SEARCH · PubMed Health

Results for “reverse engineering”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources

PubMed1992

High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions.

A highly efficient expression for human acidic fibroblast growth factor (aFGF) has been assembled to direct the synthesis of both shortened and native full-length aFGF. The full-length aFGF-154 form of the protein had not been produced before in Escherichia coli by genetic engineering, and is obtained with its initiator methionine removed. The high production of the aFGF allows one to circumvent the use of reversed-phase chromatography (RPC) during the purification procedure. Here, it is shown that RPC, routinely used to obtain pure preparations of recombinant aFGF, modifies its chemical and physical properties in an unfavorable manner.

3T3 Cells↗

PubMed1992

Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage.

Inhibitors of human neutrophil elastase were engineered by designing and producing a library of phage-displayed protease inhibitory domains derived from wild-type bovine pancreatic trypsin inhibitor and fractionating the library for binding to the target protease. The affinity of one of the engineered variants for human neutrophil elastase (Kd = 1.0 pM) is 3.6 x 10(6)-fold higher than that of the parental protein and exceeds the highest affinity reported for any reversible human neutrophil elastase inhibitor by 50-fold. Thus the display phage method has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. The technology can be applied to design high-affinity proteins for a wide variety of target molecules.

Amino Acid Sequence↗

PubMed1988

Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation.

Replacement of the putative active site Asp residue of cloned HIV-1 protease with Ala yields a molecule incapable of autocatalytic processing. Similarly, protease/reverse transcriptase and protease/reverse transcriptase/endonuclease polyproteins containing the same mutation accumulate as enzymatically inert polyproteins. Introduction of a second, wild-type, copy of protease in trans alleviates this defect, leading in the case of individually cloned protease to cleavage of the mutant protein, and with the polyprotein mutants to release of the reverse transcriptase and endonuclease polypeptides, the former of which recover enzymatic activity. In related experiments, a similar inhibition and trans-complementation of a genetically engineered gag--protease fusion protein was observed.

Amino Acid Sequence↗

PubMed1985

Visual effects with a miniature Leonardo's window: photographs and real scenes fused stereoscopically.

The drawing on a conventional Leonardo's window will be seen by both of an observer's eyes unless one of them is occluded. But, by substituting a photographic colour transparency (in a suitably compact viewer) for the drawing, one eye can view the transparency whilst the other eye gains an unobstructed direct view of the scene. The transparency can then be stereoscopically fused with the directly-viewed scene. As the perspective of the transparency depends not on the observer's viewpoint but only on the position of the camera at the time of photography, altering the viewpoint causes the effective eye-base to vary, thus enhancing, suppressing, or sometimes even reversing the stereoscopic depth. With very simple equipment several visual effects can easily be demonstrated. Practical applications of the technique have been found, particularly in those engineering and geological situations in which it is useful to detect changes since a photograph was taken.

Adult↗

PubMed2026

Systematic mapping of insertion-tolerant regions enables capsid engineering of an infectious RNA phage.

RNA phages are attractive platforms for the design of programmable bioparticles, but their development has been constrained by limited knowledge of genomic sites that can tolerate sequence insertion. Here, we combined MuA transposase-mediated in vitro insertion mutagenesis with our established reverse genetics systems to systematically identify insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. Screening of 4,555 MS2 and 2,228 PP7 random insertion clones identified 29 and 26 non-redundant ITRs, respectively. We further analyzed and compared these ITRs in the context of RNA genome organization and virion architecture. Both phages contained ITRs within the maturation protein, whereas only PP7 tolerated insertions within the coat protein (CP). On the basis of structural location and plaque-forming capacity, an ITR situated between Gly74 and Glu75 (GGC^GAG) in the PP7 CP was selected for further study. Infectious phage particles generated from complementary DNA clones retained the 15-bp insertion at both the RNA and protein levels. Engineered PP7 phages carrying an Arg-Gly-Asp motif inserted into the CP at this ITR displayed enhanced in vivo clearance in a Drosophila model, despite having in vitro stability comparable to that of the wild type. These findings provide the first example of CP engineering in an infectious RNA phage and establish a framework for engineering RNA phages for biological and biotechnological applications.IMPORTANCEA major obstacle to developing RNA phages as synthetic biology platforms is the lack of design principles for genomic insertion. Here, we address this limitation by establishing a mutagenesis-and-recovery workflow that systematically identifies insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. The resulting maps reveal distinct structural constraints in the two phages and enable rational engineering of a peptide-display site in the PP7 capsid. Using this approach, we generated an engineered infectious phage with a modified capsid, thereby providing the first demonstration of capsid engineering in an infectious RNA phage, to our knowledge. This study lays the groundwork for the rational design of live RNA phage virions as tractable and engineerable scaffolds for future biological and biotechnological applications.

Animals↗

PubMed1992

On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli.

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.

Amino Acid Sequence↗

PubMed1989

Mutants of pertussis toxin suitable for vaccine development.

Immunization with chemically detoxified pertussis toxin can prevent severe whooping cough with an efficacy similar to that of the cellular pertussis vaccine, which normally gives unwanted side effects. To avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussis toxin, inactive toxins were constructed by genetic manipulation. A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These strains produce mutant pertussis toxin molecules that are nontoxic and immunogenic and that protect mice from the intracerebral challenge with virulent Bordetella pertussis. Such molecules are ideal for the development of new and safer vaccines against whooping cough.

Animals↗

PubMedDate not supplied

Site-directed mutagenesis for quantitation of base-base interactions at defined sites.

Two alkylation products implicated in initiation of carcinogenesis are O6-alkylguanine (m6G) and O4-alkylthymine (m4T). We have used site-specific insertion of these derivatives into oligonucleotides and measured the kinetic constants of various pairings, using both prokaryotic and eukaryotic polymerases for replication. Preliminary data are also reported for another carcinogen product, N2,3-ethenodeoxyguanosine ( epsilon G). The immediate neighbor bases play an important role in determining the frequency of specific changed basepairing and subsequent elongation of the annealed primer. However, both m4T and m6G prefer to form a type of G.T pairing which would lead to the transitions: G.C----A.T or T.A----C.G. The enzymes were the Klenow fragment of E. coli DNA polymerase I (Kf), engineered 3'----5' exonuclease-free Kf (exo-free Kf), polymerase alpha-primase complex from Drosophila melanogaster or calf thymus, and human immunodeficient virus-I reverse transcriptase (HIV-I RT). All enzymes led to approximately the same frequency of transitions. It is postulated that the mutation frequency at a given site is primarily a function of the structure of the sequence around the target site.

Animals↗

PubMed2026

Mechanisms linking the gut microbiota to colorectal cancer development and progression.

Colorectal cancer remains a leading cause of global cancer mortality, with a concerning rise in early-onset cases driven by complex interactions between environmental exposures, lifestyle factors, and host genetics. Mounting evidence indicates that gut microbiota dysbiosis critically modulates this oncogenic process, acting as an active participant rather than a passive bystander. This review systematically synthesizes the dichotomous roles of the intestinal microbiome in colorectal tumorigenesis through the conceptual framework of the driver-passenger model. We discuss how early initiating driver bacteria, such as Polyketide synthase-positive Escherichia coli and enterotoxigenic Bacteroides fragilis, compromise mucosal barriers, induce chronic mucosal inflammation, and inflict direct genomic instability. As the local tumor microenvironment undergoes profound metabolic remodeling, opportunistic passenger pathogens, notably Fusobacterium nucleatum, become enriched, further promoting cellular proliferation and facilitating tumor immune evasion. Conversely, protective commensals, exemplified by Clostridium butyricum and Streptococcus thermophilus, exert robust tumor-suppressive effects through multifaceted mechanisms. These beneficial microbes actively antagonize malignant progression by redirecting tumor metabolic fluxes toward oxidative stress, orchestrating deep epigenetic reprogramming, and degrading core oncoproteins to reverse chemoresistance. Transitioning from fundamental mechanisms to clinical application, we evaluate a comprehensive spectrum of microbiota-targeted interventions, encompassing non-invasive diagnostic biomarkers, fecal microbiota transplantation, engineered bacteria, phage therapy, and postbiotics. Finally, we critically address the formidable translational challenges associated with microbial heterogeneity, long-term safety, and regulatory standardization, aiming to provide a balanced perspective on integrating microbiome-based strategies into next-generation precision oncology for colorectal cancer.

Humans↗

PubMed1989

Low-temperature unfolding of a mutant of phage T4 lysozyme. 1. Equilibrium studies.

The mutant protein I3C-C97/C54T of phage T4 lysozyme is free of sulfhydryl groups and has a genetically engineered disulfide bridge between positions 3 and 97 (Perry & Wetzel, 1986). This protein has a maximum stability at 12 degrees C in 3 M guanidinium chloride and undergoes reversible high- and low-temperature melting at 28 and -3 degrees C, respectively, in this medium. The free energy of stabilization of the protein has been studied over a range of temperature that includes both melting transitions. The stability curve fits a constant delta Cp model over the entire range, permitting an unusually complete determination of the thermodynamic parameters of the protein and demonstrating that the low-temperature unfolded form of the protein may be interpreted as an extrapolation with constant delta Cp of the high-temperature unfolded form. The free energy of unfolding is a linear function of guanidinium concentration within experimental error which permits a rough estimate of the stability of the protein at low temperatures and of the differential interaction of the unfolded protein with guanidinium chloride. These equilibrium studies provide a basis for the interpretation of the kinetic studies reported in the following paper.

Calorimetry↗

PubMed1991

Novel guanosine requirement for catalysis by the hairpin ribozyme.

THERE is much interest in the development of 'designer ribozymes' to target destruction of RNAs in vitro and in vivo. Engineering of ribozymes with novel specificities requires detailed knowledge of the ribozyme-substrate interaction, and a rigorous evaluation of sequence specificity. The hairpin ribozyme catalyses an efficient and reversible site-specific cleavage reaction. We have used mutagenesis and in vitro selection strategies to show that RNA cleavage and ligation has an absolute requirement for guanosine immediately 3' to the cleavage-ligation site. This G is not required for efficient substrate binding, rather, its 2-amino group is an essential component of the active site required for catalysis.

Base Sequence↗

PubMed2026

Protein Language Model Decoys for Target Decoy Competition in Proteomics: Quality Assessment and Benchmarks.

Large-scale proteomics relies heavily on target-decoy competition for false discovery rate estimation in peptide identification, and the performance of this strategy depends strongly on the design of the decoy database. Classical generators such as reversal and shuffling remain widely used. Here, we introduce the first protein language model-based (PLM) decoy generation for peptide identification and benchmark it against classical strategies. We evaluate these approaches using three complementary quality-control layers: sequence-based separability, search-engine-agnostic spectral-space diagnostics, and end-to-end mass spectrometry benchmarks, including pipelines with rescoring. Across these analyses, PLM-based decoys are harder for sequence-only neural networks to distinguish than most classical generators, suggesting fewer obvious sequence-level artifacts. However, this signal is only weakly informative for search performance. Spectral diagnostics further show that short peptides occupy a particularly crowded target-decoy space and are therefore especially prone to local collisions across all generators. In full search pipelines, reverse decoys remain a strong baseline, and current PLM-based generators do not yet provide a clear overall advantage. We therefore view PLM-based decoys not as universal replacements for reverse decoys but as tunable tools for benchmarking, diagnostics, stress testing, and future adaptive decoy optimization, with increasing value as search models become more expressive.

Proteomics↗

PubMed2025

Disassembly activates Retron-Septu for antiphage defense.

Retrons are antiphage defense systems that produce multicopy single-stranded DNA (msDNA) and hold promise for genome engineering. However, the mechanisms of defense remain unclear. The Retron-Septu system integrates retron and Septu antiphage defenses. Cryo-electron microscopy structures reveal asymmetric nucleoprotein complexes comprising a reverse transcriptase, msDNA (a hybrid of msdDNA and msrRNA), and two PtuAB copies. msdDNA and msrRNA are essential for assembling this complex, with msrRNA adopting a conserved lariat-like structure that regulates reverse transcription. Notably, the assembled Retron-Septu complex is inactive, with msdDNA occupying the PtuA DNA binding site. Activation occurs upon disassembly, releasing PtuAB, which degrades single-stranded DNA to restrict phage replication. This "arrest-and-release" mechanism underscores the dynamic regulatory roles of msDNA, advancing our understanding of antiphage defense strategies.

Cryoelectron Microscopy↗

PubMed2026

Landscape of retron diversity across the SPIRE microbial metagenome resource reveals candidate novel type XI-like lineages.

Retrons are bacterial genetic elements encoding a specialized reverse transcriptase (RT) that synthesizes multicopy single-stranded DNA and are increasingly recognized as components of bacterial anti-phage defense systems. However, their diversity and ecological distribution across large-scale genomic resources remain poorly characterized. Here, we surveyed retron RTs across the SPIRE representative metagenome collection, a non-redundant, species-level data set spanning diverse microbial habitats. Using a curated panel of type-specific hidden Markov models, we identified retrons representing all canonical types together with additional divergent lineages. Retron distribution showed strong taxonomic and ecological structuring, with some groups restricted to specific bacterial phyla, whereas others were broadly distributed across environmental categories. Systematic novelty assessment identified two candidate type XI-like lineages, TXI_C2like and TXI_noncan_h, characterized by protease-independent architectures and distinct accessory modules associated with WYL- and DnaB_C-containing proteins, respectively. De novo covariance-based analyses further identified candidate msr/msd-like non-coding RNA structures in both lineages, supporting conservation of the canonical RT-ncRNA organizational framework despite extensive sequence divergence. Together, these findings expand the known diversity of retron systems and identify type XI-like retrons as a dynamic and previously underexplored evolutionary group.IMPORTANCERetrons are bacterial genetic elements that are increasingly exploited as programmable tools for genome editing, molecular recording, and biosensing in addition to their natural role in anti-phage defense. Despite this growing biotechnological interest, the true diversity of retrons across the bacterial world has remained largely unmapped. By mining a resource of over 100,000 processed microbial metagenomes, we uncovered thousands of retron sequences spanning known types as well as previously unrecognized lineages and found that their distribution is strongly shaped by both bacterial taxonomy and ecological niche. Among these, we identified two candidate new lineages related to type XI retrons that lack the protease domain typical of this group but instead carry distinct accessory proteins, expanding the known architectural diversity of these systems. These findings broaden the catalog of retron diversity available for functional characterization and biotechnological engineering and provide a framework for prioritizing candidate lineages for future experimental validation.

effectors↗

PubMed1978

Reversible respiratory disease in beryllium workers.

A medical and environmental survey was carried out in a beryllium extraction and processing plant in 1971, and a follow-up study was done in 1974. Peak air concentrations of beryllium of as much as 50 times the accepted peak limit value were found in the plant in 1971. Radiographic changes of interstitial disease were found in 31 workers, hypoxemia was found in 20, and in 11 workers a combination of hypoxemia and radiographic changes of interstitial disease was seen. Follow-up in 1974 showed that air concentrations had all decreased significantly, mainly because of improvements in engineering and ventilation in the plant. Improvement in hypoxemia and decreased alveolar-arterial O2 tension difference at rest ([A-a]DO2) were noted in 13 of the 20 men who had hypoxemia in 1971 and were available for follow-up. In addition, radiographic abnormalities of interstitial disease were reversed in some workers. Our results suggest that improvement in gas exchange and radiographic findings of interstitial disease occurs when peak air concentrations of beryllium are decreased. Our approach in combining environmental and medical screening in workers exposed to beryllium enabled us to identify persons with early changes, to detect reversible abnormalities, and to correlate air concentrations with the medical status of workers.

Adult↗

PubMed2026

Bacterial R-bodies with common morphologies and unrolling dynamics are phylogenetically scattered, indicating extensive lateral gene transfer and wide application potential.

Refractile bodies (R-bodies) of gram-negative bacteria are large proteinaceous assemblies, rolled up in the form of an Archimedean spiral. They exhibit rapid rod-like reversible extension in the micrometer range when cued by chemical environmental triggers and have potential for synthetic biology and biochip applications. Initially described for the Paramecium endosymbionts Caedibacter taeniospiralis and Caedimonas varicaedens, R-bodies have since been discovered in many classes of Pseudomonadota, both in endosymbionts and in non-endosymbionts. However, despite the fact that the genetics and morphologies, as well as the unrolling kinetics of R-bodies from different species, show considerable diversity, no recent study has integrated these aspects into a single framework. The latter would be advantageous for the creation of an R-body biotechnology toolbox, where different properties determine the application area. Here, we have examined the R-bodies from six different Pseudomonadota, comprising both phylogenetically diverse endosymbionts and non-endosymbionts. Comparison of the morphologies of the rolled-up and unrolled forms, obtained using electron microscopy and high-quality images, to their corresponding genetic data indicates that extensive lateral gene transfer has occurred, which confounds a common framework based on these data. However, we have also studied the R-body extension and retraction kinetics using high frame-rate light microscopic video recordings, where we show for the first time that R-bodies can be classified into two classes, showing "fast burst" or "slow" acid-induced extension kinetics, respectively. We propose that this criterion may, in fact, be the most useful for the choice of an R-body tool for biotechnological purposes.IMPORTANCER-bodies are unique proteinaceous macromolecular structures capable of massive reversible extension in response to external environmental triggers without the input of chemical energy. They comprise only a few small polypeptides, which makes them potentially highly amenable to tuning via genetic engineering, as well as being exceptionally stable. These properties would be highly desirable in biotechnology and synthetic biology, as well as in biochip applications, where a controlled mechanical extensor might play an integral part in a nanoscale molecular machine. So far, only R-bodies from a single species, Caedibacter taeniospiralis, have been characterized extensively. However, in recent years, genomic information has revealed that a panoply of R-bodies are widely distributed among gram-negative phyla, although studies have generally not included morphological data. This study brings these two areas together to provide a holistic overview of the field and also reveals new insights into key dynamic aspects of R-body extension.

R-bodies↗

PubMedDate not supplied

[Genetic engineering of peptide hormones. III. Cloning of the swine growth hormone cDNA and construction of the gene for expression of the hormone in bacteria].

The clones containing cDNA of porcine growth hormone were obtained using poly(A)-RNA from porcine pituitary as a template for reverse transcriptase. The analysis of their nucleotide sequences revealed that these cDNAs have differences not only on the nucleotide level but also on the amino acid level, i. e. the polymorphism of mRNA and protein occurs in the case of porcine growth hormone. To create the construction for expression of porcine growth hormone in E. coli, the 5'-part of cDNA, coding the first 15 amino acids of the mature hormone, was substituted by the artificial sequence.

Amino Acid Sequence↗

PubMed1990

Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

Adenoviridae↗