Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy

BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disease resulting from mutations in the survival motor neuron 1 (SMN1) gene. Recent breakthroughs in preclinical research have highlighted several potential novel therapies for SMA, increasing the need for robust and sensitive clinical trial platforms for evaluating their effectiveness in human patient cohorts. Given that most clinical trials for SMA are likely to involve young children, there is a need for validated molecular biomarkers to assist with monitoring disease progression and establishing the effectiveness of therapies being tested. Proteomics technologies have recently been highlighted as a potentially powerful tool for such biomarker discovery. METHODS: We utilized label-free proteomics to identify individual proteins in pathologically-affected skeletal muscle from SMA mice that report directly on disease status. Quantitative fluorescent western blotting was then used to assess whether protein biomarkers were robustly changed in muscle, skin and blood from another mouse model of SMA, as well as in a small cohort of human SMA patient muscle biopsies. RESULTS: By comparing the protein composition of skeletal muscle in SMA mice at a pre-symptomatic time-point with the muscle proteome at a late-symptomatic time-point we identified increased expression of both Calreticulin and GRP75/Mortalin as robust indicators of disease progression in SMA mice. We report that these protein biomarkers were consistently modified in different mouse models of SMA, as well as across multiple skeletal muscles, and were also measurable in skin biopsies. Furthermore, Calreticulin and GRP75/Mortalin were measurable in muscle biopsy samples from human SMA patients. CONCLUSIONS: We conclude that label-free proteomics technology provides a powerful platform for biomarker identification in SMA, revealing Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers capable of reporting on disease progression in samples of muscle and skin

High quality ultrafast transmission electron microscopy using resonant microwave cavities

Ultrashort, low-emittance electron pulses can be created at a high repetition rate by using a TM$_{110}$ deflection cavity to sweep a continuous beam across an aperture. These pulses can be used for time-resolved electron microscopy with atomic spatial and temporal resolution at relatively large average currents. In order to demonstrate this, a cavity has been inserted in a transmission electron microscope, and picosecond pulses have been created. No significant increase of either emittance or energy spread has been measured for these pulses. At a peak current of $814\pm2$ pA, the root-mean-square transverse normalized emittance of the electron pulses is $\varepsilon_{n,x}=(2.7\pm0.1)\cdot 10^{-12}$ m rad in the direction parallel to the streak of the cavity, and $\varepsilon_{n,y}=(2.5\pm0.1)\cdot 10^{-12}$ m rad in the perpendicular direction for pulses with a pulse length of 1.1-1.3 ps. Under the same conditions, the emittance of the continuous beam is $\varepsilon_{n,x}=\varepsilon_{n,y}=(2.5\pm0.1)\cdot 10^{-12}$ m rad. Furthermore, for both the pulsed and the continuous beam a full width at half maximum energy spread of $0.95\pm0.05$ eV has been measured

Host microbiota dictates the proinflammatory impact of LPS in the murine liver

Gut microbiota can impact liver disease development via the gut-liver axis. Liver inflammation is a shared pathological event in various liver diseases and gut microbiota might influence this pathological process. In this study, we studied the influence of gut microbiota on the inflammatory response of the liver to lipopolysaccharide (LPS). The inflammatory response to LPS (1–10 μg/ml) of livers of specific-pathogen-free (SPF) or germ-free (GF) mice was evaluated ex vivo, using precision-cut liver slices (PCLS). LPS induced a more pronounced inflammatory response in GF PCLS than in SPF PCLS. Baseline TNF-α gene expression was significantly higher in GF slices as compared to SPF slices. LPS treatment induced TNF-α, IL-1β, IL-6 and iNOS expression in both SPF and GF PCLS, but the increase was more intense in GF slices. The anti-inflammatory markers SOCS3 and IRAK-M gene expression was significantly higher in GF PCLS than SPF PCLS at 24h with 1 µg/ml LPS treatment, and IL-10 was not differently expressed in GF PCLS than SPF PCLS. In addition, TLR-4 mRNA, but not protein, at basal level was higher in GF slices than in SPF slices. Taken together, this study shows that, in mice, the host microbiota attenuates the pro-inflammatory impact of LPS in the liver, indicating a positive role of the gut microbiota on the immune homeostasis of the liver

Mesothelial cells regulate immune responses in health and disease: Role for immunotherapy in malignant mesothelioma

Highlights: Mesothelial and immune cell interactions play a crucial role in tissue homeostasis in the serosal cavities such as the pleura. Mesothelin is viewed as an attractive target for solid tumors, including malignant mesothelioma. Checkpoint inhibitor therapy has shown variable efficacy against malignant mesothelioma. CAR T cell therapies are being evaluated for malignant mesothelioma. Treatment of malignant mesothelioma will require multimodality approaches with immunotherapy central to future therapeutic approaches. The mesothelium when first described was thought to function purely as a non-adhesive surface to facilitate intracoelomic movement of organs. However, the mesothelium is now recognized as a dynamic cellular membrane with many important functions that maintain serosal integrity and homeostasis. For example, mesothelial cells interact with and help regulate the body’s inflammatory and immune system following infection, injury, or malignancy. With recent advances in our understanding of checkpoint molecules and the advent of novel immunotherapy approaches, there has been an increase in the number of studies examining mesothelial and immune cell interaction, in particular the role of these interactions in malignant mesothelioma. This review will highlight some of the recent advances in our understanding of how mesothelial cells help regulate serosal immunity and how in a malignant environment, the immune system is hijacked to stimulate tumor growth. Ways to treat mesothelioma using immunotherapy approaches will also be discussed

Theory and particle tracking simulations of a resonant radiofrequency deflection cavity in TM110_{110} mode for ultrafast electron microscopy

We present a theoretical description of resonant radiofrequency (RF) deflecting cavities in TM$_{110}$ mode as dynamic optical elements for ultrafast electron microscopy. We first derive the optical transfer matrix of an ideal pillbox cavity and use a Courant-Snyder formalism to calculate the 6D phase space propagation of a Gaussian electron distribution through the cavity. We derive closed, analytic expressions for the increase in transverse emittance and energy spread of the electron distribution. We demonstrate that for the special case of a beam focused in the center of the cavity, the low emittance and low energy spread of a high quality beam can be maintained, which allows high-repetition rate, ultrafast electron microscopy with 100 fs temporal resolution combined with the atomic resolution of a high-end TEM. This is confirmed by charged particle tracking simulations using a realistic cavity geometry, including fringe fields at the cavity entrance and exit apertures

Idiopathic pulmonary fibrosis and a role for autoimmunity

Idiopathic Pulmonary Fibrosis (IPF) is the most common of the idiopathic interstitial pneumonias. It is typically associated with extensive and progressive fibrosis, is fatal and has limited treatment options. Characteristically IPF patients display large lymphocyte aggregates composed of CD3+ T cells and CD20+ B cells within the lung tissue that are located near sites of active fibrosis. In addition, IPF patients can have autoantibodies to a range of host antigens, suggesting a breakdown in immunological tolerance. In this review we examine the role of T and B cells in IPF pathogenesis and discuss how loss of self- tolerance to lung specific proteins could exacerbate disease progression in IPF. We discuss what these results mean in terms of future prospects for immunotherapy of IPF

Direct magneto-optical compression of an effusive atomic beam for high-resolution focused ion beam application

An atomic rubidium beam formed in a 70 mm long two-dimensional magneto-optical trap (2D MOT), directly loaded from a collimated Knudsen source, is analyzed using laser-induced fluorescence. The longitudinal velocity distribution, the transverse temperature and the flux of the atomic beam are reported. The equivalent transverse reduced brightness of an ion beam with similar properties as the atomic beam is calculated because the beam is developed to be photoionized and applied in a focused ion beam. In a single two-dimensional magneto-optical trapping step an equivalent transverse reduced brightness of $(1.0\substack{+0.8-0.4})$ $\times 10^6$ A/(m$^2$ sr eV) was achieved with a beam flux equivalent to $(0.6\substack{+0.3-0.2})$ nA. The temperature of the beam is further reduced with an optical molasses after the 2D MOT. This increased the equivalent brightness to $(6\substack{+5-2})$$\times 10^6$ A/(m$^2$ sr eV). For currents below 10 pA, for which disorder-induced heating can be suppressed, this number is also a good estimate of the ion beam brightness that can be expected. Such an ion beam brightness would be a six times improvement over the liquid metal ion source and could improve the resolution in focused ion beam nanofabrication.Comment: 10 pages, 8 figures, 1 tabl