Expression of GroES TB antigen in tobacco and potato

As the world races towards a plant-based bioeconomy, plants known to be ideal and economical bioreactors are being harnessed for the production of recombinant proteins. The major immunodominant 10 kDa GroES TB antigen (Chaperonin 10) gene from Mycobacterium tuberculosis was selected for expression in plants as a putative tuberculosis (TB) subunit vaccine candidate. Two crops, tobacco and potato, were engineered by stable plant transformation for expression of the 10 kDa GroES TB antigen using non-viral binary vectors. The integration of the GroES TB gene into the genomes of tobacco and potato was confirmed by PCR and Southern blotting. The expression of the GroES TB antigen in tobacco was 0.04–1.2 % of the total soluble protein (TSP). However, the expression of the same TB antigen in the Indian potato cv. Kufri bahar was comparatively low (0.033 % of TSP). The recombinant GroES plant derived protein was characterised and confirmed by MALDI-TOF–TOF and ELISA. This is the first report of the expression of the 10 kDa chaperonin in tobacco and potato.     

Inhibition of the JAK/STAT pathway with ruxolitinib overcomes cisplatin resistance in non-small-cell lung cancer NSCLC

This study was aimed to elucidate the roles of inhibition of related JAK/STAT pathways in regulating cytotoxicity induced by cisplatin in non-small-cell lung cancer (NSCLC) cell. We treated five non-small-cell lung cancer cell lines with cisplatin alone or with cisplatin and Jak2 inhibitor (ruxolitinib) and assessed cell viability, expression of Jak2 and STAT3 and cell apoptosis. We also investigated the effect of combination treatment inhibited tumor xenograft growth in two human NSCLC xenograft models bearing the cisplatin resistant (H1299) and sensitive (A549) cells. Different cell lines with different genetic background showed half-maximal inhibitory concentrations (IC₅₀) of cisplatin from 4.66 to 68.28 µmol/L. They could be divided into cisplatin intrinsic resistant and cisplatin sensitive cell lines. In cisplatin-resistant cells with higher Jak2 and STAT3 expression, cisplatin and ruxolitinib combination dramatically suppressed the cell growth, down-regulated the expression of phosphorylated STAT3 and induced cleaved caspase-3 expression. Moreover combination with cisplatin and ruxolitinib also significantly inhibited the growth of resistant cell H1299, A549/DDP and H2347 in soft agar model. Finally, combination group significant inhibited the tumor growth and induced the caspase-3 expression compared with either single agent alone (P < 0.05) on the resistant cell xenografts model. The present study indicates that further study is warranted to determine the effectiveness of combination treatment with cisplatin and Jak2/stat3 pathway inhibitor for platinum-resistant NSCLC.     

Implementation of Transportation Distance for Analyzing FLIM and FRET Experiments

Analysis of fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) experiments in living cells is usually based on mean lifetimes computations. However, these mean lifetimes can induce misinterpretations. We propose in this work the implementation of the transportation distance for FLIM and FRET experiments in vivo. This non-fitting indicator, which is easy to compute, reflects the similarity between two distributions and can be used for pixels clustering to improve the estimation of the FRET parameters. We study the robustness and the discriminating power of this transportation distance, both theoretically and numerically. In addition, a comparison study with the largely used mean lifetime differences is performed. We finally demonstrate practically the benefits of the transportation distance over the usual mean lifetime differences for both FLIM and FRET experiments in living cells.     

The GABAA Antagonist DPP-4-PIOL Selectively Antagonises Tonic over Phasic GABAergic Currents in Dentate Gyrus Granule Cells

GABA_A receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABA_A receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABA_A receptors. The two inhibitory current types are mediated by different subpopulations of GABA_A receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABA_A receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC₅₀ values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC₅₀ values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABA_A receptors located extrasynaptically using the antagonist, DPP-4-PIOL.     

Association of vitamin D receptor FokI and ApaI polymorphisms with lung cancer risk in Tunisian population

Many studies reported that Vitamin D Receptor (VDR) gene polymorphisms might influence the cancer risk due to their antiproliferative, antiangiogenic, and apoptotic effects. The aim of this study was to explore the genetic association of VDR polymorphisms with lung cancer risk in Tunisian population. The genotype and haplotype frequencies of four VDR polymorphisms, FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) were studied using polymerase chain reaction and restriction fragment length polymorphism analysis in 240 patients with lung cancer and 280 healthy controls. The distribution of genotype frequencies differed significantly between lung cancer subjects and controls (FokI P _adj  = 0.002; ApaI P _adj  = 0.013). Haplotype analyses revealed a significant association between G-A-C and A-C-T haplotypes and lung cancer risk (P _corr  = 0.0128, P _corr  = 0.008). When patients were stratified according to gender, age, and smoking, significant associations were detected with FokI and TaqI polymorphisms. We found a lack of association between BsmI, TaqI polymorphisms and lung cancer risk (P > 0.05). Only, the attributable proportion due to interaction and the synergic index for interaction between ApaI polymorphism and smoking were statistically significant (P _adj  = 0.74, 95 % CI = 0.38–1.20) and (P _adj  = 0.63, 95 % CI = 0.05–1.21), respectively. Both the additive interaction measures suggested the existence of a biological interaction between SNP ApaI, but not FokI, and smoking. The multiplicative interaction measure was not statistically significant (P > 0.05). This is the first study in Tunisia, which suggested that VDR FokI and ApaI polymorphisms might be risk factors for lung cancer development.     

PLK4 overexpression and its effect on centrosome regulation and chromosome stability in human gastric cancer

Polo-like kinase 4 (PLK4) is a centrosomal protein that is involved in the regulation of centrosome duplication. This study aimed to determine whether the genetic abnormality of PLK4 is involved in human gastric cancer. First, we examined the status of PLK4 mRNA expression in 7 gastric cancer cell lines and 48 primary gastric cancers using an RT-PCR analysis. The upregulation of PLK4 mRNA expression was detected in 57.1 % (4/7) of the gastric cancer cell lines, and a novel PLK4 variant with exon 4, but without exon 5, was identified. In the primary gastric cancers, the upregulation of PLK4 mRNA expression in the cancerous cells was detected in 50.0 % (24/48) of the cases, and this upregulation was statistically significant (P value = 0.0139). Next, we established AGS gastric cancer cells capable of inducibly expressing PLK4 using the piggyBac transposon vector system and showed that PLK4 overexpression induced centrosome amplification and chromosome instability using immunofluorescence and FISH analyses, respectively. Furthermore, PLK4 overexpression suppressed primary cilia formation. Our current findings suggested that PLK4 is upregulated in a subset of primary gastric cancers and that PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.     

Muscimol as an Ionotropic GABA Receptor Agonist

Muscimol, a psychoactive isoxazole from Amanita muscaria and related mushrooms, has proved to be a remarkably selective agonist at ionotropic receptors for the inhibitory neurotransmitter GABA. This historic overview highlights the discovery and development of muscimol and related compounds as a GABA agonist by Danish and Australian neurochemists. Muscimol is widely used as a ligand to probe GABA receptors and was the lead compound in the development of a range of GABAergic agents including nipecotic acid, tiagabine, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, (Gaboxadol^®) and 4-PIOL.     

A comparative study of PKH67, DiI,and BrdU labeling techniques for tracing rat mesenchymal stem cells

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1–3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3?±?1.6% PKH67+ and 98.4?±?1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2?±?4.6% 10 μM BrdU+ and 77.6?±?4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91?±?3.8% PKH67+, 90?±?1.5% DiI+, 91?±?0.8% 5 μM BrdU+, and 76.9?±?0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.     

Preparation of cinnamoyl-CoA and analysis of the enzyme activity of recombinant CCR protein from Populus tomentosa

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), which catalyzes the reduction of cinnamoyl-CoA esters to their respective cinnamaldehydes, is considered as a key enzyme in lignin formation. The substrates of CCR, cinnamoyl-CoA esters, are products of 4-Coumarate-CoA ligase (4CL, EC 6.2.1.12), which is an enzyme upstream of CCR. The PtCCR and Pt4CL were isolated from Populus tomentosa and expressed in E. coli. Results showed that 4CL can catalyze the conversion of hydroxycinnamic acids to cinnamoyl-CoA esters, with high efficiency. The purification of esters using SPE cartridges suggested that 40 % methanol with 0.1 M of acetic acid was the optimal elution buffer for cinnamoyl-CoA esters. The optimization of prokaryotic expression demonstrated that the best expression conditions for recombinant PtCCR was 6 h of 0.4 mM IPTG induction at 37 °C. PtCCR enzyme assay illustrated that the recombinant protein can catalyze the reduction of cinnamoyl-CoA esters. Kinetics analysis showed that feruloyl-CoA has higher affinity to PtCCR with faster reaction speed (Vmax), indicating that feruloyl-CoA was the most favorable substrate for PtCCR catalysis. The recombinant protein was expressed in E. coli, purified through affinity column chromatography, and characterized by SDS-PAGE. SPE cartridges were used to purify the ester products of the Pt4CL reaction. HPLC-MS was used to analyze the structure of esters and evaluate their purity or quantity. Furthermore, the enzyme activity of recombinant CCR to feruloyl-CoA at different pHs indicated that compartmentalization may be an important factor in lignin monomer formation.     

Intromitochondrial IκB/NF-κB signaling pathway is involved in amyloid β peptide-induced mitochondrial dysfunction

Mitochondrial dysfunction is a hallmark of amyloid β peptide (Aβ)-induced neuronal toxicity in Alzheimer’s disease (AD). However, the underlying mechanism (s) of Aβ-induced mitochondrial dysfunction is still not fully understood. There is evidence that nuclear factor-κB (NF-κB) is involved in Aβ-induced neurotoxicity and is present in mitochondria. Using HT22 murine hippocampal neuronal cells and isolated mitochondria, the present study investigated whether intramitochondrial inhibitor of NF-κB (IκB)/NF-κB signaling pathway was involved in mitochondrial dysfunction induced by Aβ. It was found that Aβ impaired mitochondrial function through a NF-κB-dependent signaling pathway. Intramitochondrial IκBα/NF-κB pathway, induced by Aβ, decreased the expression of cytochrome c oxidase subunit (COXIII) and inhibited COX activity. These results provide new insights into the mechanism underlying the neurotoxic effect of Aβ and open up new therapeutic perspectives for AD.     

Expression of microRNA-1, microRNA-133a and Hand2 protein in cultured embryonic rat cardiomyocytes

In this study, we investigated the expression of the pathway, SRF–microRNA-1/microRNA-133a–Hand2, in the Wistar rat embryonic ventricular cardiomyocytes under conventional monolayer culture. The morphological observation of the cultured cardiomyocytes and the mRNA expression levels of three vital constituent proteins, MLC-2v, N-cadherin, and connexin43, demonstrated the immaturity of these cultured cells, which was featured by less myofibril density, immature sarcomeric structure, and significantly lower mRNA expression of the three constituent proteins than those in neonatal ventricular samples. More importantly, results in this study suggest that the change of SRF–microRNA-1/microRNA-133a–Hand2 pathway results into the attenuation of the Hand2 repression in cultured cardiomyocytes. These outcomes are valuable to understand the cellular state as embryonic cardiomyocytes to be in vitro model and might be useful for the assessment of engineered cardiac tissue and cardiac differentiation of stem cells.     

Effects of Qufeng Xuanfei Decoction in Animal Model of Post-Infectious Cough

This study evaluated the effects and potential mechanisms of Qufeng Xuanfei decoction in animal model of post-infectious cough. Sixty SD rats were randomly divided into six groups (10 animals per group): control, disease model, low- (4.62 g kg^?1), medium- (9.24 g kg^?1), and high-dose (13.86 g kg^?1) decoction, and positive treatment groups (dextromethorphan hydrobromide, 8 mL kg^?1). To model post-infectious cough, all but control group animals were challenged with exposure to 50 g sawdust and 10 cigarette smokes for 30 min day^?1 for a total of 10 days, followed by subsequent exposures to lipopolysaccharide (20 µg) and capsaicin (10^?4 M) aerosols. The drugs were given by oral gavage for 15 days after which lung pathology, cell counts and cell differentials in bronchoalveolar lavage (BAL), and concentrations of neuropeptides [substance P (SP), neurokinins A (NKA) and B (NKB), and calcitonin gene-related peptide (CGRP)] in BAL (ELISA) were assessed. Compared with control group animals, significant inflammation and damage to bronchial epithelium were observed in the disease model group. A marked decrease in BAL percentages of all types of inflammatory cells was observed in the decoction-treated groups, with most changes in the medium-dose decoction group (p < 0.001 vs. disease model group). Further, airway inflammation and damage, as well as the levels of SP, NKA, NKB, and CGRP in BAL decreased the most in the medium-dose group (p < 0.001 vs. disease model group). In conclusion, medium-dose Qufeng Xuanfei decoction efficiently decreases the levels of neuropeptides, attenuates airway inflammation, and promotes recovery from disease.     

Characterization of a class II 5-enopyruvylshikimate-3-phosphate synthase with high tolerance to glyphosate from Sinorhizobium fredii

5-Enopyruvylshikimate-3-phosphate synthase (EPSP synthase) is an important enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants and microorganisms. A novel class II EPSP synthase AroA _{S. fredii} from Sinorhizobium fredii NGR234 was overexpressed in Escherichia coli BL21. It was purified to homogeneity and its catalytic properties were studied. The enzyme exhibited optimum catalytic activity at pH 8.0 and 50 °C. It was stable below 40 °C, and over a broad range of pH 5.0–9.0. The EPSP synthase was increasingly activated by 100 mM of the chlorides of NH₄ ⁺, K⁺, Na⁺ and Li⁺. Kinetic analysis of AroA _{S. fredii} suggested that the enzyme exhibited a high glyphosate tolerance and high level of affinity for phosphoenolpyruvate, which indicates the enzyme with a high potential for structural and functional studies and its potential usage for the generation of transgenic crops resistant to the herbicide.     

Crystal structure of tRNA m1A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-l-methionine

The N ¹-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m¹A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.     

Recognition of flavin mononucleotide,Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate

D-Ribitol-5-phosphate (Rbt-5-P) is an important metabolite in the pentose phosphate pathway and an integral part of bacterial cell wall polysaccharides, specifically as polyribosyl ribitol phosphate (PRP) in Haemophilus influenzae type b (Hib). The major objective of this study was to investigate whether an antibody specific to Rbt-5-P can recognize the PRP of Hib. D-Ribose-5-phosphate was reacted with proteins in the presence of sodium cyanoborohydride to obtain Rbt-5-P epitopes; 120 h reaction resulted in conjugation of ~30 and ~17 moles of Rbt-5-P/mole of BSA and OVA, respectively, based on decrease in amino groups, MALDI–TOF analyses, an increase in apparent molecular weight (SDS–PAGE) and glycoprotein staining. Immunization of rabbits with Rbt-5-P–BSA conjugate generated antibodies to Rbt-5-P as demonstrated by dot immunoblot and non-competitive ELISA. Homogeneous Rbt-5-P-specific antibody was purified from Rbt-5-P–BSA antiserum subjected to caprylic acid precipitation followed by hapten–affinity chromatography; its affinity constant is 7.1?×?10⁸ M^?1. Rbt-5-P antibody showed 100 % specificity to Rbt-5-P, ~230 %, 10 % and 3.4 % cross-reactivity to FMN, riboflavin and FAD, respectively; the antibody showed ~4 % cross-reactivity to D-ribitol and <3 % to other sugars/sugar alcohols. Rbt-5-P–specific antibody recognized Hib conjugate vaccines containing PRP which was inhibited specifically by Rbt-5-P, and also detected Hib cell-surface capsular polysaccharides by immunofluorescence. In conclusion, Rbt-5-P–protein conjugate used as an immunogen elicited antibodies binding to an epitope also present in PRP and Hib bacteria. Rbt-5-P–specific antibody has potential applications in the detection and quantification of free/bound Rbt-5-P and FMN as well as immunological recognition of Hib bacteria and its capsular polysaccharide.