Variation in Theileria equi drug susceptibility in vitro and the potential role of ABC transporters as mediators of parasitic drug resistance

We have identified and partially sequenced 8 ABC transporters from an EST dataset ofSarcoptes scabieivar.hominis, the causative agent of scabies. Analysis confirmed that most of the known ABC subfamilies are represented in the EST dataset including several members of the multidrug resistance protein subfamily (ABC-C). Although P-glycoprotein (ABC-B) sequences were not found in the EST dataset, a partial P-glycoprotein sequence was subsequently obtained using a degenerate PCR strategy and library screening. Thus a total of 9 potentialS. scabieiABC transporters representing the subfamilies A, B, C, E, F and H have been identified. Ivermectin is currently used in the treatment of hyper-infested (crusted) scabies, and has also been identified as a potentially effective acaricide for mass treatment programmes in scabies-endemic communities. The observation of clinical andin vitroivermectin resistance in 2 crusted scabies patients who received multiple treatments has raised serious concerns regarding the sustainability of such programmes. One possible mechanism for ivermectin resistance is through ABC transporters such as P-glycoprotein. This work forms an important foundation for further studies to elucidate the potential role of ABC transporters in ivermectin resistance ofS. scabiei.

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Role of Plasmodium vivax Dihydropteroate Synthase Polymorphisms in Sulfa Drug Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01835-15 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4453-4463 ◽

Cited By ~ 9

Author(s):

Wichai Pornthanakasem ◽

Pinpunya Riangrungroj ◽

Penchit Chitnumsub ◽

Wanwipa Ittarat ◽

Darin Kongkasuriyachai ◽

...

Keyword(s):

Drug Resistance ◽

Drug Susceptibility ◽

Sulfa Drug ◽

Content Type ◽

Genetic Sequencing ◽

Dihydropteroate Synthase ◽

Transgenic Parasite

ABSTRACTDihydropteroate synthase (DHPS) is a known sulfa drug target in malaria treatment, existing as a bifunctional enzyme together with hydroxymethyldihydropterin pyrophosphokinase (HPPK). Polymorphisms in key residues ofPlasmodium falciparumDHPS (PfDHPS) have been characterized and linked to sulfa drug resistance in malaria. Genetic sequencing ofP. vivaxdhps(Pvdhps) from clinical isolates has shown several polymorphisms at the positions equivalent to those in thePfdhpsgenes conferring sulfa drug resistance, suggesting a mechanism for sulfa drug resistance inP. vivaxsimilar to that seen inP. falciparum. To characterize the role of polymorphisms in thePvDHPS in sulfa drug resistance, various mutants of recombinantPvHPPK-DHPS enzymes were expressed and characterized. Moreover, due to the lack of a continuousin vitroculture system forP. vivaxparasites, a surrogateP. bergheimodel expressingPvhppk-dhpsgenes was established to demonstrate the relationship between sequence polymorphisms and sulfa drug susceptibility and to test the activities ofPvDHPS inhibitors on the transgenic parasites. Both enzyme activity and transgenic parasite growth were sensitive to sulfadoxine to different degrees, depending on the number of mutations that accumulated in DHPS.Kivalues and 50% effective doses were higher for mutantPvDHPS enzymes than the wild-type enzymes. Altogether, the study provides the first evidence of sulfa drug resistance at the molecular level inP. vivax. Furthermore, the enzyme inhibition assay and thein vivoscreening system can be useful tools for screening new compounds for their activities againstPvDHPS.

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Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Viruses ◽

10.3390/v13061092 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1092

Author(s):

János András Mótyán ◽

Márió Miczi ◽

Stephen Oroszlan ◽

József Tőzsér

Keyword(s):

Amino Acid ◽

Zinc Finger ◽

Cleavage Site ◽

Potential Role ◽

Binding Mode ◽

Interaction Energies ◽

Vitro Assay ◽

Hiv 1

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

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Assessment of the Drug Susceptibility of Plasmodium falciparum Clinical Isolates from Africa by Using a Plasmodium Lactate Dehydrogenase Immunodetection Assay and an Inhibitory Maximum Effect Model for Precise Measurement of the 50-Percent Inhibitory Concentration

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00367-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3343-3349 ◽

Cited By ~ 61

Author(s):

Halima Kaddouri ◽

Serge Nakache ◽

Sandrine Houzé ◽

France Mentré ◽

Jacques Le Bras

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Lactate Dehydrogenase ◽

Active Metabolite ◽

Inhibitory Concentration ◽

Drug Susceptibility ◽

Maximum Effect ◽

Malaria Parasites ◽

Effect Model

ABSTRACT The extension of drug resistance among malaria-causing Plasmodium falciparum parasites in Africa necessitates implementation of new combined therapeutic strategies. Drug susceptibility phenotyping requires precise measurements. Until recently, schizont maturation and isotopic in vitro assays were the only methods available, but their use was limited by technical constraints. This explains the revived interest in the development of replacement methods, such as the Plasmodium lactate dehydrogenase (pLDH) immunodetection assay. We evaluated a commercially controlled pLDH enzyme-linked immunosorbent assay (ELISA; the ELISA-Malaria antigen test; DiaMed AG, Cressier s/Morat, Switzerland) to assess drug susceptibility in a standard in vitro assay using fairly basic laboratory equipment to study the in vitro resistance of malaria parasites to major antimalarials. Five Plasmodium falciparum clones and 121 clinical African isolates collected during 2003 and 2004 were studied by the pLDH ELISA and the [8-3H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC50) and its confidence intervals. The two methods were observed to have similar reproducibilities, but the pLDH ELISA demonstrated a higher sensitivity. The high correlation (r = 0.98) and the high phenotypic agreement (κ = 0.88) between the two methods allowed comparison by determination of the IC50s. Recently collected Plasmodium falciparum African isolates were tested by pLDH ELISA and showed drug resistance or decreased susceptibilities of 62% to chloroquine and 11.5% to the active metabolite of amodiaquine. No decreased susceptibility to lumefantrine or the active metabolite of artemisinin was detected. The availability of this simple and highly sensitive pLDH immunodetection assay will provide an easier method for drug susceptibility testing of malaria parasites.

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Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

10.21203/rs.3.rs-22444/v3 ◽

2020 ◽

Author(s):

Nonlawat Boonyalai ◽

Brian A Vesely ◽

Chatchadaporn Thamnurak ◽

Chantida Praditpol ◽

Watcharintorn Fagnark ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Drug Susceptibility ◽

Antagonistic Activity ◽

Drug Combinations ◽

Chloroquine Resistance ◽

Phenotypic Characterization ◽

Field Isolates

Abstract Background High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 ( pfpm2 ), exonuclease ( pfexo ) and chloroquine resistance transporter ( pfcrt ) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.Methods To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.Results The characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovoquone-proguanil combinations revealed synergistic antimalarial activity.Conclusions Surveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

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High Expression of GSDMC Is Associated with Poor Survival in Kidney Clear Cell Cancer

BioMed Research International ◽

10.1155/2021/5282894 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Yun-Qian Cui ◽

Fei Meng ◽

Wen-Li Zhan ◽

Zhou-Tong Dai ◽

Xinghua Liao

Keyword(s):

Potential Role ◽

Clear Cell ◽

Cell Cancer ◽

Progression Free Survival ◽

Renal Clear Cell Carcinoma ◽

High Expression ◽

Poor Survival ◽

Free Survival

This study is aimed at exploring the potential role of GSDMC in kidney renal clear cell carcinoma (KIRC). We analyzed the expression of GSDMC in 33 types of cancers in TCGA database. The results showed that the expression of GSDMC was upregulated in most cancers. We found a significant association between high expression of GSDMC and shortened patient overall survival, progression-free survival, and disease-specific survival. In vitro experiments have shown that the expression of GSDMC was significantly elevated in KIRC cell lines. Moreover, decreased expression of GSDMC was significantly associated with decreased cell proliferation. In summary, we believe that this study provides valuable data supporting future clinical treatment.

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PEAK1 promotes invasion and metastasis and confers drug resistance in breast cancer

10.21203/rs.3.rs-549884/v1 ◽

2021 ◽

Author(s):

xingang wang ◽

YAN ZHENG ◽

YU WANG

Keyword(s):

Breast Cancer ◽

Drug Resistance ◽

Cell Growth ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Multi Drug Resistance ◽

Cancer Tissues

Abstract Background and AimsPseudopodium-enriched atypical kinase 1 (PEAK1) has reported to be upregulated in human malignancies and related with poor prognosis. Enhanced PEAK1 expression facilitates tumor cell survival, invasion, metastasis and chemoresistance. However, the role of PEAK1 in breast cancer is not clear. Here, we investigated the PEAK1 expression in breast cancer and analyzed its relation with clinicopathological status and chemotherapy resistance to the neoadjuvant chemotherapy (NAC). We also investigated the role of PEAK1 on breast cancer cells in vitro and in vivo. MethodsImmunohistochemistry (IHC) was performed in 112 surgical resected breast cancer tissues. The associations between clinicopathological status, multi-drug resistance and PEAK1 expression were determined. Effect of PEAK1 overexpression or down-expression on proliferation, colony formation, invasion, migration, metastasis and Doxorubicin sensitivity in the MCF-7 cells in vitro and in vivo was detected. ResultsPEAK1 was overexpressed in breast cancer tissues and NAC -resistant breast cancer tissues. High PEAK1 expression was related with tumor size, high tumor grade, T stage, LN metastasis, recurrence, Ki-67 expression, Her-2 expression and multi-drug resistance. Targeting PEAK1 inhibited cell growth, invasion, metastasis and reversed chemoresistance to Doxorubicin in breast cancer cells in vitro and in vivo. ConclusionHigh PEAK1 expression was associated with invasion, metastasis and chemoresistance of breast cancers. Furthermore, targeting PEAK1 could inhibit cell growth and metastasis, and reverse chemoresistance in breast cancer cells, which provides an effective treatment strategies for breast cancer.

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Ozone Therapy as Adjuvant for Cancer Treatment: Is Further Research Warranted?

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/7931849 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 11

Author(s):

Bernardino Clavo ◽

Norberto Santana-Rodríguez ◽

Pedro Llontop ◽

Dominga Gutiérrez ◽

Gerardo Suárez ◽

...

Keyword(s):

Cancer Treatment ◽

Immune Modulation ◽

Animal Studies ◽

Potential Role ◽

Randomized Clinical Trials ◽

Tumor Hypoxia ◽

Tumor Resection ◽

Ozone Therapy

Introduction. This article provides an overview of the potential use of ozone as an adjuvant during cancer treatment.Methods. We summarize the findings of the most relevant publications focused on this goal, and we include our related clinical experience.Results. Over several decades, prestigious journals have publishedin vitrostudies on the capacity of ozone to induce direct damage on tumor cells and, as well, to enhance the effects of radiotherapy and chemotherapy. Indirect effects have been demonstrated in animal models: immune modulation by ozone alone and sensitizing effect of radiotherapy by concurrent ozone administration. The effects of ozone in modifying hemoglobin dissociation curve, 2,3-diphosphoglycerate levels, locoregional blood flow, and tumor hypoxia provide additional support for potential beneficial effects during cancer treatment. Unfortunately, only a few clinical studies are available. Finally, we describe some works and our experience supporting the potential role of local ozone therapy in treating delayed healing after tumor resection, to avoid delays in commencing radiotherapy and chemotherapy.Conclusions.In vitroand animal studies, as well as isolated clinical reports, suggest the potential role of ozone as an adjuvant during radiotherapy and/or chemotherapy. However, further research, such as randomized clinical trials, is required to demonstrate its potential usefulness as an adjuvant therapeutic tool.

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Pre-Clinical Drug Testing in 2D and 3D Human In Vitro Models of Glioblastoma Incorporating Non-Neoplastic Astrocytes: Tunneling Nano Tubules and Mitochondrial Transfer Modulates Cell Behavior and Therapeutic Response

International Journal of Molecular Sciences ◽

10.3390/ijms20236017 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6017 ◽

Cited By ~ 10

Author(s):

Prospero Civita ◽

Diana M. Leite ◽

Geoffrey Pilkington

Keyword(s):

Drug Resistance ◽

Hyaluronic Acid ◽

Drug Testing ◽

Tunneling Nanotubes ◽

Mitochondrial Transfer ◽

Culture Models ◽

And Migration ◽

2D And 3D

The role of astrocytes in the glioblastoma (GBM) microenvironment is poorly understood; particularly with regard to cell invasion and drug resistance. To assess this role of astrocytes in GBMs we established an all human 2D co-culture model and a 3D hyaluronic acid-gelatin based hydrogel model (HyStem™-HP) with different ratios of GBM cells to astrocytes. A contact co-culture of fluorescently labelled GBM cells and astrocytes showed that the latter promotes tumour growth and migration of GBM cells. Notably, the presence of non-neoplastic astrocytes in direct contact, even in low amounts in co-culture, elicited drug resistance in GBM. Recent studies showed that non-neoplastic cells can transfer mitochondria along tunneling nanotubes (TNT) and rescue damaged target cancer cells. In these studies, we explored TNT formation and mitochondrial transfer using 2D and 3D in vitro co-culture models of GBM and astrocytes. TNT formation occurs in glial fibrillary acidic protein (GFAP) positive “reactive” astrocytes after 48 h co-culture and the increase of TNT formations was greater in 3D hyaluronic acid-gelatin based hydrogel models. This study shows that human astrocytes in the tumour microenvironment, both in 2D and 3D in vitro co-culture models, could form TNT connections with GBM cells. We postulate that the association on TNT delivery non-neoplastic mitochondria via a TNT connection may be related to GBM drug response as well as proliferation and migration.

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Impact of the first-line treatment shift from dihydroartemisinin/piperaquine to artesunate/mefloquine on Plasmodium vivax drug susceptibility in Cambodia

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa092 ◽

2020 ◽

Vol 75 (7) ◽

pp. 1766-1771 ◽

Cited By ~ 2

Author(s):

Camille Roesch ◽

Mélissa Mairet-Khedim ◽

Saorin Kim ◽

Dysoley Lek ◽

Jean Popovici ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Vivax ◽

Drug Susceptibility ◽

Single Mutation ◽

First Line ◽

The Past ◽

Drug Assays ◽

Number Variation ◽

In Vitro Response

Abstract Background Cambodia is the epicentre of the emergence of Plasmodium falciparum drug resistance. Much less is known regarding the drug susceptibility of the co-endemic Plasmodium vivax. Only in vitro drug assays can determine the parasite’s intrinsic susceptibility, but these are challenging to implement for P. vivax and rarely performed. Objectives To evaluate the evolution of Cambodian P. vivax susceptibility to antimalarial drugs and determine their association with putative markers of drug resistance. Methods In vitro response to three drugs used in the past decade in Cambodia was measured for 52 clinical isolates from Eastern Cambodia collected between 2015 and 2018 and the sequence and copy number variation of their pvmdr1 and pvcrt genes were analysed. pvmdr1 polymorphism was also determined for an additional 250 isolates collected in Eastern Cambodia between 2014 and 2019. Results Among the 52 cryopreserved isolates tested, all were susceptible to the three drugs, with overall median IC50s of 16.1 nM (IQR 11.4–22.3) chloroquine, 3.4 nM (IQR 2.1–5.0) mefloquine and 4.6 nM (IQR 2.7–7.0) piperaquine. A significant increase in chloroquine and piperaquine susceptibility was observed between 2015 and 2018, unrelated to polymorphisms in pvcrt and pvmdr1. Susceptibility to mefloquine was significantly lower in parasites with a single mutation in pvmdr1 compared with isolates with multiple mutations. The proportion of parasites with this single mutation genotype increased between 2014 and 2019. Conclusions P. vivax with decreased susceptibility to mefloquine is associated with the introduction of mefloquine-based treatment during 2017–18.

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