Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin.
The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.
Separable structural requirements for cDNA synthesis, non-templated extension, and template jumping by a non-LTR retroelement reverse transcriptase
- Broad evolutionary expansion of polymerase families has enabled specialization of their activities for distinct cellular roles. In addition to template-complementary synthesis, many polymerases extend their duplex products by non-templated nucleotide addition (NTA). This activity is exploited for laboratory strategies of cloning and sequencing nucleic acids and could have important biological function, although the latter has been challenging to test without separation-of-function mutations. Several retroelement and retroviral reverse transcriptases (RTs) support NTA and also template jumping, by which the RT performs continuous complementary DNA (cDNA) synthesis using physically separate templates.
- Previous studies that aimed to dissect the relationship between NTA and template jumping leave open questions about structural requirements for each activity and to what extent they are interdependent. Here, we characterize the structural requirements for cDNA synthesis, NTA, template jumping, and the unique terminal transferase activity of Bombyx mori R2 non-LTR retroelement RT. With sequence alignments and structure modeling to guide mutagenesis, we generated enzyme variants across motifs generally conserved or specific to RT subgroups. Enzyme variants had diverse NTA profiles that were not correlated with other changes in cDNA synthesis activity or template jumping.
- Using these enzyme variants and panels of activity assay conditions, we show that template jumping requires NTA. However, template jumping by NTA-deficient enzymes can be rescued using primer duplex with a specific length of 3′ overhang. Our findings clarify the relationship between NTA and template jumping as well as additional activities of non-LTR RTs, with implications for specialization of RT biological functions and laboratory applications.
Enhancing biological signals and detection rates in single-cell RNA-seq experiments with cDNA library equalization
Considerable effort has been devoted to refining experimental protocols to reduce levels of technical variability and artifacts in single-cell RNA-sequencing data (scRNA-seq). We here present evidence that equalizing the concentration of cDNA libraries prior to pooling, a step not consistently performed in single-cell experiments, improves gene detection rates, enhances biological signals, and reduces technical artifacts in scRNA-seq data. To evaluate the effect of equalization on various protocols, we developed Scaffold, a simulation framework that models each step of an scRNA-seq experiment.
Numerical experiments demonstrate that equalization reduces variation in sequencing depth and gene-specific expression variability. We then performed a set of experiments in vitro with and without the equalization step and found that equalization increases the number of genes that are detected in every cell by 17-31%, improves discovery of biologically relevant genes, and reduces nuisance signals associated with cell cycle. Further support is provided in an analysis of publicly available data.
Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus
Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified into the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Herein, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated recombinant virus (rEGFP), wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene.
The rEGFP expressed EGFP in infected cells, but showed significantly lower viral growth compared to wtBToV. Moreover, the rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in non-structural proteins (NSPs) that emerged during passages exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a similar phenotype to these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen.
Importance ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, infection pathogenesis, and BToV vaccine development.
cDNA - Plant Normal Tissue: cDNA - Plant: Corn |
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| C1634330 | Biochain | 40 reactions | 741 EUR |
cDNA - Plant Normal Tissue: cDNA - Plant: Rice |
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| C1634360 | Biochain | 40 reactions | 741 EUR |
cDNA - Plant Normal Tissue: cDNA - Plant: Wheat |
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| C1634390 | Biochain | 40 reactions | 741 EUR |
cDNA - Plant Normal Tissue: cDNA - Plant: Orange |
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| C1634340 | Biochain | 40 reactions | 741 EUR |
cDNA - Plant Normal Tissue: cDNA - Plant: Potato |
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| C1634350 | Biochain | 40 reactions | 741 EUR |
cDNA - Plant Normal Tissue: cDNA - Plant: Arabidopsis |
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| C1634310 | Biochain | 40 reactions | 741 EUR |
Oak cDNA |
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| PLD-1005 | Zyagen | 30 reactions | 498 EUR |
Fig cDNA |
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| PLD-1042 | Zyagen | 30 reactions | 498 EUR |
Oat cDNA |
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| PLD-1096 | Zyagen | 30 reactions | 498 EUR |
Rye cDNA |
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| PLD-1097 | Zyagen | 30 reactions | 498 EUR |
Corn cDNA |
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| PLD-1002 | Zyagen | 30 reactions | 498 EUR |
Rice cDNA |
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| PLD-1004 | Zyagen | 30 reactions | 498 EUR |
Pear cDNA |
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| PLD-1033 | Zyagen | 30 reactions | 498 EUR |
Pine cDNA |
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| PLD-1063 | Zyagen | 30 reactions | 498 EUR |
Apple cDNA |
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| PLD-1001 | Zyagen | 30 reactions | 498 EUR |
Beans cDNA |
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| PLD-1051 | Zyagen | 30 reactions | 498 EUR |
Grape cDNA |
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| PLD-1052 | Zyagen | 30 reactions | 498 EUR |
Lemon cDNA |
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| PLD-1062 | Zyagen | 30 reactions | 498 EUR |
Wheat cDNA |
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| PLD-1084 | Zyagen | 30 reactions | 498 EUR |
Onion cDNA |
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| PLD-1092 | Zyagen | 30 reactions | 498 EUR |
Rapid and selective detection of Bacillus cereus in food using cDNA-based up-conversion fluorescence spectrum copy and aptamer modified magnetic separation
- A sensitive luminescent bioassay for the detection of Bacillus cereus (B. cereus), a common bacterium, harmful to human health, was established based on up-conversion fluorescence and magnetic separation technology. Herein, aptamers (Apt) are modified on the surface of magnetic nanoparticles (MNPs) to form Apt-MNPs capture probes. The aptamer complementary strands (cDNA) are connected to upconversion nanoparticles (UCNPs) to form UCNPs-cDNA signal probes. In the absence of analyte, the UCNPs-cDNA-Apt-MNPs complex will be formed due to the specific binding between the aptamer and the complementary strand.
- In the presence of B. cereus, the amount of free UCNPs-cDNA increased in the system, which ultimately increased the fluorescence intensity of the solution. Hence, when the UCNPs-cDNA-Apt-MNPs system was excited by a 980 nm near-infrared light, a decrease in the fluorescence of the complex was observed at 548 nm due to the detachment of UCNPs-cDNA. Therefore, based on this principle, the calibration curve is constructed between the concentration of the analyte (B. cereus) and the fluorescence intensity.
- The results show that the method has a good quantitative ability for B. cereus in the range of 49-49 × 106 cfu/mL under the optimal conditions with a detection limit of 22 cfu/mL. Moreover, the proposed detection method also exhibits a high degree of specificity. The spiked recovery rate of the actual sample was in the range of 90.54%-111.28%, with good relative standard deviation values (2.12%-3.13%), indicating that the method has good reproducibility and stability. This study demonstrates that the constructed method can be used successfully for the rapid detection of B. cereus in food.