High-Throughput, Volume 7, Issue 4 (December 2018) – 12 articles

In the past decades, many efforts have been made to individualize medical treatments, taking into account molecular profiles and the individual genetic background. The development of molecularly targeted drugs and immunotherapy have revolutionized medical treatments but the inter-patient variability in the anti-tumor drug pharmacokinetics (PK) and pharmacodynamics can be explained, at least in part, by genetic variations in genes encoding drug metabolizing enzymes and transporters (ADME) or in genes encoding drug receptors. Here, we focus on high-throughput technologies applied for PK screening for the identification of predictive biomarkers of efficacy or toxicity in cancer treatment, whose application in clinical practice could promote personalized treatments tailored on individual’s genetic make-up. Pharmacogenomic tools have been implemented and the clinical utility of pharmacogenetic screening could increase safety in patients for the identification of drug metabolism-related biomarkers for a personalized medicine. Although pharmacogenomic studies were performed in adult cohorts, pharmacogenetic pediatric research has yielded promising results. Additionally, we discuss the current challenges and theoretical bases for the implementation of pharmacogenetic tests for translation in the clinical practice taking into account that pharmacogenomics platforms are discovery oriented and must open the way for the setting of robust tests suitable for daily practice. Full article

Precision oncologic medicine is an emerging approach for cancer treatment that has recently taken giant steps in solid clinical practice. Recent advances in molecular diagnostics that can analyze the individual tumor’s variability in genes have provided greater understanding and additional strategies to treat cancers. Although tumors can be tested by several molecular methods, the use of next-generation sequencing (NGS) has greatly facilitated our understanding of pediatric cancer and identified additional therapeutic opportunities. Pediatric tumors have a different genetic make-up, with a fewer number of actionable targets than adult tumors. Nevertheless, precision oncology in the pediatric population has greatly improved the survival of patients with leukemia and solid tumors. This review discusses the current status of pediatric precision oncology and the different clinical scenarios in which it can be effectively applied. Full article

The last few years have featured an increasing interest in the study of the human microbiome and its correlations with health status. Indeed, technological advances have allowed the study of microbial communities to reach a previously unthinkable sensitivity, showing the presence of microbes also in environments usually considered as sterile. In this scenario, microbial communities have been described in the amniotic fluid, the umbilical blood cord, and the placenta, denying a dogma of reproductive medicine that considers the uterus like a sterile womb. This prenatal microbiome may play a role not only in fetal development but also in the predisposition to diseases that may develop later in life, and also in adulthood. Thus, the aim of this review is to report the current knowledge regarding the prenatal microbiome composition, its association with pathological processes, and the future perspectives regarding its manipulation for healthy status promotion and maintenance. Full article

We present a novel approach for the molecular transformation and analysis of patient clinical phenotypes. Building on the fact that drugs perturb the function of targets/genes, we integrated data from 8.2 million clinical reports detailing drug-induced side effects with the molecular world of drug-target information. Using this dataset, we extracted 1.8 million associations of clinical phenotypes to 770 human drug-targets. This collection is perhaps the largest phenotypic profiling reference of human targets to-date, and unique in that it enables rapid development of testable molecular hypotheses directly from human-specific information. We also present validation results demonstrating analytical utilities of the approach, including drug safety prediction, and the design of novel combination therapies. Challenging the long-standing notion that molecular perturbation studies cannot be performed in humans, our data allows researchers to capitalize on the vast tomes of clinical information available throughout the healthcare system. Full article

Beta-lactam resistant bacteria, which are commonly resident in tertiary hospitals, have emerged as a worldwide health problem because of ready-to-eat vegetable intake. We aimed to characterize the genes that provide resistance to beta-lactam antibiotics in Enterobacteriaceae, isolated from five commercial salad brands for human consumption in Mexico City. In total, twenty-five samples were collected, grown in blood agar plates, and the bacteria were biochemistry identified and antimicrobial susceptibility testing was done. The carried family genes were identified by endpoint PCR and the specific genes were confirmed with whole genome sequencing (WGS) by Next Generation Sequencing (NGS). Twelve positive cultures were identified and their microbiological distribution was as follows: 8.3% for Enterobacter aerogene (n = 1), 8.3% for Serratia fonticola (n = 1), 16.7% for Serratia marcesens (n = 2), 16.7% for Klebsiella pneumoniae (n = 2), and 50% (n = 6) for Enterobacter cloacae. The endpoint PCR results showed 11 colonies positive for blaBIL (91.7%), 11 for blaSHV (91.7%), 11 for blaCTX (97.7%), 12 for blaDHA (100%), four for blaVIM (33.3%), two for blaOXA (16.7%), two for blaIMP (16.7%), one for blaKPC (8.3%), and one for blaTEM (8.3%) gen; all samples were negative for blaROB, blaCMY, blaP, blaCFX and blaLAP gene. The sequencing analysis revealed a specific genotype for Enterobacter cloacae (blaSHV-12, blaCTX-M-15, blaDHA-1, blaKPC-2); Serratia marcescens (blaSHV-1, blaCTX-M-3, blaDHA-1, blaVIM-2); Klebsiella pneumoniae (blaSHV-12, blaCTX-M-15, blaDHA-1); Serratia fonticola (blaSHV-12, blaVIM-1, blaDHA-1); and, Enterobacter aerogene (blaSHV-1, blaCTX-M-1, blaDHA-1, blaVIM-2, blaOXA-9). Our results indicate that beta-lactam-resistant bacteria have acquired integrons with a different number of genes that provide pan-resistance to beta-lactam antibiotics, including penicillins, oxacillins, cefalosporins, monobactams, carbapenems, and imipenems. Full article

We model the transmission of the hepatitis B virus (HBV) by six differential equations that represent the reactions between HBV with DNA-containing capsids, the hepatocytes, the antibodies and the cytotoxic T-lymphocyte (CTL) cells. The intracellular delay and treatment are integrated into the model. The existence of the optimal control pair is supported and the characterization of this pair is given by the Pontryagin’s minimum principle. Note that one of them describes the effectiveness of medical treatment in restraining viral production, while the second stands for the success of drug treatment in blocking new infections. Using the finite difference approximation, the optimality system is derived and solved numerically. Finally, the numerical simulations are illustrated in order to determine the role of optimal treatment in preventing viral replication. Full article

In the last decade, it has been demonstrated that long non-coding RNAs (lncRNAs) are involved in cancer development. The great majority of studies on lncRNAs report alterations, principally on their expression profiles, in several tumor types with respect to the normal tissues of origin. Conversely, since lncRNAs constitute a relatively novel class of RNAs compared to protein-coding transcripts (mRNAs), the landscape of their mutations and variations has not yet been extensively studied. However, in recent years an ever-increasing number of articles have described mutations of lncRNAs. Single-nucleotide polymorphisms (SNPs) that occur within the lncRNA transcripts can affect the structure and function of these RNA molecules, while the presence of a SNP in the promoter region of a lncRNA could alter its expression level. Also, somatic mutations that occur within lncRNAs have been shown to exert important effects in cancer and preliminary data are promising. Overall, the evidence suggests that SNPs and somatic mutation on lncRNAs may play a role in the pathogenesis of cancer, and indicates strong potential for further development of lncRNAs as biomarkers. Full article

Integration of multi-omics data from different molecular levels with clinical data, as well as epidemiologic risk factors, represents an accurate and promising methodology to understand the complexity of biological systems of human diseases, including cancer. By the extensive use of novel technologic platforms, a large number of multidimensional data can be derived from analysis of health and disease systems. Comprehensive analysis of multi-omics data in an integrated framework, which includes cumulative effects in the context of biological pathways, is therefore eagerly awaited. This strategy could allow the identification of pathway-addiction of cancer cells that may be amenable to therapeutic intervention. However, translation into clinical settings requires an optimized integration of omics data with clinical vision to fully exploit precision cancer medicine. We will discuss the available technical approach and more recent developments in the specific field. Full article

Meningitis is commonly caused by infection with a variety of bacterial or viral pathogens. Acute bacterial meningitis (ABM) can cause severe disease, which can progress rapidly to a critical life-threatening condition. Rapid diagnosis of ABM is critical, as this is most commonly associated with severe sequelae with associated high mortality and morbidity rates compared to viral meningitis, which is less severe and self-limiting. We have designed a microarray for detection and diagnosis of ABM. This has been validated using randomly amplified DNA targets (RADT), comparing buffers with or without formamide, in glass slide format or on the Alere ArrayTube^TM (Alere Technologies GmbH) microarray platform. Pathogen-specific signals were observed using purified bacterial nucleic acids and to a lesser extent using patient cerebral spinal fluid (CSF) samples, with some technical issues observed using RADT and glass slides. Repurposing the array onto the Alere ArrayTube^TM platform and using a targeted amplification system increased specific and reduced nonspecific hybridization signals using both pathogen nucleic and patient CSF DNA targets, better revealing pathogen-specific signals although sensitivity was still reduced in the latter. This diagnostic microarray is useful as a laboratory diagnostic tool for species and strain designation for ABM, rather than for primary diagnosis. Full article

An alarming increase in tuberculosis (TB) caused by drug-resistant strains of Mycobacterium tuberculosis has created an urgent need for new antituberculosis drugs acting via novel mechanisms. Phylogenomic and comparative genomic analyses reviewed here reveal that the TB causing bacteria comprise a small group of organisms differing from all other mycobacteria in numerous regards. Comprehensive analyses of protein sequences from mycobacterial genomes have identified 63 conserved signature inserts and deletions (indels) (CSIs) in important proteins that are distinctive characteristics of the TB-complex of bacteria. The identified CSIs provide potential means for development of novel diagnostics as well as therapeutics for the TB-complex of bacteria based on four key observations: (i) The CSIs exhibit a high degree of exclusivity towards the TB-complex of bacteria; (ii) Earlier work on CSIs provide evidence that they play important/essential functions in the organisms for which they exhibit specificity; (iii) CSIs are located in surface-exposed loops of the proteins implicated in mediating novel interactions; (iv) Homologs of the CSIs containing proteins, or the CSIs in such homologs, are generally not found in humans. Based on these characteristics, it is hypothesized that the high-throughput virtual screening for compounds binding specifically to the CSIs (or CSI containing regions) and thereby inhibiting the cellular functions of the CSIs could lead to the discovery of a novel class of drugs specifically targeting the TB-complex of organisms. Full article

CTCF (CCCTC-binding factor) is a transcription regulator with hundreds of binding sites in the human genome. It has a main function as an insulator protein, defining together with cohesins the boundaries of areas of the genome called topologically associating domains (TADs). TADs contain regulatory elements such as enhancers which function as regulators of the transcription of genes inside the boundaries of the TAD while they are restricted from regulating genes outside these boundaries. This paper will examine the most common genetic lesions of CTCF as well as its related protein CTCFL (CTCF-like also called BORIS) in cancer using publicly available data from published genomic studies. Cancer types where abnormalities in the two genes are more common will be examined for possible associations with underlying repair defects or other prevalent genetic lesions. The putative functional effects in CTCF and CTCFL lesions will also be explored. Full article

Cytochrome P450 (CYP) is a critical drug-metabolizing enzyme superfamily. Modulation of CYP enzyme activities has the potential to cause drug–drug/herb interactions. Drug–drug/herb interactions can lead to serious adverse drug reactions (ADRs) or drug failures. Therefore, there is a need to examine the modulatory effects of new drug entities or herbal preparations on a wide range of CYP isoforms. The classic method of quantifying CYP enzyme activities is based on high-performance liquid chromatography (HPLC), which is time- and reagent-consuming. In the past two decades, high-throughput screening methods including fluorescence-based, luminescence-based, and mass-spectrometry-based assays have been developed and widely applied to estimate CYP enzyme activities. In general, these methods are faster and use lower volume of reagents than HPLC. However, each high-throughput method has its own limitations. Investigators may make a selection of these methods based on the available equipment in the laboratory, budget, and enzyme sources supplied. Furthermore, the current high-throughput systems should look into developing a reliable automation mechanism to accomplish ultra-high-throughput screening in the near future. Full article