Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.

The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.

Proteomic Analysis Reveals Proteins Involved in the Mode of Action of ß-Citronellol Identified From Citrus hystrix DC. Leaf Against Candida albicans.

Candida albicans is a fungus that lives primarily on the mucosal surfaces of healthy humans, such as the oral cavity, vagina, and gastrointestinal tract. This commensal organism can be controlled by other microbiota, while certain conditions can increase the risk of C. albicans outgrowth and cause disease. Prevalence of the drug-resistant phenotype, as well as the severity of C. albicans infection in immunocompromised patients, presents a challenge for scientists to develop novel, effective treatment, and prevention strategies. ß-Citronellol is an intriguing active compound of several plants that has been linked to antifungal activity, but data on the mechanism of action in terms of proteomic profiling are lacking. Here, ß-citronellol identified from Citrus hystrix DC. leaf against C. albicans were evaluated. A proteomic approach was used to identify potential target proteins involved in the mode of action of ß-citronellol. This study identified and discussed three protein groups based on the 126 major proteins that were altered in response to ß-citronellol treatment, 46 of which were downregulated and 80 of which were upregulated. Significant protein groups include cell wall proteins (e.g., Als2p, Rbt1p, and Pga4p), cellular stress response enzymes (e.g., Sod1p, Gst2p, and Ddr48p), and ATP synthesis-associated proteins (e.g., Atp3p, Atp7p, Cox1p, and Cobp). Results demonstrated the complexities of protein interactions influenced by ß-citronellol treatment and highlighted the potential of antifungal activity for future clinical and drug development research.

Evaluation of Mouthwash Containing Citrus hystrix DC., Moringa oleifera Lam. and Azadirachta indica A. Juss. Leaf Extracts on Dental Plaque and Gingivitis.

Oral hygiene and control of microbial plaque biofilm formation are effective methods for preventing gingivitis. Mouthwashes containing leaf extracts of the medicinal plants Citrus hystrix DC. (KL), Moringa oleifera Lam. (MO) and Azadirachta indica A. Juss. (NE) were assessed for oral healthcare and gingivitis adjunctive treatment. Three types of mouthwash were developed; KL, a combination of KL and MO (KL + MO), and a combination of KL, and NE (KL + NE). The mouthwashes were tested in vivo on 47 subjects with gingivitis who were allocated into five groups as (i) placebo, (ii) KL, (iii) KL + MO, (iv) KL + NE, and (v) 0.12% chlorhexidine gluconate (CHX). Participants were instructed to rinse with herbal mouthwash twice daily for two weeks. Gingival index (GI), plaque index (PI), and oral microbial colonies were measured at baseline and 15 days. Results showed that GI and PI of groups (ii)-(iv) significantly decreased over the placebo group, while accumulative reduction percentages of both Staphylococcus spp. and Candida spp. were found in groups (iii) and (iv). Findings indicated that the herbal mouthwashes reduced GI and PI, and showed potential as oral healthcare products.

Phytochemical Constituents of Citrus hystrix DC. Leaves Attenuate Inflammation via NF-κB Signaling and NLRP3 Inflammasome Activity in Macrophages.

Citrus hystrix DC. (CH) is found in many countries in Southeast Asia. This plant has been reported for anti-microbial, anti-cancer and anti-inflammatory bioactivities. However, the anti-inflammatory and anti-inflammasome properties of the leaves remain poorly understood. This study aimed to investigate the effect of CH leaves on NLRP3 and NF-κB signaling pathways. CH leaves were sequentially extracted using hexane, ethyl acetate and 95% ethanol to give three crude extracts. An active compound, lupeol was fractionated from the ethanolic extract using chromatographic techniques, and its structure was identified and confirmed by spectroscopic methods. Anti-inflammatory activities were observed on both lipopolysaccharide-stimulated and NLRP3 adenosine triphosphate-induced macrophages. The release of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) was analyzed by Enzyme-Linked Immunosorbent Assay (ELISA). Real-time qRT-polymerase chain reaction (PCR) was used to measure inflammatory-associated gene expression. NF-κB protein expressions were investigated using the immunoblotting technique. The active fraction of ethanolic CH leaves and lupeol significantly reduced the release of pro-inflammatory cytokines and suppressed the expression of both inflammasome genes and NF-κB proteins. The ethanolic extract of CH leaves and lupeol showed potent anti-inflammatory activities by targeting NF-κB and NLRP3 signaling pathways.