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Herein, we report that peptide nucleic acid sequences (PNAs) have been used as the probe species for detection of RNA and that a microfluidic microarray (MMA) chip is used as the platform for detection of hybridizations between immobilized PNA probes and RNA targets. The RNA targets used are derived from influenza A sequences. This paper discusses the optimization of two probe technologies used for RNA detection and investigates how the composition of the probe buffer and the content of the hybridization solution can influence the overall results. Our data show that the PNA probe is a better choice than the DNA probe when there is low salt in the probe buffer composition. Furthermore, we show that the absence of salt (NaCl) in the hybridization buffer does not hinder the detection of RNA sequences. The results provide evidence that PNA probes are superior to DNA probes in term of sensitivity and adaptability, as PNA immobilization and PNA-RNA hybridization are less affected by salt content in the reaction buffers unlike DNA probes. Key words: peptide nucleic acid (PNA), influenza viral RNA, probe buffer, hybridization buffer, salt, formamide, microfluidic microarray (MMA). Dans le present article, nous rapportons que des sequences d'acides nucleiques peptidiques (ANP) peuvent etre utilisees comme matiere a sonde pour la detection de l'ARN, et qu'une puce microfluidique peut etre employee comme plateforme pour la detection de l'hybridation entre ces sondes a base d'ANP immobilises et les ARN cibles. Les ARN cibles que nous avons utilises sont derives de sequences d'influenza A. Cet article porte sur l'optimisation des deux technologies de sonde employees pour la detection de l'ARN et sur l'etude de l'effet de la composition du tampon de sonde et de la solution d'hybridation sur les resultats globaux. Nos donnees montrent que la sonde d'ANP constitue un meilleur choix que la sonde d'ADN lorsque la teneur en sel du tampon de sonde est faible. De plus, nous avons montre que l'absence de sel (NaCl) dans le tampon d'hybridation ne nuit pas a la detection des sequences d'ARN. Les resultats permettent de conclure que les sondes d'ANP sont superieures sur les plans de la sensibilite et de l'adaptabilite, car l'immobilisation de l'ANP et l'hybridation entre l'ANP et l'ARN sont moins influencees par la teneur en sel dans les tampons reactionnels qu'elles le sont dans les sondes d'ADN. [Traduit par la Redaction] Mots-cles : acide nucleique peptidique (ANP), ARN du virus de l'influenza, tampon de sonde, tampon d'hybridation, sel, formamide, puce microfluidique., Introduction Peptide nucleic acids (PNAs) are DNA analogs in which the negatively charged deoxyribose phosphate backbone is replaced by an electrically neutral peptide-like backbone. (1,2) Because the PNA probes are [...]

Patients with brain metastases (BM) face a 90% mortality rate within one year of diagnosis and the current standard of care is palliative. Targeting BM-initiating cells (BMICs) is a feasible strategy to treat BM, but druggable targets are limited. Here, we apply Connectivity Map analysis to lung-, breast-, and melanoma-pre-metastatic BMIC gene expression signatures and identify inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo GTP synthesis pathway, as a target for BM. We show that pharmacological and genetic perturbation of IMPDH attenuates BMIC proliferation in vitro and the formation of BM in vivo. Metabolomic analyses and CRISPR knockout studies confirm that de novo GTP synthesis is a potent metabolic vulnerability in BM. Overall, our work employs a phenotype-guided therapeutic strategy to uncover IMPDH as a relevant target for attenuating BM outgrowth, which may provide an alternative treatment strategy for patients who are otherwise limited to palliation., Competing Interests: Declaration of interests A.M.K., J.W.J., C.V., J.M., and S.K.S. are listed as co-inventors for a PCT patent that has been filed, relating to this work., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The aberrant activity of histone deacetylases (HDACs) across a broad range of cancers and other disease indications has led to the development of small-molecule inhibitors that target one or more members of the HDAC protein family. Emerging HDAC inhibitors that show promise in drug discovery programs must be assessed across a range of in vitro assays to establish an inhibitor profile for potency and cellular selectivity towards target HDAC(s) as well as preliminary absorption, distribution, metabolism, and excretion (ADME) features. Here we provide an overview of methods to determine a subset of pivotal in vitro drug-like parameters for HDAC inhibitors (HDACi). We initially describe protocols for parallel artificial membrane permeability assays (PAMPA) to evaluate the passive permeability of small molecules across lipid membranes. Subsequently, we elaborate on cytotoxicity assays using CellTiter-Blue to determine HDACi-induced cell death in healthy/diseased cellular models. We next focus on assessing the target engagement of inhibitors with the appropriate HDAC isoforms in a cellular environment via Western blotting of acetylated HDAC substrates. Finally, we provide detailed guidelines on how to assess the metabolic stability of HDACi through whole blood stability assays. Collectively, these assays provide an overview of the permeability, selectivity, and stability of the HDAC inhibitor under development., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

Here, we describe detection of single nucleotide polymorphism (SNP) in genomic DNA samples using a NanoBioArray (NBA) chip. Fast DNA hybridization is achieved in the chip when target DNAs are introduced to the surface-arrayed probes using centrifugal force. Gold nanoparticles (AuNPs) are used to assist SNP detection at room temperature. The parallel setting of sample introduction in the spiral channels of the NBA chip enables multiple analyses on many samples, resulting in a technique appropriate for high-throughput SNP detection. The experimental procedure, including chip fabrication, probe array printing, DNA amplification, hybridization, signal detection, and data analysis, is described in detail.