Kelch-like proteins in the gastrointestinal tumors.

Gastrointestinal tumors have become a worldwide health problem with high morbidity and poor clinical outcomes. Chemotherapy and surgery, the main treatment methods, are still far from meeting the treatment needs of patients, and targeted therapy is in urgent need of development. Recently, emerging evidence suggests that kelch-like (KLHL) proteins play essential roles in maintaining proteostasis and are involved in the progression of various cancers, functioning as adaptors in the E3 ligase complex and promoting the specific degradation of substrates. Therefore, KLHL proteins should be taken into consideration for targeted therapy strategy discovery. This review summarizes the current knowledge of KLHL proteins in gastrointestinal tumors and discusses the potential of KLHL proteins as potential drug targets and prognostic biomarkers.

Generation and analysis of pseudohypoaldosteronism type II knock-in mice caused by a nonsense KLHL3 mutation in the Kelch domain.

Mutations in the Kelch-like 3 (KLHL3) gene are the most common cause of inherited pseudohypoaldosteronism type II (PHAII) featuring thiazide-sensitive hypertension and hyperkalemic metabolic acidosis. Although Klhl3R528H/+ knock-in (KI) mice carrying a missense mutation in the Kelch repeat domain have been reported, nonsense KLHL3 mutations in the same domain that cause PHAII have not been fully investigated in vivo. We generated and analyzed Klhl3 KI mice harboring a nonsense W523X mutation (corresponding to the human KLHL3 W470X mutation). Both heterozygous and homozygous Klhl3W523X/+ KI mice exhibited typical PHAII with low-renin hypertension, hyperkalemia with reduced renal potassium excretion, and hyperchloremic metabolic acidosis. Their kidney tissues showed the presence of Klhl3 mRNA and increased Klhl3 protein levels along with enhanced downstream Wnk1/4-Spak/Osr1-N(k)cc phosphorylation. Increased protein expression of total Spak, phosphor(p-)Spak, total Ncc, and p-Ncc from urinary extracellular vesicles (uEVs) also confirmed the activation of the Wnk-mediated Ncc pathway. In vitro studies showed that the human KLHL3 W470X mutation resulted in increased KLHL3 protein stability and disrupted its binding affinity for WNK1/4, leading to the attenuated degradation and increased abundance of total WNKs. In conclusion, nonsense Klhl3W523X/+ mice recapitulating PHAII phenotypes exhibit Klhl3 protein stability, abrogating its binding to Wnks, with enhanced Ncc expression in the kidney tissue and even in uEVs. Activation of the WNK-mediated Na+ -Cl- co-transporter reiterated the in vivo pathogenic role of nonsense KLHL3 mutations in PHAII.

Absence of Plasmodium falciparum artemisinin resistance gene mutations eleven years after the adoption of artemisinin-based combination therapy in Nigeria.

BACKGROUND: The occurrence of artemisinin resistance (ART)-associated polymorphism of Plasmodium falciparum K13-propeller (pfk13) gene before and after the introduction of artemisinin-based combination therapy (ACT) in two regions of Nigeria was investigated in this study. Regular surveillance is necessary to make a definite conclusion on the emergence and pattern of possible resistance to ART. METHODS: This cross-sectional study was carried out in the Southwestern and Southeastern geopolitical zones of Nigeria. A total of 150, 217, and 475 participants were enrolled for the study in the Southwest (2004_Group A), Southwest (2015_Group B), and southeast (2015_Group C), respectively. Blood samples were collected from the study participants for DNA extraction and a nested PCR for P. falciparum identification. Samples that were positive for P. falciparum were genotyped for the pfk13 gene using the Sanger sequencing method. The single nucleotide polymorphisms were analysed using the Bioedit software. RESULTS: A total of 116, 125, and 83 samples were positive for P. falciparum, respectively for the samples collected from the Southwest (2004 and 2015) and southeast (2015). Parasite DNA samples collected from febrile children in 2004 (Group A; n = 71) and 2015 (Group B; n = 73) in Osogbo Western Nigeria and 2015_Group C (n = 36) in southeast Nigeria were sequenced successfully. This study did not observe mutations associated with the in vitro resistance in southeast Asia, such as Y493H, R539T, I543T, and C580Y. Two new polymorphisms V520A and V581I were observed in two samples collected in Osogbo, Southwest Nigeria. These two mutations occurred in the year 2004 (Group A) before the introduction of ACT. Six mutations were identified in 17% of the samples collected in southeast Nigeria. One of these mutations (D547G) was non-synonymous, while the remaining (V510V, R515R, Q613Q, E688E, and N458N) were synonymous. Also, one (2%) heterozygote allele was identified at codon 458 in the 2015 (Group C) samples. CONCLUSIONS: None of the mutations observed in this study were previously validated to be associated with ART resistance. These results, therefore, suggest that artemisinin is likely to remain highly effective in treating malaria in the study areas that are malarious zone.

Increase in the proportion of Plasmodium falciparum with kelch13 C580Y mutation and decline in pfcrt and pfmdr1 mutant alleles in Papua New Guinea.

BACKGROUND: The C580Y mutation in the Plasmodium falciparum kelch13 gene is the most commonly observed variant in artemisinin-resistant isolates in the Greater Mekong Subregion (GMS). Until 2017, it had not been identified outside the GMS, except for Guyana/Amazonia. In 2017, three parasites carrying the C580Y mutation were identified in Papua New Guinea (PNG). As the C580Y allele rapidly spread in the GMS, there is concern that this mutant is now spreading in PNG. METHODS: In 2020, a cross-sectional survey was conducted at two clinics in Wewak, PNG. Symptomatic patients infected with P. falciparum were treated with artemether plus lumefantrine following a national treatment policy. Blood samples were obtained before treatment, and polymorphisms in kelch13, pfcrt, and pfmdr1 were determined. Parasite positivity was examined on day 3. The results were compared with those of previous studies conducted in 2002, 2003, and 2016-2018. RESULTS: A total of 94 patients were included in this analysis. The proportion of C580Y was significantly increased (2.2% in 2017, 5.7% in 2018, and 6.4% in 2020; p = 4.2 × 10-3). A significant upward trend was observed in the wild-type proportion for pfcrt (1.9% in 2016 to 46.7% in 2020; p = 8.9 × 10-16) and pfmdr1 (59.5% in 2016 to 91.4% in 2020; p = 2.3 × 10-6). Among 27 patients successfully followed on day 3, including three with C580Y infections, none showed positive parasitaemia. CONCLUSIONS: Under the conditions of significant increases in pfcrt K76 and pfmdr1 N86 alleles in PNG, the increase in kelch13 C580Y mutants may be a warning indicator of the emergence of parasites resistant to the currently used first-line treatment regimen of artemether plus lumefantrine. Therefore, nationwide surveillance of molecular markers for drug resistance and assessment of its therapeutic effects are important.

Artemisinin combination therapy fails even in the absence of Plasmodium falciparum kelch13 gene polymorphism in Central India.

Artemisinin is the frontline fast-acting anti-malarial against P. falciparum. Emergence and spread of resistant parasite in eastern-India poses a threat to national malaria control programs. Therefore, the objective of our study is to evaluate the artesunate-sulfadoxine-pyrimethamine efficacy in Central India. 180 monoclonal P. falciparum-infected patients received standard ASSP therapy during August 2015-January 2017, soon after diagnosis and monitored over next 42-days. Artemisinin-resistance was assessed through in-vivo parasite clearance half-life (PC1/2), ex-vivo ring-stage survivability (RSA), and genome analysis of kelch13 and other candidate gene (pfcrt, pfmdr1, pfatpase 6, pfdhfr and pfdhps). Of 180 P. falciparum positive patients, 9.5% showed increased PC1/2 (> 5.5 h), among them eleven isolates (6.1%) showed reduced sensitivity to RSA. In 4.4% of cases, parasites were not cleared by 72 h and showed prolonged PC1/2(5.6 h) (P < 0.005) along with significantly higher RSA (2.2%) than cured patients (0.4%). None of day-3 positive isolates contained the pfkelch13 mutation implicated in artemisinin resistance. Parasite recrudescence was observed in 5.6% patients, which was associated with triple dhfr-dhps (A16I51R59N108I164-S436G437K540G581T613) combination mutation. Emergence of reduced sensitivity to artesunate-sulfadoxine-pyrimethamine, in central India highlighted the risk toward spread of resistant parasite across different parts of India. Day-3 positive parasite, featuring the phenotype of artemisinin-resistance without pfkelch13 mutation, suggested kelch13-independent artemisinin-resistance.

Functional domain studies uncover novel roles for the ZTL Kelch repeat domain in clock function.

The small LOV/F-box/Kelch family of E3 ubiquitin ligases plays an essential role in the regulation of plant circadian clocks and flowering time by sensing dusk. The family consists of three members, ZEITLUPE (ZTL), LOV KELCH PROTEIN 2 (LKP2), and FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1), which share a unique protein domain architecture allowing them to act as photoreceptors that transduce light signals via altering stability of target proteins. Despite intensive study of this protein family we still lack important knowledge about the biochemical and functional roles of the protein domains that comprise these unique photoreceptors. Here, we perform comparative analyses of transgenic lines constitutively expressing the photoreceptor LOV domain or the Kelch repeat protein-protein interaction domains of ZTL, FKF1, and LKP2. Expression of each domain alone is sufficient to disrupt circadian rhythms and flowering time, but each domain differs in the magnitude of effect. Immunoprecipitation followed by mass spectrometry with the ZTL Kelch repeat domain identified a suite of potential interacting partners. Furthermore, the ZTL Kelch repeat domain can interact with the ZTL homologs, LKP2 and FKF1, and the LOV domain of ZTL itself. This suggests a hypothesis that the Kelch repeat domain of ZTL may mediate inter- and intra-molecular interactions of the three LOV/F-box/Kelch proteins and provides added insight into the composition of the protein complexes and an additional role for the Kelch repeat domain.

Case Report: The First Case of Genotypically Confirmed Plasmodium falciparum Kelch 13 Propeller Mutation in Sri Lanka and Its Implications on the Elimination Status of Malaria.

This case report discusses recrudescence of imported Plasmodium falciparum malaria, in the presence of P. falciparum Kelch13 (PfK13) propeller mutation, in a patient diagnosed and fully treated with artemether-lumefantrine under direct observation in Sri Lanka. This patient presented with a history of 5 days of fever following his arrival from the Democratic Republic of Congo (DRC). He had visited Rwanda 1 week before arrival to Sri Lanka. Treatment was commenced with artemisinin-based combination therapy, artemether-lumefantrine, which is the first-line drug recommended for uncomplicated falciparum malaria. Blood smears were negative for parasites by the third day of treatment. Approximately 2 weeks later, he developed fever again and was diagnosed as having a recrudescence of falciparum malaria. He was treated and responded to the second-line antimalarial dihydroartemisinin-piperaquine. Molecular testing of blood taken from the first infection revealed the presence of amino acid substitutions K189T and R561H within the PfK13 gene. R561H mutation is associated with delayed parasite clearance in Southeast Asia. Although seldom reported from DRC, an emergence and clonal expansion of parasites harboring R561H allele has been reported from Rwanda recently; thus, it is likely that this patient may have got the infection from Rwanda. Sri Lanka eliminated malaria in 2016. However, in the backdrop of continuing imported malaria cases, early diagnosis and prompt treatment is essential to prevent the re-establishment of the disease.

Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda.

Artemisinin resistance (delayed P. falciparum clearance following artemisinin-based combination therapy), is widespread across Southeast Asia but to date has not been reported in Africa1-4. Here we genotyped the P. falciparum K13 (Pfkelch13) propeller domain, mutations in which can mediate artemisinin resistance5,6, in pretreatment samples collected from recent dihydroarteminisin-piperaquine and artemether-lumefantrine efficacy trials in Rwanda7. While cure rates were >95% in both treatment arms, the Pfkelch13 R561H mutation was identified in 19 of 257 (7.4%) patients at Masaka. Phylogenetic analysis revealed the expansion of an indigenous R561H lineage. Gene editing confirmed that this mutation can drive artemisinin resistance in vitro. This study provides evidence for the de novo emergence of Pfkelch13-mediated artemisinin resistance in Rwanda, potentially compromising the continued success of antimalarial chemotherapy in Africa.

Characterization of putative drug resistant biomarkers in Plasmodium falciparum isolated from Ghanaian blood donors.

BACKGROUND: Plasmodium falciparum parasites, which could harbour anti-malaria drug resistance genes, are commonly detected in blood donors in malaria-endemic areas. Notwithstanding, anti-malaria drug resistant biomarkers have not been characterized in blood donors with asymptomatic P. falciparum infection. METHODS: A total of 771 blood donors were selected from five districts in the Greater Accra Region, Ghana. Each donor sample was screened with malaria rapid diagnostic test (RDT) kit and parasitaemia quantified microscopically. Dried blood spots from malaria positive samples were genotyped for P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum multi-drug resistance (Pfmdr1), P. falciparum dihydropteroate-synthetase (Pfdhps), P. falciparum dihydrofolate-reductase (Pfdhfr) and Kelch 13 propeller domain on chromosome 13 (Kelch 13) genes. RESULTS: Of the 771 blood donors, 91 (11.8%) were positive by RDT. Analysis of sequence reads indicated successful genotyping of Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Kelch 13 genes in 84.6, 81.3, 86.8, 86.9 and 92.3% of the isolates respectively. Overall, 21 different mutant haplotypes were identified in 69 isolates (75.8%). In Pfcrt, CVIET haplotype was observed in 11.6% samples while in Pfmdr1, triple mutation (resulting in YFN haplotype) was detected in 8.1% of isolates. In Pfdhfr gene, triple mutation resulting in IRNI haplotype and in Pfdhps gene, quintuple mutation resulting in AGESS haplotype was identified in 17.7% parasite isolates. Finally, five non-synonymous Kelch 13 alleles were detected; C580Y (3.6%), P615L (4.8%), A578S (4.8%), I543V (2.4%) and A676S (1.2%) were detected. CONCLUSION: Results obtained in this study indicated various frequencies of mutant alleles in Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Kelch 13 genes from P. falciparum infected blood donors. These alleles could reduce the efficacy of standard malaria treatment in transfusion-transmitted malaria cases. Incorporating malaria screening into donor screening protocol to defer infected donors is therefore recommended.

Systematic Analysis of Kelch Repeat F-box (KFB) Protein Gene Family and Identification of Phenolic Acid Regulation Members in Salvia miltiorrhiza Bunge.

S. miltiorrhiza is a well-known Chinese herb for the clinical treatment of cardiovascular and cerebrovascular diseases. Tanshinones and phenolic acids are the major secondary metabolites and significant pharmacological constituents of this plant. Kelch repeat F-box (KFB) proteins play important roles in plant secondary metabolism, but their regulation mechanism in S. miltiorrhiza has not been characterized. In this study, we systematically characterized the S. miltiorrhiza KFB gene family. In total, 31 SmKFB genes were isolated from S. miltiorrhiza. Phylogenetic analysis of those SmKFBs indicated that 31 SmKFBs can be divided into four groups. Thereinto, five SmKFBs (SmKFB1, 2, 3, 5, and 28) shared high homology with other plant KFBs which have been described to be regulators of secondary metabolism. The expression profile of SmKFBs under methyl jasmonate (MeJA) treatment deciphered that six SmKFBs (SmKFB1, 2, 5, 6, 11, and 15) were significantly downregulated, and two SmKFBs (SmKFB22 and 31) were significantly upregulated. Tissue-specific expression analysis found that four SmKFBs (SmKFB4, 11, 16, and 17) were expressed preferentially in aerial tissues, while two SmKFBs (SmKFB5, 25) were predominantly expressed in roots. Through a systematic analysis, we speculated that SmKFB1, 2, and 5 are potentially involved in phenolic acids biosynthesis.

Profiles of Kelch mutations in Plasmodium falciparum across South Asia and their implications for tracking drug resistance.

Artemisinin-based combination therapy (ACT) offers highly successful treatment of malaria. Emergence and spread of Plasmodium falciparum (Pf) parasites with decreased susceptibility to ACT in South-East Asia has caused concern worldwide. The current accepted criteria to assess artemisinin (ART) resistance relies upon data on treatment failure, delayed parasite clearance at day 3 (DPC3), parasite clearance half-life (PCHL) and in-vitro/ex-vivo ring stage survival assays (RSAs). Interestingly, some studies suggest that DPC3 does not provide a distinct separation between ART sensitive/resistant strains, and RSA differences may also be inconclusive. More recently, recrudescence of ART treated Pf, independent of the presence of Kelch 13 (K13) mutation (C580Y), has been reported in the monkey malaria model suggesting that genes other than K13 like coronin, dhps, dhfr, crt, mdr1 and plasmepsin1 may contribute towards ACT failure. Here we have collated the distribution of K13 mutants from Pf strains in South Asia. A total of fifty Pf-K13 mutations have been studied for ART resistance in South Asia of which nine have been validated while eleven are potentials for ART resistance. The remaining thirty K13 mutations have been reported from various locations in South Asia but lack corroborative clinical data on ART resistance/ACT failure. Of the fifty, fourteen K13 mutations have been identified in India including four novel mutations (S549Y, G625R, N657H, D702N). Structural mapping of these K13 mutations does not offer any coherent explanation for their contribution towards ART resistance as they are scattered in the K13 structure. Thus, K13 mutations likely provide only a partial synopsis, and we propose that all suspect cases of ACT failure be assessed by: 1) DPC3, 2) PCHL, 3) in-vitro/ex-vivo RSAs and 4) GWAS data in an effort to annotate the resistance status of the parasites. These efforts may help in surveillance and containment of ART resistance/ACT failure in South Asia.

Molecular Surveillance of Pfkelch13 and Pfmdr1 Mutations in Plasmodium falciparum Isolates from Southern Thailand.

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.

Kelch 13 propeller gene polymorphism among Plasmodium falciparum isolates in Lagos, Nigeria: Molecular Epidemiologic Study.

OBJECTIVE: To assess polymorphism in Kelch 13 gene of Plasmodium falciparum isolates in Lagos, Nigeria. METHODS: 195 Plasmodium falciparum-positive dried blood spots collected from individuals that accessed diagnostic care at some health facilities and during community surveys across several Local Government Areas of Lagos State, Nigeria, were investigated for the presence of mutations in the K13 gene by nested polymerase chain reaction (PCR) using haplotype-specific probes and sequencing. RESULTS: Three mutant genotypes of K13 gene were observed: A578S in 0.5%, D464N in 0.5% and Q613H in 1.5%. The frequency of K13 polymorphism was 3.1%, while the remaining parasite population had the wild K13 propeller genes. CONCLUSION: No validated Kelch 13 polymorphism associated with artemisinin resistance was seen among P. falciparum isolates from Lagos, Nigeria. As no clinical study was done, this could not be correlated with artemisinin sensitivity.

OBJECTIF: Evaluer le polymorphisme du gène Kelch 13 dans les isolats de Plasmodium falciparum à Lagos, au Nigéria. MÉTHODES: 195 gouttes de sang séchées positives pour Plasmodium falciparum recueillies auprès d'individus ayant accédé à des soins de diagnostic dans certains centres de santé et lors d'enquêtes communautaires menées dans plusieurs zones du gouvernement local de l'Etat de Lagos, au Nigéria, ont été examinées pour rechercher la présence de mutations du gène K13 par la réaction en chaîne imbriquée de la polymérase (PCR) en utilisant des sondes spécifiques à l'haplotype et par le séquençage. RÉSULTATS: Trois génotypes mutants du gène K13 ont été observés: A578S dans 0,5%, D464N dans 0,5% et Q613H dans 1,5%. La fréquence du polymorphisme K13 était de 3,1%, alors que la population parasitaire restante avait les gènes sauvages de l'hélice K13. CONCLUSION: Aucun polymorphisme validé de Kelch 13 associé à une résistance à l'artémisinine n'a été observé parmi les isolats de P. falciparum de Lagos, au Nigéria. Aucune étude clinique n'ayant été réalisée, il n'a pas été possible d'établir une corrélation entre cette observation et la sensibilité à l'artémisinine.

Unveiling the Distinct Mechanisms by which Disease-Causing Mutations in the Kelch Domain of KLHL3 Disrupt the Interaction with the Acidic Motif of WNK4 through Molecular Dynamics Simulation.

Kelch-like 3 (KLHL3) is a substrate adaptor of an E3 ubiquitin ligase complex that regulates the degradation of its substrates, including with-no-lysine [K] kinase 4 (WNK4). Mutations in KLHL3 are associated with pseudohypoaldosteronism type II (PHAII), a hereditary form of hypertension. Many PHAII-causing mutations are located in the Kelch domain of KLHL3 that binds with WNK4; however, detailed mechanisms by which these mutations disrupt the binding are not well-understood. In the present study we use molecular dynamics simulations and Western blot analyses to examine the effects of these mutations on the interaction between the Kelch domain of KLHL3 and the acidic motif (AM) of WNK4. The simulation results correlated well with those from Western blot analyses with the exception of the L387P mutation, which led to deregulation of AM degradation by KLHL3 but not recapitulated by simulations. On the basis of the simulation results, a mutation on the binding surface of the Kelch domain affected the Kelch-AM interaction through two major mechanisms: altering the electrostatic potential of the AM binding site and disrupting the Kelch-AM hydrogen bonds. The mutations buried inside the Kelch domain were predicted by our simulations to have no or modest effects on the Kelch-AM interaction. Buried mutations R384Q and S410L disrupted intramolecular hydrogen bonds within the Kelch domain and affected the Kelch-AM interaction indirectly. No significant effect of buried mutation A340V or A494T on the AM degradation or Kelch-AM interaction was observed, implying these mutations may disrupt mechanisms other than Kelch-AM interaction.

Association of mutations in the Plasmodium falciparum Kelch13 gene (Pf3D7_1343700) with parasite clearance rates after artemisinin-based treatments-a WWARN individual patient data meta-analysis.

BACKGROUND: Plasmodium falciparum infections with slow parasite clearance following artemisinin-based therapies are widespread in the Greater Mekong Subregion. A molecular marker of the slow clearance phenotype has been identified: single genetic changes within the propeller region of the Kelch13 protein (pfk13; Pf3D7_1343700). Global searches have identified almost 200 different non-synonymous mutant pfk13 genotypes. Most mutations occur at low prevalence and have uncertain functional significance. To characterize the impact of different pfk13 mutations on parasite clearance, we conducted an individual patient data meta-analysis of the associations between parasite clearance half-life (PC1/2) and pfk13 genotype based on a large set of individual patient records from Asia and Africa. METHODS: A systematic literature review following the PRISMA protocol was conducted to identify studies published between 2000 and 2017 which included frequent parasite counts and pfk13 genotyping. Four databases (Ovid Medline, PubMed, Ovid Embase, and Web of Science Core Collection) were searched. Eighteen studies (15 from Asia, 2 from Africa, and one multicenter study with sites on both continents) met inclusion criteria and were shared. Associations between the log transformed PC1/2 values and pfk13 genotype were assessed using multivariable regression models with random effects for study site. RESULTS: Both the pfk13 genotypes and the PC1/2 were available from 3250 (95%) patients (n = 3012 from Asia (93%), n = 238 from Africa (7%)). Among Asian isolates, all pfk13 propeller region mutant alleles observed in five or more specific isolates were associated with a 1.5- to 2.7-fold longer geometric mean PC1/2 compared to the PC1/2 of wild type isolates (all p ≤ 0.002). In addition, mutant allele E252Q located in the P. falciparum region of pfk13 was associated with 1.5-fold (95%CI 1.4-1.6) longer PC1/2. None of the isolates from four countries in Africa showed a significant difference between the PC1/2 of parasites with or without pfk13 propeller region mutations. Previously, the association of six pfk13 propeller mutant alleles with delayed parasite clearance had been confirmed. This analysis demonstrates that 15 additional pfk13 alleles are associated strongly with the slow-clearing phenotype in Southeast Asia. CONCLUSION: Pooled analysis associated 20 pfk13 propeller region mutant alleles with the slow clearance phenotype, including 15 mutations not confirmed previously.

Allele-Specific Isothermal Amplification Method Using Unmodified Self-Stabilizing Competitive Primers.

Rapid and specific detection of single nucleotide polymorphisms (SNPs) related to drug resistance in infectious diseases is crucial for accurate prognostics, therapeutics and disease management at point-of-care. Here, we present a novel amplification method and provide universal guidelines for the detection of SNPs at isothermal conditions. This method, called USS-sbLAMP, consists of SNP-based loop-mediated isothermal amplification (sbLAMP) primers and unmodified self-stabilizing (USS) competitive primers that robustly delay or prevent unspecific amplification. Both sets of primers are incorporated into the same reaction mixture, but always targeting different alleles; one set specific to the wild type allele and the other to the mutant allele. The mechanism of action relies on thermodynamically favored hybridization of totally complementary primers, enabling allele-specific amplification. We successfully validate our method by detecting SNPs, C580Y and Y493H, in the Plasmodium falciparum kelch 13 gene that are responsible for resistance to artemisinin-based combination therapies currently used globally in the treatment of malaria. USS-sbLAMP primers can efficiently discriminate between SNPs with high sensitivity (limit of detection of 5 × 101 copies per reaction), efficiency, specificity and rapidness (<35 min) with the capability of quantitative measurements for point-of-care diagnosis, treatment guidance, and epidemiological reporting of drug-resistance.

UmTea1, a Kelch and BAR domain-containing protein, acts at the cell cortex to regulate cell morphogenesis in the dimorphic fungus Ustilago maydis.

The spatial organization of a cell is crucial for distribution of cell components and for cell morphogenesis in all organisms. Ustilago maydis, a basidiomycete fungus, has a yeast-like and a filamentous form. The former buds once per cell cycle at one of the cell poles, and can use the same site repeatedly or choose a new site at the same pole or opposite pole. The filamentous form consists of a long apical cell with short septate basal compartments lacking cytoplasm. It grows at the apex and can reverse growth forming a new growth zone at the basal end. We are interested in understanding how these different morphologies are generated. Here we present identification and characterization of U. maydis Tea1, a homologue of the fission yeast cell end marker Tea1. We demonstrate that UmTea1, a Kelch domain protein, interacts with itself and is an important determinant of the site of polarized growth: tea1 mutants bud simultaneously from both cell poles and form bifurcate buds. UmTea1 also regulates septum positioning, cell wall deposition, cell and neck width, coordination of nuclear division and cell separation, and localization of sterol-rich membrane domains. Some of these functions are shared with UmTea4, another cell end marker. We show that Tea1::GFP localizes to sites of polarized or potential polarized growth and to the septation site in the yeast-like form. Additionally, localization of Tea1::GFP as rings along the filament suggests that the filament undergoes septation. We hypothesize that Tea1 may act as a scaffold for the assembly of proteins that determine the site of polarized growth.

Detection of the Plasmodium falciparum Kelch-13 gene P553L mutation in sporozoites isolated from mosquito salivary glands in South-Central Vietnam.

BACKGROUND: Plasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes. METHODS: Mosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4-6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing. RESULTS: Five of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out. CONCLUSION: These results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.

Coordination of Meristem Doming and the Floral Transition by Late Termination, a Kelch Repeat Protein.

Enlargement and doming of the shoot apical meristem (SAM) is a hallmark of the transition from vegetative growth to flowering. While this change is widespread, its role in the flowering process is unknown. The late termination (ltm) tomato (Solanum lycopersicum) mutant shows severely delayed flowering and precocious doming of the vegetative SAM LTM encodes a kelch domain-containing protein, with no link to known meristem maintenance or flowering time pathways. LTM interacts with the TOPLESS corepressor and with several transcription factors that can provide specificity for its functions. A subgroup of flowering-associated genes is precociously upregulated in vegetative stages of ltm SAMs, among them, the antiflorigen gene SELF PRUNING (SP). A mutation in SP restored the structure of vegetative SAMs in ltm sp double mutants, and late flowering was partially suppressed, suggesting that LTM functions to suppress SP in the vegetative SAM In agreement, SP-overexpressing wild-type plants exhibited precocious doming of vegetative SAMs combined with late flowering, as found in ltm plants. Strong flowering signals can result in termination of the SAM, usually by its differentiation into a flower. We propose that activation of a floral antagonist that promotes SAM growth in concert with floral transition protects it from such terminating effects.

Under the superficial dichotomy pathogen and allergen are two manifestations of same immune activation and pathogenesis mechanisms.

Pathogens and allergens are deemed as two contrasting facets of host immune status, deficiency and exuberant. In silico domain analysis of a diverse panel of pathogen and allergen proteins has revealed the shortcoming of this notion. Both the pathogen and allergen proteins elicit immune activation, with the outcome of immune agitation depending on the pathogen strain, allergen exposure duration, and host factors. Pathogens can replicate within the host and constantly irritate the immune system, leading to blood coagulation, respiratory collapse and death. Allergens, being non-viable, can only provoke the immune system transiently; however, depending on the allergen dose and extent exposed to, inflammation and fatality can occur. In silico analysis of pathogen and allergen proteins showed the conserved domains to be AAA, WR1, VKc, Kelch, Hr1, HAMP, HELICc, Dak2, CHAD, CHASE2, Galanin, PKS_TE, Robl_LC7, Excalibur, DISIN, etc. This exciting discovery can have far-reaching effects in drug target identification approaches.