Just Click Here : A Brief Glance at Absurd Electronic Contracts and the Law Failing to Protect Consumers

As e-commerce explodes around the world, consumers’ rights have been left behind. Before the completion of virtually every transaction on the Internet, the onus is placed on consumers to read and agree to an onslaught of terms and conditions. Often hidden in the middle of this extremely lengthy list of terms are massive exemption and limitation of liability clauses that deny consumers most if not all of their rights as “equal” trading partners. The common law principle that all onerous clauses in a contract need to be brought to the attention of the consumer for them to be binding seems to have been lost with the invention of the “click here to agree” button for signing online contracts. As the courts in Canada have not provided clear guidance on this issue thus far, other means must be pursued in order to protect consumers from the near-tyrannical control of unencumbered electronic standard form contracts in e-commerce. This paper will describe the principle of sufficiency of notice as it applies to paper contracts, and then contrast it with the newer jurisprudence that has refused to apply the principle to electronic contracts. The reasons for the refusal will be explored, followed by an examination of why the principle of sufficiency of notice needs to be applied and strengthened to respond to the increasingly onerous provisions hidden in electronic contracts. Finally, some other options for achieving the goal of consumer protection from hidden onerous clauses will be briefly explored. These other options include introducing stiffer consumer protection legislation domestically, the creation of international treaties, developing voluntary standards of contracting, and relying on Internet self-regulation

Pyrazoleamide compounds are potent antimalarials that target Na+ homeostasis in intraerythrocytic Plasmodium falciparum

The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na+ regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na+ homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na+ homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes

A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples.

BACKGROUND:Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. RESULTS:The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest  2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to  date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. CONCLUSIONS:Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria

Contrasting ex vivo efficacies of "reversed chloroquine" compounds in chloroquine-resistant Plasmodium falciparum and P. vivax isolates.

Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance ex vivo. Between March to October 2011 and January to September 2013, two "reversed chloroquine" (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of Plasmodium falciparum (n = 41) and Plasmodium vivax (n = 45) in Papua, Indonesia, using a modified ex vivo schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant P. falciparum and P. vivax field isolates. For P. falciparum, the median 50% inhibitory concentrations (IC50s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (P < 0.001 and P = 0.036, respectively). The corresponding values for P. vivax were 19.0, 60.0, and 60.9 nM (P < 0.001 and P = 0.018, respectively). There was a significant correlation between IC50s of CQ and PL69 (Spearman's rank correlation coefficient [r s] = 0.727, P < 0.001) and PL106 (rs = 0.830, P < 0.001) in P. vivax but not in P. falciparum. Both RCQs were equally active against the ring and trophozoite stages of P. falciparum, but in P. vivax, PL69 and PL106 showed less potent activity against trophozoite stages (median IC50s, 130.2 and 172.5 nM) compared to ring stages (median IC50s, 17.6 and 91.3 nM). RCQ compounds have enhanced ex vivo activity against CQ-resistant clinical isolates of P. falciparum and P. vivax, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two Plasmodium species

Plasmodium falciparum and P. vivax demonstrate contrasting chloroquine resistance reversal phenotypes

High-grade chloroquine (CQ) resistance has emerged in both P. falciparum and P. vivax The aim of the present study was to investigate phenotypic differences of CQ resistance in both of these species and the ability of known CQ resistance reversal agents (CQRRAs) to alter CQ susceptibility.Between April 2015 and April 2016, the potential of verapamil (VP), mibefradil (MF), L703,606 (L7), and primaquine (PQ) to reverse CQ resistance was assessed in 46 P. falciparum and 34 P. vivax clinical isolates in Papua, Indonesia, where CQ resistance is present in both species, using a modified schizont maturation assay.In P. falciparum, CQ IC50s were reduced when CQ was combined with VP (1.4-fold), MF (1.2-fold), L7 (4.2 fold), or PQ (1.8 fold). The degree of CQ resistance reversal in P. falciparum was highly correlated with CQ susceptibility for all CQRRAs: VP (R(2)=0.951), (R(2)=0.852), L7 (R(2)=0.962), and PQ (R(2)=0.901), in line with observations in P. falciparum laboratory strains. In contrast, no reduction in CQ IC50s was observed with any of the CQRRAs in P. vivax, even in those isolates with high chloroquine IC50s.The differential effect of CQRRAs in P. falciparum and P. vivax suggests significant differences in CQ kinetics and potentially the likely mechanism of CQ resistance between these two species

Plasmodium falciparum and P. vivax demonstrate contrasting chloroquine resistance reversal phenotypes

High-grade chloroquine (CQ) resistance has emerged in both P. falciparum and P. vivax The aim of the present study was to investigate phenotypic differences of CQ resistance in both of these species and the ability of known CQ resistance reversal agents (CQRRAs) to alter CQ susceptibility.Between April 2015 and April 2016, the potential of verapamil (VP), mibefradil (MF), L703,606 (L7), and primaquine (PQ) to reverse CQ resistance was assessed in 46 P. falciparum and 34 P. vivax clinical isolates in Papua, Indonesia, where CQ resistance is present in both species, using a modified schizont maturation assay.In P. falciparum, CQ IC50s were reduced when CQ was combined with VP (1.4-fold), MF (1.2-fold), L7 (4.2 fold), or PQ (1.8 fold). The degree of CQ resistance reversal in P. falciparum was highly correlated with CQ susceptibility for all CQRRAs: VP (R(2)=0.951), (R(2)=0.852), L7 (R(2)=0.962), and PQ (R(2)=0.901), in line with observations in P. falciparum laboratory strains. In contrast, no reduction in CQ IC50s was observed with any of the CQRRAs in P. vivax, even in those isolates with high chloroquine IC50s.The differential effect of CQRRAs in P. falciparum and P. vivax suggests significant differences in CQ kinetics and potentially the likely mechanism of CQ resistance between these two species

Comparative ex vivo activity of novel endoperoxides in multidrug-resistant Plasmodium falciparum and P. vivax

The declining efficacy of artemisinin derivatives against Plasmodium falciparum highlights the urgent need to identify alternative highly potent compounds for the treatment of malaria. In Papua Indonesia, where multidrug resistance has been documented against both P. falciparum and P. vivax malaria, comparative ex vivo antimalarial activity against Plasmodium isolates was assessed for the artemisinin derivatives artesunate (AS) and dihydroartemisinin (DHA), the synthetic peroxides OZ277 and OZ439, the semisynthetic 10-alkylaminoartemisinin derivatives artemisone and artemiside, and the conventional antimalarial drugs chloroquine (CQ), amodiaquine (AQ), and piperaquine (PIP). Ex vivo drug susceptibility was assessed in 46 field isolates (25 P. falciparum and 21 P. vivax). The novel endoperoxide compounds exhibited potent ex vivo activity against both species, but significant differences in intrinsic activity were observed. Compared to AS and its active metabolite DHA, all the novel compounds showed lower or equal 50% inhibitory concentrations (IC(50)s) in both species (median IC(50)s between 1.9 and 3.6 nM in P. falciparum and 0.7 and 4.6 nM in P. vivax). The antiplasmodial activity of novel endoperoxides showed different cross-susceptibility patterns in the two Plasmodium species: whereas their ex vivo activity correlated positively with CQ, PIP, AS, and DHA in P. falciparum, the same was not apparent in P. vivax. The current study demonstrates for the first time potent activity of novel endoperoxides against drug-resistant P. vivax. The high activity against drug-resistant strains of both Plasmodium species confirms these compounds to be promising candidates for future artemisinin-based combination therapy (ACT) regimens in regions of coendemicit

Passively versus actively detected malaria: similar genetic diversity but different complexity of infection

The surveillance of malaria is generally undertaken on the assumption that samples passively collected at health facilities are comparable to or representative of the broader Plasmodium reservoir circulating in the community. Further characterization and comparability of the hidden asymptomatic parasite reservoir are needed to inform on the potential impact of sampling bias. This study explores the impact of sampling strategy on molecular surveillance by comparing the genetic make-up of Plasmodium falciparum and Plasmodium vivax isolates collected by passive versus active case detection. Sympatric isolates of P. falciparum and P. vivax were collected from a large community survey and ongoing clinical surveillance studies undertaken in the hypomesoendemic setting of Mimika District (Papua, Indonesia). Plasmodium falciparum isolates were genotyped at nine microsatellite loci and P. vivax at eight loci. Measures of diversity and differentiation were used to compare different patient and parasitological sample groups. The results demonstrated that passively detected cases (symptomatic) had comparable population diversity to those circulating in the community (asymptomatic) in both species. In addition, asymptomatic patent infections were as diverse as subpatent infections. However, a significant difference in multiplicity of infection (MOI) and percentage of polyclonal infections was observed between actively and passively detected P. vivax cases (mean MOI: 1.7 ± 0.7 versus 1.4 ± 1.4, respectively; P = 0.001). The study findings infer that, in hypomesoendemic settings, passive sampling is appropriate for molecular parasite surveillance strategies using the predominant clone in any given infection; however, the findings suggest caution when analyzing complexity of infection. Further evaluation is required in other endemic settings