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At least 127 records · Page 7Linked to original sources
PubMed2005

Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics.

Elongation factor (EF-) Tu.GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu.GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu.guanylyl iminodiphosphate (GDPNP).enacyloxin IIa complex at 2.3 A resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu.GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu.GDPNP.enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe.EF-Tu (Thermus aquaticus).GDPNP.enacyloxin IIa complex, solved at 3.1 A resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics.

Anti-Bacterial Agents↗
PubMed1995

Analysis of transcription control signals using artificial neural networks.

The role of the upstream region in controlling the transcription efficiency of a gene is well established. However, the question of predicting the extent of gene expressed given the upstream region has so far remained unresolved. Using an artificial neural network (ANN) to capture the internal representation associated with the transcription control signal, the present work predicts the rate of mRNA synthesis based on the pattern contained in the upstream region. Further, the model has been used to predict the transcription efficiency for all possible single base mutations associated with the beta-globin promoter. The simulation results reveal that apart from the experimental observation that alpha-79G-A and -78G-A mutation increases the efficiency of transcription, mutation in these regions by C or T also causes an increase in transcription. Furthermore the simulation results verify that mutations in the conserved region, in general, decrease the transcriptional efficiency. However, the results also show that certain sequence elements, when mutated, either cause a marginal increase in the level of transcription or have no effect on transcription levels. The simulation results can be used as a guide in designing mutation experiments since an a priori estimate of the possible outcome of a mutation can be obtained.

Animals↗
PubMed2025

In planta genome editing in citrus facilitated by co-expression of CRISPR/Cas and developmental regulators.

Recent advances in the field of genome editing offer a promising avenue for targeted trait improvements in fruit trees. However, the predominant method taken for genome editing in citrus (and other fruit trees) involves the time-consuming tissue culture approach, thereby prolonging the overall citrus breeding process and subjecting it to the drawbacks associated with somaclonal variation. In this study, we introduce an in planta approach for genome editing in soil-grown citrus plants via direct transformation of young seedlings. Our editing system, abbreviated here as IPGEC (in planta genome editing in citrus), is designed to transiently co-express three key gene groups in citrus tissue via Agrobacterium tumefaciens: (i) a genome-editing catalytic group, (ii) a shoot induction and regeneration group, and (iii) a T-DNA enhanced delivery group. This integrated system significantly improves de novo shoot induction and regeneration efficiency of edited tissue. By incorporating single-guides RNA's (sgRNA's) targeting the carotenoid biosynthetic gene PHYTOENE DESATURASE (CsPDS), the IPGEC system effectively produced mutated albino shoots, confirming its ability to generate homozygous/biallelic genome-edited plants. By using high throughput screening, we provide evidence that transgene-free genome-edited plants could be obtained following the IPGEC approach. Our findings further suggest that the efficiency of specific developmental regulators in inducing transformation and regeneration rates may be cultivar-specific and therefore needs to be optimized per cultivar. Finally, targeted breeding for specific trait improvements in already successful cultivars is likely to revolutionize fruit tree breeding and will pave the way for accelerating the development of high-quality citrus cultivars.

Citrus↗
PubMed1998

Heterologous protection of mice from a lethal human H1N1 influenza A virus infection by H3N8 equine defective interfering virus: comparison of defective RNA sequences isolated from the DI inoculum and mouse lung.

We have examined the RNAs involved in the heterologous protection of adult mice from otherwise lethal intranasal infection with mouse-adapted human A/WSN (H1N1) by defective interfering (DI) equine A/equine/Newmarket/7339/79 (H3N8: EQV) influenza virus, as well as the RNAs involved in the protection of WSN- or EQV-infected mice by their homologous DI viruses. The aim of this study was to describe the types of defective RNAs present in protected mice in order to guide the design of potentially protective DI RNAs. The interfering and mouse-protecting activity of DI virus was destroyed by prolonged UV irradiation (iDI virus) demonstrating that protection correlated with an active DI genome, and not viral antigen. Protected mice were all infected but suffered a lower degree of morbidity than those given iDI virus. The DI EQV inoculum contained defective segment 1-8 RNAs while DI WSN inoculum contained only defective segment 1-6 RNAs. However lungs of mice given EQV + DI EQV contained only defective segments 1-4 or 1-6 RNAs (mouse-to-mouse variation), while control mice given EQV or EQV + iDI EQV contained few very defective RNAs. Thus prevention of death was the result of quantitative and/or qualitative differences in defective RNAs administered to the mice. Only defective segments 1-3 RNAs were isolated from the lungs of mice given WSN + DI WSN, confirming the earlier report of Noble and Dimmock (1995). A detailed analysis showed that most defective RNAs isolated from the lungs of mice protected from a lethal WSN infection by DI EQV were EQV in origin. Thus, as no infectious EQV was present, these defective RNAs from the DI EQV inoculum must have been heterologously replicated in mouse lung by WSN. All defective segment 3-6 RNAs isolated were of EQV origin, indicating that they were replicated by WSN in preference to its own. Defective segments 1 and 2 were a mixture of EQV and WSN RNAs. Of 17 defective EQV segment 1-3 sequences from mouse lung, all but three differed in their primary central deletion from 20 defective RNAs isolated from the inoculum. No bias in the break points was evident. A number of minor deletions of 2 or more nts were also present in defective EQV and WSN RNAs in segments 1 and 2, but none in segment 3. Their 5', but not 3', breakpoints were heterogeneous, suggesting that defective RNAs were generated during positive strand synthesis. Two cloned EQV-defective segment 3 RNAs were chimeras containing a 30 nt insert from segment 1. Most defective RNAs possessed at least 178 nts from the 5' end of vRNA. The amount of 5' sequence present in those RNAs correlated with the segment of origin, suggesting that this was the minimum required for propagation of viral RNA in mouse lung and hence possibly for protection also.

Animals↗
PubMed2026

Simulation of CRISPR/Cas9-mediated gene editing for the Vitellogenin gene in Apis mellifera.

CRISPR/Cas9 genome editing provides a powerful framework for interrogating gene function in Apis mellifera. Yet, empirical application remains challenging due to biological constraints, including haplodiploid genetics, narrow embryonic injection window, and the social rearing requirements that complicate functional validation. These constraints necessitate in silico pre-screening to maximize editing success before resource-intensive wet-lab implementation. Within the omnigenic framework, which distinguishes core regulatory genes from peripheral loci buffered by network effects, vitellogenin (Vg) represents an optimal target which is ancestrally dedicated to yolk provisioning; it has been co-opted to orchestrate diverse non-reproductive functions including longevity, stress resistance, immunity, and social behavior. We developed a computational pipeline to design a list of 57 and 56 candidate guide RNAs (gRNA) for targeted Vg knockout, evaluating candidate sites in both functional exons 2 and 3 based on structural accessibility and frameshift efficiency. Comparative analysis revealed complementary strengths in two top-best candidates from initial target pool of predicted gRNAs. The gRNA targeting exon 2 exhibits weaker secondary structure (ΔG = -0.25 kcal/mol versus -2.10 kcal/mol for exon 3), aligning with empirical evidence that sites with ΔG > -1.0 kcal/mol achieve 2-5 × higher Cas9 binding efficiency. This site yielded moderate frameshift frequency (77.8%; 61.9 percentile). Conversely, the predicted editing outcome for the gRNA targeting exon 3, despite stronger structural constraints, demonstrated superior functional disruption metrics demonstrating very high frameshift frequency (88.3%; 95.2 percentile), high in silico editing precision, minimal microhomology-mediated repair bias, and reproducible outcomes wherein nearly all predicted indels disrupt the coding sequence. Protein structure and domain analyses further predict that frameshift edits will generate a truncated protein missing all downstream functional domains. We recommend parallel empirical validation of both exon 2 and exon 3 targets to resolve the trade-off between structural accessibility (favoring higher editing rates) and frameshift efficacy (favoring complete loss-of-function). This dual-target strategy accommodates uncertainty in in vivo performance while maximizing the probability of generating informative phenotypes. Our in silico framework enables rational CRISPR design in non-model organisms by computationally balancing biophysical accessibility with functional impact, accelerating functional genomics in species where empirical optimization faces substantial biological constraints.

Animals↗
PubMed2002

Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream.

The recent flood of reports using real-time Q-PCR testifies to the transformation of this technology from an experimental tool into the scientific mainstream. Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers. The range of applications of real-time Q-PCR is immense and has been fueled in part by the proliferation of lower-cost instrumentation and reagents. Successful application of real-time Q-PCR is not trivial. However, this review will help guide the reader through the variables that can limit the usefulness of this technology. Careful consideration of the assay design, template preparation, and analytical methods are essential for accurate gene quantification.

Biotechnology↗
PubMedDate not supplied

Current concepts in antiretroviral therapy failure.

Currently, the goal for the first and second, possibly the third, antiretroviral regimen is the suppression of HIV replication to plasma levels below assay detection (ie, <50 HIV-1 RNA copies/mL). In patients with advanced treatment failure and resistance to numerous drugs, the goal of therapy has been to reduce viral load as much as possible and to maintain immunologic and clinical integrity. With the recent availability of new drugs and new classes of drugs, complete suppression of viral replication may be possible even in late salvage. When designing a regimen for a patient for whom antiretroviral therapy has failed, the regimen should contain at least 3 active drugs, and regimens are best selected with assistance from genotypic and phenotypic drug resistance testing. The selection should also be guided by knowledge of potential pharmacokinetic interactions among drugs. This article summarizes a presentation on antiretroviral failure by Carlos del Rio, MD, at the 8th Annual clinical meeting for Ryan White CARE Act clinicians in New Orleans in June 2005, sponsored by the International AIDS Society-USA and includes more recent data presented at the 13th Conference on Retroviruses and Opportunistic Infections (CROI) in 2006.

Anti-HIV Agents↗
PubMed1990

External guide sequences for an RNA enzyme.

Ribonuclease P (RNase P) from Escherichia coli or its catalytic RNA subunit can efficiently cleave small RNA substrates that lack the conserved features of natural substrates of RNase P if an additional small RNA is also present. This additional RNA must contain a sequence complementary to the substrate [external guide sequence (EGS)] and a 3'-proximal CCA sequence to ensure cleavage. The aminoacyl acceptor stem and some additional 5'- and 3'-terminal sequences of a precursor transfer RNA are sufficient to allow efficient cleavage by RNAase P, and the 2'-hydroxyl group at the cleavage site is not absolutely necessary for cleavage. In principle, any RNA could be targeted by a custom-designed EGS RNA for specific cleavage by RNase P in vitro or in vivo.

Base Sequence↗
PubMed2004

The design of a potent inhibitor of the hepatitis C virus NS3 protease: BILN 2061--from the NMR tube to the clinic.

The virally encoded serine protease NS3/NS4A is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. Until very recently, the design of inhibitors for the HCV NS3 protease was limited to large peptidomimetic compounds with poor pharmacokinetic properties, making drug discovery an extremely challenging endeavor. In our quest for the discovery of a small-molecule lead that could block replication of the hepatitis C virus by binding to the HCV NS3 protease, the critical protein-polypeptide interactions between the virally encoded NS3 serine protease and its polyprotein substrate were investigated. Lead optimization of a substrate-based hexapeptide, guided by structural data, led to the understanding of the molecular dynamics and electronic effects that modulate the affinity of peptidomimetic ligands for the active site of this enzyme. Macrocyclic beta-strand scaffolds were designed that allowed the discovery of potent, highly selective, and orally bioavailable compounds. These molecules were the first HCV NS3 protease inhibitors reported that inhibit replication of HCV subgenomic RNA in a cell-based replicon assay at low nanomolar concentrations. Optimization of their biopharmaceutical properties led to the discovery of the clinical candidate BILN 2061. Oral administration of BILN 2061 to patients infected with the hepatitis C genotype 1 virus resulted in an impressive reduction of viral RNA levels, establishing proof-of-concept for HCV NS3 protease inhibitors as therapeutic agents in humans.

Carbamates↗
PubMed2006

Dual targeting of GyrB and ParE by a novel aminobenzimidazole class of antibacterial compounds.

A structure-guided drug design approach was used to optimize a novel series of aminobenzimidazoles that inhibit the essential ATPase activities of bacterial DNA gyrase and topoisomerase IV and that show potent activities against a variety of bacterial pathogens. Two such compounds, VRT-125853 and VRT-752586, were characterized for their target specificities and preferences in bacteria. In metabolite incorporation assays, VRT-125853 inhibited both DNA and RNA synthesis but had little effect on protein synthesis. Both compounds inhibited the maintenance of negative supercoils in plasmid DNA in Escherichia coli at the MIC. Sequencing of DNA corresponding to the GyrB and ParE ATP-binding regions in VRT-125853- and VRT-752586-resistant mutants revealed that their primary target in Staphylococcus aureus and Haemophilus influenzae was GyrB, whereas in Streptococcus pneumoniae it was ParE. In Enterococcus faecalis, the primary target of VRT-125853 was ParE, whereas for VRT-752586 it was GyrB. DNA transformation experiments with H. influenzae and S. aureus proved that the mutations observed in gyrB resulted in decreased susceptibilities to both compounds. Novobiocin resistance-conferring mutations in S. aureus, H. influenzae, and S. pneumoniae were found in gyrB, and these mutants showed little or no cross-resistance to VRT-125853 or VRT-752586 and vice versa. Furthermore, gyrB and parE double mutations increased the MICs of VRT-125853 and VRT-752586 significantly, providing evidence of dual targeting. Spontaneous frequencies of resistance to VRT-752586 were below detectable levels (<5.2x10(-10)) for wild-type E. faecalis but were significantly elevated for strains containing single and double target-based mutations, demonstrating that dual targeting confers low levels of resistance emergence and the maintenance of susceptibility in vitro.

Anti-Bacterial Agents↗
PubMed2005

Structure-guided discovery of novel aminoglycoside mimetics as antibacterial translation inhibitors.

We report the structure-guided discovery, synthesis, and initial characterization of 3,5-diamino-piperidinyl triazines (DAPT), a novel translation inhibitor class that targets bacterial rRNA and exhibits broad-spectrum antibacterial activity. DAPT compounds were designed as structural mimetics of aminoglycoside antibiotics which bind to the bacterial ribosomal decoding site and thereby interfere with translational fidelity. We found that DAPT compounds bind to oligonucleotide models of decoding-site RNA, inhibit translation in vitro, and induce translation misincorporation in vivo, in agreement with a mechanism of action at the ribosomal decoding site. The novel DAPT antibacterials inhibit growth of gram-positive and gram-negative bacteria, including the respiratory pathogen Pseudomonas aeruginosa, and display low toxicity to human cell lines. In a mouse protection model, an advanced DAPT compound demonstrated efficacy against an Escherichia coli infection at a 50% protective dose of 2.4 mg/kg of body weight by single-dose intravenous administration.

Aminoglycosides↗
PubMed2004

An RNA external guide sequence ribozyme targeting human interleukin-4 receptor alpha mRNA.

RNA oligonucleotides termed External Guide Sequence (EGS) and RNAi have been described that target specific gene expression by site-specific cleavage of mRNA. EGS serve as an RNA catalyst or ribozyme by directing bound mRNA to the ubiquitous cellular enzyme RNAse P. We describe an EGS targeting human interleukin (IL)-4 receptor alpha mRNA, an important cytokine receptor in the pathogenesis of asthma and allergic disease expressed in pulmonary tissues. This EGS was designed to explore pulmonary delivery of catalytic RNA oligonucleotides as a novel therapy in asthma and other atopic diseases. Inhaled DNA oligonucleotides termed Respirable Antisense OligoNucleotide Sequences (RASONS) are selectively internalized in lung tissues in a complex with endogenous lipid surfactants present in normal lung and can alter pulmonary gene expression. Potential applications of inhaled RNA oligonucleotides in therapy of pulmonary and related systemic diseases are discussed.

Administration, Inhalation↗
PubMed1999

U82, a novel snoRNA identified from the fifth intron of human and mouse nucleolin gene.

A novel snoRNA, designated as U82, was found from the sequence analysis of the 5th intron of human and mouse nucleolin gene. The snoRNA U82 has characteristic boxes C, D and D' and 11 nucleotides (nt) antisense complementarity to the 18S rRNA. Presumably U82 functions as a guide in the methylation of residue A1678 in human 18S rRNA. Northern blot analysis with various oligodeoxynucleotide probes showed that human and mouse U82 is expressed as RNA variants with length of 70 (+/- 1) and 67 (+/- 1) nt in HeLa and mouse C127 cells. Most probably, the 70 nt variant of U82 is encoded by nucleolin gene 5th intron. The 67 nt variant of U82 could be a transcript of another gene, the genomic locus of which remains unknown.

Animals↗
PubMed2026

SpacerScope: binary-vectorized, genome-wide off-target profiling for RNA-guided nucleases without prior candidate-site bias.

The precision of CRISPR/Cas systems is fundamental to their application in plant and animal biotechnology. However, comprehensive sequence-based off-target candidate discovery remains a computational bottleneck, particularly in large and complex genomes. Here we developed SpacerScope, an off-target candidate discovery framework that enables unbiased, genome-wide discovery by leveraging binary vectorization, bitwise filtering, and right-end-anchored alignment. Benchmarking against human CIRCLE-seq data demonstrated that SpacerScope recovered 100% of validated off-target sites (6142/6142), matching the sensitivity of exhaustive algorithms. Crucially, SpacerScope achieved this maximum candidate recovery while substantially reducing computational overhead. In large-genome evaluations, SpacerScope maintained low peak memory usage of 2.20 GiB and achieved substantial runtime improvements over indel-aware comparator tools, including more than 50-fold speedup relative to Cas-OFFinder 3 (544&#xa0;s versus 29&#xa0;185&#xa0;s). Furthermore, comparative analyses in polyploid species, such as the octoploid strawberry, revealed that SpacerScope identified larger sequence-compatible candidate burdens than standard web-based design platforms. Our results establish SpacerScope as a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes. The source code and program was publicly available at https://github.com/charlesqu666/SpacerScope. Short Abstract CRISPR/Cas sequence-based off-target candidate discovery remains computationally challenging in large, repetitive, and polyploid genomes. Existing tools either miss indel-containing candidate sites or incur prohibitive runtime and memory costs. We developed SpacerScope, a binary-vectorized framework that enables unbiased, genome-wide off-target candidate discovery without pre-selected candidate sites. By integrating bitwise filtering with right-end-anchored alignment, SpacerScope recovered 100% of validated off-target sites in human CIRCLE-seq data while using only 2.20 GiB of memory and achieving more than 10-fold speedup over indel-aware alternatives. Evaluation in plant genomes, including rice and octoploid strawberry, further demonstrated SpacerScope's capacity to identify larger sequence-compatible candidate burdens overlooked by standard tools. SpacerScope thus provides a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes, supporting downstream prioritization.

CRISPR-Cas Systems↗
PubMed2004

Tissue microarrays: a practical guide.

Tissue microarrays are a recent innovation in the field of pathology. They were originally designed as a high-throughput approach for researchers to assess the expression of interesting candidate disease-related genes or gene products simultaneously on hundreds of tissue samples. However, their use is becoming more widespread in routine pathology, for example for quality assurance and for the optimisation of diagnostic reagents such as monoclonal antibodies and gene probes. Several molecular and conventional pathological techniques can be performed on a single tissue array, thereby enabling morphology, DNA, RNA and protein targets to be analysed on sequential sections through multiple tissue samples. Moreover, compared with full-face tissue sections, tissue microarrays are a cost- and time-efficient, effective approach to analysing biomarker expression on a large number of samples. Whilst tissue microarrays are available from commercial sources, many pathology laboratories prefer to make in-house arrays from their often extensive pathology archive to facilitate the correlation of their findings with clinical parameters. The technical skills necessary to produce tissue arrays are well within the capacity of most laboratories. However, several pitfalls to successful array production exist. The present article describes the applications of this technique and details practical points for optimal tissue array production.

Gene Expression Profiling↗
PubMed1992

Targeted cleavage of mRNA by human RNase P.

Ribonuclease P from Escherichia coli can cleave RNAs in simple, hydrogen-bonded complexes of two oligoribonucleotides that resemble the aminoacyl stem and 5' leader sequence of tRNA precursors. RNase P from human (HeLa) cells cannot catalyze the cleavage in vitro of the 5'-proximal oligoribonucleotide that contains the leader sequence in such simple complexes but can do so when the 3'-proximal oligoribonucleotide (external guide sequence) is altered to resemble three-quarters of a tRNA molecule. In such a complex, the efficiency of cleavage of the mRNA for chloramphenicol acetyltransferase, as the 5'-proximal oligoribonucleotide, depends on the structural details of the external guide sequence and on the choice of target site within the mRNA. The presence of the appropriately designed external guide sequence in cells in tissue culture reduces chloramphenicol acetyltransferase activity and the level of the corresponding intact mRNA in the cells. Thus, it appears that the use of such external guide sequences may provide a general technique for gene inactivation.

Base Sequence↗