Two mutations in TUBB8 cause developmental arrest in human oocytes and early embryos.

RESEARCH QUESTION: How can the effect of genetic mutations that may cause primary female infertility be evaluated? DESIGN: Patients and their family members underwent whole-exome sequencing and Sanger sequencing to detect the infertility-causing gene and inheritance pattern. To study the function of mutant proteins in vitro, vectors containing wild-type or mutant TUBB8 cDNA were constructed for transient expression in HeLa cells, and in-vitro transcribed mRNA were used for microinjection in germinal vesicle-stage mouse oocytes. Immunofluorescence staining was used to observe the microtubule structure in HeLa cells or meiotic spindle in mouse oocytes. RESULTS: A maternally inherited TUBB8 (Tubulin beta 8 class VIII) mutation (NM_177987.2: c. 959G>A: p. R320H) and a previously reported (NM_177987.2: c. 161C>T: p. A54V) recessive mutation from two infertile female patients were identified. The oocytes from the patient carrying p.A54V mutation failed fertilization, whereas oocytes with p.R320H mutation could be fertilized but showed heavy fragmentation during early development. In vitro, functional assays showed that p. A54V mutant disrupted the microtubule structure in HeLa cells (49.3% of transfected cells) and caused large polar body extrusion in mouse oocytes (27.5%), whereas the p.R320H mutant caused a higher abnormal rate (69.7%) in cultured cells and arrested mouse oocytes at meiosis I (38.7%). CONCLUSION: Two TUBB8 mutations (p.A54V and p.R320H) were identified and their pathogeny was confirmed by in-vitro functional assays.

Exploring rare cellular activity in more than one million cells by a transscale scope.

In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.

Intracellular Delivery of Doxorubicin by Iron Oxide-Based Nano-Constructs Increases Clonogenic Inactivation of Ionizing Radiation in HeLa Cells.

In this study, we determined the potential of polyethylene glycol-encapsulated iron oxide nanoparticles (IONPCO) for the intracellular delivery of the chemotherapeutic doxorubicin (IONPDOX) to enhance the cytotoxic effects of ionizing radiation. The biological effects of IONP and X-ray irradiation (50 kV and 6 MV) were determined in HeLa cells using the colony formation assay (CFA) and detection of γH2AX foci. Data are presented as mean ± SEM. IONP were efficiently internalized by HeLa cells. IONPCO radiomodulating effect was dependent on nanoparticle concentration and photon energy. IONPCO did not radiosensitize HeLa cells with 6 MV X-rays, yet moderately enhanced cellular radiosensitivity to 50 kV X-rays (DMFSF0.1 = 1.13 ± 0.05 (p = 0.01)). IONPDOX did enhance the cytotoxicity of 6 MV X-rays (DMFSF0.1 = 1.3 ± 0.1; p = 0.0005). IONP treatment significantly increased γH2AX foci induction without irradiation. Treatment of HeLa cells with IONPCO resulted in a radiosensitizing effect for low-energy X-rays, while exposure to IONPDOX induced radiosensitization compared to IONPCO in cells irradiated with 6 MV X-rays. The effect did not correlate with the induction of γH2AX foci. Given these results, IONP are promising candidates for the controlled delivery of DOX to enhance the cytotoxic effects of ionizing radiation.

HPV caught in the tetraspanin web?

Tetraspanins are master organizers of the cell membrane. Recent evidence suggests that tetraspanins themselves may become crowded by virus particles and that these crowds/aggregates co-internalize with the viral particles. Using microscopy, we studied human papillomavirus (HPV) type 16-dependent aggregates on the cell surface of tetraspanin overexpressing keratinocytes. We find that aggregates are (1) rich in at least two different tetraspanins, (2) three-dimensional architectures extending up to several micrometers into the cell, and (3) decorated intracellularly by filamentous actin. Moreover, in cells not overexpressing tetraspanins, we note that obscurin-like protein 1 (OBSL1), which is thought to be a cytoskeletal adaptor, associates with filamentous actin. We speculate that HPV contact with the cell membrane could trigger the formation of a large tetraspanin web. This web may couple the virus contact site to the intracellular endocytic actin machinery, possibly involving the cytoskeletal adaptor protein OBSL1. Functionally, such a tetraspanin web could serve as a virus entry platform, which is co-internalized with the virus particle.

Probing Multidimensional Mechanical Phenotyping of Intracellular Structures by Viscoelastic Spectroscopy.

Mechanical phenotyping of complex cellular structures gives insight into the process and function of mechanotransduction in biological systems. Several methods have been developed to characterize intracellular elastic moduli, while direct viscoelastic characterization of intracellular structures is still challenging. Here, we develop a needle tip viscoelastic spectroscopy method to probe multidimensional mechanical phenotyping of intracellular structures during a mini-invasive penetrating process. Viscoelastic spectroscopy is determined by magnetically driven resonant vibration (about 15 kHz) with a tiny amplitude. It not only detects the unique dynamic stiffness, damping, and loss tangent of the cell membrane-cytoskeleton and nucleus-nuclear lamina but also bridges viscoelastic parameters between the mitotic phase and interphase. Self-defined dynamic mechanical ratios of these two phases can identify two malignant cervical cancer cell lines (HeLa-HPV18+, SiHa-HPV16+) whose membrane or nucleus elastic moduli are indistinguishable. This technique provides a quantitative method for studying mechanosensation, mechanotransduction, and mechanoresponse of intracellular structures from a dynamic mechanical perspective. This technique has the potential to become a reliable quantitative measurement method for dynamic mechanical studies of intracellular structures.

Phase-contrast 3D tomography of HeLa cells grown in PLLA polymer electrospun scaffolds using synchrotron X-rays.

Advanced imaging is useful for understanding the three-dimensional (3D) growth of cells. X-ray tomography serves as a powerful noninvasive, nondestructive technique that can fulfill these purposes by providing information about cell growth within 3D platforms. There are a limited number of studies taking advantage of synchrotron X-rays, which provides a large field of view and suitable resolution to image cells within specific biomaterials. In this study, X-ray synchrotron radiation microtomography at Diamond Light Source and advanced image processing were used to investigate cellular infiltration of HeLa cells within poly L-lactide (PLLA) scaffolds. This study demonstrates that synchrotron X-rays using phase contrast is a useful method to understand the 3D growth of cells in PLLA electrospun scaffolds. Two different fiber diameter (2 and 4 µm) scaffolds with different pore sizes, grown over 2, 5 and 8 days in vitro, were examined for infiltration and cell connectivity. After performing visualization by segmentation of the cells from the fibers, the results clearly show deeper cell growth and higher cellular interconnectivity in the 4 µm fiber diameter scaffold. This indicates the potential for using such 3D technology to study cell-scaffold interactions for future medical use.

Mechanistic basis of an epistatic interaction reducing age at onset in hereditary spastic paraplegia.

Many genetic neurological disorders exhibit variable expression within affected families, often exemplified by variations in disease age at onset. Epistatic effects (i.e. effects of modifier genes on the disease gene) may underlie this variation, but the mechanistic basis for such epistatic interactions is rarely understood. Here we report a novel epistatic interaction between SPAST and the contiguous gene DPY30, which modifies age at onset in hereditary spastic paraplegia, a genetic axonopathy. We found that patients with hereditary spastic paraplegia caused by genomic deletions of SPAST that extended into DPY30 had a significantly younger age at onset. We show that, like spastin, the protein encoded by SPAST, the DPY30 protein controls endosomal tubule fission, traffic of mannose 6-phosphate receptors from endosomes to the Golgi, and lysosomal ultrastructural morphology. We propose that additive effects on this pathway explain the reduced age at onset of hereditary spastic paraplegia in patients who are haploinsufficient for both genes.

Near-infrared light-mediated DOX-UCNPs@mHTiO2 nanocomposite for chemo/photodynamic therapy and imaging.

Recently, incorporating multiple components into one nanoplatform for anticancer theranostics has attracted most attention. Herein, a rattle-structured nanocomposite by using UCNPs (NaYF4:Yb,Tm@NaYF4) as core coated with hollow mesoporous TiO2 (UCNPs@mHTiO2) was constructed as the nanocarrier. First, UCNPs@SiO2@TiO2 was prepared, by a selective etching method to remove SiO2 shell, to make sure the hollow mesoporous structure and high surface area (347m2g-1) of UCNPs@mHTiO2. Under near-infrared (NIR) light irradiation, the UV emission can excite TiO2 to produce ROS and to realize photodynamic therapy (PDT). In addition, the hollow structure offers space to store antitumor drug molecules (doxorubicin, DOX) and this nanocomposite also exhibits the improved DOX release in mildly acidic environment, which could greatly promote chemotherapy efficiency. Moreover, the luminescence resonance energy transfer (LRET) from UCNPs to DOX, owing to the effective distance restricted by the cavity, can be used to monitor the intercellular drug release kinetics. HeLa cells were used as the model cancer cells and the detailed cell experiments show the enhanced cytotoxicity, ascribing to the synergistic effect of chemotherapy and PDT. Therefore, the novel multifunctional nanocomposite, combining with chemotherapy, PDT, and imaging, should be a potential candidate in anticancer field.

[Killing effect and its mechanism of low-temperature plasma on different human cancer cell lines].

Objective: To investigate the killing effect of low-temperature plasma (LTP) on HepG2, A549 and HeLa cell lines and explore its possible mechanism. Methods: The inhibitory effect of LTP on the proliferation of HepG2, A549 and HeLa cells was determined by MTT assay. Transmission electron microscopy was used to observe the ultrastructural changes of HepG2, A549 and HeLa cells treated with LTP. Cell apoptosis was detected by Muse cytometry. Western blot was used to detect the expression of apoptosis-related proteins. Results: The survival rates of LTP-irradiated HepG2 cells (irradiated for 107 s), HeLa cells (irradiated for 121 s) and A549 cells (irradiated for 127 s) were 50%. LTP destroyed the ultrastructure of HepG2, A549 and HeLa cells to different degrees, showing nuclear fragmentation and organelle damages. The apoptosis rates of the three cell lines were increased at 24 h after exposure to LTP for 1/6 IC50 irradiation time. Furthermore, LTP irradiation also suppressed the protein expression of Bcl-2 and XRCC1 and increased that of Bax. Conclusions: LTP has an obvious killing effect on HepG2, A549 and HeLa cancer cell lines. This effect may be related to the induction of cell apoptosis and inhibition of DNA repair.

A drug-compatible and temperature-controlled microfluidic device for live-cell imaging.

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.

Contact-dependent transfer of TiO2 nanoparticles between mammalian cells.

Cellular organelles have been shown to shuttle between cells in co-culture. We hereby show that titanium dioxide (TiO2) nanoparticles (NPs) can be transferred in such a manner, between cells in direct contact, along with endosomes and lysosomes. A co-culture system was employed for this purpose and the NP transfer was observed in mammalian cells including normal rat kidney (NRK) and HeLa cells. We found that the small GTPase Arf6 facilitates the intercellular transfer of smaller NPs and agglomerates. Spherical, anatase nano-TiO2 with sizes of 5 (Ti5) and 40 nm (Ti40) were used in this study. Humans are increasingly exposed to TiO2 NPs from external sources such as constituents of foods, cosmetics, and pharmaceuticals, or from internal sources represented by Ti-based implants, which release NPs upon abrasion. Exposure to 5 mg/l of Ti5 and Ti40 for 24 h did not affect cellular viability but modified their ability to communicate with surrounding cells. Altogether, our results have important implications for the design of nanomedicines, drug delivery and toxicity.

Fourier Transform Infrared spectroscopy discloses different types of cell death in flow cytometrically sorted cells.

Fourier Transform Infrared (FTIR) spectroscopy is a label free methodology showing promise in characterizing different types of cell death. Cervical adenocarcinoma (HeLa) and African monkey kidney (Vero) cells were treated with a necrosis inducer (methanol), novel apoptotic inducers (diphenylphosphino gold (I) complexes) and positive control, auranofin. Following treatment, cells stained with annexin-V and propidium iodide were sorted using a Fluorescence Activated Cell Sorter (FACS Aria) to obtain populations consisting of either viable, necrotic or apoptotic cells. Transmission Electron Microscopy confirmed successful sorting of all three populations. Four bands were identified which could discriminate between viable and necrotic cells namely 989 cm(-1), 2852 cm(-1), 2875 cm(-1) and 2923 cm(-1). In HeLa cells viable and induced apoptosis could be distinguished by 1294 cm(-1), while four bands were different in Vero cells namely; 1626 cm(-1), 1741 cm(-1), 2852 cm(-1) 2923 cm(-1). Principal Component Analysis showed separation between the different types of cell death and the loadings plots indicated an increase in an additional band at 1623 cm(-1) in dead cells. FTIR spectroscopy can be developed into an invaluable tool for the assessment of specific types of chemically induced cell death with notably different molecular signatures depending on whether the cells are cancerous and mechanism of cell death.

Low ozone concentrations stimulate cytoskeletal organization, mitochondrial activity and nuclear transcription.

Ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone concentrations and used in medicine as an alternative/adjuvant treatment for different diseases. However, the cellular mechanisms accounting for the positive effects of mild ozonization are still largely unexplored. To this aim, in the present study the effects of low ozone concentrations (1 to 20 µg O3/mL O2) on structural and functional cell features have been investigated in vitro by using morphological, morphometrical, cytochemical and immunocytochemical techniques at bright field, fluorescence and transmission electron microscopy. Cells exposed to pure O2 or air served as controls. The results demonstrated that the effects of ozoneadministration are dependent on gas concentration, and the cytoskeletal organization, mitochondrial activity and nuclear transcription may be differently affected. This suggests that, to ensure effective and permanent metabolic cell activation, ozone treatments should take into account the cytological and cytokinetic features of the different tissues.

Y2O3:Tm,Yb nanophosphors for correlative upconversion luminescence and cathodoluminescence imaging.

We present a phosphor nanoparticle that shows both upconversion luminescence (UCL) and cathodoluminescence (CL). With this particle, low-autofluorescence, deep-tissue and wide-field fluorescence imaging can be achieved with nanometer-order high-spatial-resolution imaging. We synthesized Y2O3:Tm,Yb nanophosphors that emit visible and near-infrared UCL under 980 nm irradiation and blue CL via electron beam excitation. The phosphors were applied to fluorescent imaging of HeLa cells. The photostability of the phosphors was superior to that of a conventional organic dye. We show that after uptake by HeLa cells, the particles can be imaged with SEM and CL contrast in a cellular section. This indicates that correlative UCL and CL imaging of biological samples could be realized.

Application of a high power Yb fiber-based laser compatible with commercial optical parametric oscillator for coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful tool for chemical analysis at a subcellular level, frequently used for imaging lipid dynamics in living cells. We report a high-power picosecond fiber-based laser and its application for optical parametric oscillator (OPO) pumping and CARS microscopy. This fiber-based laser has been carefully characterized. It produces 5 ps pulses with 0.8 nm spectral width at a 1,030 nm wavelength with more than 10 W of average power at 80 MHz repetition rate; these spectral and temporal properties can be slightly modified. We then study the influence of these modifications on the spectral and temporal properties of the OPO. We find that the OPO system generates a weakly spectrally chirped signal beam constituted of 3 ps pulses with 0.4 nm spectral width tunable from 790 to 930 nm optimal for CARS imaging. The frequency doubling unconverted part is composed of 7-8 ps pulses with 0.75 nm spectral width compatible with CARS imaging. We also study the influence of the fiber laser properties on the CARS signal generated by distilled water. In agreement with theory, we find that shorter temporal pulses allow higher peak powers and thus higher CARS signal, if the spectral widths are less than 10 cm(-1) . We demonstrate that this source is suitable for performing CARS imaging of living cells during several hours without photodamages. We finally demonstrate CARS imaging on more complex aquatic organisms called copepods (micro-crustaceans), on which we distinguish morphological details and lipid reserves.

Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution.

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

Immunocytochemistry, electron tomography, and energy dispersive X-ray spectroscopy (EDXS) on cryosections of human cancer cells doped with stimuli responsive polymeric nanogels loaded with iron oxide nanoparticles.

The cryosectioning technique is an alternative method for preparing biological material for Transmission Electron Microscopy (TEM). We have applied this technique to study the mechanism of cell internalization of stimuli-responsive polymeric nanogels exploited as cargo nanovectors. With respect to conventional TEM processing, cryosectioning technique better preserves the morphology of solvent-sensitive nanogels and enhances the visibility of membrane-bounded organelles inside the cell cytoplasm. In this chapter we describe the protocols we have established to perform Electron Microscopy (EM)-immunocytochemistry, Electron Tomography (ET), and Energy Dispersive X-ray Spectroscopy (EDXS) chemical analysis in Scanning TEM (STEM) on cryosections of HeLa cells treated with pH-responsive nanogels hosting short interference RNA (siRNAs) and iron oxide nanoparticles (IONPs).