Insights into innate immune activation via PS-ASO-protein-TLR9 interactions.

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary-in part-as a function of 2'modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO-protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.

The basic chemical substances of total alkaloids of Uncaria rhynchophylla and their anti-neuroinflammatory activities.

To clarify the chemical basis of the total alkaloids of Uncaria rhynchophylla, HPLC-VWD chromatogram of total alkaloids was established. Under its guidance, modern chromatographic and spectroscopic techniques were used to track, isolate and identify the representative principal components. As a result, one new monoterpenoid indole alkaloid, 3S,15S-N4-methoxymethyl-geissoschizine methyl ether (1), together with 20 known alkaloids (2-21), and 5 other known compounds (22-26) were obtained. Meanwhile, sixteen characteristic peaks were identified from the total alkaloids using HPLC analysis. Then, the anti-neuroinflammatory effect of compounds 1-21 was assessed through inhibiting nitric ---oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 microglial cells. Among them, compounds 1, 3, 7, 8, 11, 12, 19 and 21 showed potent inhibitory activities with IC50 values of 5.87-76.78 µM.

In Vitro Susceptibility of Plasmodium falciparum Isolates from the China-Myanmar Border Area to Piperaquine and Association with Candidate Markers.

Plasmodium falciparum from the Greater Mekong subregion has evolved resistance to the artemisinin-based combination therapy dihydroartemisinin and the partner drug piperaquine. To monitor the potential westward spread or independent evolution of piperaquine resistance, we evaluated the in vitro susceptibility of 120 P. falciparum isolates collected at the China-Myanmar border during 2007-2016. The parasite isolates displayed a relatively wide range of piperaquine susceptibility estimates. While 56.7% of the parasites showed bimodal drug response curves, all but five generated area-under-the-curve (AUC) estimates consistent with a susceptible phenotype. Using the piperaquine survival assay (PSA), 5.6% parasites showed reduced susceptibility. Of note, parasites from 2014-2016 showed the highest AUC value and the highest proportion with a bimodal curve, suggesting falling effectiveness in these later years. Unsupervised K-mean analysis of the combined data assigned parasites into three clusters and identified significant correlations between IC50, IC90, and AUC values. No parasites carried the E415G mutation in a putative exo-nuclease, new mutations in PfCRT, or amplification of the plasmepsin 2/3 genes, suggesting mechanisms of reduced piperaquine susceptibility that differ from those described in other countries of the region. The association of increased AUC, IC50, and IC90 values with major PfK13 mutations (F446I and G533S) suggests that piperaquine resistance may evolve in these PfK13 genetic backgrounds. Additionally, the Pfmdr1 F1226Y mutation was associated with significantly higher PSA values. Further elucidation of piperaquine resistance mechanisms and continuous surveillance are warranted.

Efficacy of artemether-lumefantrine for treating uncomplicated Plasmodium falciparum cases and molecular surveillance of drug resistance genes in Western Myanmar.

BACKGROUND: Currently, artemisinin-based combination therapy (ACT) is the first-line anti-malarial treatment in malaria-endemic areas. However, resistance in Plasmodium falciparum to artemisinin-based combinations emerging in the Greater Mekong Sub-region is a major problem hindering malaria elimination. To continuously monitor the potential spread of ACT-resistant parasites, this study assessed the efficacy of artemether-lumefantrine (AL) for falciparum malaria in western Myanmar. METHODS: Ninety-five patients with malaria symptoms from Paletwa Township, Chin State, Myanmar were screened for P. falciparum infections in 2015. After excluding six patients with a parasite density below 100 or over 150,000/µL, 41 P. falciparum patients were treated with AL and followed for 28 days. Molecular markers associated with resistance to 4-amino-quinoline drugs (pfcrt and pfmdr1), antifolate drugs (pfdhps and pfdhfr) and artemisinin (pfk13) were genotyped to determine the prevalence of mutations associated with anti-malarial drug resistance. RESULTS: For the 41 P. falciparum patients (27 children and 14 adults), the 28-day AL therapeutic efficacy was 100%, but five cases (12.2%) were parasite positive on day 3 by microscopy. For the pfk13 gene, the frequency of NN insert after the position 136 was 100% in the day-3 parasite-positive group as compared to 50.0% in the day-3 parasite-negative group, albeit the difference was not statistically significant (P = 0.113). The pfk13 K189T mutation (10.0%) was found in Myanmar for the first time. The pfcrt K76T and A220S mutations were all fixed in the parasite population. In pfmdr1, the Y184F mutation was present in 23.3% of the parasite population, and found in both day-3 parasite-positive and -negative parasites. The G968A mutation of pfmdr1 gene was first reported in Myanmar. Prevalence of all the mutations in pfdhfr and pfdhps genes assessed was over 70%, with the exception of the pfdhps A581G mutation, which was 3.3%. CONCLUSIONS: AL remained highly efficacious in western Myanmar. Pfk13 mutations associated with artemisinin resistance were not found. The high prevalence of mutations in pfcrt, pfdhfr and pfdhps suggests high-degree resistance to chloroquine and antifolate drugs. The pfmdr1 N86/184F/D1246 haplotype associated with selection by AL in Africa reached > 20% in this study. The detection of > 10% patients who were day-3 parasite-positive after AL treatment emphasizes the necessity of continuously monitoring ACT efficacy in western Myanmar.

Multiple relapses of Plasmodium vivax malaria acquired from West Africa and association with poor metabolizer CYP2D6 variant: a case report.

BACKGROUND: Plasmodium vivax transmission in West Africa, dominant for the Duffy-negative blood group, has been increasingly recognized from both local residents as well as international travelers who contracted P. vivax malaria there. However, the relapsing pattern and sensitivity to antimalarial treatment of P. vivax strains originated from this region are largely unknown. There is evidence that the efficacy of primaquine for radical cure of relapsing malaria depends on host factors such as the hepatic enzyme cytochrome P450 (CYP) 2D6. CASE PRESENTATION: A 49-year-old Chinese man was admitted to the Shanglin County Hospital in Guangxi Province, China, on December 19, 2016, 39 days after he returned from Ghana, where he stayed for one and a half years. He was diagnosed by microscopy as having uncomplicated P. vivax malaria. Treatment included 3 days of intravenous artesunate (420 mg total), and 3 days of chloroquine (1550 mg total), and 8 days of primaquine (180 mg total). Although parasites and symptoms were cleared rapidly and he was malaria-negative for almost two months, he suffered four relapses with relapse intervals ranging from 58 to 232 days. The last relapse occurred at 491 days from his first vivax attack. For the first three relapses, he was treated similarly with chloroquine and primaquine, sometimes supplemented with additional artemisinin combination therapies (ACTs). For the last relapse, he was treated with intravenous artesunate, 3 days of an ACT, and 7 days of azithromycin, and had remained healthy for 330 days. Molecular studies confirmed P. vivax infections for all the episodes. Although this patient was diagnosed to have normal glucose-6-phosphate dehydrogenase (G6PD) activity, his CYP2D6 genotype corresponded to a *2A/*36 allele variant suggesting of an impaired primaquine metabolizer phenotype. CONCLUSIONS: This clinical case suggests that P. vivax malaria originating from West Africa may produce multiple relapses extending beyond one year. The failures of primaquine as an anti-relapse therapy may be attributed to the patient's impaired metabolizer phenotype of the CYP2D6. This highlights the importance of knowing the host G6PD and CYP2D6 activities for effective radical cure of relapsing malaria by primaquine.

Advancing and Lengthening Genioplasty in Contouring of the Receding and Short Chin.

BACKGROUND: In East Asia, receding and short chin are common complaints of patients who do not have satisfied lower face. In most former studies, receding and short chin are considered and treated separately. But during the clinical work, the authors found that, in many patients, neither vertical elongation nor horizontal advancement of the chin is sufficient to achieve a harmonious result. In regards of this problem, the authors performed an advancing and lengthening genioplasty and the results were aesthetically satisfactory. STUDY DESIGN: Twenty-six patients with receding and short chin were involved in this study. After presurgical computed tomography (CT) scan, advancing and lengthening genioplasty with/without other osteotomy operations were performed on all the patients. All patients underwent postoperative CT scan and had at least 3-month follow-up. RESULT: All patients were satisfied with the final results. According to the postoperative CT images and 3-month follow-up, no severe complications occurred. CONCLUSION: For patients with receding and short chin, advancing and lengthening genioplasty is a reliable therapy to obtain harmonious East Asian lower face.

Meta-analysis of initial natriuretic peptides in the setting of sepsis-induced myocardial dysfunction.

Aim: To investigate the association of initial brain natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP) with the detection of sepsis-induced myocardial dysfunction (SIMD) in the setting of Sepsis 3.0. Methods: Three databases were searched to analyze initial BNP and NT-proBNP levels between SIMD and non-SIMD groups. Results: Eighteen studies were included, most of which defined SIMD based on echocardiography. The SIMD group exhibited higher initial BNP and NT-proBNP levels in blood. NT-proBNP higher than a certain cutoff value (>3000 pg/ml) was an independent risk factor for SIMD and its accuracy for SIMD diagnosis was moderate (pooled area under the curve: 0.81). Conclusion: Initial blood BNP and NT-proBNP levels are useful to assist in the detection of SIMD and further studies are warranted to determine the SIMD definition.

Phosphorylation of collagen fibrils enhances intrafibrillar mineralization and dentin remineralization.

The hierarchical assembly of nanoapatite within a type I collagen matrix was achieved through biomimetic mineralization in vitro, cooperatively regulated by non-collagenous proteins and small biomolecules. Here, we demonstrated that IP6 could significantly promote intrafibrillar mineralization in two- and three-dimensional collagen models through binding to collagen fibrils via hydrogen bonds (the interaction energy ∼10.21 kJ mol-1), as confirmed by the FTIR spectra and isothermal experimental results. In addition, we find that IP6 associated with dental collagen fibrils can also enhance the remineralization of calcium-depleted dentin and restore its mechanical properties similar to the natural dentin within 4 days. The promoting effect is mainly due to the chemical modification of IP6, which alters the interfacial physicochemical properties of collagen fibrils, strengthening the interaction of calcium phosphate minerals and mineral ions with collagen fibrils. This strategy of interfacial regulation to accelerate the mineralization of collagen fibrils is essential for dental repair and the development of a clinical product for the remineralization of hard tissue.

Correction: Phosphorylation of collagen fibrils enhances intrafibrillar mineralization and dentin remineralization.

Correction for 'Phosphorylation of collagen fibrils enhances intrafibrillar mineralization and dentin remineralization' by Bo Zheng et al., Nanoscale, 2024, https://doi.org/10.1039/d4nr00652f.

Characterization of cooperative PS-oligo activation of human TLR9.

Single-stranded phosphorothioate oligonucleotides (PS-oligos) can activate TLR9, leading to an innate immune response. This can occur with PS-oligos containing unmethylated CpG sites, the canonical motif, or PS-oligos that do not contain those motifs (non-CpG). Structural evidence shows that TLR9 contains two PS-oligo binding sites, and recent data suggest that synergistic cooperative activation of TLR9 can be achieved by adding two separate PS-oligos to cells, each engaging with a separate site on TLR9 to enhance TLR9 activation as a pair. Here, we demonstrate and characterize this cooperativity phenomenon using PS-oligos in human cell lines, and we introduce several novel PS-oligo pairs (CpG and non-CpG pairs) that show cooperative activation. Indeed, we find that cooperative PS-oligos likely bind at different sites on TLR9. Interestingly, we find that PS-oligos that generate little TLR9 activation on their own can prime TLR9 to be activated by other PS-oligos. Finally, we determine that previous models of TLR9 activation cannot be used to fully explain data from systems using human TLR9 and PS-oligos. Overall, we reveal new details of TLR9 activation, but we also find that more work needs to be done to determine where certain PS-oligos are binding to TLR9.

Systematic Analysis of Chemical Modifications of Phosphorothioate Antisense Oligonucleotides that Modulate Their Innate Immune Response.

While rare, some gapmer phosphorothioate (PS) antisense oligonucleotides (ASOs) can induce a noncanonical TLR9-dependent innate immune response. In this study, we performed systematic analyses of the roles of PS ASO backbone chemistry, 2' modifications, and sequence in PS ASO induced TLR9 signaling. We found that each of these factors can contribute to altering PS ASO induced TLR9 signaling, and in some cases the effects are quite dramatic. We also found that the positioning (5' vs. 3') of a particular backbone or 2' modification within a PS ASO can affect its TLR9 signaling. Interestingly, medicinal chemical strategies that decrease TLR9 signaling for one sequence can have opposing effects on another sequence. Our results demonstrate that TLR9 signaling is highly PS ASO sequence dependent, the mechanism of which remains unknown. Despite this, we determined that placement of two mesyl phosphoramidate linkages within the PS ASO gap is the most promising strategy to mitigate PS ASO dependent TLR9 activation to enhance the therapeutic index and, therefore, further streamline PS ASO drug development.

Effect of aspartic acid on the crystallization kinetics of ACP and dentin remineralization.

Type I collagen and non-collagen proteins are the main organic components of dentin. This study aimed to investigate the biomimetic remineralization of demineralized dentin by aspartic acid (Asp), which is abundant in non-collagenous proteins (NCPs). Asp was added to a mineralizing solution containing polyacrylic acid (PAA) to explore the mechanism of Asp regulating the pure amorphous calcium phosphate (ACP) phase transition process. The remineralization process and superstructure of the remineralized layer of demineralized dentin were evaluated and analyzed by transmission electron microscope (TEM) and scanning electron microscope (SEM), and the biological stability of the remineralized layer was investigated by collagenase degradation experiment. It demonstrated that Asp promoted the crystallization kinetics of PAA-stabilized amorphous calcium phosphate to hydroxyapatite (HAP), and shortened the remineralization time of demineralized dentin from 7 days to 2 days. The newly formed remineralized dentin had similar morphology and biological stability to the natural dentin layer. The presence of a large number of Asp residues in NCPs promoted the phase transformation of ACP, and further revealed the mechanism of action of NCPs in dentin biomineralization. This experiment also showed that Asp promoted the biomimetic remineralization of dentin; the morphology and hierarchical structure of remineralized layer was similar to that of natural teeth, and had good biological properties.

Unraveling the Complexity of Imported Malaria Infections by Amplicon Deep Sequencing.

Imported malaria and recurrent infections are becoming an emerging issue in many malaria non-endemic countries. This study aimed to determine the molecular patterns of the imported malaria infections and recurrence. Blood samples were collected from patients with imported malaria infections during 2016-2018 in Guangxi Zhuang Autonomous Region, China. Next-generation amplicon deep-sequencing approaches were used to assess parasite genetic diversity, multiplexity of infection, relapse, recrudescence, and antimalarial drug resistance. A total of 44 imported malaria cases were examined during the study, of which 35 (79.5%) had recurrent malaria infections within 1 year. The majority (91.4%) had one recurrent malaria episode, whereas two patients had two recurrences and one patient had three recurrences. A total of 19 recurrence patterns (the species responsible for primary and successive clinical episodes) were found in patients returning from malaria epidemic countries. Four parasite species were detected with a higher than usual proportion (46.2%) of non-falciparum infections or mixed-species infections. An increasing trend of recurrence infections and reduced drug treatment efficacy were observed among the cases of imported malaria. The high recurrence rate and complex patterns of imported malaria from Africa to non-endemic countries have the potential to initiate local transmission, thereby undermining efforts to eliminate locally acquired malaria. Our findings highlight the power of amplicon deep-sequencing applications in molecular epidemiological studies of the imported malaria recurrences.

First Detection in West Africa of a Mutation That May Contribute to Artemisinin Resistance Plasmodium falciparum.

Background: The spread of drug resistance has seriously impacted the effective treatment of infection with the malaria parasite, Plasmodium falciparum. Continuous monitoring of molecular marker polymorphisms associated with drug resistance in parasites is essential for malaria control and elimination efforts. Our study describes mutations observed in the resistance genes Pfkelch13, Pfcrt, and Pfmdr1 in imported malaria and identifies additional potential drug resistance-associated molecular markers. Methods: Chinese patients infected in Africa with P. falciparum were treated with intravenous (IV) injections of artesunate 240-360 mg for 3-5 days while hospitalized and treated with oral dihydroartemisinin-piperaquine (DHP) for 3 days after hospital discharge. Blood samples were collected and PCR sequencing performed on genes Pfkelch13, Pfcrt, and Pfmdr1 from all isolates. Results: We analyzed a total of 225 patients from Guangxi, China with P. falciparum malaria acquired in Africa between 2016 and 2018. All patients were cured completely after treatment. The F446I mutation of the Pfkelch13 gene was detected for the first time from samples of West African P. falciparum, with a frequency of 1.0%. Five haplotypes of Pfcrt that encode residues 72-76 were found, with the wild-type CVMNK sequence predominating (80.8% of samples), suggesting that the parasites might be chloroquine sensitive. For Pfmdr1, N86Y (13.1%) and Y184F (58.8%) were the most prevalent, suggesting that artemether-lumefantrine may not, in general, be a suitable treatment for the group. Conclusions: For the first time, this study detected the F446I mutation of the Pfkelch13 gene from Africa parasites that lacked clinical evidence of resistance. This study provides the latest data for molecular marker surveillance related to antimalarial drug resistance genes Pfkelch13, Pfcrt, and Pfmdr1 imported from Africa, in Guangxi, China from Chinese migrate workers. Clinical Trial Registration: ChiCTROPC17013106.

Positive selection at high temperature reduces gene transcription in the bacteriophage ϕX174.

BACKGROUND: Gene regulation plays a central role in the adaptation of organisms to their environments. There are many molecular components to gene regulation, and it is often difficult to determine both the genetic basis of adaptation and the evolutionary forces that influence regulation. In multiple evolution experiments with the bacteriophage ϕX174, adaptive substitutions in cis-acting regulatory sequences sweep through the phage population as the result of strong positive selection at high temperatures that are non-permissive for laboratory-adapted phage. For one cis-regulatory region, we investigate the individual effects of four adaptive substitutions on transcript levels and fitness for phage growing on three hosts at two temperatures. RESULTS: The effect of the four individual substitutions on transcript levels is to down-regulate gene expression, regardless of temperature or host. To ascertain the conditions under which these substitutions are adaptive, fitness was measured by a variety of methods for several bacterial hosts growing at two temperatures, the control temperature of 37°C and the selective temperature of 42°C. Time to lysis and doublings per hour indicate that the four substitutions individually improve fitness over the ancestral strain at high temperature independent of the bacterial host in which the fitness was measured. Competition assays between the ancestral strain and either of two mutant strains indicate that both mutants out-compete the ancestor at high temperature, but the relative frequencies of each phage remain the same at the control temperature. CONCLUSIONS: Our results strongly suggest that gene transcription plays an important role in influencing fitness in the bacteriophage ϕX174, and different point mutations in a single cis-regulatory region provided the genetic basis for this role in adaptation to high temperature. We speculate that the adaptive nature of these substitutions is due to the physiology of the host at high temperature or the need to maintain particular ratios of phage proteins during capsid assembly. Our investigation of regulatory evolution contributes to interpreting genome-level assessments of regulatory variation, as well as to understanding the molecular basis of adaptation.

Associations among Soil-Transmitted Helminths, G6PD Deficiency and Asymptomatic Malaria Parasitemia, and Anemia in Schoolchildren from a Conflict Zone of Northeast Myanmar.

In tropical areas of developing countries, the interactions among parasitic diseases such as soil-transmitted helminths (STHs) and malaria, and glucose-6-phosphate dehydrogenase deficiency (G6PDd), are complex. Here, we investigated their interactions and impact on anemia in school students residing in a conflict zone of northeast Myanmar. A cross-sectional survey was conducted between July and December 2015 in two schools located along the China-Myanmar border. Stool samples from the schoolchildren were analyzed for STH infections, whereas finger-prick blood samples were analyzed for G6PDd, hemoglobin concentrations, and Plasmodium infections. Among 988 enrolled children, Plasmodium vivax, Plasmodium falciparum, hookworm, Ascaris lumbricoides, and Trichuris trichiura infections occurred in 3.3%, 0.8%, 31.5%, 1.2%, and 0.3%, respectively. Glucose-6-phosphate dehydrogenase deficiency was present in 16.9% of the children, and there was a very high prevalence of anemia (73%). Anthropometric measures performed on all children showed that 50% of the children were stunted and 25% wasted. Moderate to severe anemia was associated with STH infections, stunting, and wasting. In addition, children had increasing odds of anemia with increasing burden of infections. This study revealed a high prevalence of G6PDd, STHs, and anemia in schools located in a conflict zone. In areas where malnutrition and STH infections are rampant, testing for both glucose-6-phosphate dehydrogenase and anemia should be considered before treating vivax malaria with 8-aminoquinolines.

Widespread resistance mutations to sulfadoxine-pyrimethamine in malaria parasites imported to China from Central and Western Africa.

BACKGROUND: Imported cases of infectious disease provide invaluable information about epidemiological conditions abroad, and should guide treatment decisions at home and abroad. Here, we examined cases of malaria imported from Africa to China for mutations eroding the efficacy of sulfadoxine-pyrimethamine (SP), sometimes used as an intermittent preventive treatment during for pregnant women and infants. METHODS: A total of 208 blood samples were collected from P. falciparum-infected workers who had returned from Western and Central Africa to Guangxi Province Frequency distribution. Samples were analyzed for the mutations in dhfr and dhps genes by PCR -sequencing. The prevalence of dhfr and dhps polymorphisms was analyzed. Among the isolates, polymorphisms were detected in mutants N51I, C59R, S108N and I164L of Pfdhfr and I431V, S436 A/F, A437G, K540 E/N, A581G and A613T of pfdhps. RESULTS: Mutations promoting drug resistance were widespread in this cohort. For pfdhfr and pfdhps, wild types were equally rare among patients returned from Western Africa and Central Africa. A triple-mutant dhfr haplotype was most prevalent (>70%). We report for the first time mutation I164L-dhfr and I431V-dhps in Ghana, and for the first time we found A581G to exceed a clinically-relevant threshold that may counter-indicate current clinical practices. For Pfdhps, the double-mutant IAGKAA was high prevalent haplotype in Ghana, Western Africa. The single-mutant ISGKAA was a majority haplotype in Cameroon. Alarmingly, a "super resistance" quintuple mutant was detected, for the first time, in parasites of West African origin (defined by IAGKAA/IRNI in combination with pfdhps 581G and dhfr I164L). This may limit the efficacy of this drug combination for even intermittent clinical applications. CONCLUSIONS: These data are cause for great concern and call for continued surveillance of the efficacy of SP in source and recipient populations, and should be considered when developing treatment policy for imported malaria cases in China and elsewhere.

Ex vivo susceptibilities of Plasmodium vivax isolates from the China-Myanmar border to antimalarial drugs and association with polymorphisms in Pvmdr1 and Pvcrt-o genes.

BACKGROUND: Vivax malaria is an important public health problem in the Greater Mekong Subregion (GMS), including the China-Myanmar border. Previous studies have found that Plasmodium vivax has decreased sensitivity to antimalarial drugs in some areas of the GMS, but the sensitivity of P. vivax to antimalarial drugs is unclear in the China-Myanmar border. Here, we investigate the drug sensitivity profile and genetic variations for two drug resistance related genes in P. vivax isolates to provide baseline information for future drug studies in the China-Myanmar border. METHODOLOGY/PRINCIPAL FINDINGS: A total of 64 P. vivax clinical isolates collected from the China-Myanmar border area were assessed for ex vivo susceptibility to eight antimalarial drugs by the schizont maturation assay. The medians of IC50 (half-maximum inhibitory concentrations) for chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate, artemether, dihydroartemisinin were 84.2 nM, 34.9 nM, 4.0 nM, 22.3 nM, 41.4 nM, 2.8 nM, 2.1 nM and 2.0 nM, respectively. Twelve P. vivax clinical isolates were found over the cut-off IC50 value (220 nM) for chloroquine resistance. In addition, sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvcrt-o (P. vivax chloroquine resistance transporter-o), and difference in pvmdr1 copy number were studied. Sequencing of the pvmdr1 gene in 52 samples identified 12 amino acid substitutions, among which two (G698S and T958M) were fixed, M908L were present in 98.1% of the isolates, while Y976F and F1076L were present in 3.8% and 78.8% of the isolates, respectively. Amplification of the pvmdr1 gene was only detected in 4.8% of the samples. Sequencing of the pvcrt-o in 59 parasite isolates identified a single lysine insertion at position 10 in 32.2% of the isolates. The pvmdr1 M908L substitutions in pvmdr1 in our samples was associated with reduced sensitivity to chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate and dihydroartemisinin. CONCLUSIONS: Our findings depict a drug sensitivity profile and genetic variations of the P. vivax isolates from the China-Myanmar border area, and suggest possible emergence of chloroquine resistant P. vivax isolates in the region, which demands further efforts for resistance monitoring and mechanism studies.

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

In vitro susceptibility of Plasmodium falciparum isolates from the China-Myanmar border area to artemisinins and correlation with K13 mutations.

Mutations in the Kelch domain of the K13 gene (PF3D7_1343700) were previously associated with artemisinin resistance in Plasmodium falciparum. This study followed the dynamics of the K13 polymorphisms in P. falciparum parasites from the China-Myanmar border area obtained in 2007-2016, and their in vitro sensitivities to artesunate (AS) and dihydroartemisinin (DHA). The 50% effective concentration (EC5072h) values of 133 culture-adapted field isolates to AS and DHA, measured by the conventional 72 h SYBR Green I-based assay, varied significantly among the parasites from different years; all were significantly higher than that of the reference strain 3D7. Compared with parasites from 2007 to 2008, ring survival rates almost doubled in parasites obtained in later years. Sequencing the full-length K13 genes identified 11 point mutations present in 85 (63.9%) parasite isolates. F446I was the predominant (55/133) variant, and its frequency was increased from 17.6% (3/17) in 2007 to 55.9% (19/34) in 2014-2016. No wild-type (WT) Kelch domain sequences were found in the 34 samples obtained from 2014 to 2016. In the 2014-2016 samples, a new mutation (G533S) appeared and reached 44.1% (15/34). Collectively, parasites with the Kelch domain mutations (after amino acid 440) had significantly higher ring survival rates than the WT parasites. Individually, F446I, G533S and A676D showed significantly higher ring survival rates than the WT. Although the drug sensitivity phenotypes measured by the RSA6h and EC5072h assays may be intrinsically linked to the in vivo clinical efficacy data, the values determined by these two assays were not significantly correlated. This study identified the trend of K13 mutations in parasite populations from the China-Myanmar border area, confirmed an overall correlation of Kelch domain mutations with elevated ring-stage survival rates, and emphasized the importance of monitoring the evolution and spread of parasites with reduced artemisinin sensitivity along the malaria elimination course.