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Recently, a method for estimating three-dimensional acoustic impedance profiles in cultured cells and human dermal organs was proposed by interpreting the reflected ultrasonic signal based on a 1-D transmission line model for acoustic impedance microscopy (AIM). However, AIM has a disadvantage that reflected signals from cells overlap with that from a reference substrate. Additionally, the amplitudes of the reflected signals from the specimens are significantly weaker than that from the substrate. In this paper, we proposed a new method for separation of those signals based on a concept of clutter filter, which had been developed for a color Doppler method in medical ultrasonic imaging. The proposed filter using singular value decomposition (SVD) could separate original signals into desired signals such as those from the substrate and cells. Additionally, an effect from a tilt of the substrate was investigated in this study. Separability of the proposed filter was evaluated by two investigations. First one was to evaluate the separability by estimating a correlation coefficient between the filtered signal and signal reflected from a position only with the substrate. Second one was to compare a slope of the substrate estimated from the original signal with that estimated from the filtered signals from the substrate. The experimental results showed that the proposed filter could separate signals from the substrate, and the compensation of the tilt of the substrate could improve the performance of the proposed filter., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10-20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

Development and study of cell-cultured constructs, such as tissue-engineering scaffolds or organ-on-a-chip platforms require a comprehensive, representative view on the cells inside the used materials. However, common characteristics of biomedical materials, for example, in porous, fibrous, rough-surfaced, and composite materials, can severely disturb low-energy imaging. In order to image and quantify cell structures in optically challenging samples, we combined labeling, 3D X-ray imaging, and in silico processing into a methodological pipeline. Cell-structure images were acquired by a tube-source X-ray microtomography device and compared to optical references for assessing the visual and quantitative accuracy. The spatial coverage of the X-ray imaging was demonstrated by investigating stem-cell nuclei inside clinically relevant-sized tissue-engineering scaffolds (5x13 mm) that were difficult to examine with the optical methods. Our results highlight the potential of the readily available X-ray microtomography devices that can be used to thoroughly study relative large cell-cultured samples with microscopic 3D accuracy.

The diagnosis of patients with malignancies relies on the results of a clinical cytological examination. To enhance the diagnostic qualities of cytological examinations, it is important to have a detailed analysis of the cell's characteristics. There is, therefore, a need for developing a new auxiliary method for cytological diagnosis. In this study, we focused on studying the charge of the cell membrane surface of fixed cells, which is one of important cell's characteristics. Although fixed cells lose membrane potential which is observed in living cells owing to ion dynamics, we hypothesized that fixed cells still have a cell membrane surface charge due to cell membrane components and structure. We used 5 cell lines in this study (ARO, C32TG, RT4, TK, UM-UC-14). After fixation with CytoRich Red, we measured the cell membrane surface charge of fixed cells in solution using zeta potential measurements and fixed cells on glass slides, visualizing it using antibody-labeled beads and positively-charged beads. Furthermore, we measured the cell membrane surface charge of fixed cells under different conditions, such as different solution of fixative, ion concentration, pH, and pepsin treatments. The zeta potential measurements and visualization using the beads indicated that the cell membrane surface of fixed cells was negatively charged, and also that the charge varied among fixed cells. The charge state was affected by the different treatments. Moreover, the number of cell-bound beads was small in interphase, anaphase, and apoptotic cells. We concluded that the negative cell membrane surface charge was influenced by the three-dimensional structure of proteins as well as the different types of amino acids and lipids on the cell membrane. Thus, cell surface charge visualization can be applied as a new auxiliary method for clinical cytological diagnosis. This is the first systematic report of the cell membrane surface charge of fixed cells., Competing Interests: The authors have declared that no competing interests exist.

To establish widespread cell therapy for type 1 diabetes mellitus, we aimed to develop an effective protocol for generating insulin-producing cells (IPCs) from adipose-derived stem cells (ADSCs). We established a 3D culture using a human recombinant peptide (RCP) petaloid μ-piece with xeno-antigen free reagents. Briefly, we employed our two-step protocol to differentiate ADSCs in 96-well dishes and cultured cells in xeno-antigen free reagents with 0.1 mg/mL RCP μ-piece for 7 days (step 1), followed by addition of histone deacetylase inhibitor for 14 days (step 2). Generated IPCs were strongly stained with dithizone, anti-insulin antibody at day 21, and microstructures resembling insulin secretory granules were detected by electron microscopy. Glucose stimulation index (maximum value, 4.9) and MAFA mRNA expression were significantly higher in 3D cultured cells compared with conventionally cultured cells (P < 0.01 and P < 0.05, respectively). The hyperglycaemic state of streptozotocin-induced diabetic nude mice converted to normoglycaemic state around 14 days after transplantation of 96 IPCs under kidney capsule or intra-mesentery. Histological evaluation revealed that insulin and C-peptide positive structures existed at day 120. Our established xeno-antigen free and RCP petaloid μ-piece 3D culture method for generating IPCs may be suitable for clinical application, due to the proven effectiveness in vitro and in vivo.

Objective: In orthopedic joint injection, the most frequently used local anesthetics are ropivacaine, bupivacaine, and 1% or 2% lidocaine. The aim of this study was to examine effects of these various anesthetics on the viability of human chondrocytes. Our hypothesis was that all local anesthetics tested damage human chondrocytes in vitro ., Methods: Primary human chondrocytes were isolated and cultured from 6 donated human knee joints (mean age of donors 61.2 years). Local anesthetics were added to these cultures. Toxicity analysis was performed by visualization of cell structure using light microscopy. Determination of vital chondrocytes was performed by use of a Casy cell counter. Chondrocytes' cell death was examined by fluorescence microscopy and an XTT ELISA assay., Results: Light microscope and fluorescence microscope data revealed a defect cell structure and increased number of dead cells after addition of 1% or 2% lidocaine and bupivacaine but not ropivacaine. We were able to show an increased level of XTT activity after treatment with bupivacaine, 2% lidocaine or ropivacaine. The count of vital chondrocytes was significantly decreased after treatment with bupivacaine, 1% or 2% lidocaine, and ropivacaine., Conclusions: The data show that treatment with local anesthetics induces cell damage of human chondrocytes in vitro . Ropivacaine seems to be a local anesthetic with the lowest toxic potential on human chondrocytes, a feature that may favor its preference for use in joint injection.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6 hours with full containers, 36 hours with full containers, 36 hours with container three quarters full of medium, and 36 hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36 hours if the container is full.

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This chapter gives insight into task-specific methodologies for the evaluation of matrix ultrastructure by light and electron microscopy. It separately considers the isolation and preparation of molecular isolates for negative staining, immunolabeling, rotary shadowing, and single particle analysis. Also considered is the preparation of whole tissues and cultured cells by chemical fixation and cryofixation methodologies. Immunoelectron microscopy for immunoidentification of matrix components may be accomplished en bloc or via section-surface protocols; the advantages and pitfalls in both methodologies are described. Correlative light and electron microscopy, particularly utilizing GFP constructs, demands special consideration in fixation and embedding protocols. TEM imaging methods, including the use of montage software and the acquisition of thick-section tilt series are discussed. The protocols presented in this chapter, with the exception of single particle analysis, are those which are continuously used in our laboratory and represent the latest modifications in our protocols., (© 2018 Elsevier Inc. All rights reserved.)

Cell mechanics is a novel label-free biomarker for indicating cell states and pathological changes. The advent of atomic force microscopy (AFM) provides a powerful tool for quantifying the mechanical properties of single living cells in aqueous conditions. The wide use of AFM in characterizing cell mechanics in the past two decades has yielded remarkable novel insights in understanding the development and progression of certain diseases, such as cancer, showing the huge potential of cell mechanics for practical applications in the field of biomedicine. In this paper, we reviewed the utilization of AFM to characterize cell mechanics. First, the principle and method of AFM single-cell mechanical analysis was presented, along with the mechanical responses of cells to representative external stimuli measured by AFM. Next, the unique changes of cell mechanics in two types of physiological processes (stem cell differentiation, cancer metastasis) revealed by AFM were summarized. After that, the molecular mechanisms guiding cell mechanics were analyzed. Finally the challenges and future directions were discussed.

Skin is the largest organ of the human body. Thus far, tissue engineering of skin has developed rapidly and has used many types of growth factors and nanofibrous scaffolds. In this study, we differentiated neonate keratinocytes for epithelialization on the polycaprolactone-Platelet gel (PCL-PG) scaffold. Fabricated PCL nanofibers prepared by electrospinning technology and coated by platelet gel. Subsequently, the structure of the scaffold was evaluated by SEM, FTIR-ATR, contact angle and tensile test assays. After seeding the neonate keratinocytes on neat PCL and PCL-PG scaffolds, the epidermal maturation was tested by detecting cytokeratin 10 and loricrin determinants by immunocytochemistry; moreover, keratinocyte genes such as keratin 14, keratin 10, and Involucrin were investigated by real-time PCR. The results of MTT assay indicated an increase in cell viability and cell proliferation of neonate keratinocytes on PCL-PG nanofiber scaffolds compared with PCL. RT-PCR and immunocytochemical analysis showed better cell differentiation on the PCL-PG scaffolds than neat PCL. Furthermore, SEM microscopy images demonstrated that neo-keratinocytes enhance adhesion and proliferation on PCL-PG nanofiber scaffolds. We found that PG increases biocompatibility and wettability of scaffold, cell adhesion, and expression of keratinocyte markers. Overall, this procedure is recommended to be employed in skin tissue engineering and wounds healing., (© 2016 Wiley Periodicals, Inc.)

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders characterized by progressive spasticity of the lower limbs due to degeneration of the corticospinal motor neurons. In a Bulgarian family with three siblings affected by complicated hereditary spastic paraplegia, we performed whole exome sequencing and homozygosity mapping and identified a homozygous p.Thr512Ile (c.1535C > T) mutation in ATP13A2. Molecular defects in this gene have been causally associated with Kufor-Rakeb syndrome (#606693), an autosomal recessive form of juvenile-onset parkinsonism, and neuronal ceroid lipofuscinosis (#606693), a neurodegenerative disorder characterized by the intracellular accumulation of autofluorescent lipopigments. Further analysis of 795 index cases with hereditary spastic paraplegia and related disorders revealed two additional families carrying truncating biallelic mutations in ATP13A2. ATP13A2 is a lysosomal P5-type transport ATPase, the activity of which critically depends on catalytic autophosphorylation. Our biochemical and immunocytochemical experiments in COS-1 and HeLa cells and patient-derived fibroblasts demonstrated that the hereditary spastic paraplegia-associated mutations, similarly to the ones causing Kufor-Rakeb syndrome and neuronal ceroid lipofuscinosis, cause loss of ATP13A2 function due to transcript or protein instability and abnormal intracellular localization of the mutant proteins, ultimately impairing the lysosomal and mitochondrial function. Moreover, we provide the first biochemical evidence that disease-causing mutations can affect the catalytic autophosphorylation activity of ATP13A2. Our study adds complicated hereditary spastic paraplegia (SPG78) to the clinical continuum of ATP13A2-associated neurological disorders, which are commonly hallmarked by lysosomal and mitochondrial dysfunction. The disease presentation in our patients with hereditary spastic paraplegia was dominated by an adult-onset lower-limb predominant spastic paraparesis. Cognitive impairment was present in most of the cases and ranged from very mild deficits to advanced dementia with fronto-temporal characteristics. Nerve conduction studies revealed involvement of the peripheral motor and sensory nerves. Only one of five patients with hereditary spastic paraplegia showed clinical indication of extrapyramidal involvement in the form of subtle bradykinesia and slight resting tremor. Neuroimaging cranial investigations revealed pronounced vermian and hemispheric cerebellar atrophy. Notably, reduced striatal dopamine was apparent in the brain of one of the patients, who had no clinical signs or symptoms of extrapyramidal involvement., (© The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

GLT-1 is the main glutamate transporter in the brain and undergoes trafficking processes that control its concentration on the cell surface thereby shaping glutamatergic neurotransmission. We have investigated how the traffic of GLT-1 is regulated by transporter activity. We report that internalization of GLT-1 from the cell surface is accelerated by transportable substrates like glutamate or aspartate, as well as by the transportable inhibitor L-trans-2,4-PDC, but not by the non-substrate inhibitor WAY 213613 in primary mixed cultures and in transiently transfected HEK293 cells. Analysis of the mechanism of endocytosis in HEK293 cells revealed that glutamate promoted the association with the transporter of the adaptor protein β-arrestin and the ubiquitin ligase Nedd4-2. The addition of glutamate is accompanied by an increase in the transporter ubiquitination, and the internalization is suppressed by an ubiquitination inhibitor (PYR41), and in a mutant defective in C-terminal lysines. The glutamate triggered endocytosis was also suppressed by siRNA for β-arrestin. This regulatory mechanism might be relevant in controlling the amount of transporter on the cell surface in conditions such as ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The etiological agent of schistosomiasis in Brazil, Schistosoma mansoni, requires an obligatory passage through Biomphalaria snails to complete its life cycle. In these intermediate hosts, interaction with the parasite is mediated by humoral factors and hemocytes by mechanisms that are not yet fully understood. Extant studies exploring these processes are usually conducted through experimental infection of Biomphalaria with S. mansoni miracidia. Thus, tissue-derived cultures of Biomphalaria may be useful in increasing the understanding of that interaction at cellular level. However, in the absence of morphological characterization of those cells in culture, the application of such models is delayed. In the present work, we cultured different tissues of B. tenagophila, the second most important host of S. mansoni in Brazil, using a strain that is naturally and absolutely resistant to S. mansoni infection. This decision was driven by the view that this strain might be provided with the most effective response against parasite infection. Primary cultures were successfully established from nine Biomphalaria tissues and the respective cells in culture were ultra structurally described. Attention was particularly devoted to cells derived from mantle cavity and kidney tissues. Although they have been considered important centers for hemocyte production in Biomphalaria, no detailed cell characterization is available in the pertinent literature. Herein, kidney-derived cells partially shared hematoblast characteristics. Moreover, under optical microscopy, kidney cells in culture were very similar to those derived from amebocyte-producing organ (APO) cultures, which have been recently shown to be capable of eliminating S. mansoni sporocysts in vitro. Based on the close resemblance of those cultures and their anatomical proximity inside the mantle cavity, we suggest the effective participation of Biomphalaria kidney cells in hematopoiesis and in host response to S. mansoni infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ∼10⁹ cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation., (© 2014 Cold Spring Harbor Laboratory Press.)

Mitochondria are complex organelles at the center of cellular metabolism, apoptosis, and signaling. They continue to be the subject of intense basic investigation to understand their composition and function, but they have also captivated the attention of clinical researchers because of the growing knowledge of the (sometimes unexpected) roles of mitochondria in human diseases and aging. A full understanding of these intriguing organelles often requires their purification from cells or tissues under specific physiological or pathological conditions. Here we provide some introductory considerations for those interested in purifying mitochondria for subsequent downstream biophysical, structural, and functional analysis., (© 2014 Cold Spring Harbor Laboratory Press.)

There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose., (© 2014 Cold Spring Harbor Laboratory Press.)

Formaldehyde is a powerful cross-linking agent that elicits protein-protein and protein-nucleic acid cross-links. This protocol describes the formaldehyde cross-linking of intact cells followed by either the preparation of a whole-cell extract by sonication or the preparation of nuclear and cytoplasmic extracts by fractionation. Cross-linked extracts are treated with high salt or sodium dodecyl sulfate (SDS) to disrupt non-cross-linked aggregates. Protein-protein and protein-nucleic acid interactions can then be studied by purifying the components of interest from the extracts (e.g., using immunoprecipitation). Formaldehyde cross-links are reversible by heat., (© 2014 Cold Spring Harbor Laboratory Press.)

Objective: Periodontal ligament (PDL) cells and their substrates play key roles in periodontal regeneration. However, there has been no report on the use of amniotic membrane (AM) as a substrate for culturing PDL cells. In the current study, we conducted an analysis of PDL cells cultivated on AM to determine the distribution of factors responsible for maintaining the characteristics of PDL., Materials and Methods: Amniotic membrane was obtained from women undergoing cesarean sections, whereas PDL tissue was obtained from human maxillary third molars. The harvested PDL cells were maintained in explant culture for three or four passages, following which they were cultured on AM., Results: After 3 weeks of culture, the PDL cells had grown well on AM. Immunofluorescence showed that these cells were capable of proliferating and potentially maintaining their PDL-like properties. In addition, strong cell-cell adhesion structures, namely desmosomes and tight junctions, were shown to be present between cells. Electron microscopy images showed that the cultured PDL cells had differentiated and proliferated on AM with lateral conjugation and adhesion to AM., Conclusion: We conclude that AM may represent a suitable substrate for culturing PDL cells and that PDL cells cultured on AM show sheet formation., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the cytokinesis-block micronucleus (CBMN) assay as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-5 Gy) as well as blind samples (0.1-6.4 Gy) were sent to the participants. The CBMN assay was performed according to protocols individually established and varying among participating laboratories. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 4 days after sample arrival. The CBMN dose estimates were reported with high accuracy (MAD values of 0.20-0.50 Gy at doses below 6.4 Gy for both manual and automated scoring procedures), but showed a limitation of the assay at the dose point of 6.4 Gy, which resulted in a clear dose underestimation in all cases. The MAD values (without 6.4 Gy) differed significantly (P = 0.03) between manual (0.25 Gy, SEM = 0.06, n = 4) or automated scoring procedures (0.37 Gy, SEM = 0.08, n = 5), but lowest MAD were equal (0.2 Gy) for both scoring procedures. Likewise, both scoring procedures led to the same allocation of dose estimates to triage categories of clinical significance (about 83% accuracy and up to 100% specificity).

Nanomaterial-biosystem interaction is emerging as a major concern hindering wide adoption of nanomaterials. Using quantum dots (Qdots) of different sizes (Qdot-440nm and Qdot-680nm) as a model system, we studied the effects of polyethylene glycol (PEG) thin-layer surface modification in attenuating Qdot-related cytotoxicity, genotoxicity perturbation and oxidative stress in a cellular system. We found that uncoated Qdots (U-Qdots) made of core/shell CdSe/ZnS could indeed induce cytotoxic effects, including the inhibition of cell growth. Also, both the neutral comet assay and γH2AX foci formation showed that U-Qdots caused significant DNA damage in a time- and dose-dependent manner. In contrast, results from cytotoxicity analysis and γH2AX generation indicate minimal impact on cells after exposure to PEG-coated Qdots. This lack of observed toxic effects from PEG-coated Qdots may be due to the fact that PEG-coating can inhibit ROS generation induced by U-Qdots. Based on these observations, we conclude that the genotoxicity of Qdots could be significantly decreased following proper surface modification, such as PEG encapsulation. In addition, PEG encapsulation may also serve as a general method to attenuate nanotoxicity for other nanoparticles., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Clathrin assembly proteins AP180 and CALM regulate the assembly of clathrin-coated vesicles (CCVs), which mediate diverse intracellular trafficking processes, including synaptic vesicle (SV) recycling at the synapse. Although studies using several invertebrate model systems have indicated a role for AP180 in SV recycling, less is known about AP180's or CALM's function in the synapse of mammalian neurons. In this study, we examined synapses of rat hippocampal neurons in which the level of AP180 or CALM had been reduced by RNA interference (RNAi). Using light microscopy, we visualized synaptic puncta in these AP180- or CALM-reduced neurons by co-expressing Synaptophysin::EGFP (Syp::EGFP). We found that neurons with reduced AP180 or reduced CALM had smaller Syp::EGFP-illuminated puncta. Using electron microscopy, we further examined the ultrastructure of the AP180- or CALM-reduced presynaptic terminals. We found that SVs became variably enlarged in both the AP180-reduced and CALM-reduced presynaptic terminals. Lower AP180 and CALM also reduced the density of SVs and the size of SV clusters. Our findings demonstrate that in the presynaptic terminals of hippocampal neurons, AP180 and CALM have a similar role in regulating synaptic vesicles. This overlapping activity may be necessary for high-precision and high-efficacy SV formation during endocytosis.

Cholesterol is an essential component of eukaryotic plasma membranes and plays an important role in membrane organization and signaling processes. It is the major lipid component of detergent resistant caveolin-1 containing rafts which previously had been reported as a platform for nerve growth factor (NGF) signaling in oligodendrocytes (OL). Surprisingly, a knockdown of caveolin-1 attenuated the process formation of OL (Schmitz et al. J Neurosci Res 88:572-588, 2010), for which a loss of cholesterol could be responsible. In the present report, we could show that a caveolin-1 knockdown resulted in an elevation of cellular cholesterol level; it may indicate an important role of caveolin-1 in cholesterol trafficking to the plasma membrane. Treatment with exogenous PEG cholesterol, which was incorporated to the plasma membrane, supported oligodendroglial process formation, in particular when OL were stimulated by NGF. In this context we have found that OL express NPC1L1 (Niemann-Pick disease type C1-Like 1) which could modulate cholesterol uptake. In contrast, depletion of membrane-bound cholesterol diminished NGF-induced process formation concomitant with a reduced activity of p42/44 mitogen-activated protein kinases.

Rationale: An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH)., Objective: Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC., Methods and Results: The nifedipine-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt)., Conclusions: The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.

The serotonin 5-HT(7) receptor has been linked to various psychiatric disorders, including schizophrenia, anxiety and depression, and is antagonized by antipsychotics such as risperidone, clozapine and lurasidone. In this study, we examined whether inhibiting the 5-HT(7) receptor could reverse behavioral abnormalities in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP), an experimental mouse model for psychiatric disorders such as schizophrenia. The selective 5-HT(7) antagonist SB-269970 effectively suppressed abnormal jumping behavior in PACAP-deficient mice. SB-269970 tended to alleviate the higher immobility in the forced swim test in PACAP-deficient mice, although SB-269970 reduced the immobility also in wild-type mice. In addition, we found that mutant mice had impaired performance in the Y-maze test, which was reversed by SB-269970. In the mutant mouse brain, 5-HT(7) protein expression did not differ from wild-type mice. In primary embryonic hippocampal neurons, the 5-HT(7) agonist AS19 increased neurite length and number. Furthermore, SB-269970 significantly inhibited the increase in neurite extension mediated by the 5-HT(1A/7) agonist 8-OH-DPAT. These results indicate that 5-HT(7) receptor blockade ameliorates psychomotor and cognitive deficits in PACAP-deficient mice, providing additional evidence that the 5-HT(7) receptor is a rational target for the treatment of psychiatric disorders.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases neurite outgrowth, although signaling via its receptor PACAP-specific receptor (PAC1R) has not been fully characterized. Because mitochondria also play an important role in neurite outgrowth, we examined whether mitochondria contribute to PACAP-mediated neurite outgrowth. When mouse primary hippocampal neurons and Neuro2a cells were exposed to PACAP, neurite outgrowth and the mitochondrial membrane potential increased in both cell types. These results were reproduced using the PAC1R-specific agonist maxadilan and the adenylate cyclase activator forskolin, whereas the protein kinase A inhibitor H89 and mitochondrial uncoupling agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) inhibited these effects. Expression levels of peroxisome proliferator-activated receptor γ coactivator 1α (Pgc1α), a master regulator of mitochondrial activation, and its downstream effectors, such as cytochrome C and cytochrome C oxidase subunit 4, increased in response to PACAP. Knocking down Pgc1α expression using small interfering RNA or treatment with CCCP significantly attenuated neurite outgrowth and reduced the mitochondrial membrane potential in PACAP-treated cells. These data suggest that mitochondrial activation plays a key role in PACAP-induced neurite outgrowth via a signaling pathway that includes PAC1R, PKA, and Pgc1α.

Atypical cadherin Celsr3, a regulator of planar cell polarity, is critical for the development of the axonal blueprint. We previously showed that expression of Celsr3 is necessary to establish forebrain connections such as the anterior commissure and thalamocortical and corticospinal tracts. The requirement for Celsr3 during hippocampal wiring and its action in the hippocampus remain largely unexplored. Here, we compared the connectivity and maturation of the hippocampal formation in Celsr3|Foxg1 and Celsr3|Dlx mice. Celsr3 is inactivated in the whole telencephalon, including the hippocampal primordium, in Celsr3|Foxg1 mice, and in the early basal telencephalon, including ganglionic eminences and ventral diencephalon, in Celsr3|Dlx mice. Behavioral tests showed that both mutants were hyperactive and had impaired learning and memory. Abnormal cytoarchitecture of CA1, CA3, and dentate gyrus was found in the Celsr3|Foxg1 mutant, in which afferent and efferent hippocampal pathways, as well as intrinsic connections, were dramatically disrupted. In Celsr3|Dlx mutant mice, hippocampal cytoarchitecture was mildly affected and extrinsic and intrinsic connectivity moderately disturbed. In both mutants, pyramidal neurons in CA1 harbored atrophic dendritic trees, with decreased synapse density and increased proportion of symmetric versus asymmetric synapses, and long-term potentiation was altered. In contrast, mutant hippocampal neurons extended neurites that were normal, even longer than those of control neurons, indicating that anomalies in vivo are secondary to defective connections. Postnatal neurogenesis was preserved and mutant interneurons were able to migrate to the hippocampus. Thus, like in neocortex, Celsr3 is required for hippocampal development, connectivity and function, and for pyramidal cell maturation.

Microtubule organization and dynamics are essential during axon and dendrite formation and maintenance in neurons. However, little is known about the regulation of microtubule dynamics during synaptic development and function in mammalian neurons. Here, we present evidence that the microtubule plus-end tracking protein CLASP2 (cytoplasmic linker associated protein 2) is a key regulator of axon and dendrite outgrowth that leads to functional alterations in synaptic activity and formation. We found that CLASP2 protein levels steadily increase throughout neuronal development in the mouse brain and are specifically enriched at the growth cones of extending neurites. The short-hairpin RNA-mediated knockdown of CLASP2 in primary mouse neurons decreased axon and dendritic length, whereas overexpression of human CLASP2 caused the formation of multiple axons, enhanced dendritic branching, and Golgi condensation, implicating CLASP2 in neuronal morphogenesis. In addition, the CLASP2-induced morphological changes led to significant functional alterations in synaptic transmission. CLASP2 overexpression produced a large increase in spontaneous miniature event frequency that was specific to excitatory neurotransmitter release. The changes in presynaptic activity produced by CLASP2 overexpression were accompanied by increases in presynaptic terminal circumference, total synapse number, and a selective increase in presynaptic proteins that are involved in neurotransmitter release. Also, we found a smaller increase in miniature event amplitude that was accompanied by an increase in postsynaptic surface expression of GluA1 receptor localization. Together, these results provide evidence for involvement of the microtubule plus-end tracking protein CLASP2 in cytoskeleton-related mechanisms underlying neuronal polarity and interplay between microtubule stabilization and synapse formation and activity.

Introduction: Epidemiological studies indicate that obesity is associated with an increased risk of 20-25% with several types of cancer., Objective: The frequency of chromosome aberrations was evaluated in lymphocytes from postmenopausal obese and non-obese women., Materials and Methods: Twenty obese and 20 non-obese women, all post-menopause, were recruited. The groups were matched according to age (± 5 years) and place of origin. After signing the consent form, women were interviewed using a structured questionnaire, and a blood sample (5 ml) was drawn into vacutainer tubes. From each sample, lymphocyte cell cultures were established with and without mitomycin C (challenge assay). Afterwards, the frequency of chromosome aberrations were recorded for each group and treatment. Data were analyzed using the statistical program SPSS, v. 14.0., Results: Obese women had a higher frequency of chromosome aberrations when compared with non-obese women. After exposing the cell cultures to mitomycin C, obese women presented an increase in the number of total chromosome aberrations in comparison to non-obese women (3.7± 0.6 vs. 2.70±0.6; p=0.001)., Conclusions: The higher frequency of chromosome aberrations in lymphocytes from postmenopausal obese women compared to non-obese women suggested differences in the DNA repair capacity. This may indicate an association between genomic instability and the higher incidence of cancer in this population.

Introduction: Mutagens contained in complex mixtures can present synergistic interactions, either additive or antagonistic. Therefore, development of experimental approaches is necessary to elucidate which is the responsible agent for the effect in the mixtures., Objective: An experimental design was developed that allowed an understanding of the processes between the compounds of complex mixtures., Materials and Methods: Human lymphocytes were exposed to binary mixtures of the mutagens B[a]P, DMBA, Trp-P-1 and MX for 1 hour with or without S9. Viability was assessed with trypan blue dye and the genotoxicity by the comet assay., Results: All of the hydrocarbon showed an effect with furanone. With and without S9, the most toxic interactions were observed between hydrocarbons. Synergistic interaction was observed without S9 between B [a] P and Trp-P-1 and between DMBA and Trp-P-1 with metabolic activity. Without S9 antagonistic interaction was observed only between Trp-P-1+DMBA, and with S9 between Trp-P-1+MX and MX+DMBA. It observed an increase dose dependent in tail length. Half the cultures showed genotoxic damage and increased cell damage. For each mixture, minimum concentrations were determined at which adverse effects are observed; for some only the maximum concentration was determined at which no adverse effects are observed., Conclusion: The processes between mutagens present in a mixture have become better understood, and the results validated an analytical model that determined which component had an effect on another. The results also showed that the type of compounds in the mixture determined whether or not a risk threshold was present.

The recent steep increase in population dose from radiation-based medical diagnostics, such as computed tomography (CT) scans, requires insight into human health risks, especially in terms of cancer development. Since the induction of genetic damage is considered a prominent cause underlying the carcinogenic potential of ionizing radiation, we quantified the induction of micronuclei and loss of heterozygosity events in human cells after exposure to clinically relevant low doses of X rays. A linear dose-response relationship for induction of micronuclei was observed in human fibroblasts with significantly increased frequencies at doses as low as 20 mGy. Strikingly, cells exposed during S-phase displayed the highest induction, whereas non S-phase cells showed no significant induction below 100 mGy. Similarly, the induction of loss of heterozygosity in human lymphoblastoid cells quantified at HLA loci, was linear with dose and reached significance at 50 mGy. Together the findings favor a linear-no-threshold model for genetic damage induced by acute exposure to ionizing radiation. We speculate that the higher radiosensitivity of S-phase cells might relate to the excessive cancer risk observed in highly proliferative tissues in radiation exposed organisms.

Polymorphonuclear neutrophils constitute the first line of defense against infections. Among their strategies to eliminate pathogens they release neutrophil extracellular traps (NETs), being chromatin fibers decorated with antimicrobial proteins. NETs trap and kill pathogens very efficiently, thereby minimizing tissue damage. Furthermore, NETs modulate inflammatory responses by activating plasmacytoid dendritic cells. In this study, we show that NETs released by human neutrophils can directly prime T cells by reducing their activation threshold. NETs-mediated priming increases T cell responses to specific Ags and even to suboptimal stimuli, which would not induce a response in resting T cells. T cell priming mediated by NETs requires NETs/cell contact and TCR signaling, but unexpectedly we could not demonstrate a role of TLR9 in this mechanism. NETs-mediated T cell activation adds to the list of neutrophil functions and demonstrates a novel link between innate and adaptive immune responses.

Transglutaminases are Ca(2+)-dependent enzymes that catalyse the covalent cross-linking of protein-bound glutamine and lysine residues, which can stabilise proteins or protein aggregates. In the brain, elevated expression levels and activity of transglutaminases are known to be linked with several neurodegenerative diseases. However, little is known about the physiological functions of transglutaminases in the central nervous system. In this study, we examined the expression and activity of transglutaminase 1 in chicken telencephalic cell cultures. We observed a cytosolic expression of transglutaminase 1 in telencephalic neurons. However, transglutaminase 1 activity was restricted to synaptic endings. Transglutaminase targets in the cultured cells were characterised via a biotinylation assay and β-actin was identified as a substrate. Furthermore, we were able to show that β-actin is a target for the activity of recombinant human transglutaminase 1 in vitro. We propose a mechanism where neuronal transglutaminase 1 is activated by synaptic activity-dependent influx of calcium ions and thereupon catalyse the formation of an intramolecular cross-link in β-actin, thereby stabilising the actin cytoskeleton against depolymerising effects. In this way, transglutaminase 1 could modulate actin-dependent plasticity events at synaptic endings.

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 10(6) protoplasts/1.0g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder characterized by progressive myopathy that is often accompanied by bone weakening and/or frontotemporal dementia. Although it is known to be caused by mutations in the gene encoding valosin-containing protein (VCP), the underlying disease mechanism remains elusive. Like IBMPFD, neurofibromatosis type 1 (NF1) is an autosomal dominant disorder. Neurofibromin, the protein encoded by the NF1 gene, has been shown to regulate synaptogenesis. Here, we show that neurofibromin and VCP interact and work together to control the density of dendritic spines. Certain mutations identified in IBMPFD and NF1 patients reduced the interaction between VCP and neurofibromin and impaired spinogenesis. The functions of neurofibromin and VCP in spinogenesis were shown to correlate with the learning disability and dementia phenotypes seen in patients with IBMPFD. Consistent with the previous finding that treatment with a statin rescues behavioral defects in Nf1(+/-) mice and providing further support for our hypothesis that there is crosstalk between neurofibromin and VCP, statin exposure neutralized the effect of VCP knockdown on spinogenesis in cultured hippocampal neurons. The data presented here demonstrate that there is a link between IBMPFD and NF1 and indicate a role for VCP in synapse formation.

Genetic variations in the DTNBP1 gene (encoding the protein dysbindin-1) have been implicated as risk factors in the pathogenesis of schizophrenia. Previous studies have indicated that dysbindin-1 functions in the regulation of synaptic activity. Recently, dysbindin-1 has also been documented to be involved in neuronal development. In this study, we identified necdin as a binding partner of dysbindin-1 using a yeast two-hybrid screen. Dysbindin-1 recruits necdin to the cytoplasm, thereby attenuating the repressive effects of necdin on p53 transcriptional activity. Knockdown of dysbindin-1, like knockdown of p53, greatly decreases the expressions of the p53 target genes coronin 1b and rab13, which are required for neurite outgrowth. Moreover, overexpression of p53 restores the neurite outgrowth blocked by dysbindin-1 knockdown. In brains of dysbindin-1 null mice (the sandy strain), p21, Coronin 1b and Rab13 levels are reduced. Furthermore, primary cultured cortical neurons from sandy mice display neurite outgrowth defects when compared with those from wild-type mice. Thus, our data provide evidence that dysbindin-1 has an important role in neurite outgrowth through its regulation of p53's transcriptional activity.

Stress triggers complex response mechanisms designed to recognize and adapt to perturbations in homeostasis. The immune system is highly responsive to stress, although the complete mechanisms linking stress and immune mediators including T lymphocytes, are not fully understood. Stress exerts its effects on immune effectors through two primary pathways: the sympathetic-adrenal-medullary pathway, and the hypothalamic-pituitary-adrenal pathway which modulate adaptive immunity and lymphocyte migration. In this report we show that stress via release of stress hormones induces early T cell activation and greatly impacts the cytoskeleton by modulating numerous actin-regulating proteins. In particular, proteomic profiling revealed significant decreases in numerous key actin-binding proteins including moesin. Although confocal microscopy showed that moesin and actin were uniformly distributed on the surface of resting T cells, a remarkable polarization and redistribution of moesin and actin was observed following treatment with stress hormones with moesin localizing at the distal pole complex. In addition, the alteration in moesin localization and eventual decrease in expression were accompanied by a loss of CD43; a receptor involved in negatively regulating T cell activation. In conclusion, we have defined a novel molecular mechanism whereby stress hormones negatively impact T cell activation and migration through regulation of key cytoskeletal and plasma membrane factors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Inter-individual variation in DNA repair capacity is thought to modulate breast cancer risk. The phenotypic mutagen sensitivity assay (MSA) measures DNA strand breaks in lymphocytes; women with familial and sporadic breast cancers have a higher mean number of breaks per cell (MBPC) than women without breast cancer. Here, we explore the relationships between the MSA and the Rad51 gene, which encodes a DNA repair enzyme that interacts with BRCA1 and BRCA2, in BRCA1 mutation carriers and women with sporadic breast cancer., Methods: Peripheral blood lymphoblasts from women with known BRCA1 mutations underwent the MSA (n = 138 among 20 families). BRCA1 and Rad51 genotyping and sequencing were performed to identify SNPs and haplotypes associated with the MSA. Positive associations from the study in high-risk families were subsequently examined in a population-based case-control study of breast cancer (n = 1170 cases and 2115 controls)., Results: Breast cancer diagnosis was significantly associated with the MSA among women from BRCA1 families (OR = 3.2 95%CI: 1.5-6.7; p = 0.004). The Rad51 5'UTR 135 C>G genotype (OR = 3.64; 95% CI: 1.38, 9.54; p = 0.02), one BRCA1 haplotype (p = 0.03) and in a polygenic model, the E1038G and Q356R BRCA1 SNPs were significantly associated with MBPC (p = 0.009 and 0.002, respectively). The Rad51 5'UTR 135C genotype was not associated with breast cancer risk in the population-based study., Conclusions: Mutagen sensitivity might be a useful biomarker of penetrance among women with BRCA1 mutations because the MSA phenotype is partially explained by genetic variants in BRCA1 and Rad51.

Feline coronavirus (FCoV) consists of two biotypes based on their growth in cell culture and their antigenicity. Infections with FCoV are highly prevalent in the cat population worldwide. In this study, Felis catus whole fetus (Fcwf-4)cell culture was infected with FCoV UPM11C/08. Virus multiplication in cell culture was monitored and examined under the transmission electron microscope. The virus particles revealed the characteristic morphology of feline FCoV represented by envelope viruses surrounded by peplomers. Virus attachment and entry into the cell occurred 15 h post-infection (pi), and the myriad of virus particles were observed both extracellularly and intracellularly after 48 h pi. Thereafter, intracellular virus particles were observed to be present in vacuoles or present freely in the cytoplasm.

As a new kind of non-contacting high-resolution scanning probe microscopy, hopping probe scanning ion conductance microscopy (HPSICM) overcomes the disadvantages of the continuous feedback-control scanning mode of the conventional scanning ion conductance microscopy (SICM), which has been restricted to imaging relatively flat biological samples. The basic operation principles of HPSICM were briefly introduced in this paper. Then the high-resolution 3D morphological images of the two live neuronal cell lines, SK-N-SH cells and NGF-differentiated neurite PC12 cells, were respectively acquired in their physiological culture environment using HPSICM. It is demonstrated that HPSICM provides a powerful microscopy for in-depth studying the microstructures and their dynamic changes of live neuronal cells in real time.

Our objective was to determine the presence of chromosomal abnormalities in primary cultures of ovarian surface epithelial cells in women of different ages with no history of cancer. Throughout conventional cytogenetic techniques, we analyzed chromosome spreads of cultured ovarian epithelial cells from 10 donors who were 50 or more years old (B) and 16 controls between 20 and 49 years old (A), belonging to the mestizo population in Bogota DC, Colombia. Of the 26 cultures that were analyzed in passage 1, 61.5% had an abnormal chromosome complement (62.5% in A, and 60% in B). Abnormalities included polyploidies, endoduplications and monosomies. Deletions in chromosomes 3 and 11 were found in just one metaphase. None of the samples showed weaknesses or breakpoints. After transforming and applying the exact student's t-test for variance heterogeneity, we found significant differences in the frequency of metaphases, that were higher in A than in B (p=0.05), and in the frequency of polyploidies, which were higher in B than in A (p=0.044). Through the application of the Mann-Whitney test, we determined that the frequency of endoduplications was higher in A than in B (p=0.126), without reaching significant differences. There were no significant differences in the frequency of monosomies. The level of significance was set at p < or = 0.05. Taking into account that polyploidization is a marker of chromosomal instability and that the risk of cancer arising from the ovarian surface epithelium augments substantially after menopause, the increase in the frequency of age-associated polyploidies could be used as a predictor of ovarian cancer in women from an ethnically homogeneous population as the mestizo one in Bogota DC.

Synapse loss induced by amyloid beta (Abeta) is thought to be a primary contributor to cognitive decline in Alzheimer's disease. Abeta is generated by proteolysis of amyloid precursor protein (APP), a synaptic receptor whose physiological function remains unclear. In the present study, we investigated the role of APP in dendritic spine formation, which is known to be important for learning and memory. We found that overexpression of APP increased spine number, whereas knockdown of APP reduced spine density in cultured hippocampal neurons. This spine-promoting effect of APP required both the extracellular and intracellular domains of APP, and was accompanied by specific upregulation of the GluR2, but not the GluR1, subunit of AMPA receptors. In an in vivo experiment, we found that cortical layers II/III and hippocampal CA1 pyramidal neurons in 1 year-old APP-deficient mice had fewer and shorter dendritic spines than wild-type littermates. In contrast, transgenic mice overexpressing mutant APP exhibited increased spine density compared to control animals, though only at a young age prior to overaccumulation of soluble amyloid. Additionally, increased glutamate synthesis was observed in young APP transgenic brains, whereas glutamate levels were decreased and GABA levels were increased in APP-deficient mice. These results demonstrate that APP is important for promoting spine formation and is required for proper spine development., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Several algorithms have been proposed for detecting fluorescently labeled subcellular objects in microscope images. Many of these algorithms have been designed for specific tasks and validated with limited image data. But despite the potential of using extensive comparisons between algorithms to provide useful information to guide method selection and thus more accurate results, relatively few studies have been performed., Results: To better understand algorithm performance under different conditions, we have carried out a comparative study including eleven spot detection or segmentation algorithms from various application fields. We used microscope images from well plate experiments with a human osteosarcoma cell line and frames from image stacks of yeast cells in different focal planes. These experimentally derived images permit a comparison of method performance in realistic situations where the number of objects varies within image set. We also used simulated microscope images in order to compare the methods and validate them against a ground truth reference result. Our study finds major differences in the performance of different algorithms, in terms of both object counts and segmentation accuracies., Conclusions: These results suggest that the selection of detection algorithms for image based screens should be done carefully and take into account different conditions, such as the possibility of acquiring empty images or images with very few spots. Our inclusion of methods that have not been used before in this context broadens the set of available detection methods and compares them against the current state-of-the-art methods for subcellular particle detection.

von Willebrand factor (VWF) is an essential mediator of platelet adhesion to the vessel wall, but little is known about its role in megakaryocytopoiesis. VWF and its platelet receptor, glycoprotein Ibalpha (GPIbalpha), are both expressed during megakaryocyte (MK) maturation. This study was designed to evaluate whether the enhanced VWF-GPIbalpha interactions typical of patients with von Willebrand disease type 2B (VWD2B) modify platelet production. Platelets from 9 patients with VWD2B with 7 different gain-of-function mutations were examined by electron microscopy (EM) and immunofluorescence labeling. For the patients with VWD2B, EM characteristically showed variable numbers of structurally abnormal giant platelets, sometimes in agglutinates. Cultures of MKs from controls performed with or without purified VWF confirmed a positive influence of VWF on platelet production with specific inhibition by an antibody blocking VWF binding to GPIbalpha. VWD2B MK cultures examined by EM showed a disorganized demarcation membrane system and abnormal granule distribution. They produced platelets with structural abnormalities typical of VWD2B. Confocal examination of MK revealed limited extension of pseudopods with few large proplatelets. These results confirm that megakaryocytopoiesis is modified by the enhanced VWF-GPIbalpha interactions. These data obtained for controls and patients with VWD2B suggest a novel regulatory role of VWF-GPIbalpha interactions in platelet production.

Rational: Vascular smooth muscle cells (VSMCs) switching from a contractile/differentiated to a synthetic/dedifferentiated phenotype has an essential role in atherosclerosis, postangioplastic restenosis and hypertension. However, how normal VSMCs maintain the differentiated state is less understood., Objective: We aimed to indentify the effect of cartilage oligomeric matrix protein (COMP), a normal vascular extracellular matrix, on modulation of VSMCs phenotype., Methods and Results: We demonstrated that COMP was associated positively with the expression of VSMC differentiation marker genes during phenotype transition. Knockdown of COMP by small interfering (si)RNA favored dedifferentiation. Conversely, adenoviral overexpression of COMP markedly suppressed platelet-derived growth factor-BB-elicited VSMC dedifferentiation, characterized by altered VSMC morphology, actin fiber organization, focal adhesion assembly, and the expression of phenotype-dependent markers. Whereas alpha(7) integrin coimmunoprecipitated with COMP in normal rat VSMCs and vessels, neutralizing antibody or siRNA against alpha(7) integrin inhibited VSMC adhesion to COMP, which indicated that alpha(7)beta(1) integrin is a potential receptor for COMP. As well, blocking or interference by siRNA of alpha(7) integrin completely abolished the effect of COMP on conserving the contractile phenotype. In accordance, ectopic adenoviral overexpression of COMP greatly retarded VSMC phenotype switching, rescued contractility of carotid artery ring, and inhibited neointima formation in balloon-injured rats., Conclusions: Our data suggest that COMP is essential for maintaining a VSMC contractile phenotype and the protective effects of COMP are mainly mediated through interaction with alpha(7)beta(1) integrin. Investigations to identify the factors affecting the expression and integrity of COMP may provide a novel therapeutic target for vascular disorders.

Fanconi anemia (FA) is a rare autosomal recessive genetic disease, associated with congenital anomalies and a predisposition to cancers. FA patients exhibit spontaneous chromosome breakage and FA cells are sensitive to DNA interstrand crosslink agents and expresses high frequency of chromosome breakage. Recently 13 genes have been shown to be involved with the FA phenotype. We have carried out a detailed study in clinically diagnosed FA patients in an Indian population. Thirty three patients were clinically diagnosed with FA and had aplastic anemia and bleeding abnormalities. The genetic analysis revealed a significantly (P<0.0001) high frequency (36.4%) of parental consanguinity in FA patients compared to controls (3.33%). Chromosomal analysis revealed spontaneous chromosome breakage in 63.64% FA patients. The mitomycin C and diepoxybutane induced cultures showed a significantly (P<0.001) high frequency of chromosome breakage and radial formation compared to controls. Among 33 patients, nine (27.27%) patients developed malignancies and chromosomal abnormalities were detected in five (55.5%) patients bone marrow cells including monosomy 5 and 7, trisomy 10, der(1q) and inv(7). Cytogenetic investigation is important in aplastic anemia to rule out FA. The clinical presentation and the associated high frequency of consanguinity in FA, and the molecular analysis are complementary in the study of an Indian population.

Objective: Megakaryocytic cells (Mks) undergo endomitosis and become polyploid. Mk ploidy correlates with platelet production. We previously showed that nicotinamide (NIC) greatly increases Mk ploidy in cultures of human mobilized peripheral blood CD34(+) cells. This study aims to examine the generality of NIC effects, NIC's impact on Mk ultrastructure, and potential mechanisms for the increased ploidy., Materials and Methods: We used electron microscopy to examine Mk ultrastructure and flow cytometry to evaluate NIC effects on Mk differentiation and ploidy in mobilized peripheral blood CD34(+) cell cultures under diverse megakaryopoietic conditions. Mk ploidy and NAD(H) content were evaluated for NIC and other NAD(+) precursors. We tested additional inhibitors of the sirtuin (or SIRT) 1 and SIRT2 histone/protein deacetylases and, after treatment with NIC, evaluated changes in the acetylation of SIRT1/2 targets., Results: NIC increased ploidy under diverse culture conditions and did not alter Mk ultrastructure; 6.25 mM NIC increased NAD(+) levels fivefold. Quinolinic acid increased NAD(+) similar to that for 1 mM NIC, but yielded a much smaller ploidy increase. Similar increases in Mk ploidy were obtained using NIC or the SIRT1/2 inhibitor cambinol, while the SIRT2 inhibitor AGK2 moderately increased ploidy. SIRT1/2 inhibition in cells treated with NIC was evidenced by increased acetylation of nucleosomes and p53. Greater p53 acetylation with NIC was associated with increased binding of p53 to its consensus DNA binding sequence., Conclusion: NIC greatly increases Mk ploidy under a wide range of conditions without altering Mk morphology. Inhibition of SIRT1 and/or SIRT2 is primarily responsible for NIC effects on Mk maturation.