The extraction of biopesticides from plants has become a promising field for agricultural development. To explore a high-efficiency and viable method for the screening of plant compounds with ...insecticidal activity, we screened for active ingredients in the insecticidal plant,
Oroxylum indicum
L
.
Vent, using Sf9 cells. A CCK-8 cytotoxicity assay kit was used for high-throughput screening of 34 compounds contained in
O. indicum
. The apoptosis-inducing effect of the highly cytotoxic compound on Sf9 cells was investigated by morphological characterization using inverted microscopy, caspase-3 activity assay, and DNA gel electrophoresis. Finally, the biological activity of compounds against aphids was evaluated using the leaf-pest dipping methods and leaf dipping methods. Results showed that among the main compounds identified, lapachol, chrysin, and baicalein had good proliferation inhibitory effects on Sf9 cells, with their recorded IC
50
being 11.53 mg/L, 38.39 mg/L, and 42.10 mg/L, respectively. Moreover, the IC
50
value of lapachol was lower than the control insecticides rotenone (18.03 mg/L) and fipronil (21.04 mg/L). Apoptosis assay further showed that lapachol promoted the production of caspase-3 and led to DNA fragmentation in Sf9 cells. Lapachol showed high biological activity against
Aphis gossypii
,
Sitobion avenae
, and
Semiaphis heraclei
, with its recorded LC
50
being 104.40, 101.80, and 110.29 mg/L, respectively, which were comparable to the activity of the control insecticide rotenone. High-throughput screening of active ingredients in the insecticidal plant
O. indicum
using Sf9 cells is feasible, and the identification of lapachol as the main aphidicidal active substance is valuable for further study.
Display omitted
•Structurally, the VP topology and capsid morphology of AAV-TT is homologous to AAV2.•AAV-TT differs by 14 amino acids from AAV2, two of which, S585R and T588R are essential for HSPG ...binding.•AAV-TT is significantly more stable than AAV2 throughout the endo/lysosomal pathway and suggests a potential to withstand destabilizing conditions during infection.•Identified residues that can be engineered, they do not affect the capsid structure, but result in vectors with improved function such as escape of neutralizing antibodies.
Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (Tm) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in Tm, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.
Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT (Gcn5-related ...N-acetyltransferase) family of HATs. In metazoans this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 acetyltransferase) and ATAC (Ada Two A-containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, whereas in the SAGA HAT module ADA2b is present instead of ADA2a. To better understand how the activity of human (h) hGCN5 is regulated in the two related, but different, HAT complexes we carried out in vitro HAT assays. We compared the activity of hGCN5 alone with its activity when it was part of purified recombinant hATAC or hSAGA HAT modules or endogenous hATAC or hSAGA complexes using histone tail peptides and full-length histones as substrates. We demonstrated that the subunit environment of the HAT complexes into which GCN5 incorporates determines the enhancement of GCN5 activity. On histone peptides we show that all the tested GCN5-containing complexes acetylate mainly histone H3K14. Our results suggest a stronger influence of ADA2b as compared with ADA2a on the activity of GCN5. However, the lysine acetylation specificity of GCN5 on histone tails or full-length histones was not changed when incorporated in the HAT modules of ATAC or SAGA complexes. Our results thus demonstrate that the catalytic activity of GCN5 is stimulated by subunits of the ADA2a- or ADA2b-containing HAT modules and is further increased by incorporation of the distinct HAT modules in the ATAC or SAGA holo-complexes.
Background: Histone acetyltransferases (HATs) incorporated in large multiprotein complexes are involved in a wide variety of cellular processes, including transcription regulation.
Results: Subunits of HAT complexes enhance the enzymatic activity of their catalytic subunits.
Conclusion: The activity, but not the specificity of HAT complexes, is stimulated by the corresponding subunits.
Significance: We gained important insights into histone acetylation function and specificity of HAT complexes.
Display omitted
In addition to vaccines, noninfectious virus-like particles (VLPs) that mimic the viral capsid show an attractive possibility of presenting immunogenic epitopes or targeting molecules ...on their surface. Here, functionalization of norovirus-derived VLPs by simple non-covalent conjugation of various molecules is shown. By using the affinity between a surface-exposed polyhistidine-tag and multivalent tris-nitrilotriacetic acid (trisNTA), fluorescent dye molecules and streptavidin–biotin conjugated to trisNTA are displayed on the VLPs to demonstrate the use of these VLPs as easily modifiable nanocarriers as well as a versatile vaccine platform. The VLPs are able to enter and deliver surface-displayed fluorescent dye into HEK293T cells via a surface-attached cell internalization peptide (VSV-G). The ease of manufacturing, the robust structure of these VLPs, and the straightforward conjugation provide a technology, which can be adapted to various applications in biomedicine.
Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell ...chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 µm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 µm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.
As a tightly controlled cell death process, apoptosis eliminates unwanted cells and plays a vital role in multicellular organisms. Previous study have demonstrated that apoptosis occurred in ...Spodoptera frugiperda cultured Sf9 cells, which triggered by diverse apoptotic stimuli, including azadirachtin, camptothecin and ultraviolet. Due to its simplicity, high sensitivity and reliable specificity, RT-qPCR has been used widespread for analyzing expression levels of target genes. However, the selection of reference genes influences the accuracy of results profoundly. In this study, eight genes were selected for analyses of their suitability as references for normalizing RT-PCR data in Sf9 cells treated with apoptotic agents. Five algorithms, including NormFinder, BestKeeper, Delta Ct method, geNorm, and RefFinder, were used for stability ranking. Based on comprehensively analysis, the expression stability of selected genes varied in cells with different apoptotic stimuli. The best choices for cells under different apoptosis conditions were listed: EF2 and EF1α for cells treated with azadirachtin; RPL13 and RPL3 for cells treated with camptothecin; EF1α and β-1-TUB for cells irradiated under ultraviolet; and EF1α and EF2 for combinational analyses of samples. Our results not only facilitate a more accurate normalization for RT-qPCR data, but also provide the reliable assurance for further studies of apoptotic mechanisms under different stimulus in Sf9 cells.
Functional genetic studies in honeybees have been limited by transformation tools that lead to a high rate of transposon integration into the germline of the queens. A high transformation rate is ...required to reduce screening efforts because each treated queen needs to be maintained in a separate honeybee colony. Here, we report on further improvement of the transformation rate in honeybees by using a combination of different procedures. We employed a hyperactive transposase protein (hyPBase
), we tripled the amount of injected transposase mRNAs and we injected embryos into the first third (anterior part) of the embryo. These three improvements together doubled the transformation rate from 19% to 44%. We propose that the hyperactive transposase (hyPBase
) and the other steps used may also help to improve the transformation rates in other species in which screening and crossing procedures are laborious.
Traditional sanitation practices remain the main strategy for controlling
infections caused by microsporidia
. This actualizes the development of new approaches to increase the silkworm resistance to ...this parasite. Here, we constructed a mouse scFv library against the outer loops of
ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of
PTP2 and
COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.
•AcMNPV-miR-3, another miRNA from AcNMPV, was identified and characterized.•AcMNPV-miR-3 down-regulates the viral gene ac101 markedly. Moreover, four additional viral genes ac23, ac25, ac86, and ac98 ...are also demonstrated to be targeted by AcMNPV-miR-3.•Administrating excessive AcMNPV-miR-3 can reduce infectious budded virions (BV) and accelerated the formation of occlusion-derived virions (ODV).
MicroRNAs (miRNAs), which are small noncoding RNAs found in plants, animals, and many viruses, regulate various biological processes. Our group has previously reported the first miRNA encoded by Autographa californica multiple Nucleopolyhedrovirus (AcMNPV), AcMNPV-miR-1, which regulates the expression of three viral genes. This study characterizes another miRNA encoded by AcMNPV, AcMNPV-miR-3. This miRNA is located on the opposite strand of the viral gene ac101 coding sequence in the AcMNPV genome, and it can be detected at 6 h post-infection and accumulated to a peak around 12 h post-infection in AcMNPV infected Sf9 cells. Five viral genes (ac101, ac23, ac25, ac86, and ac98) were verified to be regulated by AcMNPV-miR-3. Ac101 was markedly down-regulated by AcMNPV-miR-3 that may be via a siRNA-like cleavage mode. Administrating excessive AcMNPV-miR-3 resulted in decreased production of infectious budded virions (BV) and accelerated the formation of occlusion-derived virions (ODV). These results suggest that AcMNPV-miR-3 may play a regulatory role in BV and ODV production.
Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in ...immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17-36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK