Knockdown of Anopheles dirus far upstream element-binding protein gene lower oocyst numbers of Plasmodium vivax.

The modulation of gene expression levels of Anopheles dirus on Plasmodium vivax infection at the ookinete and oocyst stages was previously reported. In the present study, several upregulated An. dirus genes were selected based on their high expression levels and subcellular locations to examine their roles in P. vivax infection. Five An. dirus genes-carboxylesterase, cuticular protein RR-2 family, far upstream element-binding protein, kraken, and peptidase212-were knocked down by dsRNA feeding using dsRNA-lacZ as a control. The dsRNA-fed mosquitoes were later challenged by P. vivax-infected blood, and the oocyst numbers were determined. The expression of these five genes was examined in many organs of both male and female mosquitoes. The results showed that the decreased expression level of the far upstream element-binding protein gene could lower the oocyst numbers, whereas the others showed no effect on P. vivax infection. The expression levels of these genes in ovaries were found, and in many organs, they were similar between male and female mosquitoes. The reduction of these five gene expressions did not affect the lifespan of the mosquitoes. In addition, the malaria box compound, MMV000634, demonstrated the lowest binding energy to the far upstream element-binding protein using virtual screening. This protein might be a target to block malaria transmission.

Malaria Box Compounds against Anopheles gambiae (Diptera: Culicidae) Carboxypeptidase B Activity to Block Malaria Transmission.

Carboxypeptidase B (CPB) plays an important role in blood digestion in mosquitos, aiding the release of free amino acids. Anopheles CPB is a target to block malaria transmission because it facilitates Plasmodium invasion of the mosquito midgut. Our study aimed to discover inhibitors of Anopheles CPB to prevent Plasmodium development in the mosquito. The Anopheles gambiae cpb (Agcpb) gene without a signal sequence was cloned into the pET28b expression vector. The recombinant AgCPB protein was expressed in E. coli BL21(DE3) within inclusion bodies after induction with 0.5 mM isopropyl ß-D-1-thiogalactopyranoside at 37°C for 4 h. The protein pellet was dissolved in 6 M urea, purified by affinity chromatography, and dialyzed in reaction buffer. The refolded recombinant AgCPB could digest the hippuryl-arginine substrate similarly to that of the commercial porcine pancreas CPB. The 20 top-scoring malaria box compounds from the virtual-screening results were then chosen for an in vitro inhibition assay against AgCPB. Four of the 20 malaria box compounds could inhibit AgCPB activity. The compound MMV007591 was the most potent inhibitor with an IC50 at 0.066 µM. The results indicate that these candidate compounds may be utilized in drug development against mosquito CPB activity to curb malaria transmission.

Osteopontin induces osteogenic differentiation by human periodontal ligament cells via calcium binding domain-ALK-1 interaction.

BACKGROUND: Recently we have generated recombinant human osteopontin (rhOPN) using a plant platform (Nicotiana benthamiana) and demonstrated, when coated on culture plates, its osteogenic induction capacity of human periodontal ligament (PDL) cells. The aim of this study is to elucidate the molecular mechanism underlying the rhOPN-induced osteogenic differentiation of human PDL cells. METHODS: Full length rhOPN (FL-OPN) and three constructs of OPN containing integrin binding domain (N142), calcium binding domain (C122) and mutated calcium-binding domain (C122δ) were generated from N. benthamiana. Human PDL cells were isolated from extracted third molars and cultured on FL-OPN, N142, C122, or C122δ-coated surfaces. Real-time PCR and Western blot analyses were used to determine mRNA and protein expression. In vitro calcification was determined by Alizarin red staining. A chemical inhibitor and RNAi silencing were used to elucidate signaling pathways. In silico analyses were performed to predict the protein-protein interaction. In vivo analysis was performed using a rat calvaria defect model. RESULTS: Human PDL cells seeded on FL-OPN and C122-coated surfaces significantly increased both mRNA and protein expression of osterix (OSX) and enhanced in vitro calcification. Soluble FL-OPN as well as a surface coated with N142 did not affect OSX expression. Inhibition of activin receptor-like kinase (ALK-1) abolished the induction of osterix expression. In silico analysis suggested a possible interaction between the calcium binding domain (CaBD) of OPN and ALK-1 receptor. C122, but not C122δ coated surfaces, induced the expression of p-Smad-1 and this induction was inhibited by an ALK-1 inhibitor and RNAi against ALK-1. In vivo data showed that 3D porous scaffold containing C-122 enhanced new bone formation as compared to scaffold alone. CONCLUSION: The results suggest that next to full length OPN, the CaBD of OPN, if coated to a surface, induces osteogenic differentiation via interaction with ALK-1 receptor.

Anopheles dirus yellow-g mediates Plasmodium vivax infection.

OBJECTIVE: Our previous transcriptome analysis of Anopheles dirus revealed upregulation of the An. dirus yellow-g gene upon ingestion of Plasmodium vivax-infected blood. This gene belongs to the yellow gene family, but its role regarding P. vivax infection is not known and remains to be validated. The aim of this study was to investigate the role of the An. dirus yellow-g gene in P. vivax infection. METHODS: The qRT-PCR was used to detect the expression of the yellow-g gene in many organs of both male and female mosquitos. The yellow-g gene silencing was performed by dsRNA membrane feeding to An. dirus. These mosquitoes were later challenged by P. vivax-infected blood. The oocyst numbers were determined. RESULTS: The yellow-g transcript was detected in several organs of both male and female An. dirus mosquitoes. Successful knockdown of yellow-g was achieved and resulted in reduced P. vivax infection in the mosquitoes. The decrease in yellow-g expression had no effect on the life span of the mosquitoes. CONCLUSIONS: These results support the yellow-g gene as having an important function in Plasmodium development in Anopheles mosquitoes.

Transcriptome analysis of Anopheles dirus and Plasmodium vivax at ookinete and oocyst stages.

Malaria is transmitted by Plasmodium parasites through the bite of female Anopheles mosquitoes. One of the most important mosquito vectors in the Greater Mekong Subregion is Anopheles dirus. This study reports RNA sequencing (RNA-Seq) transcriptome analysis of An. dirus at 18 hours and 7 days after a P. vivax-infected blood meal, which represent infection at the ookinete and oocyst parasite developmental stages, respectively. Following infection, 582 An. dirus transcripts were modulated. The 2,408 P. vivax transcripts could be classified into ookinete-specific, two-stage, and oocyst-specific groups. Results were validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analysis of the vector and parasite revealed several biological pathways for both, providing a better understanding of Anopheles-Plasmodium interactions at the ookinete and oocyst stages.

Recombinant human osteopontin expressed in Nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells.

Tissue engineering aims to utilise biologic mediators to facilitate tissue regeneration. Several recombinant proteins have potential to mediate induction of bone production, however, the high production cost of mammalian cell expression impedes patient access to such treatments. The aim of this study is to produce recombinant human osteopontin (hOPN) in plants for inducing dental bone regeneration. The expression host was Nicotiana benthamiana using a geminiviral vector for transient expression. OPN expression was confirmed by Western blot and ELISA, and OPN was purified using Ni affinity chromatography. Structural analysis indicated that plant-produced hOPN had a structure similar to commercial HEK cell-produced hOPN. Biological function of the plant-produced hOPN was also examined. Human periodontal ligament stem cells were seeded on an OPN-coated surface. The results indicated that cells could grow normally on plant-produced hOPN as compared to commercial HEK cell-produced hOPN determined by MTT assay. Interestingly, increased expression of osteogenic differentiation-related genes, including OSX, DMP1, and Wnt3a, was observed by realtime PCR. These results show the potential of plant-produced OPN to induce osteogenic differentiation of stem cells from periodontal ligament in vitro, and suggest a therapeutic strategy for bone regeneration in the future.

Biofilm inhibitors targeting the outer membrane protein A of Pasteurella multocida in swine.

Pasteurella multocida (Pm) is the causative agent of atrophic rhinitis in swine. This study aimed to discover biofilm inhibitors against swine Pm to counteract antibiotic resistance and decrease virulence. The virulence factor outer membrane protein A (OmpA) was targeted. A library of drugs approved by the Food and Drug Administration (FDA) was used to perform virtual screening against PmOmpA. The top-scoring compounds had no effect on the growth of Pm serotype A or D. Mycophenolate mofetil showed the highest efficacy in inhibiting biofilm formation by Pm serotype A, with an IC50 of 7.3 nM. For Pm serotype D, indocyanine green showed the highest effect at an IC50 of 11.7 nM. Nevertheless, these compounds had no effect on an established biofilm of Pm. This study offers an alternative way to prevent biofilm formation by Pm that could also be applied to other pathogens.

Antiviral Compounds Against Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe diarrhea disease in swine that is caused by porcine epidemic diarrhea virus (PEDV). Nucleocapsid (N) protein is the RNA-binding protein of PEDV, which plays an important role for virus life cycle. The aim of this research was to screen and characterize the compounds that could inhibit the activity of PEDV N protein. The gene encoding PEDV N protein obtained from PEDV Thai isolate was cloned and expressed in E. coli. Its amino acid sequence was employed to generate the three dimensional structure by homology modeling. There were 1,286 compounds of FDA-approved drug database that could virtually bind to the RNA-binding region of N protein. Three compounds, trichlormethiazide, D-(+) biotin, and glutathione successfully bound to the N protein, in vitro, with the IC50 at 8.754 mg/mL, 0.925 mg/mL, and 2.722 mg/mL. Antiviral activity in PEDV-infected Vero cells demonstrated that the effective concentration of trichlormethiazide, D-(+) biotin, and glutathione in inhibiting PEDV replication were 0.094, 0.094 and 1.5 mg/mL. This study demonstrated a strategy applied for discovery of antiviral agents capable of inhibiting PEDV N protein and PEDV replication. The compounds identified here exhibited a potential use as therapeutic agents for controlling PEDV infection.

Active Compounds Against Anopheles minimus Carboxypeptidase B for Malaria Transmission-Blocking Strategy.

Malaria transmission-blocking compounds have been studied to block the transmission of malaria parasites, especially the drug-resistant Plasmodium. Carboxypeptidase B (CPB) in the midgut of Anopheline mosquitoes has been demonstrated to be essential for the sexual development of Plasmodium in the mosquito. Thus, the CPB is a potential target for blocking compounds. The aim of this research was to screen compounds from the National Cancer Institute (NCI) diversity dataset and U.S. Food and Drug Administration (FDA)-approved drugs that could reduce the Anopheles CPB activity. The cDNA fragment of cpb gene from An. minimus (cpbAmi) was amplified and sequenced. The three-dimensional structure of CPB was predicted from the deduced amino acid sequence. The virtual screening of the compounds from NCI diversity set IV and FDA-approved drugs was performed against CPBAmi. The inhibition activity against CPBAmi of the top-scoring molecules was characterized in vitro. Three compounds-NSC-1014, NSC-332670, and aminopterin with IC50 at 0.99 mM, 1.55 mM, and 0.062 mM, respectively-were found to significantly reduce the CPBAmi activity.

Analysis of the Vitellogenin gene of rice moth, Corcyra cephalonica Stainton.

Vitellogenin (Vg) is a precursor of the major yolk protein, an essential nutrient for the embryonic development of oviparous animals including insects. Here, the gene(CceVg [Corcyra cephalonica Vg] ) encoding the Vg (CceVg of moth, C. cephalonica, was cloned and sequenced. The gene sequence was 6,721-bp long and contained 5five introns and six exons that together formed a 5,382-bp open reading frame. The deduced protein (CceVg) consisted of 1,793 amino acid residues, including a 16-amino-acid signal peptide. The putative molecular weight of the primary Vg protein was 202.46 kDa. The CceVg contained all conserved domains and motifs that were commonly found in most insect Vgs except the presence of a polyserine tract at the C-terminal region, which had not been reported in other lepidopteran Vgs. The expression pattern showed that CceVg was first transcribed at a very low level in the early larval stage but disappeared in later stage larva. In female, the CceVg mRNA was detected in early pupal stage and throughout adult stage. Interestingly, the CceVg mRNA was detected only in mated males at low levels, not in the virgin ones. Injection of CceVg double-stranded RNA into early-emergent females caused severely abnormal ovaries.