Insect Cells Research Articles

Isolation and Characterization of Insecticidal Cyclotides from Viola communis.

Cyclotides are cysteine-rich plant-derived peptides composed of 28-37 amino acids with a head-to-tail cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Their beneficial biophysical properties make them promising molecules for pharmaceutical and agricultural applications. The Violaceae plant family is the major cyclotide-producing family, and to date, every examined plant from this family has been found to contain cyclotides. The presence of cyclotides in Viola communis was inferred by mass spectroscopy previously, but their sequences and properties had yet to be explored. In this study, the occurrence of cyclotides in this plant was investigated using proteomics and transcriptomics. Twenty cyclotides were identified at the peptide level, including two new members from the bracelet (Vcom1) and Möbius (Vcom2) subfamilies. Structural analysis of these newly identified peptides demonstrated a similar fold compared with cyclotides from the same respective subfamilies. Biological assays of Vcom1 and Vcom2 revealed them to be cytotoxic to Sf9 insect cell lines, with Vcom1 demonstrating higher potency than Vcom2. The results suggest that they could be further explored as insecticidal agents and confirm earlier general findings that bracelet cyclotides have more potent insecticidal activity than their Möbius relatives. Seven new cyclotide-like sequences were observed in the transcriptome of V. communis, highlighting the Violaceae as a rich source for new cyclotides with potential insecticidal activity. An analysis of sequences flanking the cyclotide domain in the various precursors from V. communis and other Violaceae plants revealed new insights into cyclotide processing and suggested the possibility of two alternative classes of N-terminal processing enzymes for cyclotide biosynthesis.

Drosophila Evi5 is a critical regulator of intracellular iron transport via transferrin and ferritin interactions

Vesicular transport is essential for delivering cargo to intracellular destinations. Evi5 is a Rab11-GTPase-activating protein involved in endosome recycling. In humans, Evi5 is a high-risk locus for multiple sclerosis, a debilitating disease that also presents with excess iron in the CNS. In insects, the prothoracic gland (PG) requires entry of extracellular iron to synthesize steroidogenic enzyme cofactors. The mechanism of peripheral iron uptake in insect cells remains controversial. We show that Evi5-depletion in the Drosophila PG affected vesicle morphology and density, blocked endosome recycling and impaired trafficking of transferrin-1, thus disrupting heme synthesis due to reduced cellular iron concentrations. We show that ferritin delivers iron to the PG as well, and interacts physically with Evi5. Further, ferritin-injection rescued developmental delays associated with Evi5-depletion. To summarize, our findings show that Evi5 is critical for intracellular iron trafficking via transferrin-1 and ferritin, and implicate altered iron homeostasis in the etiology of multiple sclerosis.

Dual N-linked glycosylation at residues 133 and 158 in the hemagglutinin are essential for the efficacy of H7N9 avian influenza virus like particle vaccine in chickens and mice

H7N9 subtype avian influenza virus (AIV) poses a great challenge to poultry industry. Virus-like particle (VLP) is a prospective alternative for the traditional egg-based influenza vaccines. N-linked glycosylation (NLG) regulates the efficacy of influenza vaccines, whereas the impact of NLG modifications on the efficacy of influenza VLP vaccines remains unclear. Here, H7N9 VLPs were assembled in insect cells through co-infection with the baculoviruses expressing the NLG-modified hemagglutinin (HA), neuraminidase and matrix proteins, and the VLP vaccines were assessed in chickens and mice. NLG modifications significantly enhanced hemagglutination-inhibition and virus neutralization antibody responses in mice, rather than in chickens, because different immunization strategies were used in these animal models. The presence of dual NLG at residues 133 and 158 significantly elevated HA-binding IgG titers in chickens and mice. The VLP vaccines conferred complete protection and significantly suppressed virus replication and lung pathology post challenge with H7N9 viruses in chickens and mice. VLP immunization activated T cell immunity-related cytokine response and inhibited inflammatory cytokine response in mouse lung. Of note, the presence of dual NLG at residues 133 and 158 optimized the capacity of the VLP vaccine to stimulate interleukin-4 expression, inhibit virus shedding or alleviate lung pathology in chickens or mice. Intriguingly, the VLP vaccine with NLG addition at residue 133 provided partial cross-protection against the H5Nx subtype AIVs in chickens and mice. In conclusion, dual NLG at residues 133 and 158 in HA can be potentially used to enhance the efficacy of H7N9 VLP vaccines in chickens and mammals.

Insect Cell-Expressed Major Ragweed Allergen Amb a 1.01 Exhibits Similar Allergenic Properties to Its Natural Counterpart from Common Ragweed Pollen

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Deleterious effects of the endosymbiont Rickettsiella viridis in Myzus persicae are environmentally dependent

AbstractEndosymbionts living within insect cells can modify host fitness and could provide new tools for pest control. The endosymbiont Rickettsiella viridis has been transferred experimentally into the green peach aphid, Myzus persicae, a globally important agricultural pest. This Rickettsiella spreads via vertical and horizontal transmission and induces host fitness costs which could potentially suppress pest populations. Endosymbiont prevalence can fluctuate in natural populations, and it is important to identify factors that contribute to their spread or loss. Here, we explore the effects of Rickettsiella infection when aphids are reared on eight different host plants or exposed to thermal, starvation and desiccation stresses. Rickettsiella infection reduced M. persicae fecundity and longevity across all host plants, but the magnitude of costs varied among host plants and generations. Rickettsiella was horizontally transmitted and spread in caged populations at initial ratios of 1:2 Rickettsiella (+): Rickettsiella (−) on all host plants, but with limited long-term persistence under cycling 20–30 °C. We also identified temperature-dependent costs of Rickettsiella infection on heat knockdown time, chill coma recovery, and starvation tolerance. Finally, we present evidence that Rickettsiella infection reduces host activity levels under heat stress. Our results suggest that Rickettsiella infections induce a variety of deleterious effects but with complex environment-dependent interactions. This work helps understand ecological conditions that enhance or limit the spread of these endosymbionts in aphid populations.

Development of a label‐free, impedance‐based biosensor to identify harmful effects of pesticides on insect cells

AbstractInsects are a major part of the planet´s ecosystem and their vital role as pollinators for agriculture is undisputed. Alongside factors as climate change or loss of habitats, rising use of pesticides emerges as a key threat to insect populations. For fighting this man‐made problem, development of an easy, fast, sensitive and non‐invasive biosensor for determining pesticide toxicity may help to ban harmful substances and formulations. Here, a biosensor based on Sf21 (Spodoptera frugiperda) insect cells as sensors and electric cell‐substrate impedance sensing (ECIS) as physical transducer is described. Sf21 cell suspensions and well‐defined pesticide solutions were mixed immediately before seeding on planar gold‐film electrodes. The capacitance at 20 kHz was recorded as a function of time as a measurand for cell adhesion providing dose‐response profiles of pesticide impact. For future in‐field applications, decoupling of the cell culture routines from the actual cytotoxicity assay is mandatory. Thus, suspensions of Sf21 cells were cryopreserved at –80°C in the wells of multi‐electrode arrays and thawed anytime for conducting the assays. Five pesticides were tested for their concentration dependent cytotoxicity expressed as EC50 values by ECIS and validated using the well‐established WST‐1 cell viability assay. Results were found to be in good agreement. Our studies revealed cytotoxic effects of some pesticides sold for home usage far below the recommended concentration and were found to be more toxic than formulations sold for agricultural industry only.This article is protected by copyright. All rights reserved.

Abstract PO4-28-08: Unraveling the Role of BRCA1-BARD1 E3 Ubiquitin Ligase in DNA Repair: A Promise for Enhanced Chemotherapy Outcomes

Abstract BACKGROUND: BRCA1, a central player in gynecological cancers, collaborates with BARD1 to shape a potent complex pivotal for DNA binding and ubiquitin E3 ligase activities. This complex impacts a myriad of biological pathways. Notably, BRCA1-BARD1’s role in tumor suppression and homology-directed DNA repair (HDR) has been spotlighted. Our innovative approach used RING-domain mutations to craft ligase-dead BRCA1-BARD1 mutants, hypothesizing that these mutants would offer fresh insights into the DNA repair dynamics of BRCA1-BARD1. METHODS: Full length BRCA1-BARD1 or truncated mutants, and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. Stable mammalian cell lines HeLa and MDA-MB-436 that express wild type or mutant forms of BRCA1 and BARD1 were established for a host of analyses, including cellular fractionation, foci analysis, Proximity ligation assay (PLA), comet assays and clonogenic survival assays with various DNA damage agents. RESULTS: By systematic biochemical screening and a series of in vitro assays, we generated a truly BRCA1-BARD1 E3 dead mutant, BRCA1I26A, L63A, K65A-BARD1 (BRCA1-E3d), which lacks E3 ligase activity but possesses all other known attributes such as retention of BRCA1-BARD1 heterodimeric structure formation, DNA binding, and intact RAD51-mediated recombinase activity. To our surprise, we discovered that previously described BRCA1-BARD1 RING-domain mutant (BRCA1-I26A) still possessed ubiquitylation activity not found in BRCA1-E3d. To determine the biological significance of these mutants, cells stably expressing BRCA1-E3d were treated with various DNA-damaging agents and shown to more sensitive than WT or previously identified mutants. DNA repair pathway reporter assays determined these cells were deficient in various repair pathways compared to their BRCA1-WT counterparts. Further studies demonstrate that BRCA1-BARD1 E3 ligase is required for DNA resection during HDR, as evidenced in reduced levels of DNA repair-related foci formation such as RPA, RAD51 and CtIP in BRCA1-E3d cells. Furthermore, compared to BRCA1-WT cells, BRCA1-E3d cells were more sensitive to DNA damage reagents after depletion of 53BP1, which indicates BRCA1-BARD1 E3 ligase function also contributes to later stages of DNA repair completion. CONCLUSIONS: Our work dispels prevailing ambiguities surrounding BRCA1-BARD1 E3 ligase functions, underscoring its paramountcy in genome repair. This trailblazing research not only enriches our understanding but also beckons therapeutic interventions targeting tumor suppression. The unveiling of BRCA1-BARD1 E3 ligase's intricate regulatory dynamics combined with our novel mutants paves the way for an exciting new era in cancer therapeutics, hinting at superior treatments to enhance patient recovery. Citation Format: Wenjing Li, Meiling Wang, Nozomi Tomimatsu, Jae-Hoon Ji, Salvador Alejo, Gangadhara R. Sareddy, Sandeep Burma, Rachel Klevit, Weixing Zhao. Unraveling the Role of BRCA1-BARD1 E3 Ubiquitin Ligase in DNA Repair: A Promise for Enhanced Chemotherapy Outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-08.

The innovation of Wolbachia mosquito technology to control dengue hemorrhagic fever

Wolbachia is a bacterium that generally occurs naturally in 50% of insect species, lives in insect cells, and is passed from one generation to the next through insect eggs. Wolbachia in the mosquito's body can inhibit the replication of the Dengue virus or other viruses. Based on the Decree of the Minister of Health of the Republic of Indonesia Number 1341 concerning the Implementation of the Pilot Project for the Implementation of Wolbachia as a Dengue Management Innovation, the application of this technology was carried out in five cities, namely Semarang City, West Jakarta, Bandung City, Kupang City, and Bontang City. The effectiveness of Wolbachia itself has been proven and researched since 2011, when it was conducted in Yogyakarta. As a result, Wolbachia was proven to reduce dengue fever cases by up to 77% and reduce the proportion of hospital admissions by 86%. This method of spreading Wolbachia mosquitoes in several cities will reduce local transmission of infection in those areas. This reduction in local transmission of dengue infection occurs both in the rainy and dry seasons. Scientifically, the success of the Wolbachia method is apparent, but the controversy is due to confused information circulating, giving rise to concern among the public. Vector control using Wolbachia is an alternative disease control method that uses mosquito vectors and is quite effective. However, the application of Wolbachia in the community must be monitored continuously to determine its impact on humans, local mosquitoes, and the ecosystem.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host’s cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5ʹ proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5ʹ coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Recombinant SpTransformer proteins are functionally diverse for binding and phagocytosis by three subtypes of sea urchin phagocytes.

The California purple sea urchin, Strongylocentrotus purpuratus, relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the SpTransformer (SpTrf) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine Vibrio. A recombinant (r)SpTrf-E1-Ec protein produced by E. coli also binds Vibrio but does not augment phagocytosis. To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis. These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.

Structure and Activity of Reconstructed Pseudo-Ancestral Cyclotides.

Cyclotides are cyclic peptides that are promising scaffolds for the design of drug candidates and chemical tools. However, despite there being hundreds of reported cyclotides, drug design studies have commonly focussed on a select few prototypic examples. Here, we explored whether ancestral sequence reconstruction could be used to generate new cyclotides for further optimization. We show that the reconstructed 'pseudo-ancestral' sequences, named Ancy-m (for the ancestral cyclotide of the Möbius sub-family) and Ancy-b (for the bracelet sub-family), have well-defined structures like their extant members, comprising the core structural feature of a cyclic cystine knot. This motif underpins efforts to re-engineer cyclotides for agrochemical and therapeutic applications. We further show that the reconstructed sequences are resistant to temperatures approaching boiling, bind to phosphatidyl-ethanolamine lipid bilayers at micromolar affinity, and inhibit the growth of insect cells at inhibitory concentrations in the micromolar range. Interestingly, the Ancy-b cyclotide had a higher oxidative folding yield than its comparator cyclotide cyO2, which belongs to the bracelet cyclotide subfamily known to be notoriously difficult to fold. Overall, this study provides new cyclotide sequences not yet found naturally that could be valuable starting points for the understanding of cyclotide evolution and for further optimization as drug leads.

Determination, expression and characterization of an UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) from the Pacific oyster, Crassostrea gigas

Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host’s immune response, depend on carbohydrates. Therefore, the investigation of mollusc glycosylation capacities is of high relevance to understand the interaction of parasites with their host. UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) is the key enzyme for the biosynthesis of hybrid and complex type N-glycans catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the α-1,3 Man antenna of Man5GlcNAc2. Thereby, the enzyme produces a suitable substrate for further enzymes, such as α-mannosidase II, GlcNAc-transferase II, galactosyltransferases or fucosyltransferases. The sequence of GnT- I from the Pacific oyster, Crassostrea gigas, was obtained by homology search using the corresponding human enzyme as the template. The obtained gene codes for a 445 amino acids long type II transmembrane glycoprotein and shared typical structural elements with enzymes from other species. The enzyme was expressed in insect cells and purified by immunoprecipitation using protein A/G-plus agarose beads linked to monoclonal His-tag antibodies. GnT-I activity was determined towards the substrates Man5-PA, MM-PA and GnM-PA. The enzyme displayed highest activity at pH 7.0 and 30 °C, using Man5-PA as the substrate. Divalent cations were indispensable for the enzyme, with highest activity at 40 mM Mn2+, while the addition of EDTA or Cu2+ abolished the activity completely. The activity was also reduced by the addition of UDP, UTP or galactose. In this study we present the identification, expression and biochemical characterization of the first molluscan UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.Graphical abstractIllustration of GnT-I activity. (a) Transfer of GlcNAc to Man5-PA, creating Man5GlcNAc3-PA. (b) Transfer of GlcNAc to MM-PA, creating MGn-PA. (c) Transfer of GlcNAc to GnM-PA, creating GnGn-PA. Blue squares represent N-acetylglucosamine, green cycles depict mannose. Graphic illustration of N-glycans were created using bioRENDER.

Interaction of fluralaner with binary model membranes. Potential implications in the selectivity for invertebrates/vertebrates

GABAA receptors (GABAA-R) are the target of widely used gabaergic insecticides such as fluralaner, a second generation non-competitive antagonist. Fluralaner blocks GABAA-R, binding to a pocket in the transmembrane subunit interface. Because of the location of its binding pocket, it is expected that fluralaner accesses to the receptor by previously partitioning into the bilayer. Therefore, differences in lipid composition of insect and mammal cells becomes relevant to determine the impact of membrane composition on the nonspecific selectivity of gabaergic insecticides. Insect cells show minimal concentration of cholesterol and they modulate membrane fluidity by regulating POPE:POPC ratios. On the other hand, vertebrate cholesterol concentration can be as high as 50%, depending on the type of cell. We used experimental Langmuir monolayer and Molecular Dynamics Simulations (MDS) to characterize the effect and interactions of the insecticide fluralaner with model membranes with varying concentrations of POPC:POPE and POPC:CHOL. Monolayer experiments showed that this insecticide reduces film rigidity. MSD showed that fluralaner is located at the interface between the acyl chain and the polar region of the membrane, but this location shifts at high CHOL concentrations towards the phosphate region. This is accompanied by a shift in the orientation of the fluralaner backbone, affecting fluralaner solubility. The presence of CHOL favors fluralaner aggregation, while POPE has the opposite effect, allowing its solubilization and the presence of individual insecticide molecules homogeneously dispersed. Our work suggests that differences in membrane composition could unspecifically influence the selectivity of gabaergic insecticides, modulating fluralaner solubility and fluralaner's access to GABAA-R.

The expression system affects the binding affinity between p75NTR and proNGF

ProNGF (nerve growth factor) is a precursor of NGF and a signaling peptide exerting opposite effects on neuronal cells, i.e., apoptotic or neuritogenic. The conflicting biological activity of proNGF depends on the relative levels of two membrane receptors, TrkA and p75NTR. The effect of proNGF depends on the expression levels of these receptor proteins and their affinity to proNGF. Since the affinity of proteins has been studied with various recombinant proteins, it is worth comparing the affinity of these proteins within one experiment with the same method. This study examined the affinity between a recombinant proNGF and p75NTR expressed in common systems: bacterial, insect, and mammalian cells. The extracellular domain of p75NTR expressed in the insect or mammalian systems bound to native mature NGF, with a higher affinity for the insect receptor. The uncleavable proNGF was expressed in the three systems and they showed neuritogenic activity in PC12 cells. These recombinant proteins were used to compare their binding affinity to p75NTR. The insect p75NTR showed a higher binding affinity to proNGF than the mammalian p75NTR. The insect p75NTR bound proNGF from the insect system with the highest affinity, then from the mammalian system, and the lowest from the bacterial system. Conversely, the mammalian p75NTR showed no such preference for proNGF. Because the recombinant proNGF and p75NTR from different expression systems are supposed to have the same amino acid sequences, these differences in the affinity depend likely on their post-translational modifications, most probably on their glycans. Each recombinant proNGF and p75NTR in various expression systems exhibited different mobilities on SDS-PAGE and reactivities with glycosidases and lectins.

Investigation of the insecticidal potential of curcumin derivatives that target the Helicoverpa armigera sterol carrier protein-2

Cotton bollworm (Helicoverpa armigera) is a highly polyphagous, widely prevalent, and persistent Old World insect pest that affects numerous important crops that are directly consumed by people, including tomato, cotton, pigeon pea, chickpea, rice, sorghum, and cowpea. Insects do not synthesize steroids but obtain them from their diet. Inhibition of dietary uptake of steroids by insects is a potentially effective insecticidal mechanism that should not be toxic to humans and other mammals, who synthesize their steroids. Ten curcumin derivatives were tested against H. armigera sterol carrier protein-2 (HaSCP-2) for their potential as insecticidal agents. Curcumin derivatives were initially docked at the binding site of HaSCP-2 to determine their binding affinities and plausible binding modes. The binding modes predominantly show hydrophobic interactions of derivatives with Phe53, Phe110, and Phe89 as core interacting residues in the active site. Validation of in silico results was carried out by performing a fluorescence binding and displacement assay to determine the binding affinities of curcumin derivatives. Among a collection of curcumin derivatives tested, Cur10 showed the lowest IC50 value of 9.64 μM, while Cur07 was 19.86 μM, and Cur06 was 20.79 μM. There was a significant negative correlation between the ability of the curcumin derivatives tested to displace the fluorescent probe from the sterol binding site of HaSCP-2 and to inhibit Sf9 insect cell growth in culture, which is consistent with the curcumin derivatives acting by the novel mechanism of blocking sterol uptake. Then molecular dynamics simulation studies validated the predicted binding modes and the interactions of curcumin derivatives with HaSCP-2 protein. In conclusion, these studies support the potential use of curcumin derivatives as insecticidal agents.

Intracellular localized heterogeneous protein franking by a transmembrane domain of GP64 is sufficient to be assembled on budded virions of Bombyx mori nucleopolyhedrovirus

Baculovirus has been widely used for foreign protein expression in biomedical studies, and budded virus (BV) surface display has developed into an important research tool for heterogenous membrane protein studies. The basic strategy of surface display is to construct a recombinant virus where the target gene is fused with a complete or partial gp64 gene. In this study, we further investigate and develop this BV surface displaying strategy. We constructed stable insect cell lines to express the target protein flanking with different regions of signal peptide (SP) and GP64 transmembrane domain (TMD). Subsequently, recombinant BmNPV was used to infect the cell, and the integration of heterogeneous protein into BV was detected. The results indicated that deletion of the n-region of SP (SPΔn) decreased the incorporation rate more than that of the full-length SP. However, the incorporation rate of the protein fused with h and c-region deletion of SP (SPΔh-c) was significantly enhanced by 35-40 times compare to full-length SP. Moreover, the foreign protein without SP and TMD failed to display on the BV, while the integration of foreign proteins with GP64 TMD fusion at the c-terminal was significantly enhanced by 12-26 times compared to the control. Thus, these new strategies developed the BV surface display system further.

Evaluation of immunogenicity and protective efficacy of bacteriophage conjugated haemagglutinin based subunit vaccine against equine influenza virus in a murine model.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen's eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro.

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.

The Glycan Ectodomain of SARS-CoV-2 Spike Protein Modulates Cytokine Production and Expression of CD206 Mannose Receptor in PBMC Cultures of Pre-COVID-19 Healthy Subjects.

The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.