Physicochemical properties and fatty acid profile of oil extracted from black soldier fly larvae (Hermetia illucens).

Background and Aim: Hermetia illucens, a black soldier fly, is widely recognized for sustainable recycling of organic waste. Black soldier fly larvae (BSFLs) can consume various types of biowastes and convert them into nutrient-rich biomass, including proteins, lipids, chitin, and minerals. This study investigated the best extraction method by comparing the fatty acid profiles, percentage yield, and antioxidant properties of BSFL oil extracted using different extraction methods. Materials and Methods: The physicochemical properties, fatty acid profile, and free radical scavenging ability of BSFL oil were analyzed using six extraction methods. Results: Ultrasonic extraction with hexane resulted in the highest yields compared with different extraction methods. Lauric acid (28%-37%) was the most abundant fatty acid in all extracts, followed by palmitic acid, myristic acid, oleic acid, and linoleic acid. Compared with other methods, aqueous extraction showed the highest lauric acid composition and free radical scavenging activities. In addition, high-temperature aqueous extraction resulted in higher oil yield and free radical scavenging activities than low-temperature extraction. Conclusion: High-temperature aqueous extraction is the best extraction method because it is rich in lauric acid, has antioxidant ability, and can be further developed to produce novel sustainable biomaterials for humans and animals.

Serum proteomic profile of wild stump-tailed macaques (Macaca arctoides) infected with malaria parasites in Thailand.

The number of patients infected with simian malaria is gradually increasing in many countries of Southeast Asia and South America. The most important risk factor for a zoonotic spillover event of malarial infection is mostly influenced by the interaction between humans, monkeys, and vectors. In this study, we determine the protein expression profile of a wild stump-tailed macaque (Macaca arctoides) from a total of 32 blood samples collected from Prachuap Kiri Khan Province, Thailand. The malarial parasite was analyzed using nested polymerase chain reaction (PCR) assays by dividing the samples into three groups: non-infected, mono-infected, and multiple-infected. The identification and differential proteomic expression profiles were determined using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and bioinformatics tools. A total of 9,532 proteins (total proteins) were identified with the filter-based selection methods analysis, and a subset of 440 proteins were found to be different between each group. Within these proteins, the GhostKOALA functional enrichment analysis indicated that 142 important proteins were associated with either of the organismal system (28.87%), genetic information processing (23.24%), environmental information processing (16.20%), metabolism (13.38%), cellular processes (11.97%), or causing human disease (6.34%). Additionally, using interaction network analysis, nine potential reporter proteins were identified. Here, we report the first study on the protein profiles differentially expressed in the serum of wild stump-tailed macaques between non, mono, and multiple malarial infected living in a natural transmission environment. Our findings demonstrate that differentially expressed proteins implicated in host defense through lipid metabolism, involved with TGF pathway were suppressed, while those with the apoptosis pathway, such as cytokines and proinflammation signals were increased. Including the parasite's response via induced hemolysis and disruption of myeloid cells. A greater understanding of the fundamental processes involved in a malarial infection and host response can be crucial for developing diagnostic tools, medication development, and therapies to improve the health of those affected by the disease.

Combining isothermal recombinase polymerase amplification with lateral flow assay for diagnosis of P. cynomolgi malaria infection.

BACKGROUND: Plasmodium cynomolgi is a nonhuman primate parasite that causes malaria in humans and is transmitted by the Anopheles mosquito. Macaques, the natural hosts of P. cynomolgi, are widely distributed in Asia, especially in Southeast Asia. Anthropogenic land-use changes and wildlife habitat reduction due to local environmental changes, deforestation, urban expansion, and construction increased the frequency of human-macaque-vector interactions and facilitated the emergence of zoonotic malaria, causing an exponential increase in the infection rates in this area. Although microscopic tools are the gold standard for malaria diagnosis, they have very low sensitivity. Therefore, disease control and prevention require rapid, sensitive and accurate diagnostic tests. METHODOLOGY/PRINCIPLE FINDINGS: This study aims to develop a diagnostic method using a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method to specifically diagnose P. cynomolgi. Laboratory validation determined the method's sensitivity and specificity compared to the nested PCR method. The lower limit of detection was 22.14 copies/µl of recombinant plasmid per reaction. The combination method represented 81.82% sensitivity and 94.74% specificity compared to the nested PCR. CONCLUSIONS/SIGNIFICANCE: The diagnostic testing developed in this study combines a recombinase polymerase amplification (RPA) and a lateral flow (LF) strip, offering rapid high sensitivity and specificity. Further development of this technique could make it a promising method for detecting P. cynomolgi.

Inhibitory effects of anthracyclines on partially purified 5'-3' DNA helicase of Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum has been becoming resistant to the currently used anti-malarial drugs. Searching for new drug targets is urgently needed for anti-malarial development. DNA helicases separating double-stranded DNA into single-stranded DNA intermediates are essential in nearly all DNA metabolic transactions, thus they may act as a candidate for new drug targets against malarial parasites. METHODS: In this study, a P. falciparum 5' to 3' DNA helicase (PfDH-B) was partially purified from the crude extract of chloroquine- and pyrimethamine-resistant P. falciparum strain K1, by ammonium sulfate precipitation and three chromatographic procedures. DNA helicase activity of partially purified PfDH-B was examined by measuring its ability to unwind 32P-labelled partial duplex DNA. The directionality of PfDH-B was determined, and substrate preference was tested by using various substrates. Inhibitory effects of DNA intercalators such as anthracycline antibiotics on PfDH-B unwinding activity and parasite growth were investigated. RESULTS: The native PfDH-B was partially purified with a specific activity of 4150 units/mg. The PfDH-B could unwind M13-17-mer, M13-31-mer with hanging tail at 3' or 5' end and a linear substrate with 3' end hanging tail but not blunt-ended duplex DNA, and did not need a fork-like substrate. Anthracyclines including aclarubicin, daunorubicin, doxorubicin, and nogalamycin inhibited the unwinding activity of PfDH-B with an IC50 value of 4.0, 7.5, 3.6, and 3.1 µM, respectively. Nogalamycin was the most effective inhibitor on PfDH-B unwinding activity and parasite growth (IC50 = 0.1 ± 0.002 µM). CONCLUSION: Partial purification and characterization of 5'-3' DNA helicase of P. falciparum was successfully performed. The partially purified PfDH-B does not need a fork-like substrate structure found in P. falciparum 3' to 5' DNA helicase (PfDH-A). Interestingly, nogalamycin was the most potent anthracycline inhibitor for PfDH-B helicase activity and parasite growth in culture. Further studies are needed to search for more potent but less cytotoxic inhibitors targeting P. falciparum DNA helicase in the future.

Faecal Proteomics and Functional Analysis of Equine Melanocytic Neoplasm in Grey Horses.

Equine melanocytic neoplasm (EMN) is a common disease in older grey horses. The purpose of this study was to examine the potential proteins throughout EMN stages from faecal proteomic outlining using functional analysis. Faecal samples were collected from the rectum of 25 grey horses divided into three groups; normal group without EMN (n = 10), mild EMN (n = 6) and severe EMN (n = 9). Based on the results, 5910 annotated proteins out of 8509 total proteins were assessed from proteomic profiling. We observed differentially expressed proteins (DEPs) between the normal group and the EMN group, and 109 significant proteins were obtained, of which 28 and 81 were involved in metabolic and non-metabolic functions, respectively. We found 10 proteins that play a key role in lipid metabolism, affecting the tumour microenvironment and, consequently, melanoma progression. Interestingly, FOSL1 (FOS like 1, AP-1 transcription factor subunit) was considered as a potential highly expressed protein in a mild EMN group involved in melanocytes cell and related melanoma. Diacylglycerol kinase (DGKB), TGc domain-containing protein (Tgm2), structural maintenance of chromosomes 4 (SMC4) and mastermind-like transcriptional coactivator 2 (MAML2) were related to lipid metabolism, facilitating melanoma development in the severe-EMN group. In conclusion, these potential proteins can be used as candidate biomarkers for the monitoring of early EMN, the development of EMN, further prevention and treatment.

Molecular characterization of Plasmodium falciparum DNA-3-methyladenine glycosylase.

BACKGROUND: The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf) MAG was characterized for its potential for development as an anti-malarial candidate. METHODS: Native PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized. RESULTS: PfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4-9, optimal salt concentration of 100-200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner. CONCLUSION: PfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the recombinant enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.

Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3.

BACKGROUND: Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets. METHODS: ATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function. RESULTS: Recombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its helicase activity is dependent on divalent cations (Cu2+, Mg2+, Ni+2 or Zn+2) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netropsin, known DNA helicase inhibitors. CONCLUSIONS: Purified recombinant PfRuvB3 contains both DNA helicase and ATPase activities. Differences in properties of RuvB between the malaria parasite obtained from the study and human host provide an avenue leading to the development of novel drugs targeting specifically the malaria form of RuvB family of DNA helicases.

Characterization of recombinant malarial RecQ DNA helicase.

RecQ DNA gene of multi-drug resistant Plasmodium falciparum K1 (PfRecQ1) was cloned, and the recombinant C-terminal-decahistidine-tagged PfRecQ1 was expressed in Escherichia coli. The purified enzyme could efficiently unwind partial duplex DNA substrate in a 3' to 5' direction. The malarial RecQ1 could not unwind substrates with both 5' and 3' overhangs, those with a 5' overhang, or blunt-ended DNA duplexes. Unwinding of DNA helicase activity was driven by the hydrolysis of ATP. The drug inhibitory effects of six compounds indicated that only doxorubicin and daunorubicin could inhibit the unwinding activity.