Left atrial appendage filling defect in exclusive early-phase scanning of dual-phase cardiac computed tomography: An indicator for elevated thromboembolic risk.

BACKGROUND: Dual-phase cardiac computed tomography (CCT) has been applied to detect left atrial appendage (LAA) thrombosis, which is characterized as the presence of left atrial appendage filling defects (LAADF) in both early- and delayed-phase scanning. However, the clinical implication of LAAFD in exclusive early-phase scanning (LAAFD-EEpS) of CCT in patients with atrial fibrillation (AF) is unclear. METHODS: The baseline clinical data and dual-phase CCT findings in 1183 AF patients (62.1 ± 11.6 years, 59.9% male) was collected and analyzed. A further analysis of CCT and transesophageal echocardiography (TEE) data (within 5 days) in a subgroup of 687 patients was performed. LAAFD-EEpS was defined as LAAFD present in early-phase and absent in delayed-phase scanning of dual-phase CCT. RESULTS: A total of 133 (11.2%) patients were detected with LAAFD-EEpS. Patients with LAAFD-EEpS had a higher prevalence of ischemic stroke or transient ischemic attack (TIA) (p < 0.001) and a higher predefined thromboembolic risk (p < 0.001). In multivariate analysis, a history of ischemic stroke or TIA was independently associated with LAAFD-EEpS (odds ratio [OR] 11.412, 95% confidence interval [CI] 6.561-19.851, p < 0.001). When spontaneous echo contrast in TEE was used as the reference standard, the sensitivity, specificity, positive predictive value, and negative predictive value of LAAFD-EEpS was 77.0% (95% CI 66.5-87.6%), 89.0% (95% CI 86.5-91.4%), 40.5% (95% CI 31.6-49.5%), 97.5% (96.3-98.8%), respectively. CONCLUSIONS: In AF patients, LAAFD-EEpS is not an uncommon finding in dual-phase CCT scanning, and is associated with elevated thromboembolic risk.

Talaromyces sp. Ethyl Acetate Crude Extract as Potential Mosquitocide to Control Culex pipiens quinquefasciatus.

Vector control is considered an effective approach to controlling diseases spread by mosquito bites. Entomopathogenic fungi are widely used in agriculture to control insect pests, and fungal metabolites can potentially be developed as effective mosquitocides. In this study, a high-throughput screening method was used to search for potential mosquitocides in the Global Fungal Extract Library (GFEL). We tested the larvicidal activity of 264 fungal ethyl acetate crude extracts against Culex pipiens quinquefasciatus. Nine fungal extracts caused moderate to high mortality rates (>50%), with two fungal extracts (58A7 and 101H12) causing a 100% mortality rate. The lethal concentrations for 50% of the population (LC50) were 44.27 mg/L and 31.90 mg/L, respectively. Fraction 14 had a high mortality rate, with an LC50 value of 12.13 mg/L, and was isolated from 58A7 (Fractions 1-11) and 101H12 (Fractions 12-15). Further analyses showed that Fraction 14 was made up of vermistatin and dihydrovermistatin. In a Cx. p. quinquefasciatus larvicidal bioassay, vermistatin (LC50 = 28.13 mg/L) was more toxic than dihydrovermistatin (LC50 = 83.87 mg/L). Our findings suggested that the active fungal extract 101H12 from Talaromyces sp. and its compound vermistatin could be developed as mosquitocides.

Optimization of superior vena cava isolation with aid of ablation index guidance.

INTRODUCTION: To investigate the optimal range of quantitative ablation index (AI) value during superior vena cava (SVC) electrical isolation by radiofrequency catheter ablation (RFCA). METHODS: First, in a development cohort of patients with atrial fibrillation (AF), the RFCA with 40 W was performed to complete SVC isolation guided by the conduction breakthrough point from the right atrium to SVC. Then, the range of AI value was calculated by offline analysis on different segments of SVC. Lastly, for the validation of AF patients, the safety and effectiveness of SVC isolation with the optimized target range of AI value were evaluated with an additional adenosine test. RESULTS: A total of 101 patients with AF were included in the study (44 patients in the development cohort/57 in the validation cohort). The segmental ablation strategy was applied in 70% of the patients. According to the offline analysis of the AI values in the development cohort, the target AI value range was set as 350-400. The success rate of SVC isolation in the validation cohort was significantly higher than that in the exploration cohort (100% vs. 90.9%, p = .02), and no complications occurred in the exploration cohort. During the adenosine test, the recovery rate of electrical conduction in SVC was significantly lower than that in the pulmonary vein (3.5% vs. 17.5%). CONCLUSION: The target AI value with a range from 350 to 400 is safe and effective for high-power RFCA to complete SVC isolation.

Arsinothricin Inhibits Plasmodium falciparum Proliferation in Blood and Blocks Parasite Transmission to Mosquitoes.

Malaria, caused by Plasmodium protozoal parasites, remains a leading cause of morbidity and mortality. The Plasmodium parasite has a complex life cycle, with asexual and sexual forms in humans and Anopheles mosquitoes. Most antimalarials target only the symptomatic asexual blood stage. However, to ensure malaria eradication, new drugs with efficacy at multiple stages of the life cycle are necessary. We previously demonstrated that arsinothricin (AST), a newly discovered organoarsenical natural product, is a potent broad-spectrum antibiotic that inhibits the growth of various prokaryotic pathogens. Here, we report that AST is an effective multi-stage antimalarial. AST is a nonproteinogenic amino acid analog of glutamate that inhibits prokaryotic glutamine synthetase (GS). Phylogenetic analysis shows that Plasmodium GS, which is expressed throughout all stages of the parasite life cycle, is more closely related to prokaryotic GS than eukaryotic GS. AST potently inhibits Plasmodium GS, while it is less effective on human GS. Notably, AST effectively inhibits both Plasmodium erythrocytic proliferation and parasite transmission to mosquitoes. In contrast, AST is relatively nontoxic to a number of human cell lines, suggesting that AST is selective against malaria pathogens, with little negative effect on the human host. We propose that AST is a promising lead compound for developing a new class of multi-stage antimalarials.

Plasmodium parasitophorous vacuole membrane protein Pfs16 promotes malaria transmission by silencing mosquito immunity.

With rising cases for the first time in years, malaria remains a significant public health burden. The sexual stage of the malaria parasite infects mosquitoes to transmit malaria from host to host. Hence, an infected mosquito plays an essential role in malaria transmission. Plasmodium falciparum is the most dominant and dangerous malaria pathogen. Previous studies identified a sexual stage-specific protein 16 (Pfs16) localized to the parasitophorous vacuole membrane. Here, we elucidate the function of Pfs16 during malaria transmission. Our structural analysis identified Pfs16 as an alpha-helical integral membrane protein with one transmembrane domain connecting to two regions across parasitophorous vacuole membrane. ELISA assays showed that insect cell-expressed recombinant Pfs16 (rPfs16) interacted with Anopheles gambiae midguts, and microscopy found that rPfs16 was bound to midgut epithelial cells. Transmission-blocking assays demonstrated that polyclonal antibodies against Pfs16 significantly reduced the number of oocysts in mosquito midguts. However, on the contrary, feeding rPfs16 increased the number of oocysts. Further analysis revealed that Pfs16 reduced the activity of mosquito midgut caspase 3/7, a key enzyme in the mosquito Jun-N-terminal kinase immune pathway. We conclude that Pfs16 facilitates parasites to invade mosquito midguts by actively silencing the mosquito's innate immunity through its interaction with the midgut epithelial cells. Therefore, Pfs16 is a potential target to control malaria transmission.

Targeting plasmodium α-tubulin-1 to block malaria transmission to mosquitoes.

Plasmodium ookinetes use an invasive apparatus to invade mosquito midguts, and tubulins are the major structural proteins of this apical complex. We examined the role of tubulins in malaria transmission to mosquitoes. Our results demonstrate that the rabbit polyclonal antibodies (pAb) against human α-tubulin significantly reduced the number of P. falciparum oocysts in Anopheles gambiae midguts, while rabbit pAb against human ß-tubulin did not. Further studies showed that pAb, specifically against P. falciparum α-tubulin-1, also significantly limited P. falciparum transmission to mosquitoes. We also generated mouse monoclonal antibodies (mAb) using recombinant P. falciparum α-tubulin-1. Out of 16 mAb, two mAb, A3 and A16, blocked P. falciparum transmission with EC50 of 12 µg/ml and 2.8 µg/ml. The epitopes of A3 and A16 were determined to be a conformational and linear sequence of EAREDLAALEKDYEE, respectively. To understand the mechanism of the antibody-blocking activity, we studied the accessibility of live ookinete α-tubulin-1 to antibodies and its interaction with mosquito midgut proteins. Immunofluorescent assays showed that pAb could bind to the apical complex of live ookinetes. Moreover, both ELISA and pull-down assays demonstrated that insect cell-expressed mosquito midgut protein, fibrinogen-related protein 1 (FREP1), interacts with P. falciparum α-tubulin-1. Since ookinete invasion is directional, we conclude that the interaction between Anopheles FREP1 protein and Plasmodium α-tubulin-1 anchors and orients the ookinete invasive apparatus towards the midgut PM and promotes the efficient parasite infection in the mosquito.

Prevalence, predictors and management of left atrial appendage thrombogenic milieu in atrial fibrillation with low thromboembolic risk.

BACKGROUND: The present study aimed to investigate the prevalence, predictors, and management of left atrial appendage (LAA) thrombogenic milieu (TM) identified with transesophageal echocardiography (TEE) in non-valvular atrial fibrillation (NVAF) patients with low to moderate thromboembolic (TE) risk. METHODS: We retrospectively analyzed the baseline clinical data and TEE findings in 391 NVAF patients (54.7 ± 8.9 years, 69.1% male) with low to moderate TE risk according to the CHA2DS2-VASc score. LAA TM was defined as LAA thrombus (LAAT), sludge or spontaneous echo contrast (SEC). Management of LAA TM was at the discretion of the treating physician. RESULTS: A total of 43 patients (11.0%) were detected with LAA TM, including 5 with LAAT (11.6%), 4 with LAAT + Sect. (9.3%), 3 with sludge (7.0%), and 31 with Sect. (72.1%). In multivariate model, non-paroxysmal AF (OR 3.121; 95% CI 1.205-8.083, p = 0.019), and a larger left atrial diameter (LAD) (OR 1.134; 95% CI 1.060-1.213, p < 0.001) were significantly associated with the presence of LAA TM. All LAATs or sludges effectively resolved after mean duration of 117.5 ± 20.0 days for oral anticoagulant (OAC) medication. TE events occurred in 3 patients (18.8%) among those discontinuing OAC over a mean follow-up of 26.2 ± 8.8 months, while no TE events occurred in patients with continuous OAC. CONCLUSIONS: LAA TM could be identified in 11.0% in NVAF patients with low to moderate TE risk, especially in those with non-paroxysmal AF and enlarged LAD. Short-term OAC medication could effectively resolve the LAAT or sludge.

Performance of two tools for pulmonary vein occlusion assessment with a novel navigation system in cryoballoon ablation procedure.

INTRODUCTION: Optimal occlusion of pulmonary vein (PV) is essential for atrial fibrillation (AF) cryoballoon ablation (CBA). The aim of the study was to investigate the performance of two different tools for the assessment of PV occlusion with a novel navigation system in CBA procedure. METHODS: In consecutive patients with paroxysmal AF who underwent CBA procedure with the guidance of the novel 3-dimentional mapping system, the baseline tool, injection tool and pulmonary venography were all employed to assess the degree of PV occlusion, and the corresponding cryoablation parameters were recorded. RESULTS: In 23 patients (mean age 60.0 ± 13.9 years, 56.5% male), a total of 149 attempts of occlusion and 122 cryoablations in 92 PVs were performed. Using pulmonary venography as the gold standard, the overall sensitivity, specificity of the baseline tool was 96.7% (95% confidence interval [CI] 90.0%-99.1%), and 40.5% (95% CI 26.0%-56.7%), respectively, while the corresponding value of the injection tool was 69.6% (95% CI 59.7%-78.1%), and 100.0% (95% CI 90.6%-100.0%), respectively. Cryoablation with optimal occlusion showed lower nadir temperature (baseline tool: -44.3 ± 8.4°C vs. -35.1 ± 6.5°C, p < .001; injection tool: -46.7 ± 6.4°C vs. -38.3 ± 9.2°C, p < .001) and longer total thaw time (baseline tool: 53.3 ± 17.0 s vs. 38.2 ± 14.9 s, p = .003; injection tool: 58.5 ± 15.5 s vs. 41.7 ± 15.2 s, p < .001) compared with those without. CONCLUSIONS: Both tools were able to accurately assess the degree of PV occlusion and predict the acute cryoablation effect, with the baseline tool being more sensitive and the injection tool more specific.

Long-Term prognosis of radiofrequency catheter ablation for atrial fibrillation with different subtypes of heart failure in the era of ablation index guidance.

Background: The long-term outcomes of ablation index (AI)-guided radiofrequency catheter ablation (RFCA) on atrial fibrillation (AF) and different subtypes of heart failure (HF) remain unknown. The aim of the study was to evaluate the long-term prognosis of AI-guided RFCA procedures in patients with AF and concomitant HF. Methods: We retrospectively included consecutive patients with AF and HF who underwent the initial RFCA procedure with AI guidance from March 2018 to June 2021 in our institution. The patients were categorized into two groups: HF with preserved ejection fraction (HFpEF) group and HF with mid-range ejection fraction (HFmrEF) +HF with reduced ejection fraction (HFrEF) group. Results: A total of 101 patients were included. HFpEF and HFmrEF + HFrEF groups consisted of 71 (70.3%) and 30 patients (29.7%), respectively. During a median follow-up of 32.0 (18.2, 37.6) months, no significant difference was detected in AF recurrence between groups (21.1 vs. 33.3%) after multiple procedures, whereas the incidence of the composite endpoint of all-cause death, thromboembolic events, and HF hospitalization was significantly lower in HFpEF group (9.9 vs. 25.0%, Log-rank p = 0.018). In multivariable analysis, a history of hypertension [hazard ratio (HR) 4.667, 95% confidence interval (CI) 1.433-15.203, p = 0.011], left ventricular ejection fraction (LVEF) < 50% (HR 5.390, 95% CI 1.911-15.203, p = 0.001) and recurrent AF after multiple procedures (HR 7.542, 95% CI 2.355-24.148, p = 0.001) were independently associated with the incidence of the composite endpoint. Conclusion: Long-term success could be achieved in 75% of patients with AF and concomitant HF after AI-guided RFCA procedures, irrespective of different HF subtypes. Preserved LVEF was associated with a reduction in the composite endpoint compared with impaired LVEF. Patients with recurrent AF tend to have a poorer prognosis.

Crystal Structure Determination and Hirshfeld Analysis of a New Alternariol Packing Polymorph.

A new polymorph of the mycotoxin alternariol is reported and characterized by single crystal X-ray diffraction. Structural data, Hirshfeld surface analysis, and 2D fingerprint plots are used to compare differences in the intermolecular interactions of the orthorhombic Pca21 Form I (previously reported) and the monoclinic P21/c Form II (herein reported). The polymorphs have small differences in planarity-7.55° and 2.19° between the terminal rings for Form I and Form II, respectively-that brings about significant differences in the crystal packing and O-H … H interactions.

Distinct pathological changes of osteochondral units in early OVX-OA involving TGF-ß signaling.

Introduction: Different opinions exist about the role of subchondral bone in osteoarthritis (OA), probably because subchondral bone has different effects on cartilage degeneration in OA induced by different pathologies. Animal studies to illustrate the role of subchondral bone in cartilage degeneration were mostly based on post-traumatic OA (PT-OA). Postmenopausal women experience a much higher occurrence of OA than similar-aged men. The physiological changes and pathogenesis of the osteochondral unit in ovariectomy-induced OA (OVX-OA) might be distinct from other types of OA. Methods: The osteochondral alterations of post-traumatic OA (PT-OA) and OVX-OA at week 9 after surgery were compared. Then the alterations of osteochondral units in OVX-OA rats were tracked over time for the designed groups: Sham, OVX and OVX rats treated with estrogen (OVX+E). DXA, micro-CT, and histochemical staining were performed to observe alterations in osteochondral units. Results: Rapid cartilage degeneration and increased bone formation were observed in PT-OA, while only mild cartilage erosion and significant bone loss were observed in OVX-OA at week 9 after surgery. Subchondral bone degradation preceded cartilage degeneration by 6 weeks in OVX-OA. TGF-ß expression was downregulated in the osteochondral unit of OVX rats. Estrogen supplementation inhibited subchondral bone loss, cartilage degradation and TGF-ß expression decrease. Discussion: This research demonstrated the distinct behaviors of the osteochondral unit and the critical role of subchondral bone in early OVX-OA compared with PT-OA. Inhibiting subchondral bone catabolism at the early stage of OVX-OA could be an effective treatment for post-menopausal OA. Based on the results, estrogen supplementation and TGF-ß modulation at the early stage are both potential therapies for post-menopausal OA.

Discovery of mosquitocides from fungal extracts through a high-throughput cytotoxicity-screening approach.

BACKGROUND: Mosquitoes transmit a variety of diseases. Due to widespread insecticide resistance, new effective pesticides are urgently needed. Entomopathogenic fungi are widely utilized to control pest insects in agriculture. We hypothesized that certain fungal metabolites may be effective insecticides against mosquitoes. METHODS: A high-throughput cytotoxicity-based screening approach was developed to search for insecticidal compounds in our newly established global fungal extract library. We first determined cell survival rates after adding various fungal extracts. Candidate insecticides were further analyzed using traditional larval and adult survival bioassays. RESULTS: Twelve ethyl acetate extracts from a total of 192 fungal extracts displayed > 85% inhibition of cabbage looper ovary cell proliferation. Ten of these 12 candidates were confirmed to be toxic to Anopheles gambiae Sua5B cell line, and six showed > 85% inhibition of Anopheles mosquito cell growth. Further bioassays determined a LC50, the lethal concentration that kills 50% of larval or adult mosquitoes, of 122 µg/mL and 1.7 µg/mosquito, respectively, after 24 h for extract 76F6 from Penicillium toxicarium. CONCLUSIONS: We established a high-throughput MTT-based cytotoxicity screening approach for the discovery of new mosquitocides from fungal extracts. We discovered a candidate extract from P. toxicarium that exhibited high toxicity to mosquito larvae and adults, and thus were able to demonstrate the value of our recently developed approach. The active fungal extracts discovered here are ideal candidates for further development as mosquitocides.

Sterigmatocystin Limits Plasmodium falciparum Proliferation and Transmission.

As part of our drug discovery program against malaria, the Penicillium janthinellum extract was discovered to inhibit P. falciparum proliferation in blood and transmission to mosquitoes. Bioactivity-guided fractionation of P. janthinellum extraction was carried out using chromatographic techniques. We determined the activities of fractions against Plasmodium falciparum asexual stage parasite proliferation in culture and sexual stage parasite transmission to mosquitoes using standard membrane feeding assays (SMFA). One active compound was isolated. Based on mass spectrometry and nuclear magnetic resonance profiles, the compound was structurally determined to be sterigmatocystin. Sterigmatocystin inhibited P. falciparum proliferation in the blood with an IC50 of 34 µM and limited the sexual parasites to infect mosquitoes with an IC50 of 48 µM. Meanwhile, sterigmatocystin did not show any acute toxicity to human kidney cells at a concentration of 64 µM or lower. Sterigmatocystin can be used as a drug lead for malaria control and as a probe to understand molecular mechanisms of malaria transmission.

The incidence and location of epicardial connections in the era of contact force guided ablation for pulmonary vein isolation.

BACKGROUND: The effects of epicardial connections (ECs) involving pulmonary veins (PVs) in atrial fibrillation (AF) ablation have been revealed recently. However, no systematic approaches to identify and ablate the ECs were established. METHODS: Patients with AF undergoing radiofrequency (RF) catheter ablation were retrospectively analyzed. ECs were identified when (1) PV isolation (PVI) cannot be achieved after first-pass isolation; (2) PVI was still absent although the conduction gap was detected and ablated; (3) the earliest activation area (EAA) was revealed located within the PV antrum distant from the initial ablation line using high-density mapping (HDM) technique; (4) focal ablation at the EAA was effective to achieve PVI. Relevant pacing maneuvers were performed to elucidate ECs' bidirectional conduction. RESULTS: Overall, 36 ECs were identified and ablated in 35/597 (5.86%) patients. Among the 35 patients with ECs, at least one PV insertion of ECs was located at the carina region. The most common pattern was a single breakthrough in 31 (88.6%) patients, followed by multiple breakthroughs in 3 and wide breakthroughs in 1. The median distance from EAA to the initial ablation line was 10.0 mm. The average number of RF energy delivery was 1.75 ± 1.00, and single RF delivery was adequate in 16/36 (44.4%) patients. Continuous potentials were present at the EAA in 9/34 (26.5%) patients. CONCLUSION: ECs were confirmed and ablated successfully in 5.86% (35/597) AF patients using HDM. PV insertions of ECs were mainly located at the carina region. Continuous potentials might assist in the prediction of ECs.

Studies of the Parasite-Midgut Interaction Reveal Plasmodium Proteins Important for Malaria Transmission to Mosquitoes.

Malaria transmission relies on parasite-mosquito midgut interaction. The interactive proteins are hypothesized to be ideal targets to block malaria transmission to mosquitoes. We chose 76 genes that contain signal peptide-coding regions and are upregulated and highly abundant at sexual stages. Forty-six of these candidate genes (60%) were cloned and expressed using the baculovirus expression system in insect cells. Six of them, e.g., PF3D7_0303900, PF3D7_0406200 (Pfs16), PF3D7_1204400 (Pfs37), PF3D7_1214800, PF3D7_1239400, and PF3D7_1472800 were discovered to interact with blood-fed mosquito midgut lysate. Previous works showed that among these interactive proteins, knockout the orthologs of Pfs37 or Pfs16 in P. berghei reduced oocysts in mosquitoes. Here we further found that anti-Pfs16 polyclonal antibody significantly inhibited P. falciparum transmission to Anopheles gambiae. Investigating these candidate proteins will improve our understanding of malaria transmission and discover new targets to break malaria transmission.

Efficiency, Safety, and Efficacy of High-Power Short-Duration Radiofrequency Ablation in Patients with Atrial Fibrillation.

Pulmonary vein isolation (PVI) is the cornerstone therapy of atrial fibrillation (AF). Radiofrequency catheter ablation (RFCA) is performed using a point-by-point method to achieve durable PVI. However, this procedure remains complex and time-consuming, and the long-term clinical outcomes are still not satisfactory. Recently, there has been increasing interest in the clinical application of high-power short-duration (HPSD) approaches in the field of RFCA. HPSD ablation, distinguishing it from the conventional ablation strategy, delivers RF energy at a high power and saves the dwell time at each site. It is unknown whether the HPSD approach can bring some gratifying changes in the field of RF energy ablation. A number of experimental studies and clinical studies have been conducted regarding this topic. The review aimed to summarize the research findings and evaluate the procedural efficiency, safety, and clinical outcomes of the HPSD approach based on the evidence available to date.

A novel fungal metabolite inhibits Plasmodium falciparum transmission and infection.

BACKGROUND: Malaria transmission depends on infected mosquitoes and can be controlled by transmission-blocking drugs. The recently discovered FREP1-mediated malaria transmission pathway is an excellent target to screen drugs for limiting transmission. METHODS: To identify candidate small molecules, we used an ELISA-based approach to analyze extracts from a fungal library for inhibition of the FREP1-parasite interaction. We isolated and determined one active compound by chromatography and crystallography, respectively. We measured the effects of the bioactive compound on malaria transmission to mosquitoes through standard membrane-feeding assays (SMFA) and on parasite proliferation in blood by culturing. RESULTS: We discovered the ethyl acetate extract of the fungus Purpureocillium lilacinum that inhibited Plasmodium falciparum transmission to mosquitoes. Pre-exposure to the extract rendered Anopheles gambiae resistant to Plasmodium infection. Furthermore, we isolated one novel active compound from the extract and identified it as 3-amino-7,9-dihydroxy-1-methyl-6H-benzo[c]chromen-6-one, or "pulixin." Pulixin prevented FREP1 from binding to P. falciparum-infected cell lysate. Pulixin blocked the transmission of the parasite to mosquitoes with an EC50 (the concentration that gave half-maximal response) of 11 µM based on SMFA. Notably, pulixin also inhibited the proliferation of asexual-stage P. falciparum with an EC50 of 47 nM. The compound did not show cytotoxic effects at a concentration of 116 µM or lower. CONCLUSION: By targeting the FREP1-Plasmodium interaction, we discovered that Purpureocillium lilacinum extract blocked malaria transmission. We isolated and identified the bioactive agent pulixin as a new compound capable of stopping malaria transmission to mosquitoes and inhibiting parasite proliferation in blood culture.

A diverse global fungal library for drug discovery.

BACKGROUND: Secondary fungal metabolites are important sources for new drugs against infectious diseases and cancers. METHODS: To obtain a library with enough diversity, we collected about 2,395 soil samples and 2,324 plant samples from 36 regions in Africa, Asia, and North America. The collection areas covered various climate zones in the world. We examined the usability of the global fungal extract library (GFEL) against parasitic malaria transmission, Gram-positive and negative bacterial pathogens, and leukemia cells. RESULTS: Nearly ten thousand fungal strains were isolated. Sequences of nuclear ribosomal internal transcribed spacer (ITS) from 40 randomly selected strains showed that over 80% were unique. Screening GFEL, we found that the fungal extract from Penicillium thomii was able to block Plasmodium falciparum transmission to Anopheles gambiae, and the fungal extract from Tolypocladium album was able to kill myelogenous leukemia cell line K562. We also identified a set of candidate fungal extracts against bacterial pathogens.

Analysis of blood-induced Anopheles gambiae midgut proteins and sexual stage Plasmodium falciparum interaction reveals mosquito genes important for malaria transmission.

Plasmodium invasion of mosquito midguts is a mandatory step for malaria transmission. The roles of mosquito midgut proteins and parasite interaction during malaria transmission are not clear. This study aims to identify mosquito midgut proteins that interact with and affect P. falciparum invasion. Based on gene expression profiles and protein sequences, 76 mosquito secretory proteins that are highly expressed in midguts and up-regulated by blood meals were chosen for analysis. About 61 candidate genes were successfully cloned from Anopheles gambiae and expressed in insect cells. ELISA analysis showed that 25 of the insect cell-expressed recombinant mosquito proteins interacted with the P. falciparum-infected cell lysates. Indirect immunofluorescence assays confirmed 17 of them interacted with sexual stage parasites significantly stronger than asexual stage parasites. Knockdown assays found that seven candidate genes significantly changed mosquitoes' susceptibility to P. falciparum. Four of them (AGAP006268, AGAP002848, AGAP006972, and AGAP002851) played a protective function against parasite invasion, and the other three (AGAP008138, FREP1, and HPX15) facilitated P. falciparum transmission to mosquitoes. Notably, AGAP008138 is a unique gene that only exists in Anopheline mosquitoes. These gene products are ideal targets to block malaria transmission.

Fungal Metabolite Asperaculane B Inhibits Malaria Infection and Transmission.

Mosquito-transmitted Plasmodium parasites cause millions of people worldwide to suffer malaria every year. Drug-resistant Plasmodium parasites and insecticide-resistant mosquitoes make malaria hard to control. Thus, the next generation of antimalarial drugs that inhibit malaria infection and transmission are needed. We screened our Global Fungal Extract Library (GFEL) and obtained a candidate that completely inhibited Plasmodium falciparum transmission to Anopheles gambiae. The candidate fungal strain was determined as Aspergillus aculeatus. The bioactive compound was purified and identified as asperaculane B. The concentration of 50% inhibition on P. falciparum transmission (IC50) is 7.89 µM. Notably, asperaculane B also inhibited the development of asexual P. falciparum with IC50 of 3 µM, and it is nontoxic to human cells. Therefore, asperaculane B is a new dual-functional antimalarial lead that has the potential to treat malaria and block malaria transmission.