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International Journal of Molecular... Jun 2024Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell...
Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase ( NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.
Topics: Nitroreductases; Nitroimidazoles; Metronidazole; Prodrugs; Escherichia coli Proteins; Positron-Emission Tomography; Escherichia coli; Catalytic Domain; Protein Engineering; Models, Molecular; Aziridines
PubMed: 38928299
DOI: 10.3390/ijms25126593 -
International Journal of Molecular... Jun 2024Polymerase Chain Reaction (PCR) amplification is widely used for retrieving information from DNA storage. During the PCR amplification process, nonspecific pairing...
Polymerase Chain Reaction (PCR) amplification is widely used for retrieving information from DNA storage. During the PCR amplification process, nonspecific pairing between the 3' end of the primer and the DNA sequence can cause cross-talk in the amplification reaction, leading to the generation of interfering sequences and reduced amplification accuracy. To address this issue, we propose an efficient coding algorithm for PCR amplification information retrieval (ECA-PCRAIR). This algorithm employs variable-length scanning and pruning optimization to construct a codebook that maximizes storage density while satisfying traditional biological constraints. Subsequently, a codeword search tree is constructed based on the primer library to optimize the codebook, and a variable-length interleaver is used for constraint detection and correction, thereby minimizing the likelihood of nonspecific pairing. Experimental results demonstrate that ECA-PCRAIR can reduce the probability of nonspecific pairing between the 3' end of the primer and the DNA sequence to 2-25%, enhancing the robustness of the DNA sequences. Additionally, ECA-PCRAIR achieves a storage density of 2.14-3.67 bits per nucleotide (bits/nt), significantly improving storage capacity.
Topics: Algorithms; Polymerase Chain Reaction; DNA; Information Storage and Retrieval; DNA Primers; Base Sequence
PubMed: 38928155
DOI: 10.3390/ijms25126449 -
Cancers Jun 2024The prediction of the regrowth potential of pituitary adenomas after surgery is challenging. The genome-wide DNA methylation profiling of pituitary adenomas may separate...
BACKGROUND
The prediction of the regrowth potential of pituitary adenomas after surgery is challenging. The genome-wide DNA methylation profiling of pituitary adenomas may separate adenomas into distinct methylation classes corresponding to histology-based subtypes. Specific genes and differentially methylated probes involving regrowth have been proposed, but no study has linked this epigenetic variance with regrowth potential and the clinical heterogeneity of nonfunctioning pituitary adenomas. This study aimed to investigate whether DNA methylation profiling can be useful as a clinical prognostic marker.
METHODS
A DNA methylation analysis by Illumina's MethylationEPIC array was performed on 54 pituitary macroadenomas from patients who underwent transsphenoidal surgery during 2007-2017. Twelve patients were excluded due to an incomplete postoperative follow-up, degenerated biobank-stored tissue, or low DNA methylation quality. For the quantitative measurement of the tumor regrowth rate, we conducted a 3D volumetric analysis of tumor remnant volume via annual magnetic resonance imaging. A linear mixed effects model was used to examine whether different DNA methylation clusters had different regrowth patterns.
RESULTS
The DNA methylation profiling of 42 tissue samples showed robust DNA methylation clusters, comparable with previous findings. The subgroup of 33 nonfunctioning pituitary adenomas of an SF1-lineage showed five subclusters with an approximately unbiased score of 86%. There were no overall statistically significant differences when comparing hazard ratios for regrowth of 100%, 50%, or 0%. Despite this, plots of correlated survival estimates suggested higher regrowth rates for some clusters. The mixed effects model of accumulated regrowth similarly showed tendencies toward an association between specific DNA methylation clusters and regrowth potential.
CONCLUSION
The DNA methylation profiling of nonfunctioning pituitary adenomas may potentially identify adenomas with increased growth and recurrence potential. Larger validation studies are needed to confirm the findings from this explorative pilot study.
PubMed: 38927917
DOI: 10.3390/cancers16122210 -
Bioengineering (Basel, Switzerland) Jun 2024The rapid detection of the spore form of has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for...
The rapid detection of the spore form of has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from spores via a bespoke microwave lysis platform. spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen's two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within and toxin genes of and was able to detect 800 spores of within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for .
PubMed: 38927868
DOI: 10.3390/bioengineering11060632 -
Genes Jun 2024The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated...
The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/μL in the minimum extraction volume (40 μL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R, and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event.
Topics: Humans; Real-Time Polymerase Chain Reaction; Forensic Genetics; DNA; DNA Fingerprinting; Microsatellite Repeats; Semen
PubMed: 38927695
DOI: 10.3390/genes15060759 -
Genes May 2024With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still...
With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-h), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-h on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.
Topics: Erythropoietin; Animals; Mice; Doping in Sports; Dependovirus; Humans; Genetic Vectors; Male; Genetic Therapy; Models, Animal
PubMed: 38927645
DOI: 10.3390/genes15060709 -
Biomedicines May 2024Examinations of ovarian cancer cells require the ability to identify tumor cells. Array-based comparative genome hybridization (aCGH) on 30 ovarian carcinomas (OC)...
Examinations of ovarian cancer cells require the ability to identify tumor cells. Array-based comparative genome hybridization (aCGH) on 30 ovarian carcinomas (OC) identified three genomic loci (8q24.23; 17p12; 18q22.3) over- or under-represented in OC. A fluorescence in situ hybridization (FISH) probe of these three loci is intended to identify tumor cells by their signal pattern deviating from a diploid pattern. Human DNA from these three loci is isolated from bacterial artificial chromosomes (BAC), amplified and labeled with fluorescent dyes. After a standard FISH procedure, 71 OC suspensions from primary tumors, three OC cell lines, three lymphocyte suspensions, and one mesenchymal cell line LP-3 are analyzed with a fluorescence microscope. On average, 15% of the lymphocytes deviate from the expected diploid signal pattern, giving a cut-off of 36%. If this value is exceeded, tumor cells are detected. The mesenchymal cell line LP-3 shows only 21% as a negative control. The OC cell lines as positive controls exceed this value at 38%, 67%, and 54%. Of the 71 OC primary cultures, four cases fell below this cut-off as false negatives. In the two-sample t-test, the percentages of conspicuous signal patterns differ significantly.
PubMed: 38927378
DOI: 10.3390/biomedicines12061171 -
Biology May 2024Conifers are an ecologically and economically important seed plant group that can provide significant insights into the evolution of land plants. Molecular phylogenetics...
Conifers are an ecologically and economically important seed plant group that can provide significant insights into the evolution of land plants. Molecular phylogenetics has developed as an important approach in evolutionary studies, although there have been relatively few studies of conifers that employ large-scale data sourced from multiple nuclear genes. Target enrichment sequencing (target capture, exon capture, or Hyb-Seq) has developed as a key approach in modern phylogenomic studies. However, until now, there has been no bait set that specifically targets the entire conifer clade. REMcon is a target sequence capture probe set intended for family- and species-level phylogenetic studies of conifers that target c. 100 single-copy nuclear loci. We tested the REMcon probe set using 69 species, including 44 conifer genera across six families and four other gymnosperm taxa, to evaluate the efficiency of target capture to efficiently generate comparable DNA sequence data across conifers. The recovery of target loci was high, with, on average, 94% of the targeted regions recovered across samples with high read coverage. A phylogenetic analysis of these data produced a well-supported topology that is consistent with the current understanding of relationships among conifers. The REMcon bait set will be useful in generating relatively large-scale nuclear data sets consistently for any conifer lineage.
PubMed: 38927241
DOI: 10.3390/biology13060361 -
Biomolecules Jun 2024Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography...
Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings.
Topics: Humans; Lung Neoplasms; DNA Methylation; Homeodomain Proteins; Real-Time Polymerase Chain Reaction; Biomarkers, Tumor; Sensitivity and Specificity
PubMed: 38927132
DOI: 10.3390/biom14060729 -
Biomolecules May 2024Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need...
Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.
Topics: Humans; Ovarian Neoplasms; Female; RNA, Long Noncoding; Peptide Nucleic Acids; Cyclopentanes; Cell Line, Tumor; Biomarkers, Tumor
PubMed: 38927013
DOI: 10.3390/biom14060609