Impact of Molecular Modification on the Efficiency of Recombinant Baculovirus Vector Invasion to Mammalian Cells and Its Immunogenicity in Mice

The baculovirus display system (BDS), an excellent eukaryotic surface display technology that offers the advantages of safety, efficiency, and economy, is widely used in biomedicine. A previous study using rBacmid-Δgp64-ires-gp64 expressed in low copy numbers of the gp64 gene achieved high-efficiency expression and co-display of three fluorescent proteins (GFP, YFP, and mCherry). However, low expression of GP64 in recombinant baculoviruses also reduces the efficiency of recombinant baculovirus transduction into mammalian cells. In addition, the baculovirus promoter has no expression activity in mammalian cells and thus cannot meet the application requirements of baculoviral vectors for the BDS. Based on previous research, this study first determined the expression activity of promoters in insect Spodoptera frugiperda 9 cells and mammalian cells and successfully screened the very early promoter pie1 to mediate the co-expression of multiple genes. Second, utilizing the envelope display effect of the INVASIN and VSVG proteins, the efficiency of transduction of recombinant baculovirus particles into non-host cells was significantly improved. Finally, based on the above improvement, a recombinant baculovirus vector displaying four antigen proteins with high efficiency was constructed. Compared with traditional BDSs, the rBacmid-Δgp64 system exhibited increased display efficiency of the target protein by approximately 3-fold and induced an approximately 4-fold increase in the titer of serum antibodies to target antigens in Bal B/c mice. This study systematically explored the application of a new multi-gene co-display technology applicable to multi-vaccine research, and the results provide a foundation for the development of novel BDS technologies.

G-Quadruplex Regulation of VEGFA mRNA Translation by RBM4

In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5′-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5′-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.

Expression of Heat Shock Protein HSP90 in Genomic-DNA of Chickpea (Cicer arietinum L.) Callus by Heat Shock Treatment

This study was able to detect of the expression activity of heat shock proteins HSP90 and heat transcription factors HSFs for the first time in callus cultures of chickpea, Cicer arietinum L., that exposed to abiotic shocks, grown on MS medium supplemented with 1.0 mg L-1 naphthalene acetic acid (NAA) and 2.0 mg L-1 benzyl adenine (BA). Heat shock proteins HSPs were constructed for increase of withstand long-term physical shocks, and production of resistant to heat chickpeas plants, this shock was enhancement of tolerance of chickpea callus to abiotic stresses (high - temperatures). Results enhanced the ability of chickpea callus to abiotic stresses bearing and induce of HSF genes to heat shock proteins HSP90 production quickly to removing denatured proteins, avoid apoptosis, thus, supporting tolerance to the sudden action of these shocks. Expression activity of heat shock genes and transcription factors by determined based on polymerase chain reaction qPCR, that explained the gene activity increasing at shocks intensity increased

Accelerated Methods of Determining Wheat Genotypes Primary Resistance to Extreme Temperatures

Several morphological and functional mechanisms determine the resistance of plants to extreme temperatures. Depending on the specificity of mechanisms of action, we divided them into two groups: (1) the mechanisms that ensure the avoidance/reduction of the exposure dose; (2) functional mechanisms which increase plant resistance and ability to recover damages caused by stress through regulation metabolic and genes expression activity. We developed theoretical and practical methods to appreciate the contribution of parameters from both groups on the primary and adaptive resistance of different wheat genotypes. This problem became more complicated because some properties are epigenetically inherited and can influence genotypes’ primary (initial) resistance to stressors. The article describes results obtained by the accelerated determination of the initial resistance of wheat (Triticum aestivum L.) genotypes to temperature stress and the prospects for their implementation in the selection and development of methods for rational choosing wheat varieties for cultivation under specific environmental conditions.

miR-15a-3p Protects Against Isoniazid-Induced Liver Injury via Suppressing N-Acetyltransferase 2 Expression

Isoniazid (INH), an effective first-line drug for tuberculosis treatment, has been reported to be associated with hepatotoxicity for decades, but the underlying mechanisms are poorly understood. N-acetyltransferase 2 (NAT2) is a Phase II enzyme that specifically catalyzes the acetylation of INH, and NAT2 expression/activity play pivotal roles in INH metabolism, drug efficacy, and toxicity. In this study, we systematically investigated the regulatory roles of microRNA (miRNA) in NAT2 expression and INH-induced liver injury via a series of in silico, in vitro, and in vivo analyses. Four mature miRNAs, including hsa-miR-15a-3p, hsa-miR-628-5p, hsa-miR-1262, and hsa-miR-3132, were predicted to target the NAT2 transcript, and a negative correlation was observed between hsa-miR-15a-3p and NAT2 transcripts in liver samples. Further experiments serially revealed that hsa-miR-15a-3p was able to interact with the 3′-untranslated region (UTR) of NAT2 directly, suppressed the endogenous NAT2 expression, and then inhibited INH-induced NAT2 overexpression as well as INH-induced liver injury, both in liver cells and mouse model. In summary, our results identified hsa-miR-15a-3p as a novel epigenetic factor modulating NAT2 expression and as a protective module against INH-induced liver injury, and provided new clues to elucidate the epigenetic regulatory mechanisms concerning drug-induced liver injury (DILI).

Genetic factors influence the survival rate of parotid salivary gland cancer patients

Introduction. The complex of transcriptional proteins of NF-kB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) family deservedly attracts attention as a factor capable of determining the course of malignant disease. Its promising study in combination with the expression of proinflammatory gene IL6 in patients with parotid cancer (PSG) is associated with the development of modulation of malignant disease treatment and risk assessment of the disease course. Aims — to determine the effect of the expression activity of the proinflammatory interleukin-6 gene and the NFKB1 transcriptional gene on the survival rate of patients with parotid cancer. Materials and methods. A cohort retrospective study was conducted in two groups. The epidemiological group of patients included 140 people from the cancer registry of Rostov region. The clinical part of the work was carried out on 25 patients with PSG cancer of both sexes aged 50 to 80 years. Followup period of the patients after radical surgery was 18 years. Expression activity of NFKB1 and IL6 genes was estimated by real-time PCR in tumor and conditionally healthy tissue. Patient survival rate was analyzed using Kaplan-Meier method. Results. According to the results of the survival analysis in the epidemiological group, the probability that an PSG cancer patient would survive the first year after diagnosis was 95.7%, three years — 82.4%, five years — 70.9% and 10 years — 31.2%. A comparative study of gene expression levels in tumor tissue samples compared to conditionally healthy tissue revealed an increase (p<0.001) in the relative index for both the IL6 gene (5.7 times) and the NFKB1 gene (7.9 times).><0.001) in the relative index for both the IL6 gene (5.7 times) and the NFKB1 gene (7.9 times). Discussion. Analysis of our data showed the possibility of using the complex evaluation of NFKB1 and IL6 gene expression in the cells of tumor samples of PSG cancer tissue obtained during surgery to predict the long-term survival of patients after surgical treatment. Conclusions. The expression profile of NFKB1 gene in tumor tissue was a proven prognostic factor determining the course of the disease in patients with PSG cancer, which should be taken into account when forming the prognosis of the disease. The expression of IL6 gene expression in tumor cells had no independent effect on the survival rate of PSG cancer patients, but contributed to the functional activation of NFKB1 transcription gene.

Regulation of Endoplasmic Reticulum–Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3β

Mitochondria–ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER–mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3β, previously associated with MERCs disruption.

Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins

Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER–mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.

Lilium Regale Wilson WRKY3 Modulates an Antimicrobial Peptide Gene, Lrdef1, During Response to Fusarium Oxysporum

Background: WRKY transcription factors (TFs) play vital roles in plant growth and development, secondary metabolite synthesis, and response to biotic and abiotic stresses. In a previous transcriptome sequencing analysis of Lilium regale Wilson, we identified multiple WRKY TFs that respond to exogenous methyl jasmonate treatment and lily Fusarium wilt (Fusarium oxysporum).Results: In the present study, the WRKY TF LrWRKY3 was further analyzed to reveal its function in defense response to F. oxysporum. The LrWRKY3 protein was localized in the plant cell nucleus, and LrWRKY3 transgenic tobacco lines showed higher resistance to F. oxysporum compared with wild-type (WT) tobacco. In addition, some genes related to jasmonic acid (JA) biosynthesis, salicylic acid (SA) signal transduction, and disease resistance had higher transcriptional levels in the LrWRKY3 transgenic tobacco lines than in the WT. On the contrary, L. regale scales transiently expressing LrWRKY3 RNA interference fragments showed higher sensitivity to F. oxysporum infection. Moreover, a F. oxysporum-induced defensin gene, Def1, was isolated from L. regale, and the recombinant protein LrDef1 isolated and purified from Escherichia coli possessed antifungal activity to several phytopathogens, including F. oxysporum. Furthermore, co-expression of LrWRKY3 and the LrDef1 promoter in tobacco evidently up-regulated the expression activity of the LrDef1 promoter.Conclusions: These results clearly indicate that LrWRKY3 is an important positive regulator in response to F. oxysporum infection, and one of its targets is the antimicrobial peptide gene LrDef1.