Phytochemical and biological assessment of secondary metabolites isolated from a rhizosphere strain, Sphingomonas sanguinis DM of Datura metel.

BACKGROUND: The plant roots excrete a large number of organic compounds into the soil. The rhizosphere, a thin soil zone around the roots, is a hotspot for microbial activity, making it a crucial component of the soil ecosystem. Secondary metabolites produced by rhizospheric Sphingomonas sanguinis DM have sparked significant curiosity in investigating their possible biological impacts. METHODS: A bacterial strain has been isolated from the rhizosphere of Datura metel. The bacterium's identification, fermentation, and working up have been outlined. The ethyl acetate fraction of the propagated culture media of Sphingomonas sanguinis DM was fractioned and purified using various chromatographic techniques. The characterization of the isolated compounds was accomplished through the utilization of various spectroscopic techniques, such as UV, MS, 1D, and 2D-NMR. Furthermore, the evaluation of their antimicrobial activity was conducted using the agar well diffusion method, while cytotoxicity was assessed using the MTT test. RESULTS: The extract from Sphingomonas sanguinis DM provided two distinct compounds: n-dibutyl phthalic acid (1) and Bis (2-methyl heptyl) phthalate (2) within its ethyl acetate fraction. Furthermore, the 16S rRNA gene sequence of Sphingomonas sanguinis DM has been registered under the NCBI GenBank database with the accession number PP422198. The bacterial extract exhibited its effect against gram-positive bacteria, inhibiting Streptococcus mutans (12.6 ± 0.6 mm) and Staphylococcus aureus (10.6 ± 0.6 mm) compared to standard antibiotics. Conversely, compound 1 showed a considerable effect against phytopathogenic fungi such as Alternaria alternate (56.3 ± 10.6 mm) and Fusarium oxysporum (21.3 ± 1.5 mm) with a MIC value of 17.5 µg/mL. However, it was slightly active against Klebsiella pneumonia (11.0 ± 1.0 mm). Furthermore, compound 2 was the most active metabolite, having a significant antimicrobial efficacy against Rhizoctonia solani (63.6 ± 1.1 mm), Pseudomonas aeruginosa (16.7 ± 0.6 mm), and Alternaria alternate (20.3 ± 0.6 mm) with MIC value at 15 µg/mL. In addition, compound 2 exhibited the most potency against hepatocellular (HepG-2) and skin (A-431) carcinoma cell lines with IC50 values of 107.16 µg/mL and 111.36 µg/mL, respectively. CONCLUSION: Sphingomonas sanguinis DM, a rhizosphere bacterium of Datura metel, was studied for its phytochemical and biological characteristics, resulting in the identification of two compounds with moderate antimicrobial and cytotoxic activities.

Separation of microalgae from bacterial contaminants using spiral microchannel in the presence of a chemoattractant.

Cell separation using microfluidics has become an effective method to isolate biological contaminants from bodily fluids and cell cultures, such as isolating bacteria contaminants from microalgae cultures and isolating bacteria contaminants from white blood cells. In this study, bacterial cells were used as a model contaminant in microalgae culture in a passive microfluidics device, which relies on hydrodynamic forces to demonstrate the separation of microalgae from bacteria contaminants in U and W-shaped cross-section spiral microchannel fabricated by defocusing CO2 laser ablation. At a flow rate of 0.7 ml/min in the presence of glycine as bacteria chemoattractant, the spiral microfluidics devices with U and W-shaped cross-sections were able to isolate microalgae (Desmodesmus sp.) from bacteria (E. coli) with a high separation efficiency of 92% and 96% respectively. At the same flow rate, in the absence of glycine, the separation efficiency of microalgae for U- and W-shaped cross-sections was 91% and 96%, respectively. It was found that the spiral microchannel device with a W-shaped cross-section with a barrier in the center of the channel showed significantly higher separation efficiency. Spiral microchannel chips with U- or W-shaped cross-sections were easy to fabricate and exhibited high throughput. With these advantages, these devices could be widely applicable to other cell separation applications, such as separating circulating tumor cells from blood.

Efficacy of tyrosine kinase inhibitors examined by a combination of Raman micro-spectroscopy and a deep wavelet scattering-based multivariate analysis framework.

HER2 is a crucial therapeutic target in breast cancer, and the survival rate of breast cancer patients has increased because of this receptor's inhibition. However, tumors have shown resistance to this therapeutic strategy due to oncogenic mutations that decrease the binding of several HER2-targeted drugs, including lapatinib, and confer resistance to this drug. Neratinib can overcome this drug resistance and effectively inhibit HER2 signaling and tumor growth. In the present study, we examined the efficacy of lapatinib and neratinib using breast cancer cells by Raman microscopy combined with a deep wavelet scattering-based multivariate analysis framework. This approach discriminated between control cells and drug-treated cells with high accuracy, compared to classical principal component analysis. Both lapatinib and neratinib induced changes in the cellular biochemical composition. Furthermore, the Raman results were compared with the results of several in vitro assays. For instance, drug-treated cells exhibited (i) inhibition of ERK and AKT phosphorylation, (ii) inhibition of cellular proliferation, (iii) cell-cycle arrest, and (iv) apoptosis as indicated by western blotting, real-time cell analysis (RTCA), cell-cycle analysis, and apoptosis assays. Thus, the observed Raman spectral changes are attributed to cell-cycle arrest and apoptosis. The results also indicated that neratinib is more potent than lapatinib. Moreover, the uptake and distribution of lapatinib in cells were visualized through its label-free marker bands in the fingerprint region using Raman spectral imaging. These results show the prospects of Raman microscopy in drug evaluation and presumably in drug discovery.

The subcellular arrangement of alpha-synuclein proteoforms in the Parkinson's disease brain as revealed by multicolor STED microscopy.

Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)-including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn-accumulate in Lewy bodies (LBs) in different regions of the Parkinson's disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis.

Time-resolved spectroscopic and electrophysiological data reveal insights in the gating mechanism of anion channelrhodopsin.

Channelrhodopsins are widely used in optogenetic applications. High photocurrents and low current inactivation levels are desirable. Two parallel photocycles evoked by different retinal conformations cause cation-conducting channelrhodopsin-2 (CrChR2) inactivation: one with efficient conductivity; one with low conductivity. Given the longer half-life of the low conducting photocycle intermediates, which accumulate under continuous illumination, resulting in a largely reduced photocurrent. Here, we demonstrate that for channelrhodopsin-1 of the cryptophyte Guillardia theta (GtACR1), the highly conducting C = N-anti-photocycle was the sole operating cycle using time-resolved step-scan FTIR spectroscopy. The correlation between our spectroscopic measurements and previously reported electrophysiological data provides insights into molecular gating mechanisms and their role in the characteristic high photocurrents. The mechanistic importance of the central constriction site amino acid Glu-68 is also shown. We propose that canceling out the poorly conducting photocycle avoids the inactivation observed in CrChR2, and anticipate that this discovery will advance the development of optimized optogenetic tools.

Label-free vibrational imaging of different Aß plaque types in Alzheimer's disease reveals sequential events in plaque development.

The neuropathology of Alzheimer's disease (AD) is characterized by hyperphosphorylated tau neurofibrillary tangles (NFTs) and amyloid-beta (Aß) plaques. Aß plaques are hypothesized to follow a development sequence starting with diffuse plaques, which evolve into more compact plaques and finally mature into the classic cored plaque type. A better molecular understanding of Aß pathology is crucial, as the role of Aß plaques in AD pathogenesis is under debate. Here, we studied the deposition and fibrillation of Aß in different plaque types with label-free infrared and Raman imaging. Fourier-transform infrared (FTIR) and Raman imaging was performed on native snap-frozen brain tissue sections from AD cases and non-demented control cases. Subsequently, the scanned tissue was stained against Aß and annotated for the different plaque types by an AD neuropathology expert. In total, 160 plaques (68 diffuse, 32 compact, and 60 classic cored plaques) were imaged with FTIR and the results of selected plaques were verified with Raman imaging. In diffuse plaques, we detect evidence of short antiparallel ß-sheets, suggesting the presence of Aß oligomers. Aß fibrillation significantly increases alongside the proposed plaque development sequence. In classic cored plaques, we spatially resolve cores containing predominantly large parallel ß-sheets, indicating Aß fibrils. Combining label-free vibrational imaging and immunohistochemistry on brain tissue samples of AD and non-demented cases provides novel insight into the spatial distribution of the Aß conformations in different plaque types. This way, we reconstruct the development process of Aß plaques in human brain tissue, provide insight into Aß fibrillation in the brain, and support the plaque development hypothesis.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Drug Distribution in Living Cells via Label-Free Molecular Fingerprint.

A new application of stimulated Raman scattering (SRS) uses the benefit of a label-free molecular fingerprint to image the uptake and distribution of an alkyne-based drug in living cells. This method delivers information on cellular molecular composition and drug-cell interaction, showing the potential of SRS in drug development.

High-content screening Raman spectroscopy (HCS-RS) of panitumumab-exposed colorectal cancer cells.

Raman spectroscopy can provide the biomolecular fingerprint of a cell in a label-free manner. Although a variety of clinical and biomedical applications have been demonstrated, the method remains largely a niche technology. The two main problems are the complexity of data acquisition and the complexity of data analysis. Generally, Raman measurements are performed manually and require a substantial amount of time. This, on the other hand, frequently results in a low number of samples and hence with questionable statistical evaluation. Here, we propose an automated high content screening Raman spectroscopy (HCS-RS) platform, which can perform a series of experiments without human interaction, significantly increasing the number of measured samples and making the measurement more reliable. The automated image processing of bright field images in combination with automatic spectral acquisition of the molecular fingerprint of cells exposed to different physiological conditions enables label-free high content screening applications. The performance of the developed HCS-RS platform is demonstrated by investigating the effect of panitumumab on SW48 and SW480 colorectal cancer cells with wild-type and mutated K-RAS, respectively, in a series of concentrations. Our result indicates that the increased content of panitumumab prohibits the activation of the MAP kinase of the colorectal cancer cells with wild-type K-RAS strongly, whereas there is no significant effect on the K-RAS mutated cells. Moreover, the relative amount of the panitumumab content present in the cells is determined from the Raman spectral information, which could be beneficial for personalized patient treatment.

Fast and Noninvasive Diagnosis of Cervical Cancer by Coherent Anti-Stokes Raman Scattering.

Cervical cancer is the fourth most common cancer in women worldwide, and early detection of its precancerous lesions can decrease mortality. Cytopathology, HPV testing, and histopathology are the most commonly used tools in clinical practice. However, these methods suffer from many limitations such as subjectivity, cost, and time. Therefore, there is an unmet clinical need to develop new noninvasive methods for the early detection of cervical cancer. Here, a novel noninvasive, fast, and label-free approach with high accuracy is presented using liquid-based cytology Pap smears. CARS and SHG/TPF imaging was performed at one wavenumber on the Pap smears from patients with specimens negative for intraepithelial lesions or malignancy (NILM), and low-grade (LSIL) and high-grade (HSIL) squamous intraepithelial lesions. The normal, LSIL, and HSIL cells were selected on the basis of the ratio of the nucleus to the cytoplasm and cell morphology. Raman spectral imaging of single cells from the same smears was also performed to provide integral biochemical information of cells. Deep convolutional neural networks (DCNNs) were trained independently with CARS, SHG/TPF, and Raman images, taking into account both morphotextural and spectral information. DCNNs based on CARS, SHG/TPF, or Raman images have discriminated between normal and cancerous Pap smears with 100% accuracy. These results demonstrate that CARS/SHG/TPF microscopy has a prospective use as a label-free imaging technique for the fast screening of a large number of cells in cytopathological samples.

Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes.

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.

Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2.

Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

Raman micro-spectroscopy monitors acquired resistance to targeted cancer therapy at the cellular level.

Monitoring the drug efficacy or resistance in vitro is usually carried out by measuring the response of single few proteins. However, observation of single proteins instead of an integral cell response may lead to results that are not consistent with patient's response to a drug. We present a Raman spectroscopic method that detects the integral cell response to drugs such as tyrosine kinase inhibitors (TKIs). Non-small cell lung cancer (NSCLC) patients with EGFR mutations develop acquired resistance to first (erlotinib)- and third (osimertinib)-generation TKIs. Large erlotinib-induced differences were detected by Raman micro-spectroscopy in NSCLC cells without T790M EGFR mutation but not in cells with this mutation. Additionally, Raman difference spectra detected the response of NSCLC cells with T790M EGFR mutation to second- (neratinib) and third-generation (osimertinib) TKIs, and the resistance of cells with T790M/C797S EGFR mutation to osimertinib. Thus, the in vitro Raman results indicated that NSCLC cells with T790M and T790M/C797S EGFR mutations are resistant to erlotinib- and osimertinib, respectively, consistent with the observed responses of patients. This study shows the potential of Raman micro-spectroscopy to monitor drug resistance and opens a new door to in vitro companion diagnostics for screening personalized therapies.

Hierarchical deep convolutional neural networks combine spectral and spatial information for highly accurate Raman-microscopy-based cytopathology.

Hierarchical variants of so-called deep convolutional neural networks (DCNNs) have facilitated breakthrough results for numerous pattern recognition tasks in recent years. We assess the potential of these novel whole-image classifiers for Raman-microscopy-based cytopathology. Conceptually, DCNNs facilitate a flexible combination of spectral and spatial information for classifying cellular images as healthy or cancer-affected cells. As we demonstrate, this conceptual advantage translates into practice, where DCNNs exceed the accuracy of both conventional classifiers based on pixel spectra as well as classifiers based on morphological features extracted from Raman microscopic images. Remarkably, accuracies exceeding those of all previously proposed classifiers are obtained while using only a small fraction of the spectral information provided by the dataset. Overall, our results indicate a high potential for DCNNs in medical applications of not just Raman, but also infrared microscopy.

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells.

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label-free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC-MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.

Noninvasive Diagnosis of High-Grade Urothelial Carcinoma in Urine by Raman Spectral Imaging.

The current gold standard for the diagnosis of bladder cancer is cystoscopy, which is invasive and painful for patients. Therefore, noninvasive urine cytology is usually used in the clinic as an adjunct to cystoscopy; however, it suffers from low sensitivity. Here, a novel noninvasive, label-free approach with high sensitivity for use with urine is presented. Coherent anti-Stokes Raman scattering imaging of urine sediments was used in the first step for fast preselection of urothelial cells, where high-grade urothelial cancer cells are characterized by a large nucleus-to-cytoplasm ratio. In the second step, Raman spectral imaging of urothelial cells was performed. A supervised classifier was implemented to automatically differentiate normal and cancerous urothelial cells with 100% accuracy. In addition, the Raman spectra not only indicated the morphological changes that are identified by cytology with hematoxylin and eosin staining but also provided molecular resolution through the use of specific marker bands. The respective Raman marker bands directly show a decrease in the level of glycogen and an increase in the levels of fatty acids in cancer cells as compared to controls. These results pave the way for "spectral" cytology of urine using Raman microspectroscopy.

In vitro prediction of the efficacy of molecularly targeted cancer therapy by Raman spectral imaging.

Mutational acquired resistance is a major challenge in cancer therapy. Somatic tumours harbouring some oncogenic mutations are characterised by a high mortality rate. Surprisingly, preclinical evaluation methods do not show clearly resistance of mutated cancers to some drugs. Here, we implemented Raman spectral imaging to investigate the oncogenic mutation resistance to epidermal growth factor receptor targeting therapy. Colon cancer cells with and without oncogenic mutations such as KRAS and BRAF mutations were treated with erlotinib, an inhibitor of epidermal growth factor receptor, in order to detect the impact of these mutations on Raman spectra of the cells. Clinical studies suggested that oncogenic KRAS and BRAF mutations inhibit the response to erlotinib therapy in patients, but this effect is not observed in vitro. The Raman results indicate that erlotinib induces large spectral changes in SW-48 cells that harbour wild-type KRAS and BRAF. These spectral changes can be used as a marker of response to therapy. HT-29 cells (BRAF mutated) and SW-480 cells (KRAS mutated) display a smaller and no significant response, respectively. However, the erlotinib effect on these cells is not observed when phosphorylation of extracellular-signal-regulated kinase and AKT is monitored by Western blot, where this phosphorylation is the conventional in vitro test. Lipid droplets show a large response to erlotinib only in the case of cells harbouring wild-type KRAS and BRAF, as indicated by Raman difference spectra. This study shows the great potential of Raman spectral imaging as an in vitro tool for detecting mutational drug resistance.

Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells.

Predictions about the cellular efficacy of drugs tested in vitro are usually based on the measured responses of a few proteins or signal transduction pathways. However, cellular proteins are highly coupled in networks, and observations of single proteins may not adequately reflect the in vivo cellular response to drugs. This might explain some large discrepancies between in vitro drug studies and drug responses observed in patients. We present a novel in vitro marker-free approach that enables detection of cellular responses to a drug. We use Raman spectral imaging to measure the effect of the epidermal growth factor receptor (EGFR) inhibitor panitumumab on cell lines expressing wild-type Kirsten-Ras (K-Ras) and oncogenic K-Ras mutations. Oncogenic K-Ras mutation blocks the response to anti-EGFR therapy in patients, but this effect is not readily observed in vitro. The Raman studies detect large panitumumab-induced differences in vitro in cells harboring wild-type K-Ras as seen in A in red but not in cells with K-Ras mutations as seen in B; these studies reflect the observed patient outcomes. However, the effect is not observed when extracellular-signal-regulated kinase phosphorylation is monitored. The Raman spectra show for cells with wild-type K-Ras alterations based on the responses to panitumumab. The subcellular component with the largest spectral response to panitumumab was lipid droplets, but this effect was not observed when cells harbored K-Ras mutations. This study develops a noninvasive, label-free, in vitro vibrational spectroscopic test to determine the integral physiologically relevant drug response in cell lines. This approach opens a new field of patient-centered drug testing that could deliver superior patient therapies.