Proteins In Cell Lysates Research Articles

An optimized ROP6 mRNA construct successfully expressed immunogenic Toxoplasma gondii ROP6 protein in cell culture

Toxoplasma gondii is an apicomplexan parasite infecting all mammals including humans and causes toxoplasmosis. There is no vaccine available for humans and thus vaccine development efforts continue using novel antigens and/or vaccine platforms. Since our previous microarray screening study showed that ROP6 is a suitable antigen to be used in vaccine studies, in this study, we aimed to design an optimized mRNA construct encoding the ROP6 protein and then demonstrate its efficiency and immunogenicity using in vitro methods. For this, we constructed a pT7CFE1-Chis/ROP6 vector encoding optimized ROP6 mRNA containing EMCV 5′UTR with IRES and a 20 nucleotides fragment from alpha globin 3′ UTR. Then, we generated the optimized ROP6 mRNAs with anti-reverse cap analogue (ARCA) and approximately 150 nucleotide long poly-A tail. Next, HEK293T cells were transfected with the optimized ROP6 mRNAs to show recombinant ROP6 protein expression capability. Moreover, we expressed in vitro recombinant ROP6 protein in HeLa cell lysate using the pT7CFE1-Chis/ROP6 vector to reveal the immunogenicity of recombinant ROP6 protein using sera samples collected from mice infected with PRU strain of T. gondii. The IFA and Western blot results showed that the optimized ROP6 mRNAs successfully expressed the recombinant ROP6 protein in HEK293T cells. Moreover the recombinant ROP6 protein expressed in HeLa cell lysate strongly reacted with sera samples collected from mice. The absorbance difference detected among positive and negative mice serum samples analyzed was statistically significant, indicating that the recombinant ROP6 protein was immunogenic (P = 0.0003). In conclusion, this study demonstrated that the optimized ROP6 mRNAs can be used in the development of mRNA vaccines against toxoplasmosis.

Analysis of Brain Protein Stability Changes in a Mouse Model of Alzheimer's Disease.

The stability of proteins from rates of oxidation (SPROX), thermal proteome profiling (TPP), and limited proteolysis (LiP) techniques were used to profile the stability of ∼2500 proteins in hippocampus tissue cell lysates from 2- and 8-months-old wild-type (C57BL/6J; n = 7) and transgenic (5XFAD; n = 7) mice with five Alzheimer's disease (AD)-linked mutations. Approximately 200-500 protein hits with AD-related stability changes were detected by each technique at each age point. The hit overlap from technique to technique was low, and all of the techniques generated protein hits that were more numerous and largely different from those identified in protein expression level analyses, which were also performed here. The hit proteins identified by each technique were enriched in a number of the same pathways and biological processes, many with known connections to AD. The protein stability hits included 25 high-value conformation biomarkers with AD-related stability changes detected using at least 2 techniques at both age points. Also discovered were subunit- and age-specific AD-related stability changes in the proteasome, which had reduced function at both age points. The different folding stability profiles of the proteasome at the two age points are consistent with a different mechanism for proteasome dysfunction at the early and late stages of AD.

Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli

Human interferon gamma (hIFN-γ) plays a pivotal role as a soluble cytokine with diverse functions in both innate and adaptive immunity. In a previous investigation, we pinpointed three critical amino acid residues, i.e., threonine (T) 27, phenylalanine (F) 29, and leucine (L) 30, on the IFN-γ structure, which are integral to the epitope recognized by anti-IFN-γ autoantibodies. It is crucial to impede the interaction between this epitope and autoantibodies for effective therapy in adult-onset immunodeficiency (AOID). However, the challenge arises from the diminished solubility of the T27AF29L30A mutant in Escherichia coli BL21(DE3). This study delves into a targeted strategy aimed at improving the soluble expression of IFN-γ T27AF29AL30A. This is achieved through the utilization of five chaperone plasmids: pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16. These plasmids, encoding cytoplasmic chaperones, are co-expressed with the IFN-γ mutant in E.coli BL21(DE3), and we meticulously analyze the proteins in cell lysate and inclusion bodies using SDS-PAGE and Western blotting. Our findings reveal the remarkable efficacy of pG-KJE8, which houses cytoplasmic chaperones DnaK-DnaJ-GrpE and GroEL-GroES, in significantly enhancing the solubility of IFN-γ T27AF29AL30A. Importantly, this co-expression not only addresses solubility concerns but also preserves the functional dimerized structure, as confirmed by sandwich ELISA. This promising outcome signifies a significant step forward in developing biologic strategies for AOID.

Preparation of a mouse monoclonal antibody against the NS1 protein of respiratory syncytial virus

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody's application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.

A microfiber biosensor for detection of AKT protein in human embryonic kidney cell lysate based on the vernier effect

The AKT protein is closely related to the cancer and diabetes. Rapid detection of AKT is very important in medical diagnosis. This paper proposed a parallel connected tapered seven-core fiber (PTSCF) biosensor to utilize vernier effect to improve sensitivity. Experimental results showed that 5.99 times improvements have been achieved in the refractive index sensing in the ranges of 1.333–1.337. By functionalizing AKT-Ab on the PTSCF sensor surface, a fast detection (<40 min) and an ultra-low limit of detection (LoD) of 0.044 ng/mL have been achieved for AKT protein solutions. The proposed PTSCF biosensor has been applied to detect AKT proteins in human embryonic kidney cell lysates and compared with those obtained by the western blotting (WB) method. Experimental results showed that the LoD of the PTSCF biosensor is 400-fold less than that of WB method. The proposed PTSCF biosensor holds great potential for applications in various medical fields, including intra-operative rapid detection, animal experiments, and pathological research.

Transcriptomic and proteomic study of cancer cell lines exposed to actinomycin D and nutlin-3a reveals numerous, novel candidates for p53-regulated genes

Transcriptomic analyses have revealed hundreds of p53-regulated genes; however, these studies used a limited number of cell lines and p53-activating agents. Therefore, we searched for candidate p53-target genes by employing stress factors and cell lines never before used in a high-throughput search for p53-regulated genes. We performed RNA-Seq on A549 cells exposed to camptothecin, actinomycin D, nutlin-3a, as well as a combination of actinomycin D and nutlin-3a (A + N). The latter two substances synergise upon the activation of selected p53-target genes. A similar analysis was performed on other cell lines (U-2 OS, NCI–H460, A375) exposed to A + N. To identify proteins in cell lysates or those secreted into a medium of A549 cells in control conditions or treated with A + N, we employed mass spectrometry. The expression of selected genes strongly upregulated by A + N or camptothecin was examined by RT-PCR in p53-deficient cells and their controls. We found that p53 participates in the upregulation of: ACP5, APOL3, CDH3, CIBAR2, CRABP2, CTHRC1, CTSH, FAM13C, FBXO2, FRMD8, FRZB, GAST, ICOSLG, KANK3, KCNK6, KLRG2, MAFB, MR1, NDRG4, PTAFR, RETSAT, TMEM52, TNFRSF14, TRANK1, TYSND1, WFDC2, WFDC5, WNT4 genes. Twelve of these proteins were detected in the secretome and/or proteome of treated cells. Our data generated new hypotheses concerning the functioning of p53. Many genes activated by A + N or camptothecin are also activated by interferons, indicating a noticeable overlap between transcriptional programs of p53 and these antiviral cytokines. Moreover, several identified genes code for antagonists of WNT/β-catenin signalling pathways, which suggests new connections between these two cancer-related signalling systems. One of these antagonists is DRAXIN. Previously, we found that its gene is activated by p53. In this study, using mass spectrometry and Western blotting, we detected expression of DRAXIN in a medium of A549 cells exposed to A + N. Thus, this protein functions not only in the development of the nervous system, but it may also have a new cancer-related function.

Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

Site-Specific Anchoring a Luminescent Tag in a Protein with Non-Emissive Iridium(III) Complex.

Site-specific modification of proteins with synthetic fluorescent tag effectively improves the resolution of imaging, and such a labeling method with negligible three-dimensional structural perturbations and minimal impact on the biological functions of proteins is of high interest to dissect the high-resolution activities of biomolecules in complex systems. To this end, several non-emissive iridium(III) complexes [Ir(C-N)2 (H2 O)2 ]+ OTF- (C-N denotes various cyclometalated ligands) were designed and synthesized. These complexes were tested for attaching a protein by coordinating to H/X (HisMet, HisHis, and HisCys) that are separated by i and i+4 in α-helix. Replacement of the two labile water ligands in the iridium(III) complex by a protein HisHis pair increases the luminescent intensity up to over 100 folds. This labeling approach has been demonstrated in a highly specific and efficient manner in a number of proteins, and it is also feasible for labeling target proteins in cell lysates.

Highly tunable bimane-based fluorescent probes: design, synthesis, and application as a selective amyloid binding dye.

Small molecule fluorescent probes are indispensable tools for a broad range of biological applications. Despite many probes being available, there is still a need for probes where photophysical properties and biological selectivity can be tuned as desired. Here, we report the rational design and synthesis of a palette of fluorescent probes based on the underexplored bimane scaffold. The newly developed probes with varied electronic properties show tunable absorption and emission in the visible region with large Stokes shifts. Probes featuring electron-donating groups exhibit rotor effects that are sensitive to polarity and viscosity by "intramolecular charge transfer" (ICT) and twisted intramolecular charge transfer (TICT) mechanisms, respectively. These properties enable their application as "turn-on" fluorescent probes to detect fibrillar aggregates of the α-synuclein (αS) protein that are a hallmark of Parkinson's disease (PD). One probe shows selective binding to αS fibrils relative to soluble proteins in cell lysates and amyloid fibrils of tau and amyloid-β. Finally, we demonstrate the diagnostic potential of the probe in selectively detecting αS fibrils amplified from PD with dementia (PDD) patient samples.

Hsp90α forms condensate engaging client proteins with RG motif repeats.

Hsp90α, a pivotal canonical chaperone, is renowned for its broad interaction with numerous protein clients to maintain protein homeostasis, chromatin remodeling, and cell growth. Recent studies indicate its role in modifying various components of membraneless organelles (MLOs) such as stress granules and processing bodies, suggesting its participation in the regulation of protein condensates. In this study, we found that Hsp90α possesses an inherent ability to form dynamic condensates in vitro. Utilizing LC-MS/MS, we further pinpointed proteins in cell lysates that preferentially integrate into Hsp90α condensates. Significantly, we observed a prevalence of RG motif repeats in client proteins of Hsp90α condensates, many of which are linked to various MLOs. Moreover, each of the three domains of Hsp90α was found to undergo phase separation, with numerous solvent-exposed negatively charged residues on these domains being crucial for driving Hsp90α condensation through multivalent weak electrostatic interactions. Additionally, various clients like TDP-43 and hnRNPA1, along with poly-GR and PR dipeptide repeats, exhibit varied impacts on the dynamic behavior of Hsp90α condensates. Our study spotlights various client proteins associated with Hsp90α condensates, illustrating its intricate adaptive nature in interacting with diverse clients and its functional adaptability across multiple MLOs.

Efficient delivery of the immunodominant genes of African swine fever virus by adeno-associated virus serotype 2.

Adeno-associated virus serotype 2 (AAV2) represents a promising basis for developing a virus-vector vaccine against African swine fever (ASF). This study aimed to create genetic constructs based on AAV2 to deliver the immunodominant genes of ASF virus (ASFV) and to evaluate their functionality in vitro. The efficiency and specificity of transgene expression, as well as their non-toxicity in cells of target animals, were evaluated. Bioinformatics analysis methods were used to identify the immunodominant genes of ASFV. The target genes B646L, E183L, CP204L, and CP530R were identified and subsequently cloned into the pAAV-MCS vector. Assembly of recombinant AAV2 (rAAV2) was performed by cotransfection of AAV293 cells with the following plasmids: pAAV-MCS with the gene of interest, envelope, and packaging. Quantitative polymerase chain reaction was used to determine the AAV2 titer. The functionality of the constructs was evaluated in HEK293 and SPEV cells by determining the presence of mature proteins in the cell lysate and the expression levels of messenger RNA. The specificity of the target proteins in cell lysates was confirmed by Western blotting. The proposed AAV2 assembly protocol makes it possible to achieve a concentration of mature viral particles of at least 280 billion/mL of virus-containing material. The rAAV2 could effectively transduce host SPEV cells. The expression of both cistrons was detectable during the transduction of cells; therefore, the combined expression of immunogens in the cells of target animals should be possible using this method. This study demonstrated the potential of using genetic constructs based on AAV2 for the delivery of ASFV genes in vitro.

Rational surface modification of zirconium-based metal–organic framework nanomaterials for the adsorption and recovery of coloured drugs and proteins from aqueous solutions

Coloured drugs and proteins in pharmaceutical wastewater cause environmental pollution. Metal–organic frameworks (MOFs) can be used for the adsorption and recovery of pollutants from wastewater, however selective adsorption and desorption on MOFs are challenging. Herein, UiO-66 and various polyethylene glycol (PEG)-modified MOFs (UiO-66 @mPEG-PO3 and UiO-66 @DSPE) were synthesised to selectively adsorb and separate model drugs [curcumin (CUR) and myricetin (MYR)] and proteins [Bovine serum albumin (BSA) and ovalbumin (OVA)]. Results show that UiO-66 adsorption efficiency for CUR and MYR in aqueous solution reached 93% and 98%, respectively. Additionally, CUR and MYR adsorbed on the UiO-66 surface exhibit good antioxidant activity during the recycling process. X-ray photoelectron spectroscopy and Hirshfeld surface analysis indicate that the adsorption mechanisms of both CUR and MYR by UiO-66 are belonging to the intermolecular interactions. The UiO-66 @mPEG-PO3 significantly reduces the surface area of UiO-66 (216 m2/g) but does not alter the adsorption behaviour of drugs. In the process of desorption and separation of CUR and MYR, the UiO-66 @PEG-PO3 exhibits a higher desorption rate than UiO-66. By contrast, the UiO-66 @DSPE MOFs exhibits optimal protein adsorption and selectively enhances the desorption of OVA rather than BSA in a 40% acetonitrile solution. This method can be used to capture and detect OVA proteins in cell lysates. Rational surface modification is an effective way to improve the adsorption selectivity and recovery efficiency of zirconium-based MOFs, providing an effective way to use MOFs in treating novel water pollutants.

TXNL1 has dual functions as a redox active thioredoxin-like protein as well as an ATP- and redox-independent chaperone

TXNL1 (also named TRP32, for thioredoxin related protein of 32 kDa) is a cytosolic thioredoxin-fold protein expressed in all cell types and conserved from yeast to mammals, but with yet poorly known function. Here, we expressed and purified human TXNL1 together with several Cys-to-Ser variants, characterizing their enzymatic properties. TXNL1 could reduce disulfides in insulin, cystine and glutathione disulfide (GSSG) in reactions coupled to thioredoxin reductase (TXNRD1, TrxR1) using NADPH, similarly to thioredoxin (TXN, Trx1), but with lower catalytic efficacy due to at least one order of magnitude higher Km of TrxR1 for TXNL1 compared to Trx1. However, in sharp contrast to Trx1, we found that TXNL1 also had efficient chaperone activity that did not require ATP. TXNL1 made non-covalent complexes with reduced insulin, thereby keeping it in solution, and TXNL1 provided chaperone function towards whole cell lysate proteins by preventing their aggregation during heating. The chaperone activities of TXNL1 did not require its redox activity or any dithiol-disulfide exchange reactions, as revealed using Cys-to-Ser substituted variants, as well as a maintained chaperone activity of TXNL1 also in the absence of TrxR1 and NADPH. These results reveal that TXNL1 has dual functions, supporting TrxR1-driven redox activities in disulfide reduction reactions, as well as being an ATP-independent chaperone that does not require involvement of its redox activity.

Golgi α-mannosidases regulate cell surface N-glycan type and ectodomain shedding of the transmembrane protease corin

Corin is a transmembrane protease that activates natriuretic peptides on the cell membrane. Reduced cell surface targeting or increased ectodomain shedding disrupts cell membrane homeostasis of corin, thereby impairing its cell surface expression and enzyme activity. N-glycans are essential in corin ectodomain shedding. Lack of N-glycans promotes corin ectodomain shedding in the juxtamembrane and frizzled-1 domains. The nascent N-glycans, transferred onto the polypeptide of corin, undergo multistep N-glycan processing in the endoplasmic reticulum and Golgi. It remains unclear how trimming by Golgi α-mannosidases, the critical N-glycan processing steps in N-glycan maturation, may regulate corin biosynthesis. In this study, we examined the effects of kifunensine and swainsonine, the inhibitors for α-mannosidases I and II, on corin expression and function. Western analysis of corin proteins in cell lysates and conditioned media from the inhibitor-treated corin-stable HEK293cells and AC16cells showed that both α-mannosidases I and II were required to maintain complex N-glycans on cell surface corin and protect corin from ectodomain shedding in the juxtamembrane and frizzled-1 domains. Cell viability analysis revealed that inhibition of α-mannosidase I or II sensitized cardiomyocytes to hydrogen peroxide-induced injury via regulating corin. Moreover, either one of the two coding genes was sufficient to perform Golgi α-mannosidase I trimming of N-glycans on corin. Similarly, this sufficiency was observed in Golgi α-mannosidase II-coding genes. Inhibition of ectodomain shedding restored corin zymogen activation from kifunensine- or swainsonine-induced reduction. Together, our results show the important roles of Golgi α-mannosidases in maintaining cell membrane homeostasis and biological activities of corin.

Inhibition of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and atherosclerotic lesion development in ApoE-/- mice.

Pyroptosis is a form of pro-inflammatory cell death that can be mediated by gasdermin D (GSDMD) activation induced by inflammatory caspases such as caspase-1. Emerging evidence suggests that targeting GSDMD activation or pyroptosis may facilitate the reduction of vascular inflammation and atherosclerotic lesion development. The current study investigated the therapeutic effects of inhibition of GSDMD activation by the novel GSDMD inhibitor N-Benzyloxycarbonyl-Leu-Leu-Ser-Asp(OMe)-fluoromethylketone (Z-LLSD-FMK), the specific caspase-1 inhibitor N-Benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (Z-YVAD-FMK), and a combination of both on atherosclerosis in ApoE-/- mice fed a western diet at 5weeks of age, and further determined the efficacy of these polypeptide inhibitors in bone marrow-derived macrophages (BMDMs). In vivo studies there was plaque formation, GSDMD activation, and caspase-1 activation in aortas, which increased gradually from 6 to 18weeks of age, and increased markedly at 14 and 18weeks of age. ApoE-/- mice were administered Z-LLSD-FMK (200µg/day), Z-YVAD-FMK (200µg/day), a combination of both, or vehicle control intraperitoneally from 14 to 18weeks of age. Treatment significantly reduced lesion formation, macrophage infiltration in lesions, protein levels of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and pyroptosis-related proteins such as activated caspase-1, activated GSDMD, cleaved interleukin(IL)-1β, and high mobility group box 1 in aortas. No overt differences in plasma lipid contents were detected. In vitro treatment with these polypeptide inhibitors dramatically decreased the percentage of propidium iodide-positive BMDMs, the release of lactate dehydrogenase and IL-1β, and protein levels of pyroptosis-related proteins both in supernatants and cell lysates elevated by lipopolysaccharide + nigericin. Notably however, there were no significant differences in the above-mentioned results between the Z-LLSD-FMK group and the Z-YVAD-FMK group, and the combination of both did not yield enhanced effects. These findings indicate that suppression of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and lesion development in ApoE-/- mice.

Method for quantitative analysis of the marker substrate ABCB1-protein fexofenadine in Caco-2 cell lysate

One way to analyze the activity of the ABCB1 protein is to assess the accumulation of its substrate fexofenadine (F.) inside the test cells. The goal is to develop and validate a method for the quantitative analysis of F. in Caco-2 cell lysate using HPLC-MS/MS. Materials and methods. Caco-2 cell lysate was used as a matrix. The analysis was performed on an "Ultimate 3000" chromatograph with a TSQ Fortis triple quadrupole mass detector, a UCT Selectra C18 4.6 mm*100 mm 5 µm column in a gradient elution mode. The mobile phase rate was 0.3 ml/min, the sample volume was 20 µl, the ionization mode was positive, and the internal standard was amantadine (ng/ml). Sample preparation — precipitation of cell lysate protein with acetonitrile. The method was validated for the following parameters: selectivity, linearity, lower limit of quantitation (LLOQ), correctness, precision, sample transfer and sample stability. Results. Chromatograms of the blank lysate of Caco-2 cells showed no peaks with retention times characteristic of F. (5.70 min) and amantadine (3.58 min). NPKO F. was 0.5 ng/ml. F.'s transfer did not exceed 20% of NPKO, and amantadine — 5%. Based on the results of the analysis of three series of calibration standards (0.5; 1; 1.5; 5; 10; 25; 40; 50 ng/ml), linear regression equations were obtained, the correlation coefficients exceeded 0.99. Accuracy and precision were assessed within and between cycles by analyzing F. solutions in the matrix (0.5; 1.5; 25 and 40 ng/ml) within three cycles. The parameters did not exceed 20% for LLPO and 15% for other points. The stability of F. solutions (1.5 and 40 ng/ml) in the lysate was analyzed during storage at room temperature, after 3-fold freezing-thawing, storage at -80 °C for 60 days, after sample preparation and being in the autosampler for 24 hours. The accuracy was within 15% of the nominal values. Conclusions. A method for the quantitative determination of F. in Caco-2 cell lysate using HPLC-MS/MS has been developed and validated.

Prostaglandin F2α requires activation of calcium-dependent signalling to trigger inflammation in human myometrium.

Preterm birth is one of the major causes of neonatal morbidity and mortality across the world. Both term and preterm labour are preceded by inflammatory activation in uterine tissues. This includes increased leukocyte infiltration, and subsequent increase in chemokine and cytokine levels, activation of pro-inflammatory transcription factors as NF-κB and increased prostaglandin synthesis. Prostaglandin F2α (PGF2α) is one of the myometrial activators and stimulators. Here we investigated the role of PGF2α in pro-inflammatory signalling pathways in human myometrial cells isolated from term non-labouring uterine tissue. Primary myometrial cells were treated with G protein inhibitors, calcium chelators and/or PGF2α. Nuclear extracts were analysed by TranSignal cAMP/Calcium Protein/DNA Array. Whole cell protein lysates were analysed by Western blotting. mRNA levels of target genes were analysed by RT-PCR. The results show that PGF2α increases inflammation in myometrial cells through increased activation of NF-κB and MAP kinases and increased expression of COX-2. PGF2α was found to activate several calcium/cAMP-dependent transcription factors, such as CREB and C/EBP-β. mRNA levels of NF-κB-regulated cytokines and chemokines were also elevated with PGF2α stimulation. We have shown that the increase in PGF2α-mediated COX-2 expression in myometrial cells requires coupling of the FP receptor to both Gαq and Gαi proteins. Additionally, PGF2α-induced calcium response was also mediated through Gαq and Gαi coupling. In summary, our findings suggest that PGF2α-induced inflammation in myometrial cells involves activation of several transcription factors - NF-κB, MAP kinases, CREB and C/EBP-β. Our results indicate that the FP receptor signals via Gαq and Gαi coupling in myometrium. This work provides insight into PGF2α pro-inflammatory signalling in term myometrium prior to the onset of labour and suggests that PGF2α signalling pathways could be a potential target for management of preterm labour.

Genomic DNA causes membrane fouling during sterile filtration of cell lysates

During biopharmaceutical production, efficient removal of impurities via filtration is essential. It is desirable to achieve high filter capacities along with a high degree of clearance. However, the complexity of bacterial cell lysates makes it difficult to properly estimate the filtration behavior. Here we investigated how host cell-derived impurities impacted the performance of commercially available sterile filters, with a focus on characterizing the dsDNA-induced decrease of filter capacity. Pressure flow curves at a constant flow, combined with particle analytics (e.g., nanoparticle-tracking analysis), indirectly revealed that dsDNA had a major influence on rapid membrane fouling. The observed phenomenon was more pronounced when using filters with small pore sizes (<0.2 µm). Filter capacity could be substantially increased by reducing the size of the present dsDNA fragments, or by decreasing the potential for dsDNA-driven electrostatic interactions with positively charged molecules. In general, our present findings highlight the importance of monitoring dsDNA even during the primary recovery of proteins in cell lysates.

Cell-Permeable Ubiquitin and Histone Tools for Studying Post-translational Modifications.

Protein post-translational modifications (PTMs) regulate nearly all biological processes in eukaryotic cells, and synthetic PTM protein tools are widely used to detect the activity of the related enzymes and identify the interacting proteins in cell lysates. Recently, the study of these enzymes and the interacting proteome has been accomplished in live cells using cell-permeable PTM protein tools. In this concept, we will introduce cell penetrating techniques, the syntheses of cell-permeable PTM protein tools, and offer some future perspective.

Identification of photocrosslinking peptide ligands by mRNA display

Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific, efficient crosslinker can require significant optimisation. We report a modified mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.