Autophagy and formation of tubulovesicular autophagosomes provide a barrier against nonviral gene delivery

Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5^?/? cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors.     

Fluorescence spectroscopy as a tool for monitoring solubility and aggregation behavior of beta-lactoglobulin after heat treatment

Denaturation and aggregation of whey proteins are of interest to the food and pharmaceutical industry due to the importance of final structure in functionality, impact on food texture, and the chemical stability of the final product. In this study, we demonstrate the potential of fluorescence spectrometry combined with multivariate chemometric methods for quantifying solubility and aggregation behavior of beta-lactoglobulin (beta-LG); a major whey protein and a frequent food ingredient. Heat-induced aggregation of beta-LG was studied under different conditions including pH, temperature and heating durations. Results showed very good agreement between the fluorescence-based predictions and measurements obtained by HPLC and gravimetric analysis regardless of the conditions. Standard normal variate (SNV), a signal preprocessing and filtering tool, was found to enhance the predictive accuracy and robustness of the fluorescence-based model.     

Synchronous fluorescence spectroscopy as a novel tool to enable PAT applications in bioprocesses

In this work, synchronous fluorescence spectroscopy (SFS) is evaluated as a new tool for real-time bioprocess monitoring of animal cell cultures. This technique presents several advantages over the traditional two-dimensional (2D) fluorometry since it provides data on various fluorescent compounds in a single spectrum, showing improved peak resolution and recording speed. Bioreactor cultures of three monoclonal antibody-producing CHO cell lines were followed in situ by both 2D and synchronous fluorometry techniques. The time profiles of the main spectral features in each data type present some differences, but principal component analysis indicated both as containing enough information to distinguish the cultures. Partial least squares regression models were then independently developed for viable cell density and antibody levels on the basis of the different fluorescence signals recorded, hiding half of the dataset for subsequent validation purposes. Regardless of the signal used, model predictions fit very well the off-line measurements; still, the synchronous spectra collected at a wavelength difference of 20 nm allowed comparable and superior performances for cell density and antibody titer, respectively, with validation accuracies higher than 91%. Therefore, SFS compares favorably with the traditional 2D approach, becoming an improved, faster option for real-time monitoring of cells and product titer over culture time. The readiness in data acquisition facilitates the design of process control strategies meeting the requirements of a PAT application.     

Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles

Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.     

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(beta-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.     

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.     

Fourier transform infrared spectroscopy as a tool to characterize molecular composition and stress response in foodborne pathogenic bacteria

Vibrational spectroscopy techniques have shown capacity to provide non-destructive, rapid, relevant information on microbial systematics, useful for classification and identification. Infrared spectroscopy enables the biochemical signatures from microbiological structures to be extracted and analyzed, in conjunction with advanced chemometrics. In addition, a number of recent studies have shown that Fourier Transform Infrared (FT-IR) spectroscopy can help understand the molecular basis of events such as the adaptive tolerance responses expressed by bacteria when exposed to stress conditions in the environment (e.g. those that cells confront in food and during food processing). The current review gives an overview of the published experimental techniques, data-processing algorithms and approaches used in FT-IR spectroscopy to assess the mechanisms of bacterial inactivation by food processing technologies and antimicrobial compounds, to monitor the spore and membrane properties of foodborne pathogens in changing environments, to detect stress-injured microorganisms in food-related environments, to assess dynamic changes in bacterial populations, and to study bacterial tolerance responses.     

New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking

We report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1-2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes.     

Use of [125I]4'-iodoflavone as a tool to characterize ligand-dependent differences in Ah receptor behavior

We have synthesized [(125)I]4'-iodoflavone to study Ah receptor (AhR)-ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This radioligand allows the comparison of AhR-ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin. Specific binding of [(125)I]4'-iodoflavone with the AhR was detected as a single radioactive peak ( approximately 9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A approximately 6.7 S form of radioligand-bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [(125)I]4'-iodoflavone-AhR 6 S complex, compared with [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin, was recovered in nuclear extracts. A comparison of the ability of 4'-iodoflavone and TCDD to cause time-dependent translocation of AhR-yellow fluorescent protein revealed that 4'-iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [(125)I]4'-iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR.     

Fourier transform infrared spectroscopy as a new tool to determine rosmarinic acid in situ

A new procedure has been developed for the in situ FT-IR determination of rosmarinic acid (RA) in suspension cultures of Lavandula officinalis. The method involves sample preparation on ZnSe crystals or microplates from silicon, and measuring absorbance spectra between 4000 and 700 cm(-1). First derivative spectra were analysed after normalisation using partial least square (PLS) algorithm. The correlation between spectral analysis and HPLC measurements of cell extracts shows that the FT-IR procedure is suitable for qualitative and quantitative analyses of RA in cell suspension cultures.     

Evaluation of FT-IR spectroscopy as a tool to quantify bacteria in binary mixed cultures

Fourier-transform infrared (FT-IR) spectroscopy is known as a high-resolution method for the rapid identification of pure cultures of microorganisms. Here, we evaluated FT-IR as a method for the quantification of bacterial populations in binary mixed cultures consisting of Pseudomonas putida and Rhodococcus ruber. A calibration procedure based on Principal Component Regression was developed for estimating the ratio of the bacterial species. Data for method calibration were gained from pure cultures and artificially assembled communities of known ratios of the two member populations. Moreover, to account for physiological variability, FT-IR measurements were performed with organisms sampled at different growth phases. Measurements and data analyses were subsequently applied to growing mixed cultures revealing that growth of R. ruber was almost completely suppressed in co-culture with P. putida. Population ratios obtained by fatty acid analysis as an independent reference method were in high agreement with the FT-IR derived ratios.     

Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization

To probe the complexity of the cell membrane organization and dynamics, it is important to obtain simple physical observables from experiments on live cells. Here we show that fluorescence correlation spectroscopy (FCS) measurements at different spatial scales enable distinguishing between different submicron confinement models. By plotting the diffusion time versus the transverse area of the confocal volume, we introduce the so-called FCS diffusion law, which is the key concept throughout this article. First, we report experimental FCS diffusion laws for two membrane constituents, which are respectively a putative raft marker and a cytoskeleton-hindered transmembrane protein. We find that these two constituents exhibit very distinct behaviors. To understand these results, we propose different models, which account for the diffusion of molecules either in a membrane comprising isolated microdomains or in a meshwork. By simulating FCS experiments for these two types of organization, we obtain FCS diffusion laws in agreement with our experimental observations. We also demonstrate that simple observables derived from these FCS diffusion laws are strongly related to confinement parameters such as the partition of molecules in microdomains and the average confinement time of molecules in a microdomain or a single mesh of a meshwork.     

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.     

Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals

Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.     

Attenuated total reflection IR spectroscopy as a tool to investigate the orientation and tertiary structure changes in fusion proteins

Membrane fusion proceeds via a merging of two lipid bilayers and a redistribution of aqueous contents and bilayer components. It involves transition states in which the phospholipids are not arranged in bilayers and in which the monolayers are highly curved. Such transition states are energetically unfavourable since biological membranes are submitted to strong repulsive hydration electrostatic and steric barriers. Viral membrane proteins can help to overcome these barriers. Viral proteins involved in membrane fusion are membrane associated and the presence of lipids restricts drastically the potential of methods (RMN, X-ray crystallography) that have been used successfully to determine the tertiary structure of soluble proteins. We describe here how IR spectroscopy allows to solve some of the problems related to the lipid environment.The principles of the method, the experimental setup and the preparation of the samples are briefly described. A few examples illustrate how attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy can be used to gain information on the orientation and the accessibility to the water phase of the fusogenic domain of viral proteins. Recent developments suggest that the method could also be used to detect changes located in the membrane domains and to identify intermediate structural states involved in the fusion process.     

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.     

Raman spectroscopy and PCA‐SVM as a non‐invasive diagnostic tool to identify and classify qualitatively glycated hemoglobin levels in vivo

In this study we identify and classify high and low levels of glycated hemoglobin (HbA1c) in healthy volunteers (HV) and diabetic patients (DP). Overall, 86 subjects were evaluated. The Raman spectrum was measured in three anatomical regions of the body: index fingertip, right ear lobe, and forehead. The measurements were performed to compare the difference between the HV and DP (22 well controlled diabetic patients (WCDP) (HbA1c <6.5%), and 49 not controlled diabetic patients (NCDP) (HbA1c ≥6.5%)). Multivariable methods such as principal components analysis (PCA) combined with support vector machine (SVM) were used to develop effective diagnostic algorithms for classification among these groups. The forehead of HV versus WCDP showed the highest sensitivity (100%) and specificity (100%). Sensitivity (100%) and specificity (60%), were highest in the forehead of WCDP, versus NCDP. In HV versus NCDP, the fingertip had the highest sensitivity (100%) and specificity (80%). The efficacy of the diagnostic algorithm by receiver operating characteristic (ROC) curve was confirmed. Overall, our study demonstrated that the

combination of Raman spectroscopy and PCA‐SVM are feasible non‐invasive diagnostic tool in diabetes to classify qualitatively high and low levels of HbA1c in vivo.