[Mechanism of protective effect of resveratrol on poor ovarian response in mice].

This study aims to investigate the therapeutic effects and potential mechanisms of resveratrol(Res) on poor ovarian response(POR) in mice. The common target genes shared by Res and POR were predicted by network pharmacology, used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and then validated by animal experiments. The mice with regular estrous cycle after screening were randomized into normal, POR, and low-and high-dose(20 and 40 mg·kg~(-1), respectively) Res groups. The normal group was administrated with an equal volume of 0.9% sodium chloride solution by gavage, and the mice in other groups with tripterygium glycosides suspension(50 mg·kg~(-1)) by gavage for 2 weeks. After the modeling, the mice in low-and high-dose Res groups were treated with Res by gavage for 2 weeks, and the mice in normal and POR groups with an equal volume of 0.9% sodium chloride solution by gavage. Ovulation induction and sample collection were carried out on the day following the end of treatment. Vaginal smears were collected for observation of the changes in the estrous cycle, the counting of retrieved oocytes, and the measurement of ovarian wet weight and ovarian index. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of anti-mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in the serum. The ovarian tissue morphology and granulosa cell apoptosis were observed by hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL), respectively. Western blot was employed to determine the protein levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(AKT), forkhead box O(FOXO) 3a, hypoxia-inducible factor(HIF)-1α, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). A total of 222 common targets shared by Res and POR were collected. GO annotation indicated that these targets were mainly involved in oxidative stress response. KEGG enrichment analysis revealed that Res can intervene in POR via PI3K/AKT, HIF-1, and FOXO signaling pathways. Animal experiments showed that the model group had higher rate of estrous cycle disorders, lower number and poorer morphology of normally developed follicles at all levels, more atretic follicles, higher apoptosis of ovarian granulosa cells, lower number of retrieved oocytes, lower ovarian wet weight and ovarian index, higher serum levels of FSH and LH, lower levels of AMH and E_2, higher expression levels of HIF-1α, FOXO3a and Bax, and lower expression levels of PI3K, AKT, and Bcl-2 in the ovarian tissue than the normal group. Compared with the POR group, low-and high-dose Res decreased the rate of estrous cycle disorders, improved the follicle number and morphology, reduced atretic follicles, promoted the apoptosis of ovarian granulosa cells, increased retrieved oocytes, ovarian wet weight and ovarian index, and lowered serum FSH and LH levels. Moreover, Res down-regulated the expression levels of HIF-1α, FOXO3a and Bax, and up-regulated the expression levels of PI3K, AKT and Bcl-2 in the ovarian tissue. In summary, Res can inhibit apoptosis and mitigate poor ovarian response in mice by regulating the PI3K/AKT/FOXO3a and HIF-1α pathways.

Electroacupuncture for ulcerative colitis patients: A meta-analysis and acupoints selection study.

Objective: To evaluate the efficacy and safety of electroacupuncture (EA) on ulcerative colitis (UC) and explore the influence of EA parameters and acupoint compatibility to optimize the clinical treatment plan. Methods: After searching eight databases, data were extracted and analyzed to determine the quality and bias of the study's methodological design, and randomized controlled trial (RCT) datas were meta-analyzed. Results: Twelve studies that meet the criteria were included. The results of meta-analysis indicated that, compared with the control group, experimental group had better clinical efficacy [RR = 1.27, 95%CI = (1.19, 1.36), P < 0.01], Other indicators such as cure rate [RR = 1.73, 95%CI = (1.43, 2.09), P < 0.01], effective rate of mucosal lesions under enteroscopy [RR = 1.24, 95%CI = (1.11, 1.38), P < 0.01], serum inflammatory factor TNF-α [MD = -41.11, 95%CI = (-46.01, 36.22), P < 0.01] were significantly better than those in the control group. Sixteen acupoints on the Ren, Bladder, Stomach, Spleen, and Liver meridians were used 74 times. RN4-ST25 is the most compatible acupoints. Conclusion: The clinical efficacy of EA in treating UC is superior than the control group's, and it has curative effects in terms of cure rate, efficacy of mucosal lesions under colonoscopy, serum inflammatory factors, and Traditional Chinese Medicine (TCM) syndrome scores. Combining acupoints of the Bladder, Stomach, and Ren meridians and using dense wave for 30 min each time for more than 6 weeks may be optimal for UC patients.

What exactly does the PfK13 C580Y mutation in Plasmodium falciparum influence?

BACKGROUND: The emergence and spread of artemisinin resistance threaten global malaria control and elimination goals, and encourage research on the mechanisms of drug resistance in malaria parasites. Mutations in Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance, but the unique or common mechanism which results in this resistance is unclear. METHODS: We analyzed the effects of the PfK13 mutation on the transcriptome and proteome of P. falciparum at different developmental stages. Additionally, the number of merozoites, hemozoin amount, and growth of P. falciparum 3D7C580Y and P. falciparum 3D7WT were compared. The impact of iron supplementation on the number of merozoites of P. falciparum 3D7C580Y was also examined. RESULTS: We found that the PfK13 mutation did not significantly change glycolysis, TCA, pentose phosphate pathway, or oxidative phosphorylation, but did reduce the expression of reproduction- and DNA synthesis-related genes. The reduced number of merozoites, decreased level of hemozoin, and slowed growth of P. falciparum 3D7C580Y were consistent with these changes. Furthermore, adding iron supply could increase the number of the merozoites of P. falciparum 3D7C580Y. CONCLUSIONS: These results revealed that the PfK13 mutation reduced hemoglobin ingestion, leading to artemisinin resistance, likely by decreasing the parasites' requirement for haem and iron. This study helps elucidate the mechanism of artemisinin resistance due to PfK13 mutations.

Ring-stage growth arrest: Metabolic basis of artemisinin tolerance in Plasmodium falciparum.

The emergence and spread of artemisinin-tolerant malaria parasites threatens malaria control programmes worldwide. Mutations in the propeller domain of the Kelch13 protein confer Plasmodium falciparum artemisinin resistance (ART-R). ART-R is linked to the reduced susceptibility of temporary growth-arrested ring-stage parasites, but the metabolic mechanisms remain elusive. We generated two PfKelch13 mutant lines via CRISPR-Cas9 gene editing which displayed a reduced susceptibility accompanied by an extended ring stage. The metabolome of ART-induced ring-stage growth arrest parasites carrying PfKelch13 mutations showed significant alterations in the tricarboxylic acid (TCA) cycle, glycolysis, and amino acids metabolism, pointing to altered energy and porphyrin metabolism with metabolic plasticity. The critical role of these pathways was further confirmed by altering metabolic flow or through chemical inhibition. Our findings uncover that the growth arrestment associated with ART-R is potentially attributed to the adaptative metabolic plasticity, indicating that the defined metabolic remodeling turns out to be the trigger for ART-R.

Acupuncture combined with metformin versus metformin alone to improve pregnancy rate in polycystic ovary syndrome: A systematic review and meta-analysis.

Objective: The aim of this study was to evaluate the comparison between acupuncture combined with metformin versus metformin alone in improving the pregnancy rate of people with polycystic ovary syndrome (PCOS). Methods: A literature search of eight databases resulted in nine randomized controlled trials (RCTs) that assessed the effect of acupuncture combined with metformin on pregnancy rate in PCOS patients compared with metformin alone. Subsequently, data extraction and analysis were conducted to evaluate the quality and risk of bias of the methodological design of the study, and meta-analysis was conducted on the RCT data. Results: Nine RCTs and 1,159 women were included. Acupuncture can improve pregnancy rate. It was analyzed according to the diagnostic criteria of PCOS [Z = 2.72, p = 0.007, relative risk (RR) 1.31, 95% CI 1.08 to 1.60, p = 0.15, I 2 = 41%]. Analysis was performed according to different diagnostic criteria of pregnancy (Z = 3.22, p = 0.001, RR 1.35, 95% CI 1.13 to 1.63, p = 0.12, I 2 = 42%). Acupuncture can improve ovulation rate. Subgroup analysis was performed according to the number of ovulation patients (Z = 2.67, p = 0.008, RR 1.31, 95% CI 1.07 to 1.59, p = 0.04, I 2 = 63%) and ovulation cycle (Z = 3.57; p = 0.0004, RR 1.18, 95% CI 1.08 to 1.29, p = 0.57, I 2 = 0%). Statistical analysis also showed that acupuncture combined with metformin could improve homeostatic model assessment of insulin resistance (HOMA-IR) [mean difference (MD) -0.68, 95% CI -1.01 to -0.35, p = 0.003, I 2 = 83%]. Conclusions: Based on the results of this study, compared with metformin alone, acupuncture combined with metformin has a positive effect on pregnancy rate, ovulation rate, and insulin resistance in PCOS. However, due to the limitations regarding the number and quality of the included studies, the above conclusions need to be verified by further high-quality studies. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/#myprospero.

Prevalence of polycystic ovary syndrome in patients with type 2 diabetes: A systematic review and meta-analysis.

Purpose: With type 2 diabetes mellitus (T2DM) occurring at a younger age, a greater number of women with T2DM experience reproductive health problems. The prevalence of polycystic ovary syndrome (PCOS), a common reproductive disease associated with T2DM, remains unknown in women with T2DM. This systematic review and meta-analysis aimed to determine the prevalence of PCOS in women with T2DM. Methods: Stata 15.1 was used to perform a meta-analysis on the prevalence of PCOS in patients with T2DM included in this study. Additionally, a narrative review of the effects of different diagnostic methods, obesity, state, and other factors on the prevalence of PCOS was conducted. Results: Meta-analysis showed that the overall prevalence of PCOS in women with T2DM was approximately 21%. Subgroup analysis showed that the incidence of PCOS in female patients aged 25-45 years was higher than that in female patients aged < 25 years. The prevalence of PCOS in obese women was 14%, which was lower than that in normal weight women and normal weight or overweight or obese women. Women with T2DM in Oceania had the highest incidence of PCOS, followed by those in Europe and Asia; women with T2DM in North America had the lowest incidence. In terms of PCOS diagnostic standards, the prevalence of PCOS diagnosed by the National Institutes of Health was the lowest. The prevalence of PCOS diagnosed on the basis of clinical symptoms and biochemical characteristics was the highest, and the prevalence of PCOS diagnosed on the basis of medical records was 20%. Conclusions: PCOS is a common disease in female patients with T2DM. The prevalence of PCOS in women with T2DM at childbearing age was higher than that in adolescent females. Women with T2DM at childbearing age should pay attention to the screening and prevention of PCOS to avoid the hazards of PCOS to reproductive health. Systematic review registration: PROSPERO, identifier CRD42022318657.

Genome-wide landscape of ApiAP2 transcription factors reveals a heterochromatin-associated regulatory network during Plasmodium falciparum blood-stage development.

Heterochromatin-associated gene silencing controls multiple physiological processes in malaria parasites, however, little is known concerning the regulatory network and cis-acting sequences involved in the organization of heterochromatin and how they modulate heterochromatic gene expression. Based on systematic profiling of genome-wide occupancy of eighteen Apicomplexan AP2 transcription factors by ChIP-seq analysis, we identify and characterize eight heterochromatin-associated factors (PfAP2-HFs), which exhibit preferential enrichment within heterochromatic regions but with differential coverage profiles. Although these ApiAP2s target euchromatic gene loci via specific DNA motifs, they are likely integral components of heterochromatin independent of DNA motif recognition. Systematic knockout screenings of ApiAP2 factors coupled with RNA-seq transcriptomic profiling revealed three activators and three repressors of heterochromatic gene expression including four PfAP2-HFs. Notably, expression of virulence genes is either completely silenced or significantly reduced upon the depletion of PfAP2-HC. Integrated multi-omics analyses reveal autoregulation and feed-forward loops to be common features of the ApiAP2 regulatory network, in addition to the occurrence of dynamic interplay between local chromatin structure and ApiAP2s in transcriptional control. Collectively, this study provides a valuable resource describing the genome-wide landscape of the ApiAP2 family and insights into functional divergence and cooperation within this family during the blood-stage development of malaria parasites.

5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes.

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (Plasmodium yoelii) and human (Plasmodium falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knockout of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in the knockout rodent malaria parasites. Restoration of the nsun2 gene in the knockout parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.

A cascade of transcriptional repression determines sexual commitment and development in Plasmodium falciparum.

Gametocytogenesis, the process by which malaria parasites produce sexual forms that can infect mosquitoes, is essential for the transmission of malaria. A transcriptional switch of the pfap2-g gene triggers sexual commitment, but how the complex multi-step process is precisely programed remains largely unknown. Here, by systematic functional screening of a panel of ApiAP2 transcription factors, we identify six new ApiAP2 members associated with gametocytogenesis in Plasmodium falciparum. Among these, PfAP2-G5 (PF3D7_1139300) was found to be indispensable for gametocytogenesis. This factor suppresses the transcriptional activity of the pfap2-g gene via binding to both the upstream region and exonic gene body, the latter is linked to the maintenance of local heterochromatin structure, thereby preventing initiation of sexual commitment. Removal of this repressive effect through pfap2-g5 knockout disrupts the asexual replication cycle and promotes sexual commitment accompanied by upregulation of pfap2-g expression. However, the gametocytes produced fail to mature fully. Further analyses show that PfAP2-G5 is essential for gametocyte maturation, and causes the down-regulation of pfap2-g and a set of early gametocyte genes activated by PfAP2-G prior to gametocyte development. Collectively, our findings reveal a regulation cascade of gametocyte production in malaria parasites, and provide a new target for transmission blocking interventions.

The Architectural Factor HMGB1 Is Involved in Genome Organization in the Human Malaria Parasite Plasmodium falciparum.

The three-dimensional (3D) genome organization plays a critical role in the regulation of gene expression in eukaryotic organisms. In the unicellular malaria parasite Plasmodium falciparum, the high-order chromosome organization has emerged as an important epigenetic pathway mediating gene expression, particularly for virulence genes, but the related architectural factors and underlying mechanism remain elusive. Herein, we have identified the high-mobility-group protein HMGB1 as a critical architectural factor for maintenance of genome organization in P. falciparum Genome-wide occupancy analysis (chromatin immunoprecipitation sequencing [ChIP-seq]) shows that the HMGB1 protein is recruited mainly to centromeric regions likely via a DNA-binding-independent pathway. Chromosome conformation capture coupled with next-generation sequencing (Hi-C-seq) and 3D modeling analysis show that the loss of HMGB1 disrupts the integrity of centromere/telomere-based chromosome organization accompanied with diminished interaction frequency among centromere clusters. This triggers local chromatin alteration and dysregulated gene expression. Notably, the entire repertoire of the primary virulence genes (var) was completely silenced in the absence of P. falciparum HMGB1 (PfHMGB1). Furthermore, the disrupted nuclear organization was reconstituted by complementation of HMGB1, thereby rescuing the mutually exclusive expression of the var gene family. Collectively, these data demonstrate that the architectural factor HMGB1 is associated with gene expression via mediating the high-order structure of genome organization. This finding not only contributes better understanding of the epigenetic regulation of gene expression but may also provide novel targets for antimalarial strategies.IMPORTANCE Malaria remains a major public health and economic burden currently. The mutually exclusive expression of the virulence genes is associated with the pathogenesis and immune evasion of human malaria parasites in the host. The nuclear architecture provides a well-organized environment for differential gene expression in the nucleus, but the underlying mechanism remains largely unknown. In this study, we have identified the highly conserved high-mobility-group protein HMGB1 as a key architecture regulator involved in virulence gene expression by establishing high-order genome organization in the nucleus of P. falciparum Mechanistic investigation revealed that the specific interaction of HMGB1 and centromeres constructed the precisely organized nuclear architecture, which coordinated with local chromatin structure to control the singular expression of virulence genes. Hence, this protein appears to be a critical architectural regulator for the pathogenesis of malaria infection and may be a new target for the development of an intervention strategy against malaria.

Actin-related protein Arp4 regulates euchromatic gene expression and development through H2A.Z deposition in blood-stage Plasmodium falciparum.

BACKGROUND: Malaria caused by Plasmodium spp. is still a major threat to public health globally. The various approaches to developing new antimalarial agents rely on the understanding of the complex regulatory mechanisms of dynamic gene expression in the life-cycle of these malaria parasites. The nuclear members of the evolutionarily conserved actin-related protein nuclear (ARP) superfamily are the major components of nucleosome remodelling complexes. In the human malaria parasite Plasmodium falciparum, bioinformatics analysis has predicted three ARP orthologues: PfArp1, PfArp4 and PfArp6. However, little is known about the biological functions of putative PfArp4. In this study, we aimed to investigate the function and the underlying mechanisms of PfArp4 gene regulation. METHODS: A conditional gene knockdown approach was adopted by incorporating the glucosamine-inducible glmS ribozyme sequence into the 3' UTR of the PfArp4 and PfArp6 genes. The transgenic parasites PfArp4-Ty1-Ribo, PfArp6-Ty1-Ribo and pL6-PfArp4-Ty1::PfArp6-HA were generated by the CRISPR-Cas9 technique. The knockdown effect in the transgenic parasite was measured by growth curve assay and western blot (WB) analysis. The direct interaction between PfArp4 and PfArp6 was validated by co-IFA and co-IP assays. The euchromatic gene expression mediated through H2A.Z (histone H2A variant) deposition and H3K9ac modification at promoters and regulated by PfArp4, was determined by RNA-seq and ChIP-seq. RESULTS: The inducible knockdown of PfArp4 inhibited blood-stage development of P. falciparum. PfArp4 and PfArp6 were colocalized in the nucleus of P. falciparum parasites. PfArp4 gene knockdown altered the global transcriptome. PfArp4 protein colocalized with the histone variant H2A.Z and euchromatic marker H3K9ac in intergenic regions. The inducible downregulation of PfArp4 resulted in the depletion of H2A.Z and lower H3K9ac levels at the upstream regions of eukaryotic genes, thereby repressing the transcriptional abundance of H2A.Z-dependent genes. CONCLUSIONS: Our findings suggest that PfArp4 regulates the cell cycle by controlling H2A.Z deposition and affecting centromere function, contributing to the understanding the complex epigenetic regulation of gene expression and the development of P. falciparum.

Rrp6 Regulates Heterochromatic Gene Silencing via ncRNA RUF6 Decay in Malaria Parasites.

The heterochromatin environment plays a central role in silencing genes associated with the malaria parasite's development, survival in the host, and transmission to the mosquito vector. However, the underlying mechanism regulating the dynamic chromatin structure is not understood yet. Here, we have uncovered that Plasmodium falciparum Rrp6, an orthologue of eukaryotic RNA exosome-associated RNase, controls the silencing of heterochromatic genes. PfRrp6 knockdown disrupted the singular expression of the GC-rich ncRNA RUF6 family, a known critical regulator of virulence gene expression, through the stabilization of the nascent transcripts. Mechanistic investigation showed that the accumulation of the multiple RUF6 ncRNAs triggered local chromatin remodeling in situ, which activated their adjacent var genes. Strikingly, chromatin isolation by RNA purification analysis (ChIRP-seq) revealed that a remarkable RUF6 ncRNA had interacted with distal heterochromatin regions directly and stimulated a global derepression effect on heterochromatic genes, including all variant gene families and the sexual commitment-associated regulator ap2-g gene. Collectively, Rrp6 appears to conduct the epigenetic surveillance of heterochromatic gene expression through controlling RUF6 levels, thereby securing antigenic variation and sexual commitment of malaria parasites during the infection of the host.IMPORTANCE Malaria remains a major public health and economic burden. The heterochromatin environment controls the silencing of genes associated with the fate of malaria parasites. Previous studies have demonstrated that a group of GC-rich ncRNAs (RUF6) is associated with the mutually exclusive expression of var genes, but the underlying mechanisms remain elusive. Here, through a series of genetic manipulation and genome-wide multiomics analysis, we have identified the plasmodial orthologue of RNA exosome-associated Rrp6 as an upstream regulator of RUF6 expression and revealed that the dysregulation of RUF6 upon Rrp6 knockdown triggered local chromatin alteration, thereby activating most heterochromatic genes via direct interaction of RUF6 and distal gene loci. This finding not only uncovered the in-depth mechanism of RUF6-mediated regulation of heterochromatic genes but also identified Rrp6 as a novel regulator of gene expression in human malaria parasites, which provides a new target for developing intervention strategies against malaria.

The cryptic unstable transcripts are associated with developmentally regulated gene expression in blood-stage Plasmodium falciparum.

The tight gene expression regulation controls the development and pathogenesis of human malaria parasite Plasmodium falciparum throughout the complex life cycle. Recent studies have revealed the pervasive nascent transcripts in the genome of P. falciparum, suggesting the existence of a hidden transcriptome involved in the dynamic gene expression. However, the landscape and related biological functions of nascent non-coding RNAs (ns-ncRNAs) are still poorly explored. Here we profiled the transcription dynamics of nascent RNAs by rRNA-depleted and stranded RNA sequencing over the course of 48-h intraerythrocytic developmental cycle (IDC). We identified the genome-wide sources of a total of 2252 ns-ncRNAs, mostly originating from intergenic and untranslated regions of annotated genes. By integrating the nascent RNA abundances with ATAC-seq and ChIP-seq analysis, we uncovered the euchromatic microenvironment surrounding the ns-ncRNA loci, and revealed a positive correlation between ns-ncRNAs and corresponding mRNA abundances. Finally, by gene knock-down strategy, we showed that the cooperation of RNA exosome catalytic subunit PfDis3 and PfMtr4 cofactor played a major role in ns-ncRNAs degradation. Collectively, this study contributes to understanding of the potential roles of short-lived nascent ncRNAs in regulating gene expression in malaria parasites.

Efficacy and safety of acupuncture on relieving abdominal pain and distension for acute pancreatitis: A protocol for systematic review.

INTRODUCTION: The purpose of this study is to evaluate the efficacy and safety of acupuncture on relieving abdominal pain and distension in acute pancreatitis. METHODS AND ANALYSIS: We will electronically search PubMed, MEDLINE, Embase, Web of Science, the Cochrane Central Register of Controlled Trial, China National Knowledge Infrastructure, China Biomedical Literature Database, China Science Journal Database, and Wanfang Database from their inception. Furthermore, we will manually retrieve other resources, including reference lists of identified publications, conference articles, and gray literature. The clinical randomized controlled trials or quasi-randomized controlled trials related to acupuncture treating acute pancreatitis will be included in the study. The language is limited to Chinese and English. Research selection, data extraction, and research quality assessment will be independently completed by 2 researchers. Data will be synthesized using a fixed effects model or random effects model depending on the heterogeneity test. The overall response rate and the visual analog scale score will be the primary outcomes. The time of first bowel sound, the time of first defecation, the length of hospitalization, acute physiology and chronic health evaluation II score, and the adverse events will also be assessed as secondary outcomes. RevMan 5 (version 5.3) statistical software will be used for meta-analysis, and the level of evidence will be assessed by Grading of Recommendations Assessment, Development, and Evaluation. Continuous data will be expressed in the form of weighted mean difference or standardized mean difference with 95% confidence intervals, whereas dichotomous data will be expressed in the form of risk ratios with 95% confidence intervals. ETHICS AND DISSEMINATION: The protocol of this systematic review does not require ethical approval because it does not involve humans. We will publish this article in peer-reviewed journals and present at relevant conferences. PROSPERO REGISTRATION NUMBER: CRD42019147503.

Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum.

Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.

Introduction of F446I mutation in the K13 propeller gene leads to increased ring survival rates in Plasmodium falciparum isolates.

BACKGROUND: Mutations in the Plasmodium falciparum k13 gene are associated with artemisinin (ART) resistance. However, it is unclear whether the F446I mutation, the most prevalent allele at the China-Myanmar border and north of Myanmar, is associated with ART resistance. Therefore, the aim of this study was to investigate the role of this mutation in ART resistance by generating transgenic parasites expressing the F446I mutant allele. METHODS: The transgenic parasites carrying the F446I or C580Y mutation in both 3D7 and FCC1/HN isolates were generated by single crossing-over recombination and verified using PCR and gene sequencing. The ring-stage survival assay of 0-3 h (RSA0-3 h) was used to evaluate ART susceptibility of the transgenic parasites in vitro. RESULTS: Four transgenic parasite lines named 3D7F446I mut, 3D7C580Y mut, FCC1/HNF446I mut and FCC1/HNC580Y mut were successfully generated. These parasite lines showed no changes in the expression level of k13 when compared with their parent parasite isolates. However, introduction of the F446I mutation in k13 of the 3D7 and FCC1/HN isolates led to elevated ring survival rates detected using RSA0-3 h when subjected to both 700 and 20 nM concentrations of dihydroartemisinin. The survival rates were similar to those detected in the parasite lines with the C580Y mutation. CONCLUSIONS: Insertion of the F446I mutation in k13 led to increased ring survival, suggesting that this mutation may be associated with ART resistance and could be used as a molecular marker for monitoring ART-resistant parasites. The results also highlights the importance of surveillance of F446I mutants for containing the resistant parasite.

Dual regulatory effects of non-coding GC-rich elements on the expression of virulence genes in malaria parasites.

As the primary virulence factor of falciparum malaria, var genes harboring mutually exclusive expression pattern lead to antigenic variation and immune evasion of this pathogen in human host. Although various mechanisms contribute to silence of var genes, little is known of transcriptional activation pathways of a single var gene and maintenance of its active state with other silent var loci. Here, we report a monoallelic expression pattern of the non-coding GC-elements flanking chromosomal internal var genes, and transcript from the active one was required for activation of the var gene in the same array. Meanwhile, GFP reporter assays revealed a repressive effect on the adjacent gene induced by DNA motifs of the insulator-like GC-element, which was linked to heterochromatin subnuclear localization. Taken together, these data for the first time provide experimental evidence of the dual cis- and trans-acting regulatory functions of the GC-elements in both silence and activation of var genes, which would advance our understanding of the complex regulatory network of the virulence gene family in P. falciparum.

Evaluation of the population structure and genetic diversity of Plasmodium falciparum in southern China.

BACKGROUND: Yunnan and Hainan provinces are the two major endemic regions for Plasmodium falciparum malaria in China. However, few studies have investigated the characteristics of this parasite. Therefore, this study aimed to evaluate the genetic diversity and population structure of P. falciparum to predict the geographic origin of falciparum malaria. METHODS: Thirteen highly polymorphic microsatellite loci were studied to estimate the genetic diversity and population structure of 425 P. falciparum isolates obtained from blood samples collected from Yunnan and Hainan provinces of South China. The isolates were analysed for genetic diversity, linkage disequilibrium, and population structure. The parasite populations were clustered into two subgroups (i.e., Yunnan and Hainan) and a classification algorithm was used to identify molecular markers for classifying the P. falciparum populations. RESULTS: All 13 microsatellite loci were highly polymorphic, with the number of alleles per locus varying from 5 to 20. The mean expected heterozygosity (He) in Yunnan and Hainan was 0.766 ± 0.036 and 0.677 ± 0.039, respectively, revealing a moderate high level of genetic diversity. Significant linkage disequilibrium was found for some regions of Yunnan (Lazan county and Xishuangbanna region) and Hainan (Dongfang city and Sanya city) province. According to the classification algorithm, a combination of three microsatellites could be used as a discriminatory marker to identify the origin of P. falciparum isolates. CONCLUSIONS: The results on the genetic structure of P. falciparum populations from South China provide a basis for developing a genetic marker-based tool to trace the source of the parasite infections and consequently improve malaria control and elimination strategies.