Distance-controlled surface-enhanced Raman spectroscopy of nanoparticles.

Biological particles, e.g., viruses, lipid particles, and extracellular vesicles, are attracting significant research interest due to their role in biological processes and potential in practical applications, such as vaccines, diagnostics, and therapies. Their surface and interior contain many different molecules including lipids, nucleic acids, proteins, and carbohydrates. In this Letter, we show how distance-controlled surface-enhanced Raman spectroscopy (SERS) is a promising method to extract essential information from the spatial origin of the signal. This is a highly important parameter in the analysis of these biological particles. The principle of the method is demonstrated by using polystyrene (PS) beads as a biological particle model conjugated with gold nanospheres (AuNSs) functioning as distance-controlled SERS probes via biotin-streptavidin binding. By tuning the size of AuNSs, the Raman signal from the PS beads can be weakened while the signal from the biotin-streptavidin complex is enhanced.

Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles.

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell-cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols.

Novel Human Neutral Sphingomyelinase 2 Inhibitors as Potential Therapeutics for Alzheimer's Disease.

Neutral sphingomyelinase 2 (nSMase2) catalyzes the cleavage of sphingomyelin to phosphorylcholine and ceramide, an essential step in the formation and release of exosomes from cells that is critical for intracellular communication. Chronic increase of brain nSMase2 activity and related exosome release have been implicated in various pathological processes, including the progression of Alzheimer's disease (AD), making nSMase2 a viable therapeutic target. Recently, we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate 1 (PDDC), the first nSMase2 inhibitor that possesses both favorable pharmacodynamics and pharmacokinetic (PK) parameters, including substantial oral bioavailability, brain penetration, and significant inhibition of exosome release from the brain in vivo. Herein we demonstrate the efficacy of 1 (PDDC) in a mouse model of AD and detail extensive structure-activity relationship (SAR) studies with 70 analogues, unveiling several that exert similar or higher activity against nSMase2 with favorable pharmacokinetic properties.

Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS).

Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.

Comparison of time-gated surface-enhanced raman spectroscopy (TG-SERS) and classical SERS based monitoring of Escherichia coli cultivation samples.

The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here, we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman), and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples. We investigated concentration transients of glucose, acetate, AMP, and cAMP at alternating substrate availability, from deficiency to excess. Raman and SERS signals were compared to off-line metabolite analysis of carbohydrates, carboxylic acids, and nucleotides. Results demonstrate that SERS, in almost all cases, led to a higher number of identifiable signals and better resolved spectra. Spectra derived from the TG-Raman were comparable to those of micro-Raman resulting in well-discernable Raman peaks, which allowed for the identification of a higher number of compounds. In contrast, NIR-Raman provided a superior performance for the quantitative evaluation of analytes, both with and without SERS nanoparticles when using multivariate data analysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1533-1542, 2018.

Bare laser-synthesized Au-based nanoparticles as nondisturbing surface-enhanced Raman scattering probes for bacteria identification.

The ability of noble metal-based nanoparticles (NPs) (Au, Ag) to drastically enhance Raman scattering from molecules placed near metal surface, termed as surface-enhanced Raman scattering (SERS), is widely used for identification of trace amounts of biological materials in biomedical, food safety and security applications. However, conventional NPs synthesized by colloidal chemistry are typically contaminated by nonbiocompatible by-products (surfactants, anions), which can have negative impacts on many live objects under examination (cells, bacteria) and thus decrease the precision of bioidentification. In this article, we explore novel ultrapure laser-synthesized Au-based nanomaterials, including Au NPs and AuSi hybrid nanostructures, as mobile SERS probes in tasks of bacteria detection. We show that these Au-based nanomaterials can efficiently enhance Raman signals from model R6G molecules, while the enhancement factor depends on the content of Au in NP composition. Profiting from the observed enhancement and purity of laser-synthesized nanomaterials, we demonstrate successful identification of 2 types of bacteria (Listeria innocua and Escherichia coli). The obtained results promise less disturbing studies of biological systems based on good biocompatibility of contamination-free laser-synthesized nanomaterials.

Cold Tolerance of the Male Gametophyte during Germination and Tube Growth Depends on the Flowering Time.

In temperate climates, most plants flower during the warmer season of the year to avoid negative effects of low temperatures on reproduction. Nevertheless, few species bloom in midwinter and early spring despite severe and frequent frosts at that time. This raises the question of adaption of sensible progamic processes such as pollen germination and pollen tube growth to low temperatures. The performance of the male gametophyte of 12 herbaceous lowland species flowering in different seasons was examined in vitro at different test temperatures using an easy to handle testing system. Additionally, the capacity to recover after the exposure to cold was checked. We found a clear relationship between cold tolerance of the activated male gametophyte and the flowering time. In most summer-flowering species, pollen germination stopped between 1 and 5 °C, whereas pollen of winter and early spring flowering species germinated even at temperatures below zero. Furthermore, germinating pollen was exceptionally frost tolerant in cold adapted plants, but suffered irreversible damage already from mild sub-zero temperatures in summer-flowering species. In conclusion, male gametophytes show a high adaptation potential to cold which might exceed that of female tissues. For an overall assessment of temperature limits for sexual reproduction it is therefore important to consider female functions as well.

Purine analogs as phosphatidylinositol 4-kinase IIIß inhibitors.

We report on an extensive structure-activity relationship study of novel PI4K IIIß inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIß inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.

Highly Selective Phosphatidylinositol 4-Kinase IIIß Inhibitors and Structural Insight into Their Mode of Action.

Phosphatidylinositol 4-kinase IIIß is a cellular lipid kinase pivotal to pathogenesis of various RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular membranes and use them for the construction of functional replication machinery. Selective inhibitors of this enzyme are potential broad-spectrum antiviral agents, as inhibition of this enzyme results in the arrest of replication of PI4K IIIß-dependent viruses. Herein, we report a detailed study of novel selective inhibitors of PI4K IIIß, which exert antiviral activity against a panel of single-stranded positive-sense RNA viruses. Our crystallographic data show that the inhibitors occupy the binding site for the adenine ring of the ATP molecule and therefore prevent the phosphorylation reaction.

Fluorescence suppression in Raman spectroscopy using a time-gated CMOS SPAD.

A Raman spectrometer technique is described that aims at suppressing the fluorescence background typical of Raman spectra. The sample is excited with a high power (65W), short (300ps) laser pulse and the time position of each of the Raman scattered photons with respect to the excitation is measured with a CMOS SPAD detector and an accurate time-to-digital converter at each spectral point. It is shown by means of measurements performed on an olive oil sample that the fluorescence background can be greatly suppressed if the sample response is recorded only for photons coinciding with the laser pulse. A further correction in the residual fluorescence baseline can be achieved using the measured fluorescence tails at each of the spectral points.

Synthesis and evaluation of 6-aza-2'-deoxyuridine monophosphate analogs as inhibitors of thymidylate synthases, and as substrates or inhibitors of thymidine monophosphate kinase in Mycobacterium tuberculosis.

A series of 5-substituted analogs of 6-aza-2'-deoxyuridine 5'-monophosphate, 6-aza-dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin-dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N-atom in 6-aza-dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 µM). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3-(octanamido)prop-1-ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 µM). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N-atom furnished 6-aza-dTMP (1b), which still was recognized as a substrate by TMPKmt.

Synthesis and evaluation of 5-substituted 2'-deoxyuridine monophosphate analogues as inhibitors of flavin-dependent thymidylate synthase in Mycobacterium tuberculosis.

A series of 5-substituted 2'-deoxyuridine monophosphate analogues has been synthesized and evaluated as potential inhibitors of mycobacterial ThyX, a novel flavin-dependent thymidylate synthase in Mycobacterium tuberculosis. A systematic SAR study led to the identification of compound 5a, displaying an IC(50) value against mycobacterial ThyX of 0.91 µM. This derivative lacks activity against the classical mycobacterial thymidylate synthase ThyA (IC(50) > 50 µM) and represents the first example of a selective mycobacterial FDTS inhibitor.

Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system.

BACKGROUND: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control. RESULTS: By applying on-line pO2 monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels. CONCLUSION: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.