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At least 19 recordsLinked to original sources
PubMed2024

Synthetic DNA barcodes identify singlets in scRNA-seq datasets and evaluate doublet algorithms.

Single-cell RNA sequencing (scRNA-seq) datasets contain true single cells, or singlets, in addition to cells that coalesce during the protocol, or doublets. Identifying singlets with high fidelity in scRNA-seq is necessary to avoid false negative and false positive discoveries. Although several methodologies have been proposed, they are typically tested on highly heterogeneous datasets and lack a priori knowledge of true singlets. Here, we leveraged datasets with synthetically introduced DNA barcodes for a hitherto unexplored application: to extract ground-truth singlets. We demonstrated the feasibility of our framework, "singletCode," to evaluate existing doublet detection methods across a range of contexts. We also leveraged our ground-truth singlets to train a proof-of-concept machine learning classifier, which outperformed other doublet detection algorithms. Our integrative framework can identify ground-truth singlets and enable robust doublet detection in non-barcoded datasets.

Algorithms↗
PubMed1979

Immunochemical characterization of tumor-associated surface antigens on a Moloney leukemia virus-lymphoma, MBL-2.

Tumor-associated surface antigens (TASA) on a Moloney leukemia virus (M-MuLV)-induced lymphoma, MBL-2, in C57BL/6 mice (B6) were characterized. The surface proteins of MBL-2 cells were selectively radioiodinated and then extracted by Nonidet P40. The solubilized materials were then reacted with a variety of antisera: monospecific antisera to murine leukemia viral proteins (anti-gp69/71, anti-p30, anti-p15, anti-p12 and anti-p10), sera from B6 which regressed murine sarcoma tumors induced by murine sarcoma virus (anti-MSV) and a rabbit anti-MBL-2 antiserum. The resulting radioimmune precipitates were analyzed and compared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The following results were obtained. (1) Among all anti-viral protein antisera tested only anti-gp69/71 was active and detected a protein doublet of gp69/71 and its degradation fragments of 42,000 and 35,000 daltons. (2) Radioimmune precipitates prepared with anti-MSV showed a SDS-PAGE pattern similar to that seen with anti-gp69/71. This result indicated that the surface antigen detected by the anti-MSV serum on MBL-2 tumor cell was probably a viral envelope antigen. (3) The rabbit anti-MBL-2 serum detected on the cell membrane an antigen of approximately 95,000 daltons which was tumor-associated and did not appear to be related to virion components. The anti-MBL-2-serum still reacted with the 95,000 dalton antigen after absorption with disrupted M-MuLV virus and with gp69/71 and p30 purified from the virus.

Animals↗
PubMed1979

Heterogeneity in cell-associated transformation-sensitive proteins antigenically related to fibronectin.

Antiserum to purified fibronectin has been used to investigate transformation-associated heterogeneity in surface components antigenically related to fibronectin. Examination of extracts from surface-labeled rat cells by gradient gel electrophoresis revealed in "normal" cells the presence of two species with approximate molecular weights of 250,000 and 230,000, which were decreased in wild-type transformed cells. Reaction with antifibronectin serum revealed the selective precipitation of the two transformation-sensitive surface components. A similar experiment with ts-NT3-KR that expresses a normal phenotype at 37 degrees and a transformed morphology at 33 degrees did not reveal a markedly altered surface labeling pattern at both temperatures. However, reaction with antifibronectin serum did show a weak but detectable recognition of a 230,000-dalton doublet in the cells grown at 37 degrees, and immune precipitation of components in the 100,000- and 60,000-dalton region in cells grown at 33 degrees. Experiments with normal mouse cells revealed a different ratio of two fibronectin-related external proteins with similar molecular weights to those seen in normal rat cells.

Animals↗
PubMed1977

Identification of dynein as the outer arms of sea urchin sperm axonemes.

The location of dynein, the main flagellar ATPase, within the sea urchin sperm axoneme was investigated by the use of immunofluorescence and immunoelectron microscopy, employing an antiserum against a tryptic fragment of dynein 1 (Fragment 1A) purified from sea urchin sperm flagella. The axonemes were found to be stained with the antiserum when examined by an indirect immunofluorescence technique. Immunoelectron microscopy with the antiserum and a ferritin-conjugated IgG fraction of goat antiserum to rabbit IgG revealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles. With either the normal serum or antiserum absorbed with Fragment 1A, there were no ferritin particles within the axonemes. When the outer arms were extracted with 0.5 M NaCl, leaving the inner arms intact, again no ferritin dots were detected. Furthermore, it was found that the outer arm on the no. 5 doublet microtubule, which connects with the extra arm projection backward from the no. 6 doublet, had no attached ferritin particles. From these observations, it can be concluded that the outer arm consists of dynein (at least dynein 1) and that Fragment 1A, containing the active site for ATPase activity of dynein 1, is located at the distal end of the outer arms. The significance of the present findings is considered in connection with flagellar movement.

Adenosine Triphosphatases↗
PubMed1978

Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase.

Cholera toxin, using [32P]NAD+ as substrate, specifically radiolabels at least two proteins in plasma membranes of wild type S49 mouse lymphoma cells. The toxin-specific substrates are detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as bands corresponding to molecular weights of 45,000 and a doublet of 52,000 to 53,000. Membranes of two other cell types exhibit similar patterns of radiolabeled bands specifically produced by incubation with cholera toxin: the "uncoupled" variant S49 cell, which possesses adenylate cyclase activity unresponsive to hormones, and the HTC4 rat hepatoma cell, which lacks detectable catalytic adenylate cyclase activity but contains components of the cyclase system necessary for regulation by guanyl nucleotides and NaF. Little or no toxin-specific radiolabeling is observed in membranes of a fourth cell type, the adenylate cyclase activity-deficient S49 variant, which functionally lacks components of the cyclase system involved in cholera toxin action and regulation by guanyl nucleotides and NaF. The toxin-specific labeling pattern is not observed in membranes prepared from wild type S49 cells previously treated with cholera toxin in culture. One or both of the toxin substrates thus appears to be involved in regulation of adenylate cyclase by guanyl nucleotides and fluoride ion.

Adenylyl Cyclases↗
PubMed1977

Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.

Adenoviruses, Human↗
PubMed1979

The disposition of the synthetic prostaglandin analogue cloprostenol ('Estrumate') in the rat and marmoset.

1. Following subcutaneous administration of the synthetic prostaglandin analogue [14C]cloprostenol to the rat (200 micrograms/kg), the dose was quantitatively recovered from the excreta: 52% of the dose was present in the urine and 43% in faeces. After intravaginal administration (200 micrograms/kg) 42% of the dose was recovered from the excreta, equally divided between urine and faeces, and 40% (range 25--66%) of the dose was recovered from the site of application. The radiolabelled compounds present in faeces were eliminated initially via the bile. 2. The max. observed plasma concn. of total 14C in the rat was 84 ng equiv./ml at 30 min after subcutaneous administration of cloprostenol (200 micrograms/kg). A component which co-chromatographed with cloprostenol on t.l.c. was rapidly cleared from plasma with a half-life of 54 min. After intravaginal administration of cloprostenol (200 micrograms/kg), low and persistent plasma concn. of 14C were detected. 3. The metabolic fate of cloprostenol in the rat and marmoset has been studied with radiolabelled and non-labelled drug mixed such that fragments detected by mass spectrometry exhibited characteristic 12C:14C isotope clusters. Metabolites derived from cloprostenol contained these characteristic doublets. 4. In the rat cloprostenol is metabolized by beta-oxidation to tetranor-cloprostenol. Unchanged cloprostenol and a conjugate of tetranor-cloprostenol were minor urinary metabolites. In the rat biotransformation of cloprostenol in the cyclopentane ring occurred; the tetranor acid of 9-keto-cloprostenol was identified in urine. In the marmoset unchanged cloprostenol and dinor-cloprostenol were major urinary components.

Animals↗
PubMed1978

Detection of genetic variation with radioactive ligands. III. genetic polymorphism of transcobalamin II in human plasma.

We detected genetically determined, electrophoretic variants of vitamin B12 binding proteins, most probably transcobalamin II, in human plasma. Polymorphic variants were observed in all populations tested; the two most common alleles (of at least four detected to date) attain frequencies of greater than 40% in Caucasians and Orientals. The variants are autosomally inherited and are seen as doublets in homozygotes, and four-banded patterns, the sum of two dissimilar homozygote patterns, in heterozygotes. The technique used in this survey, polyacrylamide gel electrophoresis (PAGE) autoradiography of plasma and serum labeled in vitro with 57Co-vitamin B12 is particularly applicable to the study of trace proteins such as the transcobalamins (10(-9)M). Possible functional variation in the TC II allele products is described, and the selective significance of this worldwide polymorphism is considered.

Autoradiography↗
PubMed2026

Association of MPO Expression with the Immune Microenvironment in Breast Cancer: Insights from Bioinformatics and Single-Cell Analyses.

Breast cancer remains a major cause of cancer-related mortality, and exploratory computational workflows can help prioritize immune-associated markers for further investigation. Here, we used the cancer genome atlas breast invasive carcinoma (TCGA-BRCA) bulk transcriptomic data and the public single-cell dataset GSE161529 to examine associations between myeloperoxidase (MPO) expression, clinical outcomes, immune infiltration, methylation, upstream-regulator annotations, single-cell expression patterns, virtual knockdown sensitivity outputs, drug-gene interaction retrieval, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) annotation. MPO expression was lower in breast cancer tissues than in adjacent non-tumor tissues. Higher MPO expression was associated with a longer progression-free interval, whereas its associations with overall survival and disease-specific survival were not statistically significant. Receiver operating characteristic (ROC) analysis suggested tumor-normal separation within the analyzed public dataset, but this should not be interpreted as clinical diagnostic validation. Immune deconvolution and enrichment analyses indicated that MPO expression mainly tracked with immune- and myeloid-related transcriptional features, rather than establishing tumor-intrinsic regulation of the immune microenvironment. At single-cell resolution, the MPO signal was sparse, with only 85 MPO-positive cells detected before k-nearest neighbor (KNN)-based neighborhood expansion. Detectable MPO signal and MPO-associated scores were interpreted cautiously because they may be influenced by sparse expression, cell-type annotation uncertainty, dropout, doublets, or ambient RNA. In silico virtual knockdown suggested candidate immune- and inflammatory-related transcriptional changes, but these results were considered exploratory and require validation. Drug-gene interaction database (DGIdb)-based drug-gene retrieval and ADMET annotation were used only as preliminary chemical annotations and were not interpreted as therapeutic evidence. Overall, this study provides a reproducible in silico workflow for generating hypotheses about MPO-associated immune/myeloid features in breast cancer, which require external cohort validation and experimental confirmation.

Humans↗
PubMed1975

Studies on the mechanism of adenosylcobalamin-dependent ribonucleotide reduction by the use of analogs of the coenzyme.

A series of 17 analogs of 5'-deoxy-5'-adenosylcobalamin(adenosylcobalamin) have been synthesized with modifications in the base or ribose moiety of the nucleoside ligand. These analogs have been examined for their effects on reactions catalyzed by the ribonucleotide reductase of Lactobacillus leichmannii. All the analogs are inhibitors of ATP reduction in the presence of adenosylcobalamin as coenzyme, and hence all are bound to the catalytic site. Only the 3-beta-D-ribofuranosyladenine analog (isoadenosylcobalamin) showed substantial activity as a coenzyme in ATP reduction, giving a rate of 59% of that obtained with the adenosylcobalamin. Lesser rates of reduction were obtained with nebularyl-, 2'-deoxyadenosyl-, tubercidyl-, isopropylideneadenosyl-, L-adenosyl-, and ara-adenosylcobalamin, coenzyme activity decreasing in that order. Other analogs showed no significant coenzyme activity. The rate of hydrogen exchange into water from the 5'-methylene group of the nucleoside ligand appeared to parallel the coenzyme activity in those analogs examined, but only the four cobalamins with highest coenzyme activity (adenosyl, isoadenosyl, nebularyl, 2'-deoxyadenosyl) gave detectable amounts of "active coenzyme B12," THe rapidly formed paramagnetic intermediate of ribonucleotide reduction. The enzyme system produced the slowly formed paramagnetic species characterized by a doublet EPR spectrum only with adenosyl- and isoadenosylcobalamin. By contrast the enzymic degradation of analogs to cob(II)alamin and 5'-deoxynucleoside occurred not only with those analogs active as coenzymes and in the exchange reaction but also with a number of coenzymically inactive analogs, and the rate of degradation was unrelated to the rate of ribonucleotide reduction for those analogs with coenzyme activity.

Adenosine Triphosphate↗
PubMed1979

Mechanisms of cellular adhesion. IV. Role of serum glycoproteins in fibroblast spreading on glass.

We have investigated the exogenous factors required for the transition from the round shape of suspended fibroblasts to the characteristic spread shape on serum-coated glass. Following the evidence of others that the transition is facilitated by adsorbed component(s) related to CIG/LETS (cold-insoluble globulin/large external transformation-sensitive) proteins, we have isolated 2 such preparations from chick serum. Their influence has been investigated on fibroblast adhesion, spreading and growth and they have been characterized by gel electrophoresis, immunological cross-reactivity, amino acid and carbohydrate residue analysis, sedimentation velocity behaviour and circular dichroism spectroscopy. The preparations have molecular weights of 225/215,000 and 140,000 Daltons respectively and are closely similar in composition and secondary structure. The 225/215 000 Dalton doublet is probably a product of limited proteolysis which almost certainly occurred in the avian circulation. For cells seeded on glass precoated in different ways and in different supplemented media we could detect no change in the extent of attachment but there were profound influences on cell shape following this initial adhesion. We confirm that prior adsorption of either CIG-related preparation to glass does indeed promote fibroblast spreading in the absence of other serum components and that CIG is the sole serum component with this type of activity. We now add 2 important qualifications: (i) the presence of substrate-adsorbed serum CIG is not essential for spreading when other serum components are present in the medium; and (ii) the adhesive organization shown by interference reflexion microscopy is incompletely formed unless the additional serum components are present in the medium. We therefore conclude that 16C fibroblasts have the ability when given the stimulus of soluble serum components other than CIG, but not otherwise, to synthesize all the components necessary for the highly organized contacts with glass, including endogenous CIG/LETS proteins.

Amino Acids↗
PubMedDate not supplied

Group graph of the genetic code.

The genetic code doublets can be divided into two octets of completely degenerate and ambiguous coding dinucleotides. These two octets have the algebraic property of lying on continuously connected planes on the group graph (a tesseract) of the Cartesian product of two Klein 4-groups of nucleotide exchange operators. The K X K group can also be broken into four cosets, one of which has completely degenerate coding elements, and another that has completely ambiguous coding elements. The two octets of coding doublets have the further algebraic property that the product of their internal exchange operators naturally divide into two exactly equivalent sets. These properties of the genetic code are relevant to unraveling error-detecting and error-correcting (proof-reading) aspects of the genetic code and may be helpful in understanding the context-sensitive grammar of genetic language.

Genetic Code↗
PubMed1979

An iron-containing superoxide dismutase from Anacystis nidulans.

Superoxide dismutase (SOD) was isolated and purified from Anacystis nidulans to near electrophoretic homogeneity. The enzyme has a molecular weight of 37,500, as determined by gel filtration and SDS-gel electrophoresis. The enzyme molecule consists of two subunits of identical molecular weight. Proton-induced X-ray elemental analysis (PIXE) showed that the SOD of A. nidulans is an iron-containing enzyme; the Fe:enzyme mol ratio was found to be 1. The EPR spectra indicated that the active center contains high-spin ferric ion. Based on quantitative EPR data, we conclude that eseentially all iron ions were detected in the EPR experiments and were present in the Fe3+ active center. Effective g'-values were calculated from computer-simulated spectra and analysis of the g'-value anisotropy of the +/-3/2 Kramers doublet made the calculation of crystal field parameters possible. The symmetry of the Fe3+ ion in the SOD molecule was found to be close to rhombic (E/D=0.240).

Binding Sites↗
PubMed1976

A molecular concept of the properdin pathway.

The sequential events of the properdin system were analyzed. Properdin-depleted serum allows the formation of a Factor B- and D-dependent C3 convertase. This enzyme, called the properdin-receptor-forming enzyme, was shown to utilize a novel serum component, the initiating factor. The protein is a beta-globulin in precursor form and is distinct from immunoglobulins. The function of the enzyme is to deposit C3b on the surface of activator particles. Apparently doublets of C3b are required for the formation of the properdin-activating principle. It consists of a complex containing surface-bound C3b and activated Factor B. properdin precursor is activated by binding to this complex without detectable change in molecular weight. The transition of properdin precursor to activated properdin is probably caused by a conformational change. The complex, consisting of bound C3b, properdin, and activated Factor B, represents the enzyme that acts on C5, thereby initiating self-assembly of the membrane attack system. Native C3 is not needed for the function of the enzyme. It is disassembled by soluble C3 or C3b and its formation is under the control of the properdin-receptor-destroying enzyme, which may be identical with the C3b inactivator.

Beta-Globulins↗
PubMed2026

A Novel Homozygous Mutation in ARL2BP Causes Multiple Morphological Abnormalities of the Flagella and Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) and multiple morphological abnormalities of the sperm flagella (MMAF) frequently co-occur in male infertility. However, the genetic basis of this syndromic presentation remains unclear. Using whole-exome sequencing, we identified a novel homozygous ARL2BP splice-site mutation (c.294-2A>G) in a 23-year-old infertile male from a consanguineous family who presented with syndromic PCD and MMAF. This variant causes aberrant pre-mRNA splicing and triggers nonsense-mediated mRNA decay, resulting in the complete absence of ARL2BP protein expression. Transmission electron microscopy revealed extensive disorganization of flagellar axonemes with consistent central pair (CP) microtubule depletion and disorganization of peripheral doublets. Immunofluorescence confirmed a severe deficiency of the CP protein SPAG6 in the sperm flagella. Notably, the patient presented without retinal symptoms. Given that ARL2BP-related retinitis pigmentosa generally emerges during the third decade, long-term ophthalmological follow-up is essential to detect delayed-onset retinal degeneration. In conclusion, these findings confirm ARL2BP as a causative gene for both PCD and MMAF, expanding the genotypic and phenotypic spectrum of ciliopathies.

Humans↗
PubMed1976

Intragenic mutational spectra and hot spots.

In this review we outline the various factors which may contribute to the non-randomness of intragenic mutational spectra and the occurrence of hot spots. These factors include sample size limitation, particularly for sites of low mutability, and possible regions of low recombination potential. In addition, the nature of the gene product places great restraint on the detectability of either frameshift and premature chain-terminating mutations on one hand, or of the majority of missense mutations on the other. The nature of the Genetic Code itself also limits the mutational spectrum in so far as specific base pair substitutions lead only to a limited number of detectable amino acid replacements. Mutational hot spots may be a special example of the influence of neighbouring base pairs in the mutability of any given base pair. This is apparently true for frameshift mutations which tend to occur in runs of repeated base pairs or base pair doublets. Neighbouring base effects could operate not only at the level of initial reactivity with a mutagen, but also subsequently at the levels of DNA repair, recombination or replication. In some cases rare or modified bases may be responsible for neighbour effects. We suggest specific experimental approaches which seem likely to aid in the elucidation of these problems.

Base Sequence↗
PubMed1979

Specific contact-dependent cell-to-cell communication during preconjugant interactions of the ciliate Euplotes crassus.

A system has been developed to study cellular interactions between cells of complementary mating types prior to mating in the ciliate, Euplotes crassus. The presumptive mates were distinguished by using singlet and doublet cells of appropriate mating types in the mixtures. Cells of a given mating type were prelabelled with [3H]leucine and mixed with unlabelled complementary cells. Exchange of [3H]leucine-labelled material from donor to recipient cells was monitored through the various stages of the preconjugant interaction. A label transfer between the mating type complementary cells was detected from the beginning of the visible mating reaction, which occurs after a waiting period from the time of cell mixing and involves ciliary agglutination prior to cell body fusion. Complementary cells which were prevented from physically contacting each other and cells which were not competent to mate appeared unable to take up the labelled material. It is suggested that this material consists of some substance(s) playing an important role in the preconjugant cell-to-cell interactions of E. crassus.

Animals↗
PubMed1979

Site-specific instability in Drosophila melanogaster: the origin of the mutation and cytogenetic evidence for site specificity.

During a study of delayed mutations, an unstable X chromosome (Uc) was detected. Spontaneous X-linked recessive lethal mutations were detected in 34 of 993 sperm sampled from 50 males carrying this chromosome. All but three of the 34 lethals originated as clusters in three of the 50 males Cytogenetic and complementation analyses revealed 14 intrachromosomal rearrangements: ten inversions, two reverse repeats, one deficiency and one transposition. Eight of the 14 rearrangements have one break in the 6F1-2 doublet and two rearrangements have a break in 6F1-5 of the X chromosome. The remaining four rearrangements have in addition to the aberrations a lethal point mutation between 6F1 and 6F5. Though each of the lethal lines was established from a single lethal-bearing female, chromosome polymorphism is evident in 17 of the 18 lines having rearrangements, with certain aberrations recurring in several lines. The lethal mutations revert frequently to the nonlethal state, and cytological evidence indicates that more than one mutational event may occur at the unstable locus of the chromosome during one generation. Two lethal lines had more than one type of chromosome rearrangement sharing a common breakpoint. These observations are consistent with the view that the instability of the Uc lines is caused by a transposable element capable of site-specific chromosome breaks and perpetual generation of mutations. The mutagenic and genetic properties of transposable elements can be related to the two-mutation theory of KNUDSON (1971) for cancer initiation.

Animals↗