Biomedical subjects

D Yang

Publications and source records attributed to D Yang.

At least 91 records · Page 5Linked to original sources

PubMed2000

Genetic screens in mammalian cells by enhanced retroviral mutagens.

Genetic approaches such as retrovirus-mediated mutagenesis and cDNA expression libraries have contributed greatly to our understanding of signal transduction in mammalian cells. However, previously described methods for retroviral insertional mutagenesis are hindered by low mutagenesis rates and difficulties in cloning mutated genes. cDNA expression library methods are usually cell-type dependent and bias towards abundant and short messages. With the near completion of the genome projects, alternative genetic methods are needed where large numbers of genes can be more easily isolated and biochemically studied. We have developed a novel retrovirus-mediated genetic screening method in cultured cells. To achieve efficient and regulated mutagenesis, we constructed Enhanced Retroviral Mutagen (ERM) vectors that contained several engineered sequences (e.g., an ERM Tag and a splice donor) controlled by a tetracycline-responsive promoter. Endogenous genes can thus be randomly activated and tagged in a conditional system. NIH3T3 cells were used to screen for focus-forming genes using the ERM strategy. We showed that these added sequences increased the screening efficiency by >10-fold, and allowed more direct identification of the genes targeted. Sequence analysis of approximately 10% of the >600 focus clones recovered revealed both known oncogenes and novel factors such as protein kinases and GTP/GDP exchange proteins. The ERM strategy should help to facilitate large-scale gene identification in diverse pathways and integrate both genetic (with the completion of the genome projects) and functional information more readily.

3T3 Cells↗

PubMed2000

Expression of CCR6 and CD83 by cytokine-activated human neutrophils.

Polymorphonuclear leukocytes (PMNLs) are thought to be terminally differentiated, short-lived, and unable to actively synthesize new proteins or to interact with T cells. In the current study, it was found that PMNLs incubated with supernatants of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-sup) expressed high levels of CCR6 mRNA. Neutralization with IgG against several cytokines revealed that tumor necrosis factor (TNF)-alpha was largely responsible for the PHA-sup-induced CCR6 mRNA expression. Among recombinant cytokines, TNF-alpha induced high levels of CCR6 mRNA expression, whereas interferon (IFN)-gamma induced low levels. The 2 cytokines together exhibited a considerable synergy. Cytokine-activated PMNLs expressed functional CCR6, as detected by the binding of sodium iodide I 125-labeled liver and activation-regulated chemokine (LARC) and dose-dependent migration toward LARC. The induction of CCR6 suggested that these cytokine-activated PMNLs have more similarities with dendritic cells (DCs) that express CCR6 in an immature stage. In fact, the activation of PMNLs with TNF-alpha and IFN-gamma induced the expression of CD83, a dominant cell-surface marker of DCs. When PMNLs were activated with granulocyte macrophage-colony-stimulating factor, TNF-alpha, and IFN-gamma, these cells expressed CD40 and HLA-DR in addition to CD83. Taken together, PMNLs, under appropriate conditions, can undergo a differentiation process characterized by the acquisition of new phenotypes and functions, and such differentiated PMNLs may play more active roles in the adaptive immune response. (Blood. 2000;96:3958-3963)

Antigens, CD↗

PubMed2000

The human SWI/SNF-B chromatin-remodeling complex is related to yeast rsc and localizes at kinetochores of mitotic chromosomes.

The SWI/SNF family of chromatin-remodeling complexes facilitates gene expression by helping transcription factors gain access to their targets in chromatin. SWI/SNF and Rsc are distinctive members of this family from yeast. They have similar protein components and catalytic activities but differ in biological function. Rsc is required for cell cycle progression through mitosis, whereas SWI/SNF is not. Human complexes of this family have also been identified, which have often been considered related to yeast SWI/SNF. However, all human subunits identified to date are equally similar to components of both SWI/SNF and Rsc, leaving open the possibility that some or all of the human complexes are rather related to Rsc. Here, we present evidence that the previously identified human SWI/SNF-B complex is indeed of the Rsc type. It contains six components conserved in both Rsc and SWI/SNF. Importantly, it has a unique subunit, BAF180, that harbors a distinctive set of structural motifs characteristic of three components of Rsc. Of the two mammalian ATPases known to be related to those in the yeast complexes, human SWI/SNF-B contains only the homolog that functions like Rsc during cell growth. Immunofluorescence studies with a BAF180 antibody revealed that SWI/SNF-B localizes at the kinetochores of chromosomes during mitosis. Our data suggest that SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling complexes conserved from yeast to human, and could participate in cell division at kinetochores of mitotic chromosomes.

Adenosine Triphosphatases↗

PubMed2000

Human DNA polymerase kappa synthesizes DNA with extraordinarily low fidelity.

Escherichia coli DNA polymerase IV encoded by the dinB gene is involved in untargeted mutagenesis. Its human homologue is DNA polymerase kappa (Polkappa) encoded by the DINB1 gene. Our recent studies have indicated that human Polkappa is capable of both error-free and error-prone translesion DNA synthesis in vitro. However, it is not known whether human Polkappa also plays a role in untargeted mutagenesis. To examine this possibility, we have measured the fidelity of human Polkappa during DNA synthesis from undamaged templates. Using kinetic measurements of nucleotide incorporations and a fidelity assay with gapped M13mp2 DNA, we show that human Polkappa synthesizes DNA with extraordinarily low fidelity. At the lacZalpha target gene, human Polkappa made on average one error for every 200 nucleotides synthesized, with a predominant T-->G transversion mutation at a rate of 1/147. The overall error rate of human Polkappa is 1.7-fold lower than human Poleta, but 33-fold higher than human Polbeta, a DNA polymerase with very low fidelity. Thus, human Polkappa is one of the most inaccurate DNA polymerases known. These results support a role for human Polkappa in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage.

Base Pair Mismatch↗

PubMed2000

Sinoatrial node pacemaker activity requires Ca(2+)/calmodulin-dependent protein kinase II activation.

Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 micromol/L), or by KN-93 (0.1 to 3.0 micromol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 micromol/L AIP and 3 micromol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca(2+) current (I(Ca, L)), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 micromol/L), but not its inactive analog, KN-92, decreased I:(Ca, L) amplitude from 12+/-2 to 6+/-1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of I(Ca, L) from inactivation. Similar results were observed using the fast Ca(2+) chelator BAPTA, whereas the slow Ca(2+) chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca(2+) transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating I(Ca, L) inactivation and reactivation, and local Ca(2+) is critically involved in these processes.

Action Potentials↗

PubMed2000

Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos.

BACKGROUND: RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants. RESULTS: We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction. CONCLUSIONS: Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.

Animals↗

PubMed2000

Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines.

BACKGROUND: HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA. METHODS: We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided. RESULTS: The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA. CONCLUSIONS: Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.

Animals↗

PubMed2000

Highly beta-selective epoxidation of delta5-unsaturated steroids catalyzed by ketones.

A general catalytic and environmentally friendly method for beta-epoxidation of delta5-unsaturated steroids has been developed, which uses ketones as the catalysts and Oxone as the terminal oxidant. A whole range of delta5-unsaturated steroids, which bear different functional groups such as hydroxyl, carbonyl, acetyl, or ketal, as well as different side chains, were conveniently converted to the corresponding synthetically and biologically interesting 5beta,5beta-epoxides with excellent beta-selectivities and high yields.

Catalysis↗

PubMed2000

Differential regulation of responsiveness to fMLP and C5a upon dendritic cell maturation: correlation with receptor expression.

The trafficking of immature and mature dendritic cells (DCs) to different anatomical sites in vivo is critical for fulfilling their roles in the induction of Ag-specific immune responses. Although this process is complex and regulated by many mediators, the capacity of DCs to migrate is predominantly dependent on the expression of particular chemotactic receptors on the surface of DCs that enable them to move along chemotactic gradients formed by the corresponding chemokines and/or classical chemoattractants. Here we show that immature DCs (iDCs) respond to both fMLP and C5a as determined by chemotaxis and Ca2+ mobilization, whereas mature DCs (mDCs) respond to C5a, but not fMLP. Additionally, iDCs express the receptors for both fMLP and C5a at mRNA and protein levels. Upon maturation of DCs, fMLP receptor expression is almost completely absent, whereas C5a receptor mRNA and protein expression is maintained. Concomitantly, mDCs migrate chemotactically and mobilize intracellular Ca2+ in response to C5a, but not fMLP. Thus the interaction between C5a and its receptor is likely involved in the regulation of trafficking of both iDCs and mDCs, whereas fMLP mobilizes only iDCs. The differential responsiveness to fMLP and C5a of iDCs and mDCs suggests that they play different roles in the initiation of immune responses.

Animals↗

PubMed2000

Host gene regulation during coxsackievirus B3 infection in mice: assessment by microarrays.

Host genetic responses that characterize enteroviral myocarditis have not yet been determined. The injurious and inflammatory process in heart muscle may reflect host responses of benefit to the virus and ultimately result in congestive heart failure and dilated cardiomyopathy. On the other hand, host responses within the myocardium may secure the host against acute or protracted damage. To investigate the nature of modified gene expression in comparison with normal tissue, mRNA species were assessed in myocardium using cDNA microarray technology at days 3, 9, and 30 after infection. Of 7000 clones initially screened, 169 known genes had a level of expression significantly different at 1 or more postinfection time points as compared with baseline. The known regulated genes were sorted according to their functional groups and normalized expression patterns and, subsequently, interpreted in the context of viremic, inflammatory, and healing phases of the myocarditic process.

Animals↗

PubMed2000

Immunoregulation and blocking antibodies induced by interferon beta treatment in MS.

OBJECTIVE: To examine the in vivo immunoregulatory properties of interferon beta-1a (IFN beta-1a) on the T cell responses to myelin basic protein (MBP) and to evaluate the occurrence of the blocking antibodies characterized by the ability to reverse the effects of IFN beta on T cells in MS patients treated with IFN beta. METHODS: The precursor frequency of T cells recognizing MBP and control antigens was estimated in a microwell culture system. The cytokine profile of T cell lines was measured in ELISA. The binding antibodies were determined in ELISA and Western blot. Cytopathic test and the T cell functional assays were used to determine the blocking effects of the binding antibodies. RESULTS: Treatment with IFN beta resulted in a substantial reduction in the precursor frequency of MBP-reactive T cells in MS patients. The cytokine profile of MBP-reactive T cells that sustained the treatment was altered toward an increased production of interleukin (IL)-10 and decreased production of tumor necrosis factor (TNF)alpha and IFN-gamma. The immunoregulatory properties of IFN beta on T cells could be blocked by the binding antibodies derived from a proportion of patients treated with IFN beta (4 of 64, 6.25%). The blocking antibodies also neutralized anti-viral activity of IFN beta in cytopathic assays, corresponding to previously described neutralizing antibodies. CONCLUSIONS: Treatment with IFN beta alters the cytokine profile by enhancing the production of IL-10 and downregulating Th1 cytokines, which may contribute to clinical benefit in MS. The treatment also induces blocking antibodies that impair the immunoregulatory properties of IFN beta in some individuals.

Adjuvants, Immunologic↗

PubMed2000

Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein.

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.

Amino Acid Motifs↗

PubMed2000

Regioselective intramolecular oxidation of phenols and anisoles by dioxiranes generated in situ

A novel method for regioselective oxidation of phenols and anisoles has been developed in which dioxiranes, generated in situ from ketones and Oxone, oxidize phenol derivatives in an intramolecular fashion. A series of ketones with electron-withdrawing groups, such as CF(3), COOMe, and CH(2)Cl, were attached to phenols, anisoles, or aryl rings via a C(2) or C(3) methylene linker. In a homogeneous solvent system of CH(3)CN and H(2)O, oxidation of phenol derivatives 1-10 afforded spiro 2-hydroxydienones in 24-55% yields regardless of the presence of other substituents (ortho Me, meta Me or Br) on the aryl ring and the length of the linker. Experimental evidences were provided to support the mechanism that involves a regioselective pi bond epoxidation of aryl rings followed by epoxide rearrangement and hemiketal formation.

Journal Article↗

PubMed2000

Synthesis of B-ring homologated estradiol analogues that modulate tubulin polymerization and microtubule stability.

2-Methoxyestradiol is a cytotoxic human metabolite of estradiol with the ability to bind to the colchicine site of tubulin and inhibit its polymerization, and its 2-ethoxy analogue is even more potent. On the basis of a hypothetical relationship between the structures of colchicine and 2-methoxyestradiol, a B-ring-expanded 2-ethoxyestradiol analogue was synthesized in which the B-ring of the steroid is replaced by the B-ring of colchicine. The synthesis relied on the B-ring expansion of available 6-keto estradiol derivatives as opposed to a total synthesis of the homologated steroid framework. The relative configurations of the acetamido substituents in both epimers of the final product were determined by NOESY NMR and confirmed by X-ray crystallography. The epimer having the 6alpha-acetamido substituent was more active as an inhibitor of tubulin polymerization, and it was also more cytotoxic than the 6beta-epimer. These results are consistent with the proposed structural resemblance of 2-methoxyestradiol and colchicine. Several of the synthetic intermediates proved to be potent inhibitors of tubulin polymerization. On the other hand, a 3,17beta-diacetylated, B-ring-expanded analogue of 2-ethoxyestradiol having a ketone at C-6 resembled paclitaxel (Taxol) in its ability to enhance tubulin polymerization and stabilize microtubules. The corresponding 3-acetate and the 17beta-acetate were both synthesized, and it was determined that the 17beta-acetate, but not the 3-acetate, conferred on the steroid derivative its paclitaxel-like activity.

Animals↗

PubMed2000

Examination of novel non-phosphorus-containing phosphotyrosyl mimetics against protein-tyrosine phosphatase-1B and demonstration of differential affinities toward Grb2 SH2 domains.

Inhibitory potencies were compared of several mono- and dicarboxy-based pTyr mimetics in Grb2 SH2 domain versus PTP1B assays. Although in both systems pTyr residues provide critical binding elements, significant differences in the manner of recognition exist between the two. This is reflected in the current study, where marked variation in relative potencies was observed between the two systems. Of particular note was the poor potency of all monocarboxy-based pTyr mimetics against PTP1B when incorporated into a hexapeptide platform. The recently reported high PTP1B inhibitory potency of similar phenylphosphate mimicking moieties displayed in small molecule, non-peptide structures, raises questions on the limitations of using peptides as platforms for pTyr mimetics in the discovery of small molecule inhibitors.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Enantioselective total synthesis of (-)-triptolide, (-)-triptonide, (+)-triptophenolide, and (+)-triptoquinonide.

The first enantioselective total synthesis of (-)-triptolide (1), (-)-triptonide (2), (+)-triptophenolide (3), and (+)-triptoquinonide (4) was completed. The key step involves lanthanide triflate-catalyzed oxidative radical cyclization of (+)-8-phenylmenthyl ester 30 mediated by Mn(OAc)3, providing intermediate 31 with good chemical yield (77%) and excellent diastereoselectivity (dr 38:1). (+)-Triptophenolide methyl ether (5) was then prepared in > 99% enantiomeric excess (> 99% ee), and readily converted to natural products 1-4. In addition, transition state models were proposed to explain the opposite chiral induction observed in the oxidative radical cyclization reactions of chiral beta-keto esters 17 (without an alpha-substituent) and 17a (with an alpha-chloro substituent).

Crystallography, X-Ray↗

PubMed2000

Eosinophil-specific regulation of gp91(phox) gene expression by transcription factors GATA-1 and GATA-2.

The glycoprotein gp91(phox) is an essential component of the phagocyte NADPH oxidase and is expressed in eosinophils, neutrophils, monocytes, and B-lymphocytes. We previously suggested an eosinophil-specific mechanism of gp91(phox) gene expression. To elucidate the mechanism, we performed functional assays on deletion mutants of the gp91(phox) promoter in various types of gp91(phox)-expressing cells. A 10-base pair (bp) region from bp -105 to -96 of the promoter activated transcription of the gene in eosinophilic cells, but not in neutrophilic, monocytic, or B-lymphocytic cells. A 2-bp mutation introduced into the GATA site spanning bp -101 to -96 (-98GATA site) of the fragment abolished its activity. Gel shift assays using a GATA competitor and specific antibodies demonstrated that both GATA-1 and GATA-2 specifically bound to the -98GATA site with similar affinities. Individual transfection of GATA-1 and GATA-2 into Jurkat cells, which have neither endogenous GATA-1 nor GATA-2, activated the -105/+12 construct in a -98GATA site-dependent manner. Combined transfection of GATA-1 and GATA-2 activated the promoter less than transfection of GATA-1 alone. These results suggest that GATA-1 is an activator and that GATA-2 is a relative competitive inhibitor of GATA-1 in the expression of the gp91(phox) gene in human eosinophils.

Base Sequence↗

PubMed2000

Inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands. 2. 4-(2-Malonyl)phenylalanine as a potent phosphotyrosyl mimetic.

Nonhydrolyzable phosphotyrosyl (pTyr) mimetics serve as important components of many competitive Grb2 SH2 domain inhibitors. To date, the most potent of these inhibitors have relied on phosphonate-based structures to replace the 4-phosphoryl group of the parent pTyr residue. Reported herein is the design and evaluation of a new pTyr mimetic, p-malonylphenylalanine (Pmf), which does not contain phosphorus yet, in Grb2 SH2 domain binding systems, approaches the potency of phosphonate-based pTyr mimetics. When incorporated into high affinity Grb2 SH2 domain-directed platforms, Pmf is 15-20 times more potent than the closely related previously reported pTyr mimetic, O-malonyltyrosine (OMT). Pmf-containing inhibitors show inhibition constants as low as 8 nM in extracellular Grb2 binding assays and in whole cell systems, effective blockade of both endogenous Grb2 binding to cognate erbB-2, and downstream MAP kinase activation. Evidence is provided that use of an N(alpha)()-oxalyl auxiliary enhances effectiveness of Pmf and other inhibitors in both extracellular and intracellular contexts. As one of the most potent Grb2 SH2 domain-directed pTyr mimetics yet disclosed, Pmf may potentially have utility in the design of new chemotherapeutics for the treatment of various proliferative diseases, including breast cancer.

Adaptor Proteins, Signal Transducing↗