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PubMed2024

Critically unwell infants and children with mitochondrial disorders diagnosed by ultrarapid genomic sequencing.

PURPOSE: To characterize the diagnostic and clinical outcomes of a cohort of critically ill infants and children with suspected mitochondrial disorders (MD) undergoing ultrarapid genomic testing as part of a national program. METHODS: Ultrarapid genomic sequencing was performed in 454 families (genome sequencing: n = 290, exome sequencing +/- mitochondrial DNA sequencing: n = 164). In 91 individuals, MD was considered, prompting analysis using an MD virtual gene panel. These individuals were reviewed retrospectively and scored according to modified Nijmegen Mitochondrial Disease Criteria. RESULTS: A diagnosis was achieved in 47% (43/91) of individuals, 40% (17/43) of whom had an MD. Seven additional individuals in whom an MD was not suspected were diagnosed with an MD after broader analysis. Gene-agnostic analysis led to the discovery of 2 novel disease genes, with pathogenicity validated through targeted functional studies (CRLS1 and MRPL39). Functional studies enabled diagnosis in another 4 individuals. Of the 24 individuals ultimately diagnosed with an MD, 79% had a change in management, which included 53% whose care was redirected to palliation. CONCLUSION: Ultrarapid genetic diagnosis of MD in acutely unwell infants and children is critical for guiding decisions about the need for additional investigations and clinical management.

Humans↗

PubMed2004

Characterization of (2R, 3S)-2-([[4-(2-butynyloxy)phenyl]sulfonyl]amino)-N,3-dihydroxybutanamide, a potent and selective inhibitor of TNF-alpha converting enzyme.

TNF-alpha converting enzyme (TACE) is a validated therapeutic target for the development of oral tumor necrosis factor-alpha (TNF-alpha) inhibitors. Here we report the pre-clinical results and characterization of a selective and potent TACE inhibitor, (2R, 3S)-2-([[4-(2-butynyloxy)phenyl]sulfonyl]amino)-N,3-dihydroxybutanamide (TMI-2), in various in vitro and in vivo assays. TMI-2 is a potent TACE inhibitor in an enzymatic FRET assay (IC50=2 nM). It is more than 250-fold selective over MMP-1, -7, -9, -14, and ADAM-10 in vitro. In cell-based assays and human whole blood, TMI-2 inhibits lipopolysaccharide (LPS)-induced TNF secretion with IC50s<1 uM. Importantly, TMI-2 inhibits the spontaneous release of TNF-alpha in human synovium tissue explants of rheumatoid arthritis patients with an IC50 of 0.8 microM. In vivo, TMI-2 potently inhibits LPS-induced TNF-alpha production in mice (ED50=3 mg/kg). In the adjuvant-induced arthritis (AIA) model in rats, treatment with TMI-2 at 30 mg/kg and 100 mg/kg p.o. b.i.d. was highly effective in reducing joint arthritis scores. In a semi-therapeutic collagen-induced arthritis (CIA) model in mice, TMI-2 is highly effective in reducing disease severity scores after oral treatment at 100 mg/kg twice per day. In summary, TMI-2 is a potent and selective TACE inhibitor that inhibits TNF-alpha production and reduces the arthritis scores in pre-clinical models. TMI-2 represents a novel class of TACE inhibitors that may be effective and beneficial in the treatment of rheumatoid arthritis as well as other TNF-mediated inflammatory autoimmune diseases.

ADAM Proteins↗

PubMed2026

Nanopore-based epigenomic profiling reveals the absence of widespread CpG methylation in the African swine fever virus genome.

DNA methylation is a critical epigenetic mechanism implicated in regulating replication and transcription in DNA viruses. However, the epigenetic landscape of African swine fever virus (ASFV), a large double-stranded DNA virus infecting pigs, remains controversial. Here, we systematically profiled the DNA methylome of the first ASFV strain isolated in Hong Kong (HK_NT_202103) using Oxford Nanopore Technologies (ONT) R10.4.1 sequencing. We employed a paired design: native whole-genome sequencing (WGS) against a methylation-free whole-genome amplification (WGA) control. Using conservative thresholds, we found no evidence of 5-methylcytosine (5mC), especially typical CpG methylation, across the viral genome. Importantly, clear CpG methylation signals were successfully detected in the host genome from WGS data, confirming the functionality of the workflow to detect 5mC at CG sites. While widespread 5mC seems absent, a small number of putative N6-methyladenine (6mA) loci were identified. A specific 6mA candidate exhibited raw ionic current disruptions and gene-level intersection with another ASFV isolate (CAS19-01/2019), although it lacked single-base consensus across different methylation callers or between the two isolates. Although our biological findings are restricted to a single isolate under specific experimental conditions, this study introduces a novel, highly rigorous ONT framework for viral epigenomics research. Furthermore, the absence of ASFV CpG methylation indicates that host CpG-depletion remains a viable strategy for viral metagenomic enrichment. Ultimately, our work offers a critical methodological baseline for ASFV surveillance and highlights the necessity of targeted experimental validation for rare viral modifications.

African Swine Fever Virus↗

PubMed2005

The plastidic DNA replication enzyme complex of Plasmodium falciparum.

The replication and repair of organellar genomes in the malaria parasite Plasmodium falciparum is poorly understood. We have assessed the properties of an open reading frame Pfprex (formerly known as pom1) and confirm that it specifies a multi-domain polypeptide with DNA primase, DNA helicase, DNA polymerase and 3'-5' exonuclease activities. The sequence of the primase/helicase domain is phylogenetically related to the T7-bacteriophage gene 4 product and mammalian mitochondrial helicase, Twinkle. Despite that, the N-terminal sequence of this multi-domain polypeptide directs a green fluorescent protein reporter specifically to the P. falciparum apicoplast and not to the mitochondrion. Phylogenetic analysis placed the DNA polymerase sequence with the family A bacterial polymerases, most closely to those of the thermophilic Aquifex species. Notably, the malarial enzyme was optimally active at 75 degrees C. Pfprex is the first example of a gene encoding contiguous DNA polymerase, DNA primase and DNA helicase components. We propose it has a key role in replication of the malarial plastid genome, a validated drug target.

Animals↗

PubMed2026

A novel biocontrol Pseudomonas species with broad-spectrum antagonistic activity against phytopathogens.

Bacterial and fungal diseases cause significant losses in horticultural crops, and biocontrol using beneficial microorganisms offers a sustainable alternative to chemical pesticides. In this study, a novel Pseudomonas strain D3 was isolated from Actinidiae rhizosphere. D3 exhibited strong antibacterial activity in LB medium but showed no activity against fungi or oomycetes. However, when cultured in KIDO medium, it demonstrated potent antifungal activity. Phylogenetic analysis based on 16S rRNA gene showed that D3 was most closely related to Pseudomonas mosselii CIP_105259T, while whole-genome sequencing revealed ANI values below 95% with eight known P. mosselii strains. Digital DNA-DNA hybridization (dDDH) further confirmed its genomic distinctiveness, with the highest dDDH value (58.2%) against the type strain P. mosselii DSM 17497T, well below the 70% species delineation threshold, supporting D3 as a novel Pseudomonas species. Functional validation via targeted gene knockout revealed a dichotomy in the antagonistic mechanisms of D3. Knockout of individual biosynthetic gene clusters (BGCs) only partially reduced antibacterial activity against Pseudomonas syringae pv. actinidiae, indicating that multiple BGCs contribute to this activity in a partially redundant manner. In contrast, disruption of a specific lipopeptide synthase cluster completely abolished antifungal activity against Valsa mali. LC-MS/MS analysis confirmed that this lipopeptide was produced exclusively in KIDO medium, consistent with the observed medium-dependent antifungal activity. Detached leaf and twig assays showed that D3 provides strong preventive biocontrol against both pathogens. Collectively, strain D3 employs a dual biocontrol mechanism, combining antibacterial activity mediated by multiple BGCs with lipopeptide-dependent antifungal activity, positioning it as a promising agent for sustainable disease management in horticultural crops.

Pseudomonas↗

PubMed2006

Acoustic power measurement of high intensity focused ultrasound in medicine based on radiation force.

How to measure the acoustic power of HIFU is one of the most important tasks in its medical application. In the paper a whole series of formula for calculating the radiation force related to the acoustic power radiated by a single element focusing transducer and by the focusing transducer array were given. Various system of radiation force balance (RFB) to measure the acoustic power of HIFU in medicine were designed and applied in China. In high power experiments, the dependence of radiation force acting the absorbing target on the target position at the beam axis of focusing transducer was fined. There is a peak value of "radiation force" acting the absorbing target in the focal region when the acoustic power through the focal plane exceeds some threshold. In order to avoid this big measurement error caused by the 'peak effect' in focal region, the distance between the absorbing target of RFB and the focusing transducer or transducer array was defined to be equal to or less than 0.7 times of the focal length in the National Standard of China for the measurements of acoustic power and field characteristics of HIFU. More than six different therapeutic equipments of HIFU have been examined by RFB for measuring the acoustic power since 1998. These results show that RFB with the absorbing target is valid in the acoustic power range up to 500W with good linearity for the drive voltage squared of focusing transducer or array. The uncertainty of measurement is within +/-15%.

Acoustics↗

PubMed2006

Expression profiling of glial genes during Drosophila embryogenesis.

In the central nervous system of Drosophila, the induction of the glial cell fate is dependent on the transcription factor glial cells missing (gcm). Though a considerable number of other genes have been shown to be expressed in all or in subsets of glial cells, the course of glial cell differentiation and subtype specification is only poorly understood. This prompted us to design a whole genome microarray approach comparing gcm gain-of-function and, for the first time, gcm loss-of-function genetics to wildtype in time course experiments along embryogenesis. The microarray data were analyzed with special emphasis on the temporal profile of differential regulation. A comparison of both experiments enabled us to identify more than 300 potential gcm target genes. Validation by in situ hybridization revealed expression in glial cells, macrophages, and tendon cells (all three cell types depend on gcm) for 70 genes, of which more than 50 had been unknown to be under gcm control. Eighteen genes are exclusively expressed in glial cells, and their dependence on gcm was confirmed in situ. Initial considerations regarding the role of the newly discovered glial genes are discussed based on gene ontology and the temporal profile and subtype specificity of their expression. This collection of glial genes provides an important basis for the clarification of the genetic network controlling various aspects of glial development and function.

Animals↗

PubMed2006

Progress towards in vivo use of siRNAs.

RNA interference (RNAi) has become the method of choice to suppress gene expression in vitro. It is also emerging as a powerful tool for in vivo research with over 90 studies published using synthetic small interfering RNAs in mammals. These reports demonstrate the potential for use of synthetic small interfering RNAs (siRNAs) as therapeutic agents, especially in the areas of cancer and viral infection. The number of reports using siRNAs for functional genomics applications, for validation of targets for small-molecule drug development programs, and to address questions of basic biology will rapidly grow as methods and protocols for use in animals become more established. This review will first discuss aspects of RNAi biochemistry and biology that impact in vivo use, especially as relates to experimental design, and will then provide an overview of published work with a focus on methodology.

Animals↗

PubMed2002

Identification and mapping of small-molecule binding sites in proteins: computational tools for structure-based drug design.

The number of protein structures is currently increasing at an impressive rate. The growing wealth of data calls for methods to efficiently exploit structural information for medicinal and pharmaceutical purposes. Given the three-dimensional (3D) structure of a validated protein target, the identification of functionally relevant binding sites and the analysis ('mapping') of these sites with respect to molecular recognition properties are important initial tasks in structure-based drug design. To address these tasks, a variety of computational tools have been developed. Approaches to identify binding pockets include geometric analyses of protein surfaces, comparisons of protein structures, similarity searches in databases of protein cavities, and docking scans to reveal areas of high ligand complementarity. In the context of binding-site analysis, powerful data mining tools help to retrieve experimental information about related protein-ligand complexes. To identify interaction hot spots, various potential functions and knowledge-based approaches are available for mapping binding regions. The results may subsequently be used to guide virtual screenings for new ligands via pharmacophore searches or docking simulations.

Artificial Intelligence↗

PubMed2001

Automatic orienting of visuospatial attention in Parkinson's disease.

The basal ganglia are involved in not only motor behavior, but also other more cognitive processes, such as attention. We tested Parkinson's disease (PD) patients in a task that measures reflexive orienting of spatial attention. Seven patients with idiopathic PD and eight control subjects performed a covert orienting task where spatial attention was directed by means of exogenous cues (luminance increments) with no predictive validity for target position. The subjects' task was to make a speeded saccade to a visual target, which appeared a variable time after onset of the cue either in the cued or an uncued spatial position. There was no overall difference between PD patients and control subjects in terms of the initial facilitation following reflexive cues, and later inhibition of return (IOR). However, PD patients differed from control subjects in two important respects. First, they were significantly faster than were control subjects on this reflexive visual-orienting task. Second, disease severity correlated with attentional performance; more advanced patients showed less initial facilitation but greater IOR. Thus PD patients show better performance on a reflexive saccade task and, for more advanced patients, greater IOR than control subjects. These findings are consistent with the possibility that reflexive attentional processes in PD patients may be more active.

Aged↗

PubMed2006

RNAi therapy for neurodegenerative diseases.

RNA interference (RNAi) mediates gene silencing in a sequence-specific manner and has proven to be an exceptionally valuable discovery for bench scientists. In the laboratory, RNAi technologies provide efficient means for validating drug targets and for performing reverse genetics to study gene function (Friedman and Perrimon, 2004). Patients may also benefit from RNAi as applications extend to potential human therapies. RNAi-based treatments are being investigated and may provide hope for patients suffering from cancer, viral infections, or genetic diseases for which effective therapies are currently lacking. Notably, several independent studies have demonstrated that RNAi therapy can improve disease phenotypes in various mouse models of human disease. In this chapter, we focus on the potential of RNAi in treating neurologic diseases for which reduction of mutant or toxic gene expression may provide therapeutic benefit. We discuss approaches to achieving RNAi in vivo, progress in the field, and the potential pitfalls associated with RNAi-based therapies.

Animals↗

PubMedDate not supplied

Protein kinases as targets for anticancer agents: from inhibitors to useful drugs.

Many components of mitogenic signaling pathways in normal and neoplastic cells have been identified, including the large family of protein kinases, which function as components of signal transduction pathways, playing a central role in diverse biological processes, such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases caused by abnormalities in these signaling pathways is widely considered a promising approach for drug development. Because of their deregulation in human cancers, protein kinases, such as Bcr-Abl, those in the epidermal growth factor-receptor (HER) family, the cell cycle regulating kinases such as the cyclin-dependent kinases, as well as the vascular endothelial growth factor-receptor kinases involved in the neo-vascularization of tumors, are among the protein kinases considered as prime targets for the development of selective inhibitors. These drug-discovery efforts have generated inhibitors and low-molecular weight therapeutics directed against the ATP-binding site of various protein kinases that are in various stages of development (up to Phase II/III clinical trials). Three examples of inhibitors of protein kinases are reviewed, including low-molecular weight compounds targeting the cell cycle kinases; a potent and selective inhibitor of the HER1/HER2 receptor tyrosine kinase, the pyrollopyrimidine PKI166; and the 2-phenyl-aminopyrimidine STI571 (Glivec(R), Gleevec) a targeted drug therapy directed toward Bcr-Abl, the key player in chronic leukemia (CML). Some members of the HER family of receptor tyrosine kinases, in particular HER1 and HER2, have been found to be overexpressed in a variety of human tumors, suggesting that inhibition of HER signaling would be a viable antiproliferative strategy. The pyrrolo-pyrimidine PKI166 was developed as an HER1/HER2 inhibitor with potent in vitro antiproliferative and in vivo antitumor activity. Based upon its clear association with disease, the Bcr-Abl tyrosine kinase in CML represents the ideal target to validate the clinical utility of protein kinase inhibitors as therapeutic agents. In a preclinical model, STI571 (Glivec(R), Gleevec) showed potent in vitro and in vivo antitumor activity that was selective for Abl, c-Kit, and the platelet-derived growth factor-receptor. Phase I/II studies demonstrated that STI571 is well tolerated, and that it showed promising hematological and cytogenetic responses in CML and clinical responses in the c-Kit-driven gastrointestinal tumors.

Animals↗

PubMed2001

An approach to guaranteeing generalisation in neural networks.

A novel approach to generalisation is presented that is able, under certain circumstances, to guarantee the generalisation to binary-output data for which no targets have been given. The basis of the guarantee is the recognition of a persistent global minimum error solution. An empirical test for whether the guarantee holds is provided which uses a technique called target reversal. The technique employs two neural networks whose convergence using opposing targets signals validity of the guarantee.

Bayes Theorem↗

PubMed2002

Imatinib and chronic myeloid leukemia: validating the promise of molecularly targeted therapy.

The Bcr-Abl tyrosine kinase inhibitor imatinib (Glivec, formerly STI571, Novartis Pharma AG, Basel, Switzerland) produces complete hematologic and cytogenetic responses in a substantial percentage of chronic myeloid leukemia patients. Imatinib is effective in chronic phase, accelerated phase and blast crisis, with lower response rates in patients with more advanced disease. Although responses have been durable in chronic phase patients, relapses have been common in blast crisis. Relapse has been associated with reactivation of Bcr-Abl kinase activity. The clinical development of imatinib illustrates the effectiveness of targeting molecular pathogenetic events. Hopefully, this example can be extended to other malignancies.

Antineoplastic Agents↗

PubMed2003

Chemical genetics: tailoring tools for cell biology.

Chemical genetics is a research approach that uses small molecules as probes to study protein functions in cells or whole organisms. Here, I review the parallels between classical genetic and chemical-genetic approaches and discuss the merits of small molecules to dissect dynamic cellular processes. I then consider the pros and cons of different screening approaches and specify strategies aimed at identifying and validating cellular target proteins. Finally, I highlight the impact of chemical genetics on our current understanding of cell biology and its potential for the future.

Animals↗

PubMed2003

Synthesis and biological evaluation of substrate-based inhibitors of 6-phosphogluconate dehydrogenase as potential drugs against African trypanosomiasis.

The synthesis and biological evaluation of three series of 6-phosphogluconate (6PG) analogues is described. (2R)-2-Methyl-4,5-dideoxy, (2R)-2-methyl-4-deoxy and 2,4-dideoxy analogues of 6PG were tested as inhibitors of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver and also Trypanosoma brucei where the enzyme is a validated drug target. Among the three series of analogues, seven compounds were found to competitively inhibit 6PGDH from T. brucei and sheep liver enzymes at micromolar concentrations. Six inhibitors belong to the (2R)-2-methyl-4-deoxy series (6, 8, 10, 12, 21, 24) and one is a (2R)-2-methyl-4,5-dideoxy analogue (29b). The 2,4-dideoxy analogues of 6PG did not inhibit both enzymes. The trypanocidal effect of the compounds was also evaluated in vitro against T. brucei rhodesiense as well as other related trypanosomatid parasites (i.e., Trypanosoma cruzi and Leishmania donovani).

Animals↗

PubMed2002

Novel chemical genetic approaches to the discovery of signal transduction inhibitors.

Concurrent advances in both high-throughput chemistry and genomics have given rise to the field of chemical genetics as a discipline for elucidating and validating drug targets, and generating novel therapeutics. Indeed, chemical genetic approaches to drug discovery have now been applied to several important drug target classes, especially those involved in signal transduction. Chemical genetics is distinct from the broader term "chemogenomics" which is defined as the description of all possible drugs against all possible targets (reviewed in [1]). This review covers several "orthogonal" chemical genetic approaches and focuses on a unique analog sensitive kinase technology and its applications to kinase drug discovery.

Animals↗

PubMed2004

Picomolar transition state analogue inhibitors of human 5'-methylthioadenosine phosphorylase and X-ray structure with MT-immucillin-A.

Methythioadenosine phosphorylase (MTAP) functions solely in the polyamine pathway of mammals to remove the methylthioadenosine (MTA) product from both spermidine synthase (2.5.1.16) and spermine synthase (2.5.1.22). Inhibition of polyamine synthesis is a validated anticancer target. We designed and synthesized chemically stable analogues for the proposed transition state of human MTAP on the basis of the known ribooxacarbenium character at all reported N-ribosyltransferase transition states [Schramm, V. L. (2003) Acc. Chem. Res. 36, 588-596]. Methylthio-immucillin-A (MT-ImmA) is an iminoribitol tight-binding transition state analogue inhibitor with an equilibrium dissociation constant of 1.0 nM. The immucillins resemble the ribooxacarbenium ion transition states of N-ribosyltransferases and are tightly bound as the N4' cations. An ion pair formed between the iminoribitol cation and phosphate anion mimics the ribooxacarbenium cation-phosphate anion pair formed at the transition state and is confirmed in the crystal structure. The X-ray crystal structure of human MTAP with bound MT-Imm-A also reveals that the 5'-methylthio group lies in a flexible hydrophobic pocket. Substitution of the 5'-methylthio group with a 5'-phenylthio group gives an equilibrium binding constant of 1.0 nM. Methylthio-DADMe-immucillin-A is a pyrrolidine analogue of the transition state with a methylene bridge between the 9-deazaadenine group and the pyrrolidine ribooxacarbenium mimic. It is a slow-onset inhibitor with a dissociation constant of 86 pM. Improved binding energy with DADMe-immucillin-A suggests that the transition state is more closely matched by increasing the distance between leaving group and ribooxacarbenium mimics, consistent with a more dissociative transition state. Increasing the hydrophobic volume near the 5'-position at the catalytic site with 5'-phenylthio-DADMe-immucillin-A gave a dissociation constant of 172 pM, slightly weaker than the 5'-methylthio group. p-Cl-phenylthio-DADMe-immucillin-A binds with a dissociation constant of 10 pM (K(m)/K(i) value of 500000), the tightest binding inhibitor reported for MTAP. These slow-onset, tight-binding transition state analogue inhibitors are the most powerful reported for MTAP and have sufficient affinity to be useful in inhibiting the polyamine pathway.

Binding Sites↗