A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in the rpoB, katG, inhA, ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 for rpoB, katG, inhA, ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Characterization of TRIB2-mediated resistance to anti-cancer drugs

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Sub-epidemics explain localized high prevalence of reduced susceptibility to Rilpivirine in treatment-naive HIV-1-infected patients: subtype and geographic compartmentalization of baseline resistance mutations

This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Pharmacists’ knowledge and perception of topical antibacterial drug dispensing in community pharmacy setting in Kedah, Malaysia

Purpose: To assess Pharmacists’ Perceptions and Experiences of Topical Antibacterial Drug Dispensing in Community Pharmacy Setting in Kedah State, Malaysia in order to minimize drug resistance issues. Methods: A cross-sectional study involving a pre-validated questionnaire was conducted in community pharmacies within Kedah State, Malaysia. Descriptive statistics and Spearman’s correlation coefficient were used for data analysis. The collected were analysed using statistical package for social sciences (SPSS) version 18.0. Results: The result shows that, 53.4 % of CPs in Kedah State perceived that topical antibacterial is not necessary for every topical bacterial infection. Fusidic acid was the most frequently dispensed topical antibacterial drug while superficial wound was reported to be the most frequently encountered topical bacterial infection. CPs (12.60 %) encountered antibacterial resistance cases but none reported them. The drug that had resistance issue was neomycin. Conclusion: CPs in Kedah State, Malaysia generally have the right perceptions on the dispensing of topical antibacterial drugs. However, their knowledge on the rational use of topical antibacterial drugs and vigilance on antibacterial resistance issue need improvement.

Bioenergetics of cancer cells in anoxia and role of the miRNAs in melanoma resistance to targeted therapies

Tumours are characterized by a metabolic rewiring that helps transformed cells to survive in harsh conditions. The endogenous inhibitor of the ATP-synthase IF1 is overexpressed in several tumours and it has been proposed to drive metabolic adaptation. In ischemic normal-cells, IF1 acts limiting the ATP consumption by the reverse activity of the ATP-synthase, activated by ΔΨm collapse. Conversely, IF1 role in cancer cells is still unclear. It has been proposed that IF1 favours cancer survival by preventing energy dissipation in low oxygen availability, a frequent condition in solid tumours. Our previous data proved that in cancer cells hypoxia does not abolish ΔΨm, avoiding the ATP-synthase reversal and IF1 activation. In this study, we investigated the bioenergetics of cancer cells in conditions mimicking anoxia to evaluate the possible role of IF1. Data obtained indicate that also in cancer cells the ΔΨm collapse induces the ATP-synthase reversal and its inhibition by IF1. Moreover, we demonstrated that upon uncoupling conditions, IF1 favours cancer cells growth preserving ATP levels and energy charge. We also showed that in these conditions IF1 favours the mitochondrial mass renewal, a mechanism we proposed driving apoptosis-resistance. Cancer adaptability is also associated with the onset of therapy resistance, the major challenge for melanoma treatment. Recent studies demonstrated that miRNAs dysregulation drive melanoma progression and drug-resistance by regulating tumour-suppressor and oncogenes. In this context, we attempted to identify and characterize miRNAs driving resistance to vemurafenib in patient-derived metastatic melanoma cells BRAFV600E-mutated. Our results highlighted that several oncogenic pathways are altered in resistant cells, indicating the complexity of both drug-resistance phenomena and miRNAs action. Profiling analysis identified a group of dysregulated miRNAs conserved in vemurafenib-resistance cells from distinct patients, suggesting that they ubiquitously drive drug-resistance. Functional studies performed with a first miRNA confirmed its pivotal role in resistance towards vemurafenib.

TAZ role in lung cancer: resistance to BET inhibitors and functional interaction with lncRNA

Despite extensive research and introduction of innovative therapy, lung cancer prognosis remains poor, with a five years survival of only 17%. The success of pharmacological treatment is often impaired by drug resistance. Thus, the characterization of response mechanisms to anti-cancer compounds and of the molecular mechanisms supporting lung cancer aggressiveness are crucial for patient’s management. In the first part of this thesis, we characterized the molecular mechanism behind resistance of lung cancer cells to the Inhibitors of the Bromodomain and Extraterminal domain containing Proteins (BETi). Through a CRISPR/Cas9 screening we identified three Hippo Pathway members, LATS2, TAOK1 and NF2 as genes implicated in susceptibility to BETi. These genes confer sensitivity to BETi inhibiting TAZ activity. Conversely, TAZ overexpression increases resistance to BETi. We also displayed that BETi downregulate both YAP, TAZ and TEADs expression in several cancer cell lines, implying a novel BETi-dependent cytotoxic mechanism. In the second part of this work, we attempted to characterize the crosstalk between the TAZ gene and its cognate antisense long-non coding RNA (lncRNA) TAZ-AS202 in lung tumorigenesis. As for TAZ downregulation, TAZ-AS202 silencing impairs NSCLC cells proliferation, migration and invasion, suggesting a pro-tumorigenic function for this lncRNA during lung tumorigenesis. TAZ-AS202 regulates TAZ target genes without altering TAZ expression or localization. This finding implies an uncovered functional cooperation between TAZ and TAZ-AS202. Moreover, we found that the EPH-ephrin signaling receptor EPHB2 is a downstream effector affected by both TAZ and TAZ-AS202 silencing. EPHB2 downregulation significantly attenuates cells proliferation, migration and invasion, suggesting that, at least in part, TAZ-AS202 and TAZ pro-oncogenic activity depends on EPH-ephrin signaling final deregulation. Finally, we started to dissect the mechanism underlying the TAZ-AS202 regulatory activity on EPHB2 in lung cancer, which may involve the existence of an intermediate transcription factor and is the object of our ongoing research.

New chemical modalities for leishmaniasis drug discovery

Leishmaniasis is one of the major parasitic diseases among neglected tropical diseases with a high rate of morbidity and mortality. Human migration and climate change have spread the disease from limited endemic areas all over the world, also reaching regions in Southern Europe, and causing significant health and economic burden. The currently available treatments are far from ideal due to host toxicity, elevated cost, and increasing rates of drug resistance. Safer and more effective drugs are thus urgently required. Nevertheless, the identification of new chemical entities for leishmaniasis has proven to be incredibly hard and exacerbated by the scarcity of well-validated targets. Trypanothione reductase (TR) represents one robustly validated target in Leishmania that fulfils most of the requirements for a good drug target. However, due to the large and featureless active site, TR is considered extremely challenging and almost undruggable by small molecules. This scenario advocates the development of new chemical entities by unlocking new modalities for leishmaniasis drug discovery. The classical toolbox for drug discovery has enormously expanded in the last decade, and medicinal chemists can now strategize across a variety of new chemical modalities and a vast chemical space, to efficiently modulate challenging targets and provide effective treatments. Beyond others, Targeted p Protein Degradation (TPD) is an emerging strategy that uses small molecules to hijack endogenous proteolysis systems to degrade disease-relevant proteins and thus reduce their abundance in the cell. Based on these considerations, this thesis aimed to develop new strategies for leishmaniasis drug discovery while embracing novel chemical modalities and navigating the chemical space by chasing unprecedented chemotypes. This has been achieved by four complementary projects. We believe that these next-generation chemical modalities for leishmaniasis will play an important role in what was previously thought to be a drug discovery landscape dominated by small molecules.

Anticancer drug discovery using artificial intelligence: an application in pharmacological activity prediction

Hematological cancers are a heterogeneous family of diseases that can be divided into leukemias, lymphomas, and myelomas, often called “liquid tumors”. Since they cannot be surgically removable, chemotherapy represents the mainstay of their treatment. However, it still faces several challenges like drug resistance and low response rate, and the need for new anticancer agents is compelling. The drug discovery process is long-term, costly, and prone to high failure rates. With the rapid expansion of biological and chemical "big data", some computational techniques such as machine learning tools have been increasingly employed to speed up and economize the whole process. Machine learning algorithms can create complex models with the aim to determine the biological activity of compounds against several targets, based on their chemical properties. These models are defined as multi-target Quantitative Structure-Activity Relationship (mt-QSAR) and can be used to virtually screen small and large chemical libraries for the identification of new molecules with anticancer activity. The aim of my Ph.D. project was to employ machine learning techniques to build an mt-QSAR classification model for the prediction of cytotoxic drugs simultaneously active against 43 hematological cancer cell lines. For this purpose, first, I constructed a large and diversified dataset of molecules extracted from the ChEMBL database. Then, I compared the performance of different ML classification algorithms, until Random Forest was identified as the one returning the best predictions. Finally, I used different approaches to maximize the performance of the model, which achieved an accuracy of 88% by correctly classifying 93% of inactive molecules and 72% of active molecules in a validation set. This model was further applied to the virtual screening of a small dataset of molecules tested in our laboratory, where it showed 100% accuracy in correctly classifying all molecules. This result is confirmed by our previous in vitro experiments.

Tadalafil-induced Improvement In Left Ventricular Diastolic Function In Resistant Hypertension.

Left ventricular hypertrophy and diastolic dysfunction (LVDD) remain highly frequent markers of cardiac damage and risk of progression to symptomatic heart failure, especially in resistant hypertension (RHTN). We have previously demonstrated that administration of sildenafil in hypertensive rats improves LVDD, restoring phosphodiesterase type 5 (PDE-5) inhibition in cardiac myocytes. We hypothesized that the long-acting PDE-5 inhibitor tadalafil may be clinically useful in improving LVDD in RHTN independently of blood pressure (BP) reduction. A single blinded, placebo-controlled, crossover study enrolled 19 patients with both RHTN and LVDD. Firstly, subjects received tadalafil (20 mg) for 14 days and after a 2-week washout period, they received placebo orally for 14 days. Patients were evaluated by office BP and ambulatory BP monitoring (ABPM), endothelial function (FMD), echocardiography, plasma brain natriuretic peptide (BNP-32), cyclic guanosine monophosphate (cGMP) and nitrite levels. No significant differences were detected in BP measurements. Remarkably, at least four echocardiographic parameters related with diastolic function improved accompanied by decrease in BNP-32 in tadalafil use. Although increasing cGMP, tadalafil did not change endothelial function or nitrites. There were no changes in those parameters after placebo. The current findings suggest that tadalafil improves LV relaxation through direct effects PDE-5-mediated in the cardiomyocytes with potential benefit as an adjunct to treat symptomatic subjects with LVDD such as RHTN patients.

Deregulation Of Adipokines Related To Target Organ Damage On Resistant Hypertension.

Resistant hypertension (RHTN) includes patients with controlled blood pressure (BP) (CRHTN) and uncontrolled BP (UCRHTN). In fact, RHTN patients are more likely to have target organ damage (TOD), and resistin, leptin and adiponectin may affect BP control in these subjects. We assessed the relationship between adipokines levels and arterial stiffness, left ventricular hypertrophy (LVH) and microalbuminuria (MA). This cross-sectional study included CRHTN (n=51) and UCRHTN (n=38) patients for evaluating body mass index, ambulatory blood pressure monitoring, plasma adiponectin, leptin and resistin concentrations, pulse wave velocity (PWV), MA and echocardiography. Leptin and resistin levels were higher in UCRHTN, whereas adiponectin levels were lower in this same subgroup. Similarly, arterial stiffness, LVH and MA were higher in UCRHTN subgroup. Adiponectin levels negatively correlated with PWV (r=-0.42, P<0.01), and MA (r=-0.48, P<0.01) only in UCRHTN. Leptin was positively correlated with PWV (r=0.37, P=0.02) in UCRHTN subgroup, whereas resistin was not correlated with TOD in both subgroups. Adiponectin is associated with arterial stiffness and renal injury in UCRHTN patients, whereas leptin is associated with arterial stiffness in the same subgroup. Taken together, our results showed that those adipokines may contribute to vascular and renal damage in UCRHTN patients.

Nek5 Interacts With Mitochondrial Proteins And Interferes Negatively In Mitochondrial Mediated Cell Death And Respiration.

Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2μM). Nek5 silenced cells as well as cells expressing a kinase dead version of Nek5, displayed an increase in ROS formation after 4h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of hNek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro. Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells.

Glicoproteína-P, resistência a múltiplas drogas (MDR) e relação estrutura-atividade de moduladores

Multidrug resistance, MDR is a major obstacle for cancer chemotherapy. MDR can be reversed by drugs that vary in their chemical structure and main biological activity. Many efforts have been done to overcome MDR based on studies of structure-activity relationships and in this review we summarize some aspects of MDR mediated by P-glycoprotein (P-gp), as the most experimentally and clinically tested form of drug resistance. The most significant MDR mechanisms revealed until now are shortly discussed. Physicochemical and structural properties of MDR modulators, measures of the MDR reversal, and QSAR studies are included.

Carbapenem stewardship: positive impact on hospital ecology

INTRODUCTION: Excessive group 2 carbapenem use may result in decreased bacterial susceptibility. OBJECTIVE: We evaluated the impact of a carbapenem stewardship program, restricting imipenem and meropenem use. METHODS: Ertapenem was mandated for ESBL-producing Enterobacteriaceae infections in the absence of non-fermenting Gram-negative bacilli (GNB) from April 2006 to March 2008. Group 2 carbapenems were restricted for use against GNB infections susceptible only to carbapenems and suspected GNB infections in unstable patients. Cumulative susceptibility tests were done for nosocomial pathogens before and after restriction using Clinical and Laboratory Standards Institute (CLSI) guide-lines.Vitek System or conventional identification methods were performed and susceptibility testing done by disk diffusion according to CLSI.Antibiotic consumption (t-test) and susceptibilities (McNemar's test) were determined. RESULTS: The defined daily doses (DDD) of group 2 carbapenems declined from 61.1 to 48.7 DDD/1,000 patient-days two years after ertapenem introduction (p = 0.027). Mean ertapenem consumption after restriction was 31.5 DDD/1,000 patient-days. Following ertapenem introduction no significant susceptibility changes were noticed among Gram-positive cocci. The most prevalent GNB were P. aeruginosa, Klebsiella pneumoniae, and Acinetobacter spp. There was no change in P. aeruginosa susceptibility to carbapenems. Significantly improved P. aeruginosa and K. pneumoniae ciprofloxacin susceptibilities were observed, perhaps due to decreased group 2 carbapenem use. K. pneumoniae susceptibility to trimethoprim-sulfamethoxazole improved. CONCLUSION: Preferential use of ertapenem resulted in reduced group 2 carbapenem use, with a positive impact on P. aeruginosa and K. pneumoniae susceptibility.

Molecular markers and genetic diversity of Plasmodium vivax

Enhanced understanding of the transmission dynamics and population genetics for Plasmodium vivax is crucial in predicting the emergence and spread of novel parasite phenotypes with major public health implications, such as new relapsing patterns, drug resistance and increased virulence. Suitable molecular markers are required for these population genetic studies. Here, we focus on two groups of molecular markers that are commonly used to analyse natural populations of P. vivax. We use markers under selective pressure, for instance, antigen-coding polymorphic genes, and markers that are not under strong natural selection, such as most minisatellite and microsatellite loci. First, we review data obtained using genes encoding for P. vivax antigens: circumsporozoite protein, merozoite surface proteins 1 and 3α, apical membrane antigen 1 and Duffy binding antigen. We next address neutral or nearly neutral molecular markers, especially microsatellite loci, providing a complete list of markers that have already been used in P. vivax populations studies. We also analyse the microsatellite loci identified in the P. vivax genome project. Finally, we discuss some practical uses for P. vivax genotyping, for example, detecting multiple-clone infections and tracking the geographic origin of isolates.

A closer look at multiple-clone Plasmodium vivax infections: detection methods, prevalence and consequences

The naturally occurring clonal diversity among field isolates of the major human malaria parasite Plasmodium vivax remained unexplored until the early 1990s, when improved molecular methods allowed the use of blood samples obtained directly from patients, without prior in vitro culture, for genotyping purposes. Here we briefly review the molecular strategies currently used to detect genetically distinct clones in patient-derived P. vivax samples, present evidence that multiple-clone P. vivax infections are commonly detected in areas with different levels of malaria transmission and discuss possible evolutionary and epidemiological consequences of the competition between genetically distinct clones in natural human infections. We suggest that, when two or more genetically distinct clones are present in the same host, intra-host competition for limited resources may select for P. vivax traits that represent major public health challenges, such as increased virulence, increased transmissibility and antimalarial drug resistance.