Intracellular Structural Changes Research Articles

Correlative light and electron microscopy at defined cell cycle stages in a controlled environment.

Cells are dynamic machines that continuously change their architecture to adapt and respond to extracellular and intracellular stimuli. Deciphering dynamic processes with nanometer-scale resolution inside cells is critical for mechanistic understanding. Here, we present a protocol that enables the in situ study of dynamic changes in intracellular structures under close-to-native conditions at high spatiotemporal resolution. Importantly, the cells are grown, transported, and imaged in a chamber in which environmental conditions such as temperature and gas (e.g., carbon dioxide or oxygen) concentration can be controlled. We demonstrate this protocol to quantify ultrastructural changes that occur during the cell cycle of cultured mammalian cells. The environment control system opens up the possibility of applying this method to primary cells, tissues, and organoids by adjusting environmental conditions.

Antibacterial and anti-biofilm activities of probiotic Lactobacillus plantarum against Listeria monocytogenes isolated from milk, chicken and pregnant women.

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that poses significant risks to public health and food safety. The present study aimed to identify the presence of Listeria spp. in various samples, including pasteurized milk, chicken fillets, and stool samples from pregnant women in Sharkia Governorate, Egypt. Additionally, the study identified the serotypes, virulence-associated genes, antimicrobial resistance patterns, and biofilm formation in L. monocytogenes isolates. Moreover, the antibacterial and anti-biofilm activity of Lactobacillus plantarum ATCC 14917 (L. plantarum) against L. monocytogenes isolates was investigated. A cross-sectional study was conducted from August 2021 to January 2022 to collect 300 samples of pasteurized milk, chicken fillets, and stool from pregnant women admitted to outpatient clinics of hospitals. The results showed that 32.7% of the samples were positive for Listeria spp., including L. innocua (48.9%), L. monocytogenes (26.5%), L. ivanovii (14.3%), L. grayi (5.1%), and L. welshimeri (5.1%). Among all L. monocytogenes isolates, hlyA, actA, inlC, and inlJ virulence-associated genes were detected. However, the virulence genes plcB, iap, and inlA were found in 10 (38.5%), 8 (30.8%), and 25 (96.2%) isolates, respectively. The L. monocytogenes isolates classified into four serotypes (1/2a, 1/2b, 1/2c, and 4b), with 1/2a and 4b each identified in 30.8% of the isolates, while 1/2b and 1/2c were identified in 19.2% of the isolates. All L. monocytogenes isolates showed 100% resistance to streptomycin, kanamycin, and nalidix acid, and 92.3% of isolates showed gentamicin resistance. However, all isolates were susceptible to ampicillin and ampicillin/sulbactam. Multidrug resistance (MDR) was observed in 20 (76.9%) L. monocytogenes isolates. The biofilm formation ability of 26 L. monocytogenes isolates was evaluated at different incubation temperatures. At 4°C, 25°C, and 37°C, 53.8, 69.2, and 80.8% of the isolates, respectively, were biofilm producers. Furthermore, 23.1% were strong biofilm producers at both 4°C and 25°C, while 34.6% were strong biofilm formers at 37°C. Treating L. monocytogenes isolates with L. plantarum cell-free supernatant (CFS) reduced the number of biofilm-producing isolates to 15.4, 42.3, and 53.8% at 4°C, 25°C, and 37°C, respectively. L. plantarum's CFS antibacterial activity was tested against six virulent, MDR, and biofilm-forming L. monocytogenes isolates. At a concentration of 5 μg/mL of L. plantarum CFS, none of the L. monocytogenes isolates exhibited an inhibition zone. However, an inhibition zone was observed against L. monocytogenes strains isolated from pasteurized milk and pregnant women's stools when using a concentration of 10 μg/mL. Transmission electron microscopy (TEM) revealed that L. plantarum CFS induced morphological and intracellular structural changes in L. monocytogenes. In conclusion, this study identified virulent MDR L. monocytogenes isolates with strong biofilm-forming abilities in food products in Egypt, posing significant risks to food safety. Monitoring the prevalence and antimicrobial resistance profile of L. monocytogenes in dairy and meat products is crucial to enhance their safety. Although L. plantarum CFS showed potential antibacterial and anti-biofilm effects against L. monocytogenes isolates, further research is needed to explore its full probiotic potential.

Antibacterial and Antibiofilm Efficacy and Mechanism of Ginger (Zingiber officinale) Essential Oil against Shewanella putrefaciens.

Ginger (Zingiber officinale) has unique medicinal value and can be used to treat colds and cold-related diseases. The chemical composition and antibacterial activity of ginger essential oil (GEO) against Shewanella putrefaciens were determined in the present study. Zingiberene, α-curcumene, and zingerone were the main active compounds of GEO. GEO displayed significant antibacterial activity against S. putrefaciens, with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 2.0 and 4.0 μL/mL, respectively. Changes in intracellular ATP content, nucleic acid and protein structure, exopolysaccharides (EPS) content, and extracellular protease production indicated that GEO disrupted the membrane integrity of S. putrescens. At the same time, changes in biofilm metabolic activity content and the growth curve of biofilm showed that GEO could destroy the biofilm. Both scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) observations confirmed that GEO destroyed the cell membrane and lead to the leakage of the constituents. The above results indicate that GEO entered the cells via contact with bacterial membranes, and then inhibited the growth of S. putrefaciens and its biofilms by increasing membrane permeability and inhibiting various virulence factors such as EPS. The findings showed that GEO could destroy the structure of cell membrane and biofilm of tested S. putrefaciens, indicating its potential as a natural food preservative.

Protective effect of bacterial melanin in the substantia nigra in a rat model of Parkinson's disease

The major cause of Parkinson's disease is progressive disturbance of dopaminergic neurons of the substantia nigra, as well as their nerve endings located in the striatum. Therefore, therapy aimed at slowing the death of dopaminergic neurons can be effective. In recent decades, various types of neuroprotective agents have been successfully used in the treatment of neurodegenerative diseases, after which the restoration of structural disfunctions of the CNS is accelerated. In the present study, we used bacterial melanin, the therapeutic effect of which is due to the improvement of the trophic effects of the brain tissue, inhibition of the inflammatory process and gliosis. The comparative study of morphological and functional state of the substantia nigra pars compacta in a rotenone model of Parkinson's disease and combined with introduction of bacterial melanin was carried out. For morphological and histochemical study, the method of detecting the activity of Ca2 + - dependent acid phosphatase was used. The analysis of the data obtained showed that during rotenone intoxication, neuronal death and substantia nigra depigmentation are observed, as well as sharp morphological changes in intracellular structures occur, which indicates gross metabolic and morphological disorders. With the introduction of bacterial melanin, there is a tendency to preserve the normal morphological picture of the neurons of the substantia nigra pars compacta, in comparison with the Parkinson's disease model. Thus, bacterial melanin acts as a neuroprotective agent and has a supportive role in Parkinson's disease. 
 Keywords: bacterial melanin, Parkinson's Disease, substantia nigra

Characterization of Intracellular Structure Changes of Microcystis under Sonication Treatment by Polarized Light Scattering.

Cyanobacterial bloom is one of the most urgent global environmental issues, which eventually could threaten human health and safety. Sonication treatment (ST) is a potential effective method to control cyanobacteria blooms in the field. Currently, the bottleneck of extensive application of ST is the difficulty to estimate the ST effect on the cyanobacterial cells and then determine suitable ST times in the field. In this study, cyanobacterial Microcystis samples sonicated at different times were first measured by a spectrophotometer to calculate the removal efficiency of Microcystis cells. Additionally, they were observed by TEM to reveal the intracellular structure changes of the cells. Then the samples were measured by an experimental setup based on polarized light scattering to measure the polarization parameters. Experimental results indicated that the polarization parameters can effectively characterize the intracellular structural changes of Microcystis cells with different ST times, which is quite consistent with the results for removal efficiency and TEM images. Further, the optimal ST time can be inferred by the polarization parameters. These results demonstrate that polarized light scattering can be a potentially powerful tool to explore suitable times for sonication treatment of cyanobacteria blooms.

FTIR microspectroscopic study of biomacromolecular changes in As2O3 induced MGC803 cells apoptosis

It is well-known that As2O3 has significant anticancer effects, however, little is known regarding its mechanism for treating gastric cancer. Thus, we investigated biomacromolecular (DNA, proteins and lipids) changes of human gastric cancer cell line MGC803 to further understand As2O3-induced apoptosis. Conventional methods showed the increase of the apoptosis rate, the decrease of mitochondrial membrane potential (MMP), the accumulation of reactive oxygen species (ROS) and the changes of apoptotic proteins, etc. Fourier transform infrared (FTIR) microspectroscopy sensitively recognized overall biomacromolecular changes caused by the above: Peak-area ratios indicated the content/structure changes in DNA, proteins and lipids. Principle component analysis (PCA) revealed significant changes in intracellular DNA concentration and structure. This study suggests that As2O3 may exert anti-gastric cancer effect by altering intracellular biomacromolecules especially DNA.

Analysis of changes in intracellular structures of vascular smooth muscle cells under cyclic stretch stimulation

Analysis of changes in intracellular structures of vascular smooth muscle cells under cyclic stretch stimulation

Mitochondrial and Metabolic Dynamics of Endometrial Stromal Cells During the Endometrial Cycle.

The endometrial cycle in response to hormonal stimulation is essential for implantation. The female has endometrium that repeats this cycle through about half of a lifetime. The cycle includes three phases, proliferative, secretory, and menstrual, and each phase has distinct characteristics. The endometrial stromal cells (EnSCs) in each phase also have specialized characteristics, including cell cycle, morphologies, and cellular metabolic state. So we hypothesized that the cells in each phase have unique mitochondrial morphologies because they are generally linked to cellular metabolic state. To investigate the metabolic characteristics in each phase, we investigated the mitochondrial morphologies by transmission electron microscopy, oxygen consumption rate (OCR), and intracellular adenosine triphosphate (ATP) production. The decidualized EnSCs have shorter mitochondria than those in the proliferative phase. Besides, they also displayed distinct intracellular structural characteristics compared with the proliferative phase, such as ribosome-rich endoplasmic reticulum and increased formation of vesicles. OCR and luminescent ATP detection assay revealed that the basal respiration and ATP production in the decidualized EnSCs were lower than those in the proliferative phase. Thus, we concluded that morphological and intracellular structural changes were induced during the decidualization. Moreover, the decreased mitochondrial length was shown to correlate with decreased dependency on oxidative phosphorylation and ATP concentration in EnSCs.

MRI of tumor T cell infiltration in response to checkpoint inhibitor therapy.

BackgroundImmune checkpoint inhibitors, the most widespread class of immunotherapies, have demonstrated unique response patterns that are not always adequately captured by traditional response criteria such as the Response Evaluation Criteria in Solid Tumors or even immune-specific response criteria. These response metrics rely on monitoring tumor growth, but an increase in tumor size and/or appearance after starting immunotherapy does not always represent tumor progression, but also can be a result of T cell infiltration and thus positive treatment response. Therefore, non-invasive and longitudinal monitoring of T cell infiltration are needed to assess the effects of immunotherapies such as checkpoint inhibitors. Here, we proposed an innovative concept that a sufficiently large influx of tumor infiltrating T cells, which have a smaller diameter than cancer cells, will change the diameter distribution and decrease the average size of cells within a volume to a degree that can be quantified by non-invasive MRI.MethodsWe validated our hypothesis by studying tumor response to combination immune-checkpoint blockade (ICB) of anti-PD-1 and anti-CTLA4 in a mouse model of colon adenocarcinoma (MC38). The response was monitored longitudinally using Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED), a diffusion MRI-based method which has been previously shown to non-invasively map changes in intracellular structure and cell sizes with the spatial resolution of MRI, in cell cultures and in animal models. Tumors were collected for immunohistochemical and flow cytometry analyzes immediately after the last imaging session.ResultsImmunohistochemical analysis revealed that increased T cell infiltration of the tumors results in a decrease in mean cell size (eg, a 10% increase of CD3+ T cell fraction results a ~1 µm decrease in the mean cell size). IMPULSED showed that the ICB responders, mice with tumor volumes were less than 250 mm3 or had tumors with stable or decreased volumes, had significantly smaller mean cell sizes than both Control IgG-treated tumors and ICB non-responder tumors.ConclusionsIMPULSED-derived cell size could potentially serve as an imaging marker for differentiating responsive and non-responsive tumors after checkpoint inhibitor therapies, a current clinical challenge that is not solved by simply monitoring tumor growth.

Expression and purification of recombinant puroindoline A protein in Escherichia coli and its antifungal effect against Aspergillus flavus.

Aspergillus flavus is the main cause of postharvest agricultural commodity loss. In this study, puroindoline A (PINA) protein was expressed in Escherichia coli, purified, and its antifungal properties against A. flavus were characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the molecular weight of the recombinant PINA protein was approximately 44 kDa. PINA exerted a powerful antifungal effect against A. flavus at 42.42 μg/mL on potato dextrose agar culture medium. Flow cytometry and scanning electron microscopy revealed that the spore morphology was damaged by PINA exposure; spores were depressed and broken, suggesting that the cell wall was impaired. Transmission electron microscopy and propidium iodide staining illustrated significant changes in intracellular spore structure, indicating cell membrane damage. 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining indicated decreased mitochondrial membrane potential. Large nuclear condensation and DNA fragmentation were detected by 4',6-diamidino-2-phenylindole staining. The expression of genes related to the cell wall, cell membrane, and spore germination significantly changed in PINA-treated cells; this illustrated the probable mode of PINA action on A. flavus through cell wall destruction and triggered cell membrane, mitochondrial, and DNA damage leading to cell death. The antifungal mechanism of wheat PINA protein on A. flavus has been demonstrated in this study, and has potential application in preventing postharvest loss in the agricultural industry.

Nanomaterial-assisted sensitization of oncotherapy

Globally, cancer is growing at an alarming pace, which calls for development of more efficient cancer treatments. Conventional chemotherapy and radiotherapy have become crucial first-line clinical treatments for cancer. However, along with their wide usage, limited therapeutic effects, severe adverse reactions, unaffordable costs, and complicated operations lead to failures of these treatments. Moreover, the emergence of multidrug resistance inhibits the longtime usage of chemotherapeutics. One of the major causes of treatment failure is the insufficient sensitivity of cancer cells to therapeutic drugs or treatments. With the rigorous development of nanotechnology, tailored nanoparticles can efficiently sensitize malignant cells by inducing intracellular structural and functional changes, which could affect vital intracellular processes such as metabolism, signal conduction, proliferation, cell death as well as intracellular drug delivery. Here, we review recent advances in nanomaterial-assisted sensitization of oncotherapy, and challenges and strategies in the development of nanomedical approaches.

Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis

BackgroundPhotosynthetic organisms utilize carotenoids for photoprotection as well as light harvesting. Our previous study revealed that high-intensity light increases the expression of the gene for phytoene synthase (EgcrtB) in Euglena gracilis (a unicellular phytoflagellate), the encoded enzyme catalyzes the first committed step of the carotenoid biosynthesis pathway. To examine carotenoid synthesis of E. gracilis in response to light stress, we analyzed carotenoid species and content in cells grown under various light intensities. In addition, we investigated the effect of suppressing EgcrtB with RNA interference (RNAi) on growth and carotenoid content.ResultsAfter cultivation for 7 days under continuous light at 920 μmol m−2 s−1, β-carotene, diadinoxanthin (Ddx), and diatoxanthin (Dtx) content in cells was significantly increased compared with standard light intensity (55 μmol m−2 s−1). The high-intensity light (920 μmol m−2 s−1) increased the pool size of diadinoxanthin cycle pigments (i.e., Ddx + Dtx) by 1.2-fold and the Dtx/Ddx ratio from 0.05 (control) to 0.09. In contrast, the higher-intensity light treatment caused a 58% decrease in chlorophyll (a + b) content and diminished the number of thylakoid membranes in chloroplasts by approximately half compared with control cells, suggesting that the high-intensity light-induced accumulation of carotenoids is associated with an increase in both the number and size of lipid globules in chloroplasts and the cytoplasm. Transient suppression of EgcrtB in this alga by RNAi resulted in significant decreases in cell number, chlorophyll, and total major carotenoid content by 82, 82 and 86%, respectively, relative to non-electroporated cells. Furthermore, suppression of EgcrtB decreased the number of chloroplasts and thylakoid membranes and increased the Dtx/Ddx ratio by 1.6-fold under continuous illumination even at the standard light intensity, indicating that blocking carotenoid synthesis increased the susceptibility of cells to light stress.ConclusionsOur results indicate that suppression of EgcrtB causes a significant decrease in carotenoid and chlorophyll content in E. gracilis accompanied by changes in intracellular structures, suggesting that Dtx (de-epoxidized form of diadinoxanthin cycle pigments) contributes to photoprotection of this alga during the long-term acclimation to light-induced stress.

Mechanisms Involved in the Development of Chemotherapy-Induced Neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and painful condition seen in patients undergoing treatment with common agents such as vincristine, paclitaxel, oxaliplatin and bortezomib. The mechanisms of this condition are diverse, and include an array of molecular and cellular contributions. Current research implicates genetic predispositions to this condition, which then may influence cellular responses to chemotherapy. Processes found to be influenced during CIPN include increased expression of inflammatory mediators, primarily cytokines, which can create cascading effects in neurons and glia. Changes in ion channels and neurotransmission, as well as changes in intracellular signaling and structures have been implicated in CIPN. This review explores these issues and suggests considerations for future research.

Inhibition of hypertrophy, per se, may not be a good therapeutic strategy in ventricular pressure overload: other approaches could be more beneficial.

Cardiac hypertrophy was initially defined by a directly observed increase in cardiac mass that was then assumed to be mainly linked to an increase in cardiomyocyte mass. When submitted to a physiological or pathological stress, the heart undergoes a process of remodeling that involves cardiomyocyte growth but also changes in other cell types: changes in intracellular and extracellular structure, protein expression, signaling pathways, energy metabolism, vascularization, and so forth. Although hypertrophy is only one manifestation of the multiparameter remodeling of the myocardium, progressively, this term has, by oversimplification, included all others aspects of the cardiac response to stress. Classically, hypertrophy induced by pressure overload was considered as adaptive via a decreased stress allowed by a thicker myocardium. The currently accepted concept is that of pathological hypertrophy with depressed ventricular function that leads to heart failure, as opposed to physiological hypertrophy induced by exercise, for example. In this view, hypertrophy development should be prevented during pressure overload. This was proposed 10 years ago in an editorial in Circulation .1 A large number of experimental studies have been published since then, and it appears that an evaluation of the results is necessary. Response by Schiattarella and Hill on p 1457 Many studies showed a beneficial effect of hypertrophy blockade. However, some studies showed the opposite. Before reviewing them in detail, we rapidly present the evolution over time of the concept of adaptive hypertrophy that varied during the last 30 years. This reminds us of some important elements of the previous facts and interpretations that were forgotten as new ones appeared. This also allows understanding of why inhibition of hypertrophy, per se, may not be the best therapeutic strategy in the management of pressure overload–induced hypertrophy. One of the first complete reports of the initiation and evolution of the hypertrophic process toward …

Bubble-Like Response of Living Blood Cells and Microparticles in an Ultrasound Field

The bilayer sonophore model suggests that ultrasound induces a pulsating structure in the intra-membrane hydrophobic space between the two lipid monolayer lea ets of the cell membrane, assembled by dissolved gas of the surrounding area, which absorbs acoustic energy and transforms it by creating intra-cellular structural changes. This void has been referred to as a bilayer sonophore. The bilayer sonophore in ates and de ates periodically when exposed to ultrasound and may itself radiate acoustic pressure pulses in the surrounding medium in the same way a gas bubble does: once exposed to ultrasound the bilayer sonophore becomes a mechanical oscillator and a source of intracellular cavitation activity. In this paper, we describe observations of the clustering behaviour of living cells and several other particles in a standing sound eld generated inside a ring transducer. Upon sonication, blood cells and monodisperse polystyrene particles were observed to have been trapped in the same locations, corresponding to nodes of the ultrasound eld. Because polystyrene is hydrophobic, it behaves like a particle trapped inside a thin gas shell. In fact, the sonophore model treats biological cells in a similar way. Microbubbles that form the ultrasound contrast agent Quantison— behave di erently, however. These microbubbles accumulated in circles faster than blood cells and polystyrene particles. In addition, they form tightly packed clusters at the nodes, indicating very strong secondary Bjerknes forces. Cluster formation is not to be expected in cells with predicted sonophore sizes on the order of 10 100 nm.

Imidazoacridinone Derivatives as Efficient Sensitizers in Photoantimicrobial Chemotherapy

The objective of this study was to investigate a new potential photosensitizer (PS) in the photodynamic inactivation (PDI) of microorganisms in vitro (11 reference strains and 13 clinical isolates, representing common Gram-positive and Gram-negative human pathogens), with special emphasis on Candida albicans. We studied the light-induced cytotoxicity of the imidazoacridinone derivative C1330 toward fungal cells grown in planktonic form. We examined the influence of various parameters (time of incubation, PDI quencher effect, and C1330 accumulation in C. albicans cells) on the efficacy of light-dependent cytotoxicity. Additionally, we checked for the potential cyto- and phototoxic activity of C1330 against human dermal keratinocytes. In our research, we used a broadband incoherent blue light source (380 to 470 nm) with an output power of 100 mW/cm(2). In vitro studies showed that the C1330 action against C. albicans was a light-dependent process. C1330 was an efficient photosensitizer in the photodynamic inactivation of C. albicans, which reduced the growth of planktonic cells by 6.1 log10 units. Efficient accumulation of PS in the nucleus and vacuoles was observed after 30 min of incubation, which correlated with the highest photokilling efficacy. Significant changes in intracellular structure were observed upon illumination of C1330-incubated C. albicans cells. In the case of the human HaCaT cell line, approximately 40% of cells survived the treatment, which indicates the potential benefit of further study of the application of C1330 in photoantimicrobial chemotherapy. These data suggest that PDI may be a viable approach for the treatment of localized C. albicans infections.

Changes in Cytosolic ATP Levels and Intracellular Morphology during Bacteria-Induced Hypersensitive Cell Death as Revealed by Real-Time Fluorescence Microscopy Imaging

Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection.

Inhibition of cytosolic Phospholipase A2prevents prion peptide-induced neuronal damage and co-localisation with Beta III Tubulin

BackgroundActivation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites.ResultsExposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3) reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death.ConclusionsCollectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.

Statistical organelle dissection of Arabidopsis guard cells using image database LIPS

To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.

Multilayer Mie Scattering Model for Investigation of Intracellular Structural Changes in the Nucleolus and Cytoplasm

Light scattering from biological cells has been used for many years as a diagnostic tool. Several simulation methods of the scattering process were developed in the last decades in order to understand and predict the scattering patterns. We developed an analytical model of a multilayer spherical scattering cell. Here, we describe the model and show that the results obtained within this simple method are similar to those obtained with far more complicated methods such as finite-difference time-domain (FDTD). The multilayer model is then used to study the effects of changes in the distribution of internal cell structures like mitochondria distribution or nucleus internal structures that exist in biological cells. Such changes are related with cancerous processes within the cell as well as other cell pathologies. Results show the ability to discriminate between different cell stages related to the mitochondria distributions and to internal structure of the nucleolus.