[Mutations of Plasmodium falciparum Multidrug Resistance 1 Gene in Imported Plasmodium falciparum in Wuhan].

Objective: To identify Plasmodium falciparum multidrug resistance 1（Pfmdr1） point mutations in imported Plasmodium falciparum in Wuhan. Methods: Blood samples were collected from returnees infected with P. falciparum in endemic areas of Africa and Myanmar during 2010-2015 in Wuhan City. Nested PCR primers were specifically designed for Pfmdr1 gene loci 86, 1042 and 1246 of P. falciparum. The Pfmdr1 gene was then amplified by nested PCR, and the products were digested by restriction enzyme ApoⅠ, AseⅠ and EcoRⅤ, respectively. The mutation rate for loci 86, 1042 and 1246 was analyzed. Results A total of 187 patients with falciparum malaria were involved in the study. Pfmdr1 was amplified from all the blood samples. Restriction enzyme digestion revealed mutation rate of 19.3%（36/187）, 4.3% （8/187） and 9.6%（18/187） for loci 86, 1042 and 1246, respectively. In detail, the mutation rate for loci 86, 1042 and 1246 was 20.6%（36/175）, 2.9%（5/175） and 10.3%（18/175） respectively in the 175 samples from Africa, and only 3 cases with locus 1042 mutation were found in the 12 samples from Myanmar. Results: A total of 187 patients with falciparum malaria were involved in the study. Pfmdr1 was amplified from all the blood samples. Restriction enzyme digestion revealed mutation rate of 19.3%（36/187）, 4.3% （8/187） and 9.6%（18/187） for loci 86, 1042 and 1246, respectively. In detail, the mutation rate for loci 86, 1042 and 1246 was 20.6%（36/175）, 2.9%（5/175） and 10.3%（18/175） respectively in the 175 samples from Africa, and only 3 cases with locus 1042 mutation were found in the 12 samples from Myanmar. Conclusion: The loci 86, 1042 and 1246 mutations of Pfmdr1 have all been found in the samples from Africa, with only one point mutation （locus 1042） found in samples from Myanmar.

[Analysis of MSP1 Allelotypes in Imported Cases of Plasmodium falciparum Malaria from Africa].

OBJECTIVE: To understand the allelotype characteristics of the merozoite surface protein 1 (MSP1) in imported Plasmodium falciparum. METHODS: Blood samples were collected from P. falciparum-infected patients returning from African malaria endemic areas. Nested PCR was used to amplify gene fragments of MSP1 coding for block 2 and block 3 motifs of MSP1 of P. falciparum by using the MSP1-specific primers. Then the allelotype of MSPI was analyzed. RESULTS: The MSP1 allelotype was detected in 117 of 135 blood samples, with a detection rate of 86.7%. In the 117 cases with successful PCR amplification, the detection rates for MAD20, K1, RO33, MAD20+K1, MAD20+RO33, K1+RO33 and MAD20+K1+R033 were 6.0%(7/117), 36.8%(43/117), 20.5%(24/117), 6.8% (8/117), 3.4% (4/117), 17.1% (20/117) and 9.4% (11/117), respectively, wherein the mixed infection accounted for 36.8%(43/135). The mean multiplicity of infection(MOI) of MSP1 allelotype was 1.46. There was no significant difference in the proportion of patients with major severity of illness among the MAD20, K1 and RO33 genotypes. The proportions of patients with major severity of illness were 25.7%(19/74) and 32.6%(14/43) in 74 cases of singular infection and 43 cases of mixed infection, respectively. The two infection types of patients had 241 ± 176 days and 285 ± 216 days of stay abroad, with no significant difference between them. CONCLUSION: The three genotypes of MSP1 and their four types of combination exist in imported cases of P. falciparum malaria from Africa. K1 and RO33 are the dominant genotves.

ATP selection in a random peptide library consisting of prebiotic amino acids.

Based upon many theoretical findings on protein evolution, we proposed a ligand-selection model for the origin of proteins, in which the most ancient proteins originated from ATP selection in a pool of random peptides. To test this ligand-selection model, we constructed a random peptide library consisting of 15 types of prebiotic amino acids and then used cDNA display to perform six rounds of in vitro selection with ATP. By means of next-generation sequencing, the most prevalent sequence was defined. Biochemical and biophysical characterization of the selected peptide showed that it was stable and foldable and had ATP-hydrolysis activity as well.

Reconstructing a flavodoxin oxidoreductase with early amino acids.

Primitive proteins are proposed to have utilized organic cofactors more frequently than transition metals in redox reactions. Thus, an experimental validation on whether a protein constituted solely by early amino acids and an organic cofactor can perform electron transfer activity is an urgent challenge. In this paper, by substituting "late amino acids (C, F, M, T, W, and Y)" with "early amino acids (A, L, and V)" in a flavodoxin, we constructed a flavodoxin mutant and evaluated its characteristic properties. The major results showed that: (1) The flavodoxin mutant has structural characteristics similar to wild-type protein; (2) Although the semiquinone and hydroquinone flavodoxin mutants possess lower stability than the corresponding form of wild-type flavodoxin, the redox potential of double electron reduction Em,7 (fld) reached -360 mV, indicating that the flavodoxin mutant constituted solely by early amino acids can exert effective electron transfer activity.

Improved annotation of a plant pathogen genome Xanthomonas oryzae pv. oryzae PXO99A.

Many bacterial genomes have been sequenced and stored in public databases now, of which Reference Sequence (RefSeq) is the most widely used one. However, the annotation in RefSeq is still unsatisfactory. The present analysis is focused on the re-annotation of an important plant pathogen genome Xanthomonas oryzae pv. oryzae PXO99A (Xoo PXO99A), which is the causal agent of bacterial blight on rice. Based on the parameters of 28 nucleotide frequencies and support vector machine algorithm, 41 originally annotated hypothetical genes were recognized as noncoding sequences, which were further supported by principal component analysis and other evidence. Ten of them were tested with reverse transcription-polymerase chain reaction experiments (RT-PCR), and all of them were confirmed to be noncoding sequences. Furthermore, 197 potential new genes not annotated in RefSeq were both recognized by two ab initio gene finding programs. Most of them only have sequence similarities with part of the known genes in other species, so they are unlikely to be protein-coding genes. Twelve potential new genes have high full-length sequence similarities with function-known genes, which are very likely to be true protein-coding genes. All the 12 potential genes were tested with RT-PCR, and 11 of them (92%) were successfully amplified in cDNA template. The RT-PCR experiments confirm that our theoretical prediction has high accuracy. The improvement of Xoo PXO99A annotation is helpful for the research of lifestyle, metabolism, and pathogenicity of this important plant pathogen. The improved annotation can be obtained from http://211.69.128.148/Xoo .