peptide occurred after Ala-21 in the primary translation product. Mass spectral and carbohydrate analysis of the protein following Endo H treatment indicated that the preparation was still 15% (w/w) carbohydrate, probably <i>O</i>-linked. Kinetic analysis of the <i>in vitro</i> ferroxidase reaction catalyzed by this soluble Fet3p yielded precise kinetic constants. The<i>K</i> _m values for Fe(II) and O₂ were 4.8 and 1.3 μm, respectively, while<i>k</i> _cat values for Fe(II) and O₂turnover were 9.5 and 2.3 min⁻¹, consistent with an Fe(II):O₂ reaction stoichiometry of 4:1.

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.

Cancer ranks among the leading causes of human mortality. Cancer becomes intractable when it spreads from the primary tumor site to various organs (such as bone, lung, liver, and then brain). Unlike solid tumor cells, cancer stem cells and metastatic cancer cells grow in a non-attached (suspension) form when moving from their source to other locations in the body. Due to the non-attached growth nature, metastasis is often first detected in the circulatory systems, for instance in a lymph node near the primary tumor. Cancer research over the past several decades has primarily focused on treating solid tumors, but targeted therapy to treat cancer stem cells and cancer metastasis has yet to be developed. Because cancers undergo faster metabolism and consume more glucose than normal cells, glucose was chosen in this study as a reagent to target cancer cells. In particular, by covalently binding gold nanoparticles (GNPs) with thio-PEG (polyethylene glycol) and thio-glucose, the resulting functionalized GNPs (Glu-GNPs) were created for targeted treatment of cancer metastasis and cancer stem cells. Suspension cancer cell THP-1 (human monocytic cell line derived from acute monocytic leukemia patients) was selected because it has properties similar to cancer stem cells and has been used as a metastatic cancer cell model for in vitro studies. To take advantage of cancer cells' elevated glucose consumption over normal cells, different starvation periods were screened in order to achieve optimal treatment effects. Cancer cells were then fed using Glu-GNPs followed by X-ray irradiation treatment. For comparison, solid tumor MCF-7 cells (breast cancer cell line) were studied as well. Our irradiation experimental results show that Glu-GNPs are better irradiation sensitizers to treat THP-1 cells than MCF-7 cells, or Glu-GNPs enhance the cancer killing of THP-1 cells 20% more than X-ray irradiation alone and GNP treatment alone. This finding can help oncologists to design therapeutic strategies to target cancer stem cells and cancer metastasis.

Cardiovascular diseases (CVDs) are a significant burden globally and are especially prevalent in obese and/or diabetic populations. Epicardial adipose tissue (EAT) surrounding the heart has been implicated in the development of CVDs as EAT can shift from a protective to a maladaptive phenotype in diseased states. In diabetic and obese patients, an elevated EAT mass both secretes pro-fibrotic/pro-inflammatory adipokines and forms intramyocardial fibrofatty infiltrates. This narrative review considers the proposed pathophysiological roles of EAT in CVDs. Diabetes is associated with a disordered energy utilization in the heart, which promotes intramyocardial fat and structural remodeling. Fibrofatty infiltrates are associated with abnormal cardiomyocyte calcium handling and repolarization, increasing the probability of afterdepolarizations. The inflammatory phenotype also promotes lateralization of connexin (Cx) proteins, undermining unidirectional conduction. These changes are associated with conduction heterogeneity, together creating a substrate for atrial fibrillation (AF). EAT is also strongly implicated in coronary artery disease (CAD); inflammatory adipokines from peri-vascular fat can modulate intra-luminal homeostasis through an "outside-to-inside" mechanism. EAT is also a significant source of sympathetic neurotransmitters, which promote progressive diastolic dysfunction with eventual cardiac failure. Further investigations on the behavior of EAT in diabetic/obese patients with CVD could help elucidate the pathogenesis and uncover potential therapeutic targets.

Analysis of genetic interactions has been extensively exploited to study gene functions and to dissect pathway structures. One such genetic interaction is synthetic lethality, in which the combination of two non-lethal mutations leads to loss of organism viability. We have developed a dSLAM (heterozygote diploid-based synthetic lethality analysis with microarrays) technology that effectively studies synthetic lethality interactions on a genome-wide scale in the budding yeast Saccharomyces cerevisiae. Typically, a query mutation is introduced en masse into a population of approximately 6000 haploid-convertible heterozygote diploid Yeast Knockout (YKO) mutants via integrative transformation. Haploid pools of single and double mutants are freshly generated from the resultant heterozygote diploid double mutant pool after meiosis and haploid selection and studied for potential growth defects of each double mutant combination by microarray analysis of the "molecular barcodes" representing each YKO. This technology has been effectively adapted to study other types of genome-wide genetic interactions including gene-compound synthetic lethality, secondary mutation suppression, dosage-dependent synthetic lethality and suppression.

The exact molecular mechanisms by which the environmental pollutant arsenic works in biological systems are not completely understood. Using an unbiased chemogenomics approach in Saccharomyces cerevisiae, we found that mutants of the chaperonin complex TRiC and the functionally related prefoldin complex are all hypersensitive to arsenic compared to a wild-type strain. In contrast, mutants with impaired ribosome functions were highly arsenic resistant. These observations led us to hypothesize that arsenic might inhibit TRiC function, required for folding of actin, tubulin, and other proteins postsynthesis. Consistent with this hypothesis, we found that arsenic treatment distorted morphology of both actin and microtubule filaments. Moreover, arsenic impaired substrate folding by both bovine and archaeal TRiC complexes in vitro. These results together indicate that TRiC is a conserved target of arsenic inhibition in various biological systems.

Key Points PS can be overlooked in the differential diagnosis of children with severe congenital anemia. mtDNA deletion testing should be included in the genetic evaluation of patients with congenital anemia of unclear etiology.

Abstract The biochemistry of human nutritional zinc deficiency remains poorly defined. To characterize in genetic terms how cells respond to zinc deprivation, zinc-regulated genes (ZRG's) were identified in yeast. Gene expression was probed using random lacZ reporter gene fusions, integrated by transposon tagging into a diploid genome as previously described. About half of the genome was examined. Cells exhibiting differences in lacZ expression on low or moderate (~0.1 vs. 10 μm) zinc media were isolated and the gene fusions were sequenced. Ribonuclease protection assays demonstrated four- to eightfold increases for the RNAs of the ZAP1, ZRG17 (YNR039c), DPP1, ADH4, MCD4, and YEF3B genes in zinc-deficient cells. All but YEF3B were shown through reporter gene assays to be controlled by a master regulator of zinc homeostasis now known to be encoded by ZAP1. ZAP1 mutants lacked the flocculence and distended vacuoles characteristic of zinc-deficient cells, suggesting that flocculation and vacuolation serve homeostatic functions in zinc-deficient cells. ZRG17 mutants required extra zinc supplementation to repress these phenotypes, suggesting that ZRG17 functions in zinc uptake. These findings illustrate the utility of transposon tagging as an approach for studying regulated gene expression in yeast.

A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3-6% and 15-18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens.

(The American Journal of Human Genetics 103, 930–947; December 6, 2018) Reference 76 was incorrect in the original version of this paper, published online on November 29, 2018. The correct reference is Gagne, K.E., Ghazvinian, R., Yuan, D., Zon, R.L., Storm, K., Mazur-Popinska, M., Andolina, L., Bubala, H., Golebiowska, S., Higman, M.A., et al. (2014). Pearson marrow pancreas syndrome in patients suspected to have Diamond-Blackfan anemia. Blood 17, 437–440. The authors apologize for this error. The Genetic Landscape of Diamond-Blackfan AnemiaUlirsch et al.The American Journal of Human GeneticsNovember 29, 2018In BriefDiamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Full-Text PDF Open Archive

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with ribosomal protein (RP) gene mutations. Recent studies have also demonstrated an increased risk of cancer predisposition among DBA patients. In this study, we report the formation of soft tissue sarcoma in the Rpl5 and Rps24 heterozygous mice. Our observation suggests that even though one wild-type allele of the Rpl5 or Rps24 gene prevents anemia in these mice, it still predisposes them to cancer development.

Gestational diabetes mellitus (GDM) is defined as glucose intolerance with onset or first diagnosis during pregnancy, but not to the level of being diagnostic for diabetes in a nonpregnant adult. In GDM, whole-body insulin-dependent glucose disposal decreases by 40%-60% which necessitates a 200%-250% increase in insulin secretion to maintain normoglycaemia. GDM develops when a pregnant woman does not produce sufficient insulin to compensate for the reduced glucose disposal. Fibroblast growth factor 21 (FGF21) is a hormone that is expressed predominantly in the liver, but also in other metabolically active tissues such as pancreas, skeletal muscle and adipose tissue. In animals, FGF21 lowers blood glucose levels and inhibits glucagon secretion. In humans, circulating FGF21 levels are increased in insulin-resistant morbidities such as obesity and type 2 diabetes mellitus (T2DM). An elevated FGF21 level is also an independent predictor of T2DM. GDM and T2DM are proposed to have similar underlying pathophysiologies, raising the question of whether a similar relationship exists between FGF21 and GDM as it does with T2DM. There are a limited number of studies investigating FGF21 levels in patients with GDM. Moreover, recent clinical trials investigating the therapeutic potential of FGF21 have highlighted a major gap in our understanding of the biology of FGF21. This review evaluates what is currently known about FGF21 and GDM and highlights important gaps that warrant further research.

To determine the effect of glucagon-like peptide 1 receptor agonists (GLP-1RAs) on albuminuria in adult patients with type 2 diabetes mellitus (T2DM).Medline Ovid, Scopus, Web of Science, EMCARE and CINAHL databases from database inception until 27 January 2022. Studies were eligible for inclusion if they were randomized controlled trials that involved treatment with a GLP-1RA in adult patients with T2DM and assessed the effect on albuminuria in each treatment arm. Data extraction was conducted independently by three individual reviewers. The PRISMA guidelines were followed regarding data extraction and quality assessment. Data were pooled using a random effects inverse variance model and all analysis was carried out with RevMan 5.4 software. The Jadad scoring tool was employed to assess the quality of evidence and risk of bias in the randomized controlled trials.The initial search revealed 2419 articles, of which 19 were included in this study. An additional three articles were identified from hand-searching references of included reviews. Therefore, in total, 22 articles comprising 39 714 patients were included. Meta-analysis suggested that use of GLP1-RAs was associated with a reduction in albuminuria in patients with T2DM (weighted mean difference -16.14%, 95% CI -18.42 to -13.86%; p < .0001) compared with controls.This meta-analysis indicates that GLP-1RAs are associated with a significant reduction in albuminuria in adult patients with T2DM when compared with placebo.

Despite the integral role of virus-receptor interactions during viral infection, key mechanistic questions about binding, unbinding, and viral mobility remain unelucidated for many viruses. Sendai virus (SeV), the prototypical respirovirus studied here, binds to sialic acid conjugated glycolipids and glycoproteins in the target host cell. This receptor binding is mediated by the viral HN protein, which possesses both receptor-binding (hemagglutinin) and receptor-cleaving (neuraminidase) activities. Our prior work suggests a rolling mechanism for SeV mobility on supported lipid bilayers (SLBs), governed at least in part by the interplay between stochastic binding and unbinding of individual HN-receptor pairs, and eventually leading to virus detachment from the target membrane. However, the role of the HN neuraminidase activity in this process has been unclear in our data. Here, we studied the impact of neuraminidase activity on SeV mobility, binding, and unbinding by using single virus assays and SLBs as well as a fluorescence spectroscopy-based neuraminidase activity assay. We identified small molecule neuraminidase inhibitors and environmental conditions to modulate neuraminidase activity, and then used them to elucidate the contributions of neuraminidase activity to SeV mobility, binding, and unbinding. These results also provide some insight into whether the Sendai virus HN has one or two binding sites. Finally, we present progress toward the development of supported lipid bilayers composed of physiological membrane components, to be used in similar viral binding and mobility studies.

The bacterial non-homologous end-joining (NHEJ) apparatus is a two-component system that uses Ku and LigD to repair DNA double-strand breaks. Although the reaction mechanism has been extensively studied, much less is known about the physiological role of bacterial NHEJ. Recent studies suggest that NHEJ acts under conditions where DNA replication is reduced or absent (such as in a spore or stationary phase). Interestingly, genes encoding Ku and LigD have been identified in a wide range of bacteria that can chronically infect eukaryotic hosts. Strikingly, Sinohizobium meliloti, an intracellular symbiont of legume plants, carries four genes encoding Ku homologues (sku1 to sku4). Deletion analysis of the sku genes indicated that all Ku homologues are functional. One of these genes, sku2, is strongly expressed in free-living cells, as well as in bacteroid cells residing inside of the host plant. To visualize the NHEJ apparatus in vivo, SKu2 protein was fused to yellow fluorescent protein (YFP). Ionizing radiation (IR) induced focus formation of SKu2-YFP in free-living cells in a dosage-dependent manner. Moreover, SKu2-YFP foci formed in response to IR in non-dividing bacteroids, indicating that NHEJ system is functional even during the chronic infection phase of symbiosis.

To determine if implementing the operational definition of interictal epileptiform discharges (IEDs) proposed by the International Federation of Clinical Neurophysiology (IFCN) improves expert diagnostic performance and interrater reliability (IRR).

Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix) adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft progression was determined as follows: cleft cell contractility, cleft region cell-cell adhesion strength, epithelial cell mitosis rate, and cell-matrix adhesion strength.

Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle.

This study considers the problem of describing and predicting cleft formation during the early stages of branching morphogenesis in mouse submandibular salivary glands (SMG) under the influence of varied concentrations of epidermal growth factors (EGF). Given a time-lapse video of a growing SMG, first we build a descriptive model that captures the underlying biological process and quantifies the ground truth. Tissue-scale (global) and morphological features related to regions of interest (local features) are used to characterize the biological ground truth. Second, we devise a predictive growth model that simulates EGF-modulated branching morphogenesis using a dynamic graph algorithm, which is driven by biological parameters such as EGF concentration, mitosis rate, and cleft progression rate. Given the initial configuration of the SMG, the evolution of the dynamic graph predicts the cleft formation, while maintaining the local structural characteristics of the SMG. We determined that higher EGF concentrations cause the formation of higher number of buds and comparatively shallow cleft depths. Third, we compared the prediction accuracy of our model to the Glazier-Graner-Hogeweg (GGH) model, an on-lattice Monte-Carlo simulation model, under a specific energy function parameter set that allows new rounds of de novo cleft formation. The results demonstrate that the dynamic graph model yields comparable simulations of gland growth to that of the GGH model with a significantly lower computational complexity. Fourth, we enhanced this model to predict the SMG morphology for an EGF concentration without the assistance of a ground truth time-lapse biological video data; this is a substantial benefit of our model over other similar models that are guided and terminated by information regarding the final SMG morphology. Hence, our model is suitable for testing the impact of different biological parameters involved with the process of branching morphogenesis in silico, while reducing the requirement of in vivo experiments.

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTSpectroscopic Characterization of the Cu(II) Sites in the Fet3 Protein, the Multinuclear Copper Oxidase from Yeast Required for High-Affinity Iron UptakeDaniel J. Kosman, Richard Hassett, Daniel S. Yuan, and John McCrackenView Author Information Department of Biochemistry, SUNY Buffalo, New York 14214 Department of Pediatrics The Johns Hopkins University School of Medicine Baltimore, Maryland 21205 Department of Chemistry, Michigan State University East Lansing, Michigan 48824 Cite this: J. Am. Chem. Soc. 1998, 120, 16, 4037–4038Publication Date (Web):April 14, 1998Publication History Received3 December 1997Published online14 April 1998Published inissue 1 April 1998https://doi.org/10.1021/ja974104uCopyright © 1998 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views129Altmetric-Citations24LEARN 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 (41 KB) Get e-AlertsSUBJECTS:Copper,Electron paramagnetic resonance spectroscopy,Fungi,Iron,Peptides and proteins Get e-Alerts

Non-canonical intronic variants are a poorly characterized yet highly prevalent class of alterations associated with Mendelian disorders. Here, we report the first RNA expression and splicing analysis from a family whose members carry a non-canonical splice variant in an intron of RPL11 (c.396 +3A>G). This mutation is causative for Diamond Blackfan Anemia (DBA) in this family despite incomplete penetrance and variable expressivity. Our analyses revealed a complex pattern of disruptions with many novel junctions of RPL11. These include an RPL11 transcript that is translated with a late stop codon in the 3′ untranslated region (3'UTR) of the main isoform. We observed that RPL11 transcript abundance is comparable among carriers regardless of symptom severity. Interestingly, both the small and large ribosomal subunit transcripts were significantly overexpressed in individuals with a history of anemia in addition to congenital abnormalities. Finally, we discovered that coordinated expression between mitochondrial components and RPL11 was lost in all carriers, which may lead to variable expressivity. Overall, this study highlights the importance of RNA splicing and expression analyses in families for molecular characterization of Mendelian diseases.

Abstract Motivation: Microarray data are susceptible to a wide-range of artifacts, many of which occur on physical scales comparable to the spatial dimensions of the array. These artifacts introduce biases that are spatially correlated. The ability of current methodologies to detect and correct such biases is limited. Results: We introduce a new approach for analyzing spatial artifacts, termed ‘conditional residual analysis for microarrays’ (CRAM). CRAM requires a microarray design that contains technical replicates of representative features and a limited number of negative controls, but is free of the assumptions that constrain existing analytical procedures. The key idea is to extract residuals from sets of matched replicates to generate residual images. The residual images reveal spatial artifacts with single-feature resolution. Surprisingly, spatial artifacts were found to coexist independently as additive and multiplicative errors. Efficient procedures for bias estimation were devised to correct the spatial artifacts on both intensity scales. In a survey of 484 published single-channel datasets, variance fell 4- to 12-fold in 5% of the datasets after bias correction. Thus, inclusion of technical replicates in a microarray design affords benefits far beyond what one might expect with a conventional ‘n = 5’ averaging, and should be considered when designing any microarray for which randomization is feasible. Availability: CRAM is implemented as version 2 of the hoptag software package for R, which is included in the Supplementary information. Contact: dyuan@jhmi.edu Supplementary information: Supplementary Data are available at Bioinformatics online.

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.

We present FEATS (Feature Extraction and Tracking Simulator), that synthesizes feature tracks using a camera trajectory and scene geometry (e.g. CAD, multi-view stereo). We introduce 2D feature and matching noise models that can be controlled using a few parameters. We also provide a new dataset of images and ground truth camera pose. We process this data (and a synthetic version) with several current SfM algorithms and show that the synthetic tracks are representative of the real tracks. We then show two practical uses of FEATS: (1) we generate hundreds of trajectories with varying noise and show that COLMAP is more robust to noise than OpenSfM and VisualSfM; and (2) we calculate 3D point error and show that accurate camera pose estimates do not guarantee accurate 3D maps.

Comprehensive collections of open reading frame (ORF) deletion mutant strains exist for the budding yeast Saccharomyces cerevisiae. With great prescience, these strains were designed with short molecular bar codes or TAGs that uniquely mark each deletion allele, flanked by shared priming sequences. These features have enabled researchers to handle yeast mutant collections as complex pools of ∼6000 strains. The presence of any individual mutant within a pool can be assessed indirectly by measuring the relative abundance of its corresponding TAG(s) in genomic DNA prepared from the pool. This is readily accomplished by wholesale polymerase chain reaction (PCR) amplification of the TAGs using fluorescent oligonucleotide primers that recognize the common flanking sequences, followed by hybridization of the labeled PCR products to a TAG oligonucleotide microarray. Here we describe a method—diploid-based synthetic lethality analysis by microarray (dSLAM)—whereby such pools can be manipulated to rapidly construct and assess the fitness of 6000 double-mutant strains in a single experiment. Analysis of double-mutant strains is of growing importance in defining the spectrum of essential cellular functionalities and in understanding how these functionalities interrelate.

Tissue engineering scaffolds are used in conjunction with stem cells for the treatment of various diseases. A number of factors provided by the scaffolds affect the differentiation of stem cells. Mechanical cues that are part of the natural cellular microenvironment can both accelerate the differentiation toward particular cell lineages or induce differentiation to an alternative cell fate. Among such factors, there are externally applied strains and mechanical (stiffness and relaxation time) properties of the extracellular matrix. Here, the mechanics of a fibrous-porous scaffold is studied by applying a coordinated modeling and experimental approach. A force relaxation experiment is used, and a poroelastic model associates the relaxation process with the fluid diffusion through the fibrous matrix. The model parameters, including the stiffness moduli in the directions along and across the fibers as well as fluid diffusion time, are estimated by fitting the experimental data. The time course of the applied force is then predicted for different rates of loading and scaffold porosities. The proposed approach can help in a reduction of the technological and experimental efforts to produce 3-D scaffolds for regenerative medicine as well as in a higher accuracy of the estimation of the local factors sensed by stem cells.

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow disorder with mutations in ribosomal protein genes. Several animal models have been developed to study the pathological mechanism of DBA. Previously, we reported that the complete knock-out of both Rpl5 and Rps24 alleles were lethal, while heterozygous Rpl5+/- and Rps24+/- mice showed normal phenotype. To establish a more efficient mouse model for mimicking DBA symptoms, we have taken advantage of RNAi technology to generate an inducible mouse model utilizing tetracycline-induced down-regulation of Rpl5. After two weeks of treatment with doxycycline in drinking water, a subset of treated shRNA Rpl5+/- adult mice developed mild anemia while control mice had normal complete blood counts. Similarly, treated shRNA Rpl5+/- mice developed reticulocytopenia and bone marrow erythroblastopenia. Detection of DBA symptoms in these mice make them a valuable DBA model for studying the pathological mechanism underlying DBA and for further assessment of the disease and drug testing for novel therapies.

In this paper, we introduce a biologically motivated dynamic graph-based growth model to describe and predict the stages of cleft formation during the process of branching morphogenesis in the submandibular mouse gland (SMG) from 3 hrs after embryonic day E12 to 8 hrs after embryonic day E12, which can be considered as E12.5. Branching morphogenesis is the process by which many mammalian exocrine and endocrine glands undergo significant morphological transformations, from a primary bud to an adult organ. Although many studies have investigated the cellular and molecular mechanisms driving branching morphogenesis, it is not clear how the shape changes that are inherent to establishing organ structure are produced. Using morphological features extracted from sequential images of SMG organ cultures we were able to develop a dynamic graph-based predictive model that is able to mimic the process of cleft formation and predict the final state. In addition, we compare our model to a state-of-the-art Glazier-Graner-Hogeweg (GGH) simulative tool, and demonstrate that the dynamic graph-based predictive model has comparable accuracy in modeling growth of clefts across SMG developmental stages, as well as faster convergence to the target SMG morphology.

Abstract Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia inherited in an autosomal dominant pattern caused by mutations in ribosomal protein (RP) genes and in an X-linked recessive pattern by GATA1 mutations. Heterozygous mutations and large deletions in 11 RP genes, RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A, RPS7, RPS10, RPS26, RPL26, and RPL15, are present in ∼65% of DBA patients. DBA is associated with congenital abnormalities in ∼50% of patients and with increased risk of malignancy. To investigate the molecular pathogenesis of RPL5 and RPS24 gene mutations, we generated two murine lines of heterozygous mice, Rpl5 and Rps24, by knocking-out exons 1-8 in the Rpl5 gene and exons 2-3 in the Rps24 gene in C57BL/6 mice. Knock-out of both alleles of Rpl5 and Rps24 genes are embryonic lethal. In contrast, heterozygous mice exhibited normal hematological phenotype, as well as normal Rpl5 and Rps24 RNA and protein levels in their tissues, suggesting that the presence of one allele was sufficient to support the normal function of ribosomal proteins L5 and S24 in mice. To evaluate the risk of cancer development in Rpl5 +/- and Rps24 +/- mice, we monitored these mice and wild type mice until late age. Out of 21 Rpl5 +/- mice (between the ages of 14 and 26 months), two mice developed tumors at 22 months of age and two mice were euthanized due to severe dermatitis at the same age. Similarly, we have been monitoring 23 Rps24 +/- mice between 15 and 26 months of age. One of these mice developed a tumor at 17 months of age, five mice were euthanized due to severe dermatitis between the ages of 17 and 19 months, and two mice were euthanized due to injuries at ages 15 and 29 months. We also monitored 20 control wild-type mice ranging from 13 to 26 months of age. To this date, no tumors have been detected in wild-type mice, although nine of these mice developed severe dermatitis and were euthanized. Histological and immunohistochemical studies were performed to determine the nature of tumors in Rpl5 +/- and Rps24 +/- mice. Comparison of tumor tissues with normal skin from wild-type or Rpl5 +/- and Rps24 +/- with no detected tumors showed that all tissues had normal epidermis and underlying dermis, but connective tissues from tumor sections consisted of a densely cellular neoplasms composed of predominantly atypical spindle shaped cells arranged in intersecting fascicles. The tumor cells had strong cytoplasmic reactivity for vimentin and negative staining for S100, CD45, and pan-keratin, consistent with a high-grade spindle cell sarcoma. Recent studies conducted by the DBA Registry of North America revealed that out of 608 DBA patients, 18 with median age of 41 years developed various types of cancer including sarcomas, colon cancer, and acute myeloid leukemia. The relative risk of cancer in DBA was increased 5.4 fold compared to general population (Vlachos, et al., 2012). Our studies also suggest the correlation between ribosomal protein gene mutations and cancer. However, further studies are required to better understand the underlying molecular mechanism. Disclosures: No relevant conflicts of interest to declare.

Regeneration of sensory nerves is challenging in dental pulp regeneration. Schwann cells (SCs) are essential glial cells conducive to regenerating dental pulp sensory nerve, but their source is scarce. This study aimed to explore whether Schwann-like cells derived from dental pulp stem cells (SC-DPSCs) are conducive to the growth of dental pulp sensory nerve axons. In the present study, primary human dental pulp stem cells (hDPSCs) were cultured and their multidirectional differentiation ability was detected. hDPSCs were induced into SC-DPSCs using the 3 steps protocol. The expression of MBP, S-100, and P75NTR, and dynamic expression and distribution of BDNF/NT-3 in SC-DPSCs were assessed by quantitative real-time PCR, immunofluorescence and ELISA. Primary trigeminal neurons were cultured and the central neuronal markers NF200, TUBB3 and MAP2 were detected by cellular immunofluorescence. Trigeminal neurons and SC-DPSCs were indirected co-cultured, after that the growth activity of trigeminal neurons was detected by CCK-8. One key factor during the differentiation process of hDPSCs into SC-DPSCs were screened by mRNA sequencing. The results showed that SC-DPSCs significantly expressed characteristic SCs markers and promoted the axon growth of rat trigeminal nerve. High-throughput sequencing found that PTGS2 maybe the key gene participate in the induction process. Taken together, our findings demonstrate the potential of SC-DPSCs in sensory nerve regeneration in dental pulp and dentin complexes. After differentiation into SC-DPSCs, hDPSCs secreted more nerve growth factors BDNF and NT-3, which significantly promoted the growth of trigeminal nerve axons.

Successful deployment of future cognitive networks consisting of tens to thousands of IoT devices requires both fundamental research on wireless networks and an understanding of the applications that will be supported by these future networks. Our Intelligent Digital Communications Vertically Integrated Project (VIP) Team thus focuses on research on, applications for, deployment of, and experiments with cognitive networks in a large-scale testbed. The testbed is the football stadium at Georgia Tech and the intense ISM band traffic in the stadium that is generated during games by: 50,000+ fans that have smart phones; medical, security, media, and venue personnel; and, the teams' coaches, players and support staff. The applications we are currently researching and implementing include: security en-hancement by localization of jammers and unauthorized drones; collecting data on spectrum usage and congestion; and machine learning to enable real-time characterization of RF spectrum usage. This paper reports on the research that motivates this work, the progress to-date on the testbed, and goals for the future.

Stem cells play a key role in the healthy development and maintenance of organisms. They are also critically important in medical treatments of various diseases. It has been recently demonstrated that the mechanical factors such as forces, adhesion, stiffness, relaxation, etc. have significant effects on stem cell functions. Under physiological conditions, cells (stem cells) in muscles, heart, and blood vessels are under the action of externally applied strains. We consider the stem cell microenvironment and performance associated with their conversion (differentiation) into skeletal muscle cells. Two problems are studied by using mathematical models whose parameters are then optimized by fitting experiments. First, we present our analysis of the process of stem cell differentiation under the application of cyclic unidirectional strain. This process is interpreted as a transition through several (six) stages where each of them is defined in terms of expression of a set of factors typical to skeletal muscle cells. The stem cell evolution toward muscle cells is described by a system of nonlinear ODEs. The parameters of the model are determined by fitting the experimental data on the time course of expression of the factors under consideration. Second, we analyse the mechanical (relaxation) properties of a scaffold that serves as the microenvironment for stem cells differentiation into skeletal muscle cells. This scaffold (surrounded by a liquid solution) is composed of unidirectional fibers with pores between them. The relaxation properties of the scaffold are studied in an experiment where a long cylindrical specimen is loaded by the application of ramp displacement until the strain reaches a prescribed value. The magnitude of the corresponding load is recorded. The specimen is considered as transversely isotropic poroelastic cylinder whose force relaxation is associated with liquid diffusion through the pores. An analytical solution for the total force applied to the cylinder in terms of the mechanical properties of the scaffold (longitudinal and lateral Young's moduli, two Poisson's ratios, and typical time of liquid diffusion) is used. The number of constant is then reduced to three by estimating the longitudinal Young's modulus and one of Poisson's ratios from an earlier experiment. Finally, three remaining parameters are estimated by fitting the relaxation curve corresponding to strain rate of loading of 0.01 s−1. The developed mathematical solution is then tested by comparing the theoretical and experimental results for another strain rate of 0.0025 s−1. The scaffold relaxation properties can be important for differentiation of stem cells inside the pores.

Abstract Congenital hypoplastic or Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder characterized by a paucity of red blood cells and their precursors in the bone marrow. The majority of cases of DBA are due to haploinsufficient mutations in ribosomal protein genes and in rare cases result from GATA1 mutations. However, nearly half of the DBA cases do not have an identified genetic etiology. While analyzing whole exome sequencing data from a cohort of over 450 patients with a clinical diagnosis of DBA, we encountered the case of a male child of a first cousin consanguineous union who was diagnosed with DBA as an infant and remained transfusion dependent. The patient responded to corticosteroid therapy for a year as a toddler, but this treatment was discontinued due to side effects. The patient subsequently remained transfusion dependent and at 6 years of age an allogeneic bone marrow transplant from a matched maternal aunt was performed. Surprisingly, despite achievement of robust donor chimerism, the patient remained transfusion dependent. Unfortunately the patient developed severe graft-versus-host disease and died of resultant complications. Both the potential recessive nature of the mutation, given parental consanguinity, and the lack of anemia correction following transplant made this case extremely unusual. Thus we evaluated this patient's whole exome sequencing data. We identified a homozygous recessive mutation in the erythropoietin gene (EPO), which resulted in an R150Q substitution in the mature EPO protein. This mutation was absent from a cohort of 60,706 individuals depleted for Mendelian disease and fit the model of complete penetrance in the family. The R150Q mutation was expected to disrupt the high-affinity binding site to the EPO receptor (EPOR). However, we found by producing recombinant proteins that the EPO R150Q mutation only reduced the EPOR binding affinity by 3-fold. Surprisingly, the patient had an over 100-fold elevation in their serum EPO levels, suggesting that this mutation did not cause disease through altered affinity. Rather we observed altered EPOR binding kinetics by this mutant ligand. There was a slightly increased on-rate with a much faster dissociation rate (t1/2 of 10 seconds for the mutant vs. 6 minutes for the wild type). Using human erythroid cells and primary hematopoietic stem and progenitor cells, we could show that this mutant ligand never reached the same efficacy as the wild type (WT) EPO in promoting erythroid differentiation and proliferation. To better characterize this abnormal activity, we examined downstream signaling responses. We found identical phosphorylation of STAT5 at maximally potent concentrations of the WT (1 nM) and R150Q mutant (100 nM) EPO. By surveying a broad array of &gt;120 phosphorylation events using intracellular flow cytometry, we demonstrated that maximal levels of STAT3 and STAT1 phosphorylation were reduced by 30% and 25%, respectively, with the R150Q (100 nM) compared to WT (1 nM) EPO. To determine the mechanistic basis for variation in downstream effector activation by the R150Q mutant ligand, we used inhibitors of both the JAK2 kinase and the SHP1/2 phosphatases that are respectively up- and downstream of STAT phosphorylation. While SHP1/2 inhibition did not alter STAT phosphorylation, JAK2 inhibition by ruxolitinib more potently inhibited STAT1/3 phosphorylation compared to STAT5. Interestingly, treatment with a low dose of ruxolitinib (40 nM) reduced erythroid proliferation to the same extent at maximally potent concentrations of the WT or R150Q EPO, demonstrating that the impairment in signaling by the R150Q EPO was primarily due to reduced JAK2 activity. Finally, we utilized single molecule fluorescent imaging of EPOR dimerization at the intact cell surface to directly show that the kinetically-biased R150Q EPO has a reduced ability to promote productive dimerization as compared to the WT EPO, even at maximally potent concentrations. Collectively, our results demonstrate how the R150Q mutant EPO - the first pathogenic mutation in EPO identified in humans - results in biased agonism of EPOR signaling through reduced receptor dimerization and consequently impaired JAK2 activation. More broadly our findings reveal how variation of cytokine-receptor binding kinetics can be used to tune downstream responses, which has broad implications for modulating the activity of numerous hematopoietic cytokines. Disclosures No relevant conflicts of interest to declare.

(Cell 124, 1069–1081; March 10, 2006) This paper employed a high-throughput microarray-based screening process (diploid synthetic lethal analysis by microarray [dSLAM]) as an approach for identifying functional modules involved in DNA metabolism. The main conclusions of the paper pertain to the DNA integrity network identified, and these were based on follow-up validation of synthetic lethal or fitness interactions between individually tested pairs of mutations, many of which have subsequently been reproduced by others. It has come to our attention that the Experimental Procedures section did not include sufficient information regarding how mutant pairs were selected for individual follow-up validation. Mutants with 635 nm/532 nm ratios ≥ 2.0 with either uptags or downtags, as well as those with ratios ≥ 1.5 with both tags, were selected. Mutants with only one tag ratio ≥ 1.5 were also selected for genes involved in DNA metabolism or functionally related to the query. In addition, a subset of the interactions validated was not chosen for such validation, based solely on the primary microarray data. Though our original Table S1 provided an indication of this fact, we realize that the statement therein was incomplete. A re-analysis of the original data reveals that the total percentage of interacting pairs selected for validation, based strictly on primary dSLAM data, was ∼75% of the total reported. The rationale for individually testing the remaining validated interaction reported in the paper is as follows: An additional 11% were derived from analysis of dSLAM data of two query mutations known to be related due to either phenotypic similarity or similar patterns of interactors as revealed by dSLAM. Another 11% were detected based on a phenotypic miniarray test (similar to an eMAP) done on a set of genes that were indicated by the dSLAM data and other screens to be part of the larger DNA damage network. About 3% were tested based on other prior knowledge. We regret the omission and note that the subset of interactions whose identification was not based directly on the dSLAM screen cannot be used to support the efficacy of the dSLAM method itself. A revised Table S1 and a supporting data file have been prepared to indicate in which cases information beyond the original dSLAM data was used to select candidates for individual validation. Download .xlsx (.33 MB) Help with xlsx files Table S1. Individually Confirmed Synthetic Lethality or Fitness Defect Interactions, Related to Experimental Procedures Download .xlsx (.04 MB) Help with xlsx files Data S1. Supporting Data for Table S1 A DNA Integrity Network in the Yeast Saccharomyces cerevisiaePan et al.CellMarch 10, 2006In BriefA network governing DNA integrity was identified in yeast by a global genetic analysis of synthetic fitness or lethality defect (SFL) interactions. Within this network, 16 functional modules or minipathways were defined based on patterns of global SFL interactions. Modules or genes involved in DNA replication, DNA-replication checkpoint (DRC) signaling, and oxidative stress response were identified as the major guardians against lethal spontaneous DNA damage, efficient repair of which requires the functions of the DNA-damage checkpoint signaling and multiple DNA-repair pathways. Full-Text PDF Open Archive

Earlier use of a metaphor in explaining genetic redundancy in a news article has triggered a commentary and a competing metaphor by Dr Stephen Cooper, who went on to conclude that genetic redundancies are relatively unimportant for microorganisms. We argue here that the new metaphor is flawed and that genetic redundancies are integral to buffering all organisms against environmental and genetic damage.

ABSTRACT Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 1 in 100,000 to 200,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this genetically heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole exome sequencing (WES). Overall, we identified rare and predicted damaging mutations in likely causal genes for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and in one of 19 previously reported genes encoding for a diverse set of ribosomal proteins (RPs). Using WES exon coverage estimates, we were able to identify and validate 31 deletions in DBA associated genes. We also observed an enrichment for extended splice site mutations and validated the diverse effects of these mutations using RNA sequencing in patientderived cell lines. Leveraging the size of our cohort, we observed several robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. In addition to comprehensively identifying mutations in known genes, we further identified rare mutations in 7 previously unreported RP genes that may cause DBA. We also identified several distinct disorders that appear to phenocopy DBA, including 9 individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain &gt; 5% of DBA cases. Overall, this comprehensive report should not only inform clinical practice for DBA patients, but also the design and analysis of future rare variant studies for heterogeneous Mendelian disorders.

Abstract BCG vaccine protects children but does not induce long-term immunity against tuberculosis. Rapamycin has been found to directly act on MTOR of T cells and extend the longevity of CD8 T cell response to viral infection in mice. We hypothesized that rapamycin co-administration during BCG vaccination may alter the quality of CD8 function to enable long-term immunity. Methods: C57Bl/6 mice were given one dose of BCG vaccine (1 million per mouse; s.c.) or BCG with doses of rapamycin (75 microgram/mg given daily for 30 days) followed by aerosol infection with virulent Mycobacterium tuberculosis (Mtb). At 4 and 8 weeks, antigen specific CD8 T cells, memory specific markers and tetramers specific for Mtb antigens-ESAT6 and TB10.4 were analysed using flow cytometry. Log10 decline of Mtb counts in lungs was determined to correlate T cell phenotype with protection after vaccination. Results: Rapamycin coadministration with BCG enhanced the expansion of antigen-specific CD8 T cells in mice that correlated with a decline in the counts of Mtb in lungs and spleens of mice. Rapamycin increased both CD8 and CD4 T cells in the lungs and spleens and enhanced central memory (Tcm) CD8 T cells in lymphoid organs. Tcm levels, in turn, correlated with an increase in expression of Eomes transcription factor by CD8 T cells. Since Eomes is a determinant of Tcm lineage, we suggest that the long-term efficacy of BCG vaccine can be enhanced through modulation of MTOR dependent pathways of T cells.

Abstract Pearson marrow pancreas syndrome (PS) is a congenital multisystem disorder characterized by sideroblastic anemia, pancreatic insufficiency, metabolic acidosis, and other defects, and is caused by mitochondrial DNA (mtDNA) deletions. Diamond Blackfan anemia (DBA) is a congenital hypoproliferative anemia with associated physical malformations, and in which mutations in ribosomal protein (RP) genes and GATA1 have been implicated. The clinical presentation of both of these bone marrow failure (BMF) syndromes shares several features including early onset of severe anemia, sporadic genetic inheritance, variable penetrance and manifestations, and episodes of spontaneous hematologic improvement. PS is less frequently occurring than DBA, with estimated incidences of &lt; 1/1,000,000 versus 1/100,000 respectively, and therefore less often encountered by hematologists. We hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. To test this hypothesis, we retrospectively evaluated DNA samples from a cohort of patients that were submitted to a research study for DBA genetic testing. The study cohort consists of clinical samples and/or data from 362 patients, with a primary inclusion criterion of known or suspected congenital anemia. Prior genetic studies from this cohort have yielded the novel identification or confirmation of mutations and deletions in several genes implicated in DBA (e.g. RP genes, GATA1), which are to date identifiable in 175/362 samples (48%), a proportion consistent with that found in independent DBA registries. We screened peripheral blood DNA samples available from 173 genetically uncharacterized patients using a long PCR strategy, and found that 8 samples (4.6%) contained large mtDNA deletions. Deletion mapping and Southern blot analysis on DNA from these 8 patients confirmed the presence of a single deletion event within each patient, ranging in size from 2.3 - 7.0 kb of the 16.6 kb mitochondrial genome, existing as monomer or multimer mtDNA species, and in a proportion ranging from 55-80% of total mtDNA, all of which are consistent with the molecular diagnosis of PS. Follow-up with referring providers in the 1 month to 8 year time span since sample submission revealed that 2 of the 8 patients (25%) were subsequently diagnosed with PS. Of the remaining 6 undiagnosed patients, 2 had died from complications of bone marrow transplantation, performed for worsening cytopenias and concern for myelodysplasia; one patient died from bacterial sepsis; and 3 were alive with the provisional diagnosis of DBA. One of the 3 patients had become transfusion-independent. Review of bone marrow examinations revealed that the pathological hallmarks of ringed sideroblasts and/or vacuolization of precursors described in PS were inconsistently present or reported in the diagnostic evaluation. We conclude that PS is frequently overlooked in the diagnostic evaluation of children with congenital anemia. Establishing the diagnosis of PS, as distinct from DBA and other BMF disorders, holds important implications for patient management and family counseling. mtDNA deletion testing should be performed in the initial genetic evaluation of all patients with congenital anemia. Disclosures: Szczepanski: Octapharma AG: Investigator Other.

Branching morphogenesis is a complex developmental process requiring coordination of cell proliferation, cell migration, actomyosin‐mediated cell contractility, and adhesion formation, which is not understood. We utilized computer modeling to simulate cleft progression in the primary bud during salivary gland development. Using computer simulations where individual or multiple parameters were varied in specific subsets of cells and each repeated 100 times, we examined the function of multiple cellular processes in cleft formation. We developed quantitative cleft detection algorithms to report on cleft quality and used confocal imaging of salivary gland organ explant cultures to confirm predictions of the simulations, when possible. Manipulations of the percentage of cells undergoing mitosis and the location of those cells are not currently possible to perform experimentally. Of multiple conditions, computer simulations predicted that a low proliferation rate and equal division of proliferative events between the outer and inner cells in the epithelium produces ideal clefts more than other conditions. This work demonstrates that computer modeling can be used to make predictions regarding cellular mechanisms of branching morphogenesis when experimental manipulation is not technically feasible. Supported by NIH R01DE019244 (M.L. and B.Y.)

The pancreatic receptor for cholecystokinin (CCK) typifies many G protein-coupled receptors in that its ability to bind agonist can be reduced by GTP or the solubilization of membranes. We found, however, that a dye, cibacron blue, caused up to a 6-fold increase in binding of the CCK receptor agonist, 125I-CCK-8, to rat pancreatic membranes solubilized with digitonin. Binding optimally enhanced in this manner was comparable to binding of 125I-CCK-8 to native membranes with respect to time-course, maximal amount bound, reversibility, and sensitivity to inhibition by various CCK receptor ligands. Increases in affinity of the CCK receptor for CCK-8 accounted fully for the enhancement of binding of 125I-CCK-8. Cibacron blue did not enhance binding of 125I-CCK-8 to native membranes, and also failed to enhance binding of the CCK receptor antagonist, [3H]L-364,718, to solubilized or native membranes. The ability of cibacron blue to enhance binding of agonist but not that of antagonist suggests that this dye may mimic or perhaps stimulate the effects of G protein on CCK receptors. Such a phenomenon may provide new insights into the mechanisms by which receptors distinguish agonists from antagonists.

OBJECTIVE: Primordial follicle (PMF) decrement after cyclophosphamide (cyclo) has been shown. Nobox (newborn homeobox-encoding) gene is crucial in folliculogenesis, specifically in PMF activation. To determine whether chemotherapy-induced PMF decrement is related to regulation of folliculogenesis, we compared decrease of PMFs in Nobox (+/-) heterozygote (N-het) mice with wildtype(Wt) after giving cyclo. DESIGN: Animal model laboratory study. MATERIALS AND METHODS: We compared the following groups (N=8-14): Wt control (no cyclo), N-het control, Wt 75 (75 mg/kg cyclo), N-het 75. We gave 2-week old mice cyclo intraperitoneally. Ovaries were paraffin embedded after 1 week. Immunohistochemistry was performed using rabbit- derived antiSohlh1 and composite count of the ovary obtained. Results were analyzed using ANOVA and general linear model with Bonferroni correction. RESULTS: There was no difference in PMFs in untreated conditions (Wt control mean PMFs = 4715, N-het control mean PMFs = 4701; p = .99) Significant decrease in PMFs after 75 mg/kg cyclo was found in Wt 75 condition (mean = 1842, p .0022), while N-het genotype showed no significant difference between the untreated and 75 mg/kg condition (mean =2698; p=.1397). CONCLUSIONS: N-het ovaries appear to have normal follicular development. Thus, no difference between Wt and N-het genotype PMFs was found in untreated conditions. Also, rates of PMF decrement in Wt animals after cyclo were similar to those in previous studies. The Nobox heterozygote genotype seems to be protective against chemotherapy-induced PMF damage, as there was no significant difference in PMFs in untreated N-hets compared to N-hets treated with cyclo. Moreover, N- hets have 1.46 times the number of PMFs remaining after cyclo, compared to Wt. While this was not statistically significant in this small study, this difference could be clinically significant. Since Nobox is involved in PMF activation to primary follicles, Nobox haploinsufficiency may protect PMFs by diminishing their activation potential.

Binding to the host membrane is the initial infection step for animal viruses. Sendai virus (SeV), the model respirovirus studied here, utilizes sialic-acid-conjugated glycoproteins and glycolipids as receptors for binding. In a previous report studying single virus binding to supported lipid bilayers (SLBs), we found a puzzling mechanistic difference between the binding of SeV and influenza A virus (strain X31, IAVX31). Both viruses use similar receptors and exhibit similar cooperative binding behavior, but whereas IAVX31 binding was altered by SLB cholesterol concentration, which can stabilize receptor nanoclusters, SeV was not. Here, we propose that differences in viral size distributions can explain this discrepancy; viral size could alter the number of virus-receptor interactions in the contact area and, therefore, the sensitivity to receptor nanoclusters. To test this, we compared the dependence of SeV binding on SLB cholesterol concentration between size-filtered and unfiltered SeV. At high receptor density, the unfiltered virus showed little dependence, but the size-filtered virus exhibited a linear cholesterol dependence, similar to IAVX31. However, at low receptor densities, the unfiltered virus did exhibit a cholesterol dependence, indicating that receptor nanoclusters enhance viral binding only when the number of potential virus-receptor interactions is small enough. We also studied the influence of viral size and receptor nanoclusters on viral mobility following binding. Whereas differences in viral size greatly influenced mobility, the effect of receptor nanoclusters on mobility was small. Together, our results highlight the mechanistic salience of both the distribution of viral sizes and the lateral distribution of receptors in a viral infection.

<h3>Background</h3> Regulatory T cells (T_reg) -targeted therapy exhibit clinical benefit and has been reported as a promising strategy. However, many gaps remain in our understanding of T_reg biology within the context of tumor microenvironment (TME). The autoimmune toxicity and restricted efficacy are major limitations of T_reg therapies in the clinic, when T_reg depletion occurred not only in the tumor but in other organ systems, or concurrent downregulation of antitumor effector T cells.^{1, 2} <h3>Methods</h3> We profiled 51,195 single-cell transcriptomes of CD4⁺ T cells in tumors and peripheral blood from patients with head and neck squamous cell carcinomas (HNSCC)³, in inflamed tonsil tissues and in healthy peripheral blood. Canonical genes, gene sets and RNA Velocity⁴ were used to define cell states of T_reg. Cibersortx⁵ and bulk RNA sequencing data in The Cancer Genome Atlas were used to infer the association between the enrichment of T_reg subpopulations and progression-free survival of patients with solid tumors. SCENIC⁶ and Causal mixed graphical modeling⁷ were used to reconstruct the gene regulatory network (GRN). Knockout of <i>BATF</i> with CRISPR-Cas9⁸ in conjunction with bulk RNA sequencing, immunophenotyping and in vitro functional assays were used to interrogate the roles of BATF in human activated T_reg. <h3>Results</h3> We identified an activated subpopulation of T_reg expressing multiple tumor necrosis factor receptor (TNFR) genes, including <i>OX40</i> and <i>4-1BB</i>, which is highly enriched in solid TME compared with non-tumor tissues. These TNFR-activated T_reg were associated with worse prognosis across solid tumors. Notably, we found <i>BATF</i> is a central component of a GRN that controls the transcriptional signature of TNFR-activated T_reg. Consistent with single-cell analyses, BATF was co-expressed with 4-1BB, OX40, CD96 and CD39 that highly enriched in HNSCC intratumoral T_reg at protein level. CRISPR-editing results revealed an enhancement of immunosuppression in <i>BATF</i> KO T_reg and activation in <i>BATF</i> KO T_reg accompanied with increased expression of genes including 4-1BB, OX40, ICOS, LAG3 and neuropilin-1, indicating that BATF functions as a transcriptional nexus in human activated T_reg that essential for T_reg activation, function and stability. <h3>Conclusions</h3> We identify a unique intertumoral subpopulation of T_reg characterized by BATF-driven expression of tumor necrosis factor receptor family expression and associated with survival across solid tumors, suggesting a possibility to target suppressive intratumoral T_reg without causing overt autoimmunity in normal tissues. A deeper understanding of transcriptional network in T_reg biology will provide novel mechanisms for immunotherapies in cancer, but also for T_reg engineering in autoimmunity. <h3>Acknowledgements</h3> We thank the Vignali, Bruno and Benos Labs for all their constructive comments and feedback. <h3>References</h3> Zappasodi R, Sirard C, Li Y, Budhu S, Abu-Akeel M, Liu C, <i>et al</i>. Rational design of anti-GITR-based combination immunotherapy. <i>Nature Medicine</i> 2019;<b>25</b>(5):759–66. Jacobs JF, Punt CJ, Lesterhuis WJ, Sutmuller RP, Brouwer HM, Scharenborg NM, <i>et al</i>. Dendritic cell vaccination in combination with anti-CD25 monoclonal antibody treatment: a phase I/II study in metastatic melanoma patients. <i>Clin Cancer Res</i> 2010;<b>16</b>(20):5067–78. Cillo AR, Kürten CHL, Tabib T, Qi Z, Onkar S, Wang T, <i>et al</i>. Immune Landscape of Viral- and Carcinogen-Driven Head and Neck Cancer. <i>Immunity</i> 2020;<b>52</b>(1):183–99. Bergen V, Lange M, Peidli S, Wolf FA, Theis FJ. Generalizing RNA velocity to transient cell states through dynamical modeling. <i>Nature Biotechnology</i> 2020;<b>38</b>(12):1408–14. Newman AM, Steen CB, Liu CL, Gentles AJ, Chaudhuri AA, Scherer F, <i>et al</i>. Determining cell type abundance and expression from bulk tissues with digital cytometry. <i>Nature Biotechnology</i> 2019;<b>37</b>(7):773–82. Van de Sande B, Flerin C, Davie K, De Waegeneer M, Hulselmans G, Aibar S, <i>et al</i>. A scalable SCENIC workflow for single-cell gene regulatory network analysis. <i>Nature Protocols</i> 2020;<b>15</b>(7):2247–76. Sedgewick AJ, Buschur K, Shi I, Ramsey JD, Raghu VK, Manatakis DV, <i>et al</i>. Mixed graphical models for integrative causal analysis with application to chronic lung disease diagnosis and prognosis. <i>Bioinformatics</i> 2019;<b>35</b>(7):1204–12. Schumann K, Raju SS, Lauber M, Kolb S, Shifrut E, Cortez JT, et al. Functional CRISPR dissection of gene networks controlling human regulatory T cell identity. <i>Nature Immunology</i> 2020;<b>21</b>(11):1456–66. <h3>Ethics Approval</h3> All patients provided informed written consent, and this study was approved by our Institutional Review Board (University of Pittsburgh Cancer Institute, Tissue Collection Protocol 99-069).

Germline ANKRD26 mutations cause autosomal dominant thrombocytopenia with MDS/leukemia predisposition (ANKRD26-RT). ANKRD26 is highly expressed in hematopoietic stem (HSCs)/progenitor cells, but is silenced during megakaryocyte (Mk) maturation. Mutations in ANKRD26-RT cluster within a transcriptional silencer region and cause a failure to fully repress ANKRD26 during terminal megakaryopoiesis. Yet the normal function of ANKRD26 and the mechanism(s) by which dysregulated ANKRD26 expression cause thrombocytopenia and leukemia predisposition remain poorly understood. We have generated a mouse overexpression model of ANKRD26-RT by inserting the human ANKRD26 cDNA, preceded by a transcriptional stopper cassette flanked by loxP sites, into the Rosa26 locus. Activation within HSCs (Mx1-Cre) or Mks (PF4-Cre) leads to thrombocytopenia and small Mks with hypolobulated nuclei, similar to what is observed in ANKRD26-RT patients. ANKRD26 was recently reported to be a centriole component and to activate the PIDDosome complex when cells become aneuploid, leading to cell cycle arrest and/or apoptosis. We hypothesize that ANKRD26 silencing is necessary for mature Mks to avoid triggering the aneuploidy checkpoint as they undergo polyploidization. This is being tested in our mouse model and human systems. ANKRD26 has also been shown to be phosphorylated by ATM/ATR following cell radiation. We found that ANKRD26 over expression causes a failure of cell cycle arrest, delayed clearance of g-H2AX nuclear foci, and impaired p21 up regulation following irradiation. We also find that primary CD34+ bone marrow cells from ANKRD26-RT patients contain increased g-H2AX foci compare to healthy controls. We propose a model in which dysregulated ANKRD26 expression alters the stoichiometry of DNA damage response complexes leading to MDS/leukemia predisposition. Germline ANKRD26 mutations cause autosomal dominant thrombocytopenia with MDS/leukemia predisposition (ANKRD26-RT). ANKRD26 is highly expressed in hematopoietic stem (HSCs)/progenitor cells, but is silenced during megakaryocyte (Mk) maturation. Mutations in ANKRD26-RT cluster within a transcriptional silencer region and cause a failure to fully repress ANKRD26 during terminal megakaryopoiesis. Yet the normal function of ANKRD26 and the mechanism(s) by which dysregulated ANKRD26 expression cause thrombocytopenia and leukemia predisposition remain poorly understood. We have generated a mouse overexpression model of ANKRD26-RT by inserting the human ANKRD26 cDNA, preceded by a transcriptional stopper cassette flanked by loxP sites, into the Rosa26 locus. Activation within HSCs (Mx1-Cre) or Mks (PF4-Cre) leads to thrombocytopenia and small Mks with hypolobulated nuclei, similar to what is observed in ANKRD26-RT patients. ANKRD26 was recently reported to be a centriole component and to activate the PIDDosome complex when cells become aneuploid, leading to cell cycle arrest and/or apoptosis. We hypothesize that ANKRD26 silencing is necessary for mature Mks to avoid triggering the aneuploidy checkpoint as they undergo polyploidization. This is being tested in our mouse model and human systems. ANKRD26 has also been shown to be phosphorylated by ATM/ATR following cell radiation. We found that ANKRD26 over expression causes a failure of cell cycle arrest, delayed clearance of g-H2AX nuclear foci, and impaired p21 up regulation following irradiation. We also find that primary CD34+ bone marrow cells from ANKRD26-RT patients contain increased g-H2AX foci compare to healthy controls. We propose a model in which dysregulated ANKRD26 expression alters the stoichiometry of DNA damage response complexes leading to MDS/leukemia predisposition.

April 26, 2018April 10, 2018Free AccessVision impairment and papilledema as the initial manifestation of neurosyphilis in a young immunocompetent patient (P5.130)Doyle Yuan, Fabio Nascimento, Laila Woc-Colburn, Rod Foroozan, and Jacob MandelAuthors Info & AffiliationsApril 10, 2018 issue90 (15_supplement) Letters to the Editor

Therapy-related Myelodysplastic Syndrome (t-MDS) accounts for up to 20% of all MDS cases and its prevalence is expected to grow as more cancer patients survive. Unfortunately, the prognosis for t-MDS remains poor with 5-year overall survival rates <10%. Gaps in understanding the molecular basis of t-MDS have hampered development of new treatments. Important clues regarding t-MDS pathogenesis come from studies of Hiroshima atomic bombing survivors and Soviet nuclear test site residents who developed MDS. About 40-50% of these cases had acquired heterozygous loss-of-function (or dominant negative) mutations in the gene encoding the key hematopoietic transcription factor RUNX1. Likewise, acquired RUNX1 mutations occur in a high proportion patients with chemotherapy-induced MDS (∼16-40%). There is increasing evidence that RUNX1 plays a role in the normal DNA damage response (DDR). This has led to the hypothesis that genotoxic stress selects for a small population of pre-existing hematopoietic stem/progenitor cells (HSPCs) containing RUNX1 mutations. We engineered a mouse model to test this hypothesis under native hematopoietic conditions. This involves FGD5-Cre-ERT2, RUNX1fl/+, Rosa26-lox-stopper-lox-red fluorescent protein (RFP) mice in which one RUNX1 allele can be inducibly deleted in hematopoietic stem cells by a brief pulse of tamoxifen. We titrated the dose to generate mice with ∼1-10% of RUNX1-/+, RFP+ peripheral blood cells. Treatment of these mice with low-dose irradiation leads to significant and sustained expansion of RFP+ multilineage peripheral blood cells to 30-40% of the total population. In contrast, mock-treated control mice maintain ∼1-10% peripheral blood RFP+ cells. From a mechanistic standpoint, we show that RUNX1 deficiency reduces p53 protein half-life. Treatment of RUNX1 deficient human cells with the MDM2 antagonists Nutlin3a and Idasanutlin increases p53 protein levels and induces apoptosis in a subset of cells. Surviving cells have evidence of resolved DNA damage. We propose that MDM2 inhibitors have the potential to selectively target the mutant clone in RUNX1-mutated t-MDS patients by restoring their DDR. Therapy-related Myelodysplastic Syndrome (t-MDS) accounts for up to 20% of all MDS cases and its prevalence is expected to grow as more cancer patients survive. Unfortunately, the prognosis for t-MDS remains poor with 5-year overall survival rates <10%. Gaps in understanding the molecular basis of t-MDS have hampered development of new treatments. Important clues regarding t-MDS pathogenesis come from studies of Hiroshima atomic bombing survivors and Soviet nuclear test site residents who developed MDS. About 40-50% of these cases had acquired heterozygous loss-of-function (or dominant negative) mutations in the gene encoding the key hematopoietic transcription factor RUNX1. Likewise, acquired RUNX1 mutations occur in a high proportion patients with chemotherapy-induced MDS (∼16-40%). There is increasing evidence that RUNX1 plays a role in the normal DNA damage response (DDR). This has led to the hypothesis that genotoxic stress selects for a small population of pre-existing hematopoietic stem/progenitor cells (HSPCs) containing RUNX1 mutations. We engineered a mouse model to test this hypothesis under native hematopoietic conditions. This involves FGD5-Cre-ERT2, RUNX1fl/+, Rosa26-lox-stopper-lox-red fluorescent protein (RFP) mice in which one RUNX1 allele can be inducibly deleted in hematopoietic stem cells by a brief pulse of tamoxifen. We titrated the dose to generate mice with ∼1-10% of RUNX1-/+, RFP+ peripheral blood cells. Treatment of these mice with low-dose irradiation leads to significant and sustained expansion of RFP+ multilineage peripheral blood cells to 30-40% of the total population. In contrast, mock-treated control mice maintain ∼1-10% peripheral blood RFP+ cells. From a mechanistic standpoint, we show that RUNX1 deficiency reduces p53 protein half-life. Treatment of RUNX1 deficient human cells with the MDM2 antagonists Nutlin3a and Idasanutlin increases p53 protein levels and induces apoptosis in a subset of cells. Surviving cells have evidence of resolved DNA damage. We propose that MDM2 inhibitors have the potential to selectively target the mutant clone in RUNX1-mutated t-MDS patients by restoring their DDR.