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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↗
PubMed1995

Control of protein-ligand recognition using a stimuli-responsive polymer.

Stimuli-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling. Here we show that conjugating a temperature-sensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable site-specific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 degrees C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.

Acrylic Resins↗
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↗
PubMed2026

Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.

Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.

CRISPR-Cas9↗
PubMed1987

Control of oligomeric enzyme thermostability by protein engineering.

The ability to control the resistance of an enzyme to inactivation due to exposure to elevated temperatures is essential for the understanding of thermophilic behavior and for developing rational approaches to enzyme stabilization. By means of site-directed mutagenesis, point mutations have been engineered in the dimeric enzyme yeast triosephosphate isomerase that improve its thermostability. Cumulative replacement of asparagine residues at the subunit interface by residues resistant to heat-induced deterioration and approximating the geometry of asparagine (Asn-14----Thr-14 and Asn-78----Ile-78) nearly doubled the half-life of the enzyme at 100 degrees C, pH 6. Moreover, in an attempt to model the deleterious effects of deamidation, we show that replacement of interfacial Asn-78 by an aspartic acid residue increases the rate constant of irreversible thermal inactivation, drastically decreases the reversible transition temperature, and reduces the stability against dilution-induced dissociation.

Carbohydrate Epimerases↗
PubMed1992

[Genetically engineered human insulin. 1. HPLC in analyzing products of basic production stages].

Application of some variants of HPLC for the step-by-step analysis of recombinant human insulin production was studied. Chromatographic columns with commercial and specially developed supports for size-exclusion, ion-exchange and reverse phase HPLC were used. Effective combinations of the chromatographic techniques for analysis of products and intermediates at every technological step were found and used for production of insulin. The authenticity of insulin obtained in the Shemyakin Institute of Bio-organic Chemistry by the scheme described in the present paper was confirmed by means of some physical and chemical methods and biological activity analysis.

Amino Acid Sequence↗
PubMed1988

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering.

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.

Amino Acid Sequence↗
PubMed1991

Chemotherapy and chemosensitization of transgenic mice which express the human multidrug resistance gene in bone marrow: efficacy, potency, and toxicity.

A common form of multidrug resistance in human cancer results from expression of the MDR1 gene which encodes a plasma membrane energy-dependent multidrug efflux pump. We have engineered transgenic mice which express this multidrug transporter in their bone marrow cells and demonstrated that peripheral WBC of these animals provide a rapid and reliable system for assessing the bioactivity of agents that reverse multidrug resistance. Immunocytochemical analysis of bone marrow smears suggests that the activation of the MDR1 transgene has probably occurred at a very early stage of bone marrow differentiation since most bone marrow cells express the transporter. Expression of this transgene in bone marrow produces about 10-fold resistance to leukopenia induced by taxol compared to normal bone marrow. Chemosensitization of MDR1 mice to daunomycin and taxol, measured by a fall in WBC, is detectable at a dose as low as 0.01 mg/kg R-verapamil. A dose of 0.5 mg/kg R-verapamil reduces the WBC by nearly 50%. Chemosensitization of MDR-transgenic mice with 5 mg/kg R-verapamil, which is highly effective in reversing MDR and readily tolerated by mice, necessitates a reduction of the maximum tolerated dose of most chemotherapeutic agents by only 20%. In addition, detailed histopathological examination shows that treatment of mice with chemotherapeutic drugs and R-verapamil does not change the organ-related toxicity pattern but only moderately accentuates inherent toxic side effects of the chemotherapeutic agents. We conclude that MDR1-transgenic mice represent a valid model for evaluating efficacy, potency, and toxicity associated with chemotherapy and chemosensitization of multidrug-resistant cells in animals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1987

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure.

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.

Allosteric Regulation↗