Chlamydia psittaci infected cell studies by 4Pi Raman and atomic force microscopy.

Chlamydia psittaci is an avian bacterial pathogen that can cause atypical pneumonia in humans via zoonotic transmission. It is a Gram-negative intracellular bacterium that proliferates inside membrane bound inclusions in the cytoplasm of living eukaryotic cells. The study of such cells with C. psittaci inside without destroying them poses a significant challenge. We demonstrated in this work the utility of a combined multitool approach to analyze such complex samples. Atomic force microscopy was applied to obtain high-resolution images of the surface of infected cells upon entrance of bacteria. Atomic force microscopy scans revealed the morphological changes of the cell membrane of Chlamydia infected cells such as changes in roughness of cell membrane and the presence of micro vesicles. 4Pi Raman microscopy was used to image and probe the molecular composition of intracellular bacteria inside intact cells. Information about the structure of the inclusion produced by C. psittaci was obtained and it was found to have a similar molecular fingerprint as that of an intracellular lipid droplet but with less proteins and unsaturated lipids. The presented approach demonstrates complementarity of various microscopy-based approaches and might be useful for characterization of intracellular bacteria.

Alginate-CaCO3 hybrid colloidal hydrogel with tunable physicochemical properties for cell growth.

Biomaterials composed of food polysaccharides are of great interest for future biomedical applications due to their great biocompatibility, tunable mechanical properties, and complex architectural designs that play a crucial role in the modulation of cell adhesion and proliferation. In this work, a facile approach was designed to obtain novel 3D alginate-CaCO3 hybrid hydrogel particles in situ. Controlling the gel concentration from 3 to 20 mg·mL-1 allows us to control the alginate-CaCO3 hydrogel particles' size and density (size variation from 1.86 to 2.34 mm and density from 1.22 to 1.29 mg/mm3). This variable also has a considerable influence on the mineralization process resulting in CaCO3 particles with varied sizes and amounts within the hydrogel beads. The measurements of Young's modulus showed that the inclusion of CaCO3 particles into the alginate hydrogel improved its mechanical properties, and Young's modulus of these hybrid hydrogel particles had a linear relationship with alginate content and hydrogel particle size. Cell experiments indicated that alginate-CaCO3 hybrid hydrogel particles can support osteoblastic cell proliferation and growth. In particular, the amount of hydroxyapatite deposition on the cell membrane significantly increased after the treatment of cells with hybrid hydrogel particles, up to 20-fold. This work offers a strategy for constructing inorganic particle-doped polysaccharide hybrid hydrogel scaffolds that provide the potential to support cell growth.

Structural Analysis and Characterization of an Antiproliferative Lectin from Canavalia villosa Seeds.

Cells use glycans to encode information that modulates processes ranging from cell-cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin's specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.

High-throughput mechano-cytometry as a method to detect apoptosis, necroptosis, and ferroptosis.

In recent years, the importance of the investigation of regulated cell death (RCD) has significantly increased and different methods are proposed for the detection of RCD including biochemical as well as fluorescence assays. Researchers have shown that early stages of cell death could be detected by using AFM. Although AFM offers a high single-cell resolution and sensitivity, the throughput (<100 cells/h) limits a broad range of biomedical applications of this technique. Here, a microfluidics-based mechanobiology technique, named shear flow deformability cytometry (sDC), is used to investigate and distinguish dying cells from viable cells purely based on their mechanical properties. Three different RCD modalities (i.e., apoptosis, necroptosis, and ferroptosis) are induced in L929sAhFas cells and analysed using sDC. Using machine learning on the extracted parameters, it was possible to predict the dead or viable state with 92% validation accuracy. A significant decrease in elasticity can be noticed for each of these RCD modalities by analysing the deformation of the dying cells. Analysis of morphological characteristics such as cell size and membrane irregularities also indicated significant differences in the RCD induced cells versus control cells. These results highlight the importance of mechanical properties during RCD and the significance of label-free techniques, such as sDC, which can be used to detect regulated cell death and can be further linked with sorting of live and dead cells.

Cell Behavior Changes and Enzymatic Biodegradation of Hybrid Electrospun Poly(3-hydroxybutyrate)-Based Scaffolds with an Enhanced Piezoresponse after the Addition of Reduced Graphene Oxide.

This is the first comprehensive study of the impact of biodegradation on the structure, surface potential, mechanical and piezoelectric properties of poly(3-hydroxybutyrate) (PHB) scaffolds supplemented with reduced graphene oxide (rGO) as well as cell behavior under static and dynamic mechanical conditions. There is no effect of the rGO addition up to 1.0 wt% on the rate of enzymatic biodegradation of PHB scaffolds for 30 d. The biodegradation of scaffolds leads to the depolymerization of the amorphous phase, resulting in an increase in the degree of crystallinity. Because of more regular dipole order in the crystalline phase, surface potential of all fibers increases after the biodegradation, with a maximum (361 ± 5 mV) after the addition of 1 wt% rGO into PHB as compared to pristine PHB fibers. By contrast, PHB-0.7rGO fibers manifest the strongest effective vertical (0.59 ± 0.03 pm V-1 ) and lateral (1.06 ± 0.02 pm V-1 ) piezoresponse owing to a greater presence of electroactive ß-phase. In vitro assays involving primary human fibroblasts reveal equal biocompatibility and faster cell proliferation on PHB-0.7rGO scaffolds compared to pure PHB and nonpiezoelectric polycaprolactone scaffolds. Thus, the developed biodegradable PHB-rGO scaffolds with enhanced piezoresponse are promising for tissue-engineering applications.

Dual-mode vehicles with simultaneous thermometry and drug release properties based on hollow Y2O3:Er,Yb and Y2O2SO4:Er,Yb spheres.

Employing luminescence thermometry in the biomedical field is undeniably appealing as many health conditions are accompanied by temperature changes. In this work, we show our ongoing efforts and results at designing novel vehicles for dual-mode thermometry and pH-dependent drug release based on hollow spheres. Hereby for that purpose, we exploit the hollow Y2O3 and Y2O2SO4 host materials. These two inorganic hollow phosphors were investigated and showed to have excellent upconversion Er3+-Yb3+ luminescence properties and could be effectively used as optical temperature sensors in the physiological temperature range when induced by near-infrared CW light (975 nm). Further, doxorubicin was exploited as a model anti-cancer drug to monitor the pH-dependent drug release of these materials showing that they can be used for simultaneous thermometry and drug delivery applications.

The type-1 ribosome-inactivating protein OsRIP1 triggers caspase-independent apoptotic-like death in HeLa cells.

Ribosome-inactivating proteins (RIPs) are capable of removing a specific adenine from 28S ribosomal RNA, thus inhibiting protein biosynthesis in an irreversible manner. In this study, recombinant OsRIP1, a type 1 RIP from rice (Oryza sativa L.), was investigated for its anti-proliferative properties. Human cervical cancer HeLa cells were incubated in the presence of OsRIP1 for 24-72 h. OsRIP1 treatment yielded an anti-proliferation response of the HeLa cells and resulted in apoptotic-like blebbing of the plasma membrane without causing DNA fragmentation. OsRIP1 labeled with FITC accumulated at the cell surface. Pull-down assays identified ASPP1 (Apoptosis-Stimulating Protein of p53 1) and IFITM3 (interferon-induced transmembrane protein 3) as potential interaction partners for OsRIP1. Transcript levels for several critical genes related to different signaling pathways were quantified by RT-qPCR. OsRIP1 provoked HeLa cells to undergo caspase-independent cell death, associated with a significant transcriptional upregulation of the apoptotic gene PUMA, interferon regulatory factor 1 (IRF1) and the autophagy-related marker LC3. No changes in caspase activities were observed. Together, these data suggest that apoptotic-like events were involved in OsRIP1-driven caspase-independent cell death that might trigger the IRF1 signaling pathway and LC3-mediated autophagy.

Deep learning with digital holographic microscopy discriminates apoptosis and necroptosis.

Regulated cell death modalities such as apoptosis and necroptosis play an important role in regulating different cellular processes. Currently, regulated cell death is identified using the golden standard techniques such as fluorescence microscopy and flow cytometry. However, they require fluorescent labels, which are potentially phototoxic. Therefore, there is a need for the development of new label-free methods. In this work, we apply Digital Holographic Microscopy (DHM) coupled with a deep learning algorithm to distinguish between alive, apoptotic and necroptotic cells in murine cancer cells. This method is solely based on label-free quantitative phase images, where the phase delay of light by cells is quantified and is used to calculate their topography. We show that a combination of label-free DHM in a high-throughput set-up (~10,000 cells per condition) can discriminate between apoptosis, necroptosis and alive cells in the L929sAhFas cell line with a precision of over 85%. To the best of our knowledge, this is the first time deep learning in the form of convolutional neural networks is applied to distinguish-with a high accuracy-apoptosis and necroptosis and alive cancer cells from each other in a label-free manner. It is expected that the approach described here will have a profound impact on research in regulated cell death, biomedicine and the field of (cancer) cell biology in general.

AFM Analysis Enables Differentiation between Apoptosis, Necroptosis, and Ferroptosis in Murine Cancer Cells.

Regulated cell death (RCD) has a fundamental role in development, pathology, and tissue homeostasis. In order to understand the RCD mechanisms, it is essential to follow these processes in real time. Here, atomic force microscopy (AFM) is applied to morphologically and mechanically characterize four RCD modalities (intrinsic and extrinsic apoptosis, necroptosis, and ferroptosis) in murine tumor cell lines. The nano-topographical analysis revealed a distinct surface morphology in case of necroptosis, ∼ 200 nm membrane disruptions are observed. Using mechanical measurements, it is possible to detect the early onset of RCD. Combined elasticity and microrheology analysis allowed for a clear distinction between apoptotic and regulated necrotic cell death. Finally, immunofluorescence analysis of the cytoskeleton structure during the RCD processes confirm the measured mechanical changes. The results of this study not only demonstrate the possibility of early real-time cell death detection but also reveal important differences in the cytoskeletal dynamics between multiple RCD modalities.