3D7 Strain Research Articles

Multicomponent Syntheses Enable the Discovery of Novel Quisinostat-Derived Chemotypes as Histone Deacetylase Inhibitors

In this study, we synthesized and evaluated novel histone deacetylase (HDAC) inhibitors derived from the clinical candidate quisinostat. A library of 16 compounds categorized in three novel chemotypes was rapidly generated using multicomponent reactions (MCRs), enabling efficient structure-activity relationship studies. First, the compounds were evaluated for their activity against the Plasmodium falciparum strains 3D7 and Dd2, the main malaria-causing parasite, identifying compound 18b of the type C series as the most potent. It demonstrated low nanomolar IC50 values (IC50 (3D7) = 0.023 μM; IC50 (Dd2) = 0.047 μM) and high parasite selectivity (SIMRC-5/Pf3D7 > 2174). HDAC inhibition assays confirmed substantial inhibition of the P. falciparum enzyme PfHDAC1 (IC50 = 0.037 μM) as well as of human HDAC1 (IC50 = 0.021 μM) and HDAC6 (IC50 = 0.25 μM). Docking studies suggested distinct binding modes of 18b in P. falciparum and human HDAC1. Additionally, the in vitro anticancer activity was evaluated in Cal27 (head-neck carcinoma), HepG2 (hepatocellular carcinoma), A2780 (ovarian carcinoma), and U87 (glioblastoma) cell lines. Compounds 9b, 9d, and 13f showed potent antiproliferative activity and caspase 3/7 activation, in contrast to 18b. Furthermore, these compounds caused hyperacetylation of histone H3 and α-tubulin, indicating robust cellular target engagement. Overall, in this work we have identified the HDAC inhibitor 18b with selective antiplasmodial and 9b, 9d, and 13f with selective anticancer activities, providing valuable hits for further drug development efforts aimed at creating derivatives with reduced cytotoxicity against non-cancer cells compared to quisinostat.

044. Unlocking The Potential: Comparing The Effects of Probiotics Alone and in Combination with Capecitabine Against Colorectal Cancer in Rats

Background: Colorectal cancer (CRC) is a leading cause of cancer deaths globally. While capecitabine is an effective chemotherapy for CRC, its use is limited by side effects and resistance. This has sparked interest in combining capecitabine with natural therapies, such as probiotics from dadiah, a traditional West Sumatra fermented buffalo milk. Lactococcus lactis strains D4 in dadiah produce nisin and butyrate, both known for their anti-cancer properties. To compare the effects of Lactococcus lactis D4, capecitabine, and their combination on CRC progression in a rat model induced with 12-Dimethylhydrazine (DMH). Methods: A total of 25 male Sprague-Dawley rats were divided into five groups: negative control, CRC-induced positive control, probiotics, capecitabine, and combination. After CRC induction, the rats received treatment for 14 days. Nuclear factor kappa B (NFKB) (inflammation), tumor protein 53 (P53) (apoptosis), and Cyclin D (proliferation) expressions were measured using immunohistochemistry. Results: The combination group showed the greatest reduction in NFKB expression compared probiotics-only and capecitabine-only (12.99 ± 4.91 vs 24.15 ± 5.50 vs 16.07 ± 3.79) (p < 0.001); P53 expression increased in the probiotic and capecitabine groups but decreased in the combination group (3.60 ± 3.50 vs 4.40 ± 4.27 vs 3.00 ± 2.00); and Cyclin D was lower in the combination group compared both the probiotic and capecitabine groups (11.68 ± 4.78 vs 21.98 ± 6.05 vs 12.16 ± 4.65). Conclusion: Lactococcus lactis D4 combined with capecitabine synergistically reduces inflammation and cell proliferation in CRC, indicating potential as an adjunct therapy to enhance chemotherapy efficacy.

Ex-vivo evaluation of Plasmodium falciparum-induced cytokine expression in chronic hepatitis B virus-infected and uninfected individuals

Background Chronic Hepatitis B virus (CHB) infection remains a significant public health problem. Exhaustion of T cells usually contribute to the progression of HBV infection to the chronic state, which can impact the induction of immune responses to other pathogens like Plasmodium falciparum. This, in turn, may affect the effectiveness of malaria vaccines when deployed. This case-control longitudinal study sought to determine how early this T cell exhaustion state establishes, since most of the available data relates to CHB cases that have persisted for much longer times. Methods This longitudinal case-control study compared the expression of 13 cytokines between cases and controls at four time points over one year. These cytokines were induced using whole blood ex-vivo stimulation with three Plasmodium falciparum (3D7 strain) antigens. Results Hepatitis B virus-negative and CHB individuals had comparable levels of TNF-α, IL-1β, IL-6 and IL-10. Interleukin 6 which is important for the elimination of HBV was produced in very high amounts by the two groups. There was no significant difference between the groups in their ability to produce pro-inflammatory cytokines in response to malaria antigens. Conclusion Cytokine responses to the vaccine candidates from both groups were similar, indicating no impairment to the effective immune responses to malaria vaccines and probably parasites in this category of early CHB infected individuals.

Exploring the Antimalarial Potential of Entandrophragma Utile and Melochia Umbellata Extracts.

Resistance remains the fundamental problem with antiplasmodial treatments. The study investigated the antiplasmodial, cytotoxic, and antioxidant properties of two Cameroonian medicinal plants, Entandrophragma utile and Melochia umbellata. Antimalarial activity against Plasmodium falciparum strains 3D7 and Dd2 was assessed using the SYBR Green I assay. Cytotoxicity was assessed against Raw and Vero cells using the resazurin assay, and against red blood cells using a hemoglobin release quantification assay. Antioxidant potential was determined using DPPH (2, 2-diphenyl-1-picrylhydrazine), ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), and FRAP (ferric-reducing antioxidant power) assays. The most bioactive extract was further analyzed using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) to determine its phytochemical composition. Extracts from E. utile exhibited moderate anti-malarial activity against Plasmodium falciparum (IC50 =32.81-100 μg/mL), while M. umbellata leaf extract (MULAE) showed strong activity (IC50 =11.62 and 11.91 μg/mL). All extracts demonstrated antioxidant activity (9.22 to 135.8 μg/mL), were selective for Raw and Vero cells, and were not toxic to red blood cells. UHPLC-MS analysis annotated potential pyrimidones, phenolic compounds, and terpenoids in MULAE. MULAE exhibited better antiplasmodial activity, suggesting the presence of unique bioactive compounds. Further research, including in vivo investigations, is needed to develop safe and effective antiplasmodial therapies.

Effect of Sprouting, Fermentation and Cooking on Antioxidant Content and Total Antioxidant Activity in Quinoa and Amaranth.

The study of different processing techniques, such as sprouting, cooking and fermentation, can help to develop new products for human health. In this work, raw, cooked and fermented seeds and germinated seeds of Chenopodium quinoa Willd. var. Tunkahuan and Amaranthus caudatus L. var. Alegrìa were compared for the content of antioxidant molecules, total antioxidant capacity and mineral elements. Fermentation was induced spontaneously, with the yeast Saccharomyces cerevisiae, with the bacterium Lactobacillus plantarum and with both microorganisms, for 24 and 48 h. The increase in antioxidant molecules and antioxidant activity was induced by germination, by 24 h of spontaneous fermentation (polyphenols and flavonoids) and by 24 h of L. plantarum fermentation (total antioxidant activity) for both species. Germinated seeds of the two plants showed higher values in respect to seeds of macroelements and microelements. No genotoxic but rather protective effects were determined for seed and germinated seed extracts using the D7 strain of S. cerevisiae, a good tool for the evaluation of protection from oxidative damage induced by radical oxygen species (ROS) in cells and tissues. Therefore, the two varieties could be very suitable for their use in human diet and in supplements, especially as germinated seeds or as fermented foods.

Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon.

Resistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing. P lasmodium falciparum S treamlined M ultiplex A ntimalarial R esistance and R elatedness T esting ( Pf -SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach. We evaluated Pf -SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf -SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf -SMARRT's hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed. Overall, Pf -SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible. Malaria remains a critical global public health problem. Antimalarial drug resistance has repeatedly undermined control and the emergence of artemisinin partial resistance in Africa is the latest major challenge. Malaria molecular surveillance (MMS) has emerged as a powerful tool to monitor molecular markers of resistance and changes in the parasite population. Streamlined methods are needed that can be readily adopted in endemic countries. We developed P lasmodium falciparum S treamlined M ultiplex A ntimalarial R esistance and R elatedness T esting ( Pf -SMARRT), a multiplex amplicon deep sequencing approach that uses easily accessible products without proprietary steps and can be sequenced on any Illumina sequencer. We validated this tool using controls, including mocked dried blood spots, and then implemented it to evaluate resistance and parasite relatedness among 100 samples from Cameroon. The assay was able to reliably assess the within-sample allele frequency of antimalarial resistance markers and discriminate strains within and between individuals. We also evaluated a more cost-effective surveillance approach for antimalarial resistance polymorphisms using pooled samples. While within-pool frequencies of mutations were accurate in pools with higher numbers of samples, this resulted in the loss of the ability to detect variants uncommon in the pool. Overall Pf- SMARRT provides a new protocol for conducting MMS that is easily implementable in Africa.

Exploring the Antibacterial and Antiparasitic Activity of Phenylaminonaphthoquinones-Green Synthesis, Biological Evaluation and Computational Study.

Organic compounds with antibacterial and antiparasitic properties are gaining significance for biomedical applications. This study focuses on the solvent-free synthesis (green synthesis) of 1,4-naphthoquinone or 2,3-dichloro-1,4-naphthoquinone with different phenylamines using silica gel as an acid solid support. The study also includes in silico PASS predictions and the discovery of antibacterial and antiparasitic properties of phenylaminonaphthoquinone derivatives 1-12, which can be further applied in drug discovery and development. These activities were discussed in terms of molecular descriptors such as hydrophobicity, molar refractivity, and half-wave potentials. The in vitro antimicrobial potential of the synthesized compounds 1-12 was evaluated against a panel of six bacterial strains (three Gram-positive: Staphylococcus aureus, Proteus mirabilis, and Enterococcus faecalis; and three Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae). Six compounds (1, 3, 5, 7, 10, and 11) showed better activity toward S. aureus with MIC values between 3.2 and 5.7 μg/mL compared to cefazolin (MIC = 4.2 μg/mL) and cefotaxime (MIC = 8.9 μg/mL), two cephalosporin antibiotics. Regarding in vitro antiplasmodial activity, compounds 1 and 3 were the most active against the Plasmodium falciparum strain 3D7 (chloroquine-sensitive), displaying IC50 values of 0.16 and 0.0049 μg/mL, respectively, compared to chloroquine (0.33 μg/mL). In strain FCR-3 (chloroquine-resistant), most of the compounds showed good activity, with compounds 3 (0.12 μg/mL) and 11 (0.55 μg/mL) being particularly noteworthy. Additionally, docking studies were used to better rationalize the action and prediction of the binding modes of these compounds. Finally, absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions were performed.

Structure and Biosynthesis of Perochalasins A-C, Open-Chain Merocytochalasans Produced by the Marine-Derived Fungus Peroneutypa sp. M16.

Novel open-chain merocytochalasans, perochalasins A-C (1-3), containing an unusual N-O six-membered heterocyclic moiety, were isolated from cultures of the marine-derived Peroneutypa sp. M16 fungus, along with cytochalasin Z27 (4), cytochalasin Z28 (5), [12]-cytochalasin (6), and phenochalasin B (7). The structures of compounds 1-3 were established by analysis of the spectroscopic data. Full genome sequencing of Peroneutypa sp. M16 enabled the identification of a cytochalasan biosynthetic gene cluster and a proposal for the biosynthetic assembly of perochalasins. The proposal is supported by the nonenzymatic conversion of phenochalasin B (7) into 1-3, based on isotope-labeled hydroxylamine (15NH2OH and ND2OD) feeding studies in vivo and in vitro. In contrast to other merocytochalasans, these are the first cytochalasans confirmed to arise via nucleophilic addition and at a distinct location from the reactive macrocycle olefin, potentially expanding further the range of merocytochalasans to be discovered or engineered. Cytochalasin Z27 (4) exhibited antiplasmodial activities in the low micromolar range against the chloroquine-sensitive Plasmodium falciparum 3D7 strain as well as against resistant strains of the parasite (Dd2, TM90C6B, and 3D7r_MMV848).

Antimalarial mechanism of action of the natural product 9-methoxystrobilurin G.

The natural product 9-methoxystrobilurin G (9MG) from Favolaschia spp basidiomycetes is a potent and selective antimalarial. The mechanism of action of 9MG is unknown. We induced 9MG resistance in Plasmodium falciparum 3D7 and Dd2 strains and identified mutations associated with resistance by genome sequencing. All 9MG-resistant clones possessed missense mutations in the cytochrome b (CYTB) gene, a key component of mitochondrial complex III. The mutations map to the quinol oxidation site of CYTB, which is also the target of antimalarials such as atovaquone. In a complementary approach to identify protein targets of 9MG, a photoactivatable derivative of 9MG was synthesized and applied in chemoproteomic-based target profiling. Three components of mitochondrial complex III (QCR7, QCR9, and COX15) were specifically enriched consistent with 9MG targeting CYTB and complex III function in P. falciparum. Inhibition of complex III activity by 9MG was confirmed by ubiquinone cytochrome c reductase assay using P. falciparum extract. The findings from this study may be useful for developing novel antimalarials targeting CYTB.

Conformational variability in the D2 loop of Plasmodium Apical Membrane antigen 1

Apical Membrane Antigen 1 (AMA1) plays a vital role in the invasion of the host erythrocyte by the malaria parasite, Plasmodium. It is thus an important target for vaccine and anti-malaria therapeutic strategies that block the invasion process. AMA1, present on the surface of the parasite, interacts with RON2, a component of the parasite’s rhoptry neck (RON) protein complex, which is transferred to the erythrocyte membrane during invasion. The D2 loop of AMA1 plays an essential role in invasion as it partially covers the RON2-binding site and must therefore be displaced for invasion to proceed. Several structural studies have shown that the D2 loop is very mobile, a property that is probably important for the function of AMA1. Here we present three crystal structures of AMA1 from P. falciparum (strains 3D7 and FVO) and P. vivax (strain Sal1), in which the D2 loop could be largely traced in the electron density maps. The D2 loop of PfAMA1-FVO and PvAMA1 (as a complex with a monoclonal antibody Fab) has a conformation previously noted in the P. knowlesi AMA1 structure. The D2 loop of PfAMA1-3D7, however, reveals a novel conformation. We analyse the conformational variability of the D2 loop in these structures, together with those previously reported. Three different conformations can be distinguished, all of which are highly helical and show some similarity in their secondary structure organisation. We discuss the significance of these observations in the light of the flexible nature of the D2 loop and its role in AMA1 function.

Design and synthesis of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids as effective antimalarial compounds

In this work, a series of nineteen novel pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids were synthesized as potent antimalarial agents by covalently linking the scaffolds of 4-aminoquinoline and pyrano[2,3-c]pyrazoles via an ethyl linker and characterized using Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Molecular docking was used to test each hybrid's and standard chloroquine's ability to bind to Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH), an important enzyme in the parasite's glycolytic pathway. The hybrid compounds had a stronger binding affinity than the standard chloroquine (CQ). The schizontical antimalarial test of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrid compound shows that all nineteen hybrid compounds were potent with the IC50 values ranging from 0.0151 to 0.301 μM against the CQ-sensitive 3D7 P. falciparum strain, and were active against the CQ-resistant K1 P. falciparum strain with the IC50 values ranging from 0.01895 to 2.746 μM. All the tested hybrid compounds were less potent than the standard drug chloroquine dipaspate (CQDP) against the CQ-sensitive 3D7 strain. In contrast, nine of the nineteen hybrids (16d, 16g, 16h, 16i, 16l, 16n, 16o, 16r, and 16s) displayed superior antimalarial activity than the CQDP against the CQ-resistant K1 P. falciparum strain. Among all the tested hybrids, 16c against the 3D7 strain and 16h against the K1 strain were the most promising antimalarial agents with 0.0151 and 0.01895 μM of IC50 values, respectively. In addition, the compounds were selective, showing moderate to low cytotoxic activity against a human normal liver WRL68 cell line. The synthesis of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids introduces new chemical entities that have the potential to exhibit potent antimalarial activity. It could address the ongoing challenge of drug resistance in malaria treatment.

Bacterial Volatile Organic Compounds as a Strategy to Increase Drought Tolerance in Maize (Zea mays L.): Influence on Plant Biochemistry.

Maize is highly susceptible to drought, which affects growth and yield. This study investigated how bacterial volatile organic compounds (BVOCs) affect maize drought tolerance. Drought reduced shoot size but increased root length, an adaptation for accessing deeper soil moisture. BVOCs from strain D12 significantly increased root length and shoot growth under drought conditions. Drought also altered root biochemistry, decreasing enzyme activity, and increased osmolyte levels. BVOCs from strains F11 and FS4-14 further increased osmolyte levels but did not protect membranes from oxidative damage, while BVOCs from strains D12 and D7 strains reduced osmolyte levels and cell damage. In shoots, drought increased the levels of osmolytes and oxidative stress markers. BVOCs from FS4-14 had minimal effects on shoot biochemistry. BVOCs from D12 and F11 partially restored metabolic activity but did not reduce cell damage. BVOCs from D7 reduced metabolic activity and cell damage. These results suggest that BVOCs can modulate the biochemical response of maize to drought, with some strains evidencing the potential to enhance drought tolerance.

One-pot three-component synthesis of 2,4-dimethoxy-5,8,9,10-tetrahydropyrimido[4,5-b]quinolinone derivatives along with in silico and in vitro investigation of their antimalarial activity

Pyrimidoquinolinone-derived compounds have exhibited a broad spectrum of biological activities and have been acknowledged as a potential antimalarial scaffold through multiple conventional strategies. In the present study, a novel series of 2,4-dimethoxy-5,8,9,10-tetrahydropyrimido[4,5-b]quinolinones 4(a-m) were synthesized via a one-pot three-component reaction of cyclohexane-1,3‑dione, 4-amino-2,6-dimethoxypyrimidine and various aldehyde with l-proline as the catalyst and acetic acid as the solvent under reflux condition. In vitro antimalarial activity toward P. falciparum 3D7 strain cultures were investigated. Compounds 4b and 4m demonstrated significant efficacy against strains of P. falciparum, with IC50 values of 0.63 and 0.65 μg/mL, respectively. The results were also supported by molecular docking and ADME assays.

Bactofencin YH, a novel bacteriocin with high inhibitory activity against clinical Streptococcus species.

Strain Lactiplantibacillus plantarum D1 with bacteriocin producing ability was found in the intestine of Gambusia affinis. The bacteriocin was found to have high inhibitory activity against multiple Streptococcus species and several other Gram-positive and Gram-negative bacteria. Bacteriocin was purified from culture supernatant by ion-exchange chromatography, Sep-Pak C18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectral analysis determined that purified bacteriocin has a molecular mass of 2,731 Da. A partial N-terminal sequence KRKKHKXQIYNNGM was obtained from the Edman analysis. The N-terminal sequence was employed to search against a translation of the draft genome of strain D1. The translated full amino acid sequence of the mature peptide is as follows: NH2- KRKKHKCQIYNNGMPTGQYRWC, which has a molecular weight of 2738 Da. A BLAST search revealed that this bacteriocin was most similar to bactofencin A but differed from it with three amino acid residues. No identical peptide or protein has been previously reported, and this peptide, termed bactofencin YH, was therefore considered to be a new bacteriocin produced by Lactiplantibacillus plantarum D1.

Noscapine shows antimalarial activity against Plasmodium falciparum 3D7, its clinical isolate Pf140/SS, and Plasmodium berghei ANKA

Objective: To evaluate the antimalarial activity of noscapine against Plasmodium falciparum 3D7 strain (Pf3D7), its clinical isolate (Pf140/SS), and Plasmodium berghei ANKA (PbA). Methods: Using ring-stage survival assay, phenotypic assessments, and SYBR-green-based fluorescence assay, the antimalarial activities of noscapine were assessed compared with dihydroartemisinin (DHA) in in vivo and in vitro studies. In addition, hemolysis and cytotoxicity tests were carried out to evaluate its safety. RT-PCR assay was also conducted to determine the effect of noscapine on papain-like cysteine protease Plasmodium falciparum falcipain-2 (PfFP-2). Results: The antimalarial efficacy of noscapine against Pf3D7 and Pf140/SS was comparable to DHA, with IC50 values of (7.68±0.88) and (5.57±0.74) nM/mL, respectively, and >95% inhibition of PbA infected rats. Noscapine also showed a safe profile, as evidenced by low hemolysis and cytotoxicity even at high concentrations. Moreover, PfFP-2 expression was significantly inhibited in both noscapine-treated Pf3D7 and Pf140/SS (P<0.01). Conclusions: Noscapine has antimalarial properties comparable to standard antimalarial DHA with better safety profiles, which may be further explored as a therapeutic candidate for the treatment of malaria.

Expression patterns of immune checkpoint proteins and Plasmodium falciparum-induced cytokines in chronic hepatitis B virus-infected and uninfected individuals: A cross-sectional study.

Chronic hepatitis B virus (CHB) infection remains a major public health problem. The American Association for the Study of Liver Diseases (AASLD) 2018 Hepatitis B Guidelines provide that CHB individuals not requiring antiviral therapy yet are monitored to determine the need for antiviral therapy in the future; however, these tests do not include measurement of cytokines and immune cell characterization. This case-control study compared the cytokine and immune checkpoint protein expression profiles between CHB individuals not yet on antiviral treatment and hepatitis B virus (HBV)-negative individuals. CD4 and CD8 T cells from CHB and HBV-negative individuals were characterized for immune checkpoint proteins programmed cell death-1 (PD1), T cell Immunoglobulin domain and mucin domain-containing protein 3 (TIM-3), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (CD152), and a memory marker CXCR3 (CD183) using flow cytometry. Malaria-induced cytokine expression levels were determined by stimulating their blood cells with Plasmodium falciparum 3D7 strain antigens (CSP, AMA-1, and TRAP) in whole blood assays, and cytokine levels were measured using a 13-plex Luminex kit. HBV-negative and CHB individuals had comparable levels of CD4+ and CD8+ T cells. However, a proportion of the CD4+ and CD8+ populations from both groups, which were CXCR3+, expressed PD-1 and CD152. The ability to produce cytokines in response to malaria antigen stimulation was not significantly different between the groups. These findings support excluding CHB individuals from antiviral therapy at this stage of infection. However, CHB individuals require regular monitoring to determine the need for later antiviral treatment.

In vitro, in vivo and in silico antiplasmodial profiling of the aqueous extract of Hibiscus asper HOOK F. Leaf (Malvaceae)

Ethnopharmacological relevancePlasmodium resistance to antimalarial drugs raises the urgent need to seek for alternative treatments. Aqueous extract of Hibiscus asper leaves is currently used in malaria management but remains less documented.Aim of the study: The study aims to evaluate antimalarial effects of the aqueous extract of Hibiscus asper. UHPLC/MS, was used to identify some likely compounds present in the plant that were thereafter docked to some malaria parasite proteins. Study designIn vitro anti-plasmodium and antioxidant, UHPLC/Ms analysis, in vivo antimalarial of the plant extract, and in silico molecular docking prediction of some identified compounds were performed to investigate the pharmacological effects of H. asper. Material and methodsThe in vitro antiplasmodial activity of the extract was carried out on Plasmodium falciparum strains using SYBR-green dye; then, the curative antimalarial activity was conducted on Plasmodium berghei NK65-infected male Wistar rats. The UHPLC/MS analysis was used to identify plant compounds, followed by interactions (docking affinity) between some compounds and parasitic enzymes such as P. falciparum purine nucleoside phosphorylase (2BSX) and 6-phosphogluconate dehydrogenase (6FQY) to explore potential mechanisms of action at the molecular level. ResultsNo hemolysis effect of the extract was observed at concentrations up to 100 mg/mL. In vitro test of the aqueous leaves extract of H. asper showed inhibitory activity against P. falciparum Dd2 and 3D7 strains with IC50 values of 19.75 and 21.97 μg/mL, respectively. The curative antimalarial test of the H. asper extract in infected rats exhibited significant inhibition of the parasite growth (p < 0.001) with inhibition percentage of 95.11%, 97.68% and 95.59% at all the doses (50, 100 and 200 mg/kg) respectively. The extract corrected major physiological alterations such as liver and kidney impairments, oxidative stress and architectural disorganization in liver, spleen and kidneys tissues. The UHPLC/MS analysis identified 7 compounds, namely chlorogenic acid, azulene, quercetin, rhodine, 1-ethyl-2,4-dimethyl benzene and phthalan. Out of seven compounds identified in the extract quercetin and phthalan showed higher in silico inhibitory activity against P. falciparum purine nucleoside phosphorylase and Plasmodium falciparum 6-phosphosgluconate dehydrogenase parasite enzymes. ConclusionThese findings indicate that H. asper could be a promising complementary medicine to manage malaria. Meanwhile, the affinity of annoted compounds with these enzymes should be further confirmed.

Sclerotinia rot of Zephyranthes candida caused by Sclerotinia sclerotiorum and Sclerotinia minor.

Sclerotinia rot is a serious disease that occurs on Zephyranthes candida. A thorough understanding of the pathogenic fungal species and understanding the biological characteristics are important for controlling sclerotinia. Fungal strains were isolated from the diseased leaves of Z. candida through tissue isolation. Koch's hypothesis screened pathogenic strains by pathogenicity of healthy leaves, including re-isolation and identification. A multigene phylogenetic tree was constructed by extracting genomic DNA from pathogenic strains and measuring the nucleotide sequences at four sites, including the internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and heat shock protein 60 (HSP60). Morphological characteristics of the fungal structures were evaluated through microscopic analysis. The growth of pathogens was observed and recorded under different pH, different temperatures, different carbon sources and different nitrogen sources to clarify their biological characteristics. Representative strains D7, D13, X4, and X15 infected Z. candida and caused sclerotinia rot. At the beginning of the culture, white flocculent fungal hyphae appeared on the potato dextrose agar (PDA) medium, and black spherical to irregular-shaped sclerotia appeared at the edge of the colony after 7 days. The diameter of the sclerotia was 2.4-8.6 mm and 0.4-0.9 mm, respectively. One sclerotium was able to germinate from 1 to 5 apothecia. Ascus were cylindrical or spindle-shaped, with a size of 110.0-120.0 × 9.2-11.6 μm. One ascus contained eight colorless, oval ascospores, with a size of 8.4-12.0 × 4.5-5.5 μm. Based on the phylogenetic tree constructed with the gene sequences for ITS, G3PDH, HSP60, and RPB2, D7 and D13 shared 99% homology with sclerotinia sclerotiorum, whereas X4 and X15 shared 99% homology with sclerotinia minor. S. sclerotiorum growth was more suitable when the culture temperature was 15°C-25°C, pH 5.0, carbon source was maltose and nitrogen source was yeast powder. S. minor growth was more suitable when the culture temperature was 15°C, pH 5.0, the carbon source was glucose, and the nitrogen source was yeast powder. The results identified the pathogens as S. sclerotiorum and S. minor. To the best of our knowledge, this is the first report of S. sclerotiorum and S. minor causing sclerotinia rot on Z. candida. We herein aimed to identify the causal agent of sclerotinia rot of Z. candida in China based on morphological characteristics, molecular identification, and pathogenicity tests. Performed the experiments on the biological characteristics, to understand the law of disease occurrence. We also evaluated methods for the effective control of this disease. Our findings provide support for further studies on the pathogenesis mechanism of sclerotinia rot.

2D/3D-QSAR Model Development Based on a Quinoline Pharmacophoric Core for the Inhibition of Plasmodium falciparum: An In Silico Approach with Experimental Validation.

Malaria is an infectious disease caused by Plasmodium spp. parasites, with widespread drug resistance to most antimalarial drugs. We report the development of two 3D-QSAR models based on comparative molecular field analysis (CoMFA), comparative molecular similarity index analysis (CoMSIA), and a 2D-QSAR model, using a database of 349 compounds with activity against the P. falciparum 3D7 strain. The models were validated internally and externally, complying with all metrics (q2 > 0.5, r2test > 0.6, r2m > 0.5, etc.). The final models have shown the following statistical values: r2test CoMFA = 0.878, r2test CoMSIA = 0.876, and r2test 2D-QSAR = 0.845. The models were experimentally tested through the synthesis and biological evaluation of ten quinoline derivatives against P. falciparum 3D7. The CoMSIA and 2D-QSAR models outperformed CoMFA in terms of better predictive capacity (MAE = 0.7006, 0.4849, and 1.2803, respectively). The physicochemical and pharmacokinetic properties of three selected quinoline derivatives were similar to chloroquine. Finally, the compounds showed low cytotoxicity (IC50 > 100 µM) on human HepG2 cells. These results suggest that the QSAR models accurately predict the toxicological profile, correlating well with experimental in vivo data.

Molecular Surveillance of Artemisinin Combination Therapy Resistance Markers in Kelch13 Gene of Plasmodium falciparum Isolates Collected From Yobe State Nigeria

Background: Despite the discovery of artemisinin and artemisinin combination therapies (ACTs), resistance emerged due to single-nucleotide polymorphisms (SNPs) at the Kelch13 propeller domain of Plasmodium falciparum; the predominant parasite causing malaria. This research aimed at surveying the gene responsible for reduced parasite clearance of the first-line antimalarial ACT in Yobe State, Nigeria. Methods: This study analyzed the blood samples of 300 patients (18-50 years) from 3 different hospitals in Yobe State for malaria using a rapid diagnostic test (malarial strip test). The Giemsa microscopy protocol was performed on the samples that tested positive in order to increase specificity for high parasitemia. Positive samples from microscopy were confirmed by COXIII gene polymerase chain reaction amplification. The fragments of the P. falciparum kelch13 gene encompassing the propeller domains were amplified and sequenced to detect polymorphism. Results: The result of multiple sequence alignment of 16 successfully sequenced samples performed alongside the reference 3D7 strain revealed nine novel mutations that have not been reported elsewhere. Two were synonymous (S485S and A675A), and seven were non-synonymous (E461V, S485R, D648H, E668Q, S679L, H697Y, and W706M). The genetic diversity parameters obtained in this study indicated non-severe mutations with haplotype diversity [Hd=0.662, D (Tadjima)=-2.16692 and D* (Fu and Li) -3.08435]. Seven different haplotypes were discovered, and the result of the haplotype network demonstrated Hap_1 to be the predominant haplotype in the population. Conclusion: The absence of the WHO-validated markers may suggest that ACTs are still effective in Yobe.