Photodynamic antibacterial effect of graphene quantum dots

Synthesis of new antibacterial agents is becoming increasingly important in light of the emerging antibiotic resistance. In the present study we report that electrochemically produced graphene quantum dots (GQD), a new class of carbon nanoparticles, generate reactive oxygen species when photoexcited (470 nm, 1 W), and kill two strains of pathogenic bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli. Bacterial killing was demonstrated by the reduction in number of bacterial colonies in a standard plate count method, the increase in propidium iodide uptake confirming the cell membrane damage, as well as by morphological defects visualized by atomic force microscopy. The induction of oxidative stress in bacteria exposed to photoexcited GQD was confirmed by staining with a redox-sensitive fluorochrome dihydrorhodamine 123. Neither GQD nor light exposure alone were able to cause oxidative stress and reduce the viability of bacteria. Importantly, mouse spleen cells were markedly less sensitive in the same experimental conditions, thus indicating a fairly selective antibacterial photodynamic action of GQD.     

Inhibition of apoptosis in human induced pluripotent stem cells during expansion in a defined culture using angiopoietin-1 derived peptide QHREDGS

Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival, self-renewal, and differentiation. Thus, hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture. Soluble angiopoeitin-1 (Ang-1)-derived peptide QHREDGS added to defined serum-free media increased hPSC colony cell number and size during long- and short-term culture when grown on feeder cell layers or Matrigel, i.e. on standard substrates, without affecting hPSC morphology, growth rate or the ability to differentiate into multiple lineages both in vitro and in vivo. Importantly, QHREDGS treatment decreased hPSC apoptosis during routine passaging and single-cell dissociation. Mechanistically, the interaction of QHREDGS with β₁-integrins increased expression of integrin-linked kinase (ILK), increased expression and activation of extracellular signal-regulated kinases 1/2 (ERK1/2), and decreased caspase-3/7 activity. QHREDGS immobilization to polyethylene glycol hydrogels significantly increased cell adhesion in a dose-dependent manner. We propose QHREDGS as a small molecule inhibitor of hPSC apoptosis and the basis of an affordable defined substrate for hPSC maintenance.     

Reshaping of T-lymphocyte compartment in adult prepubertaly ovariectomised rats: A putative role for progesterone deficiency

This study explores the role of ovarian hormones in the phenotypic shaping of peripheral T-cell pool over the reproductive lifespan of rats. For this purpose, 2-month-old prepubertally ovariectomised (Ox) rats, showing oestrogen and progesterone deficiency, and 11-month-old Ox rats, exhibiting only progesterone deficiency, were examined for thymus output, and cellularity and composition of major TCRαβ+ peripheral blood lymphocyte (PBL) and splenocyte subsets. Although ovariectomy increased thymic output in both 2- and 11-month-old rats, the count of both CD4+ and CD8+ PBLs and splenocytes increased only in the former. In the blood and spleen of 11-month-old Ox rats only the count of CD8+ cells increased. Although ovariectomy affected the total CD4+ count in none of the examined compartments from the 11-month-old rats, it increased CD4+FoxP3+ PBL and splenocyte relative proportions over those in the age-matched controls. The age-related differences in the cellularity and the major subset composition in Ox rats were linked to the differences in the ovarian steroid hormone levels registered in 2- and 11-month-old rats. The administration of progesterone to Ox rats during the seven days before the sacrificing confirmed contribution of this hormone deficiency to the ovariectomy-induced changes in the TCRαβ+ PBL and splenocyte pool from 11-month-old rats. The expansion of the CD8+ splenocyte subset in the 11-month-old Ox rats reflected increases in cellularity of memory and, particularly, naïve cells. This was due to greater thymic output of CD8+ cells and homeostatic proliferation than apoptosis in 11-month-old Ox rats when compared with age-matched sham-Ox control rats. The homeostatic changes within CD8+ splenocyte pool from 11-month-old Ox rats, most likely, reflected the enhanced splenic IL-7 and TGF-β mRNA expression. Overall, in adult female rats, circulating oestrogen and progesterone provide maintenance of T-cell counts, a diversity of T-cell repertoire, and the main T-cell subset composition in the periphery. Progesterone deficiency affects mainly the CD8+ lymphocyte compartment through increasing thymic CD8+ cell export and upsetting homeostatic regulation within the CD8+ splenocyte pool. These alterations were reversible through progesterone supplementation.     

Central nervous system-infiltrated immune cells induce calcium increase in astrocytes via astroglial purinergic signaling

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell–astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca²⁺ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca²⁺ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca²⁺ increase is specifically triggered by the autoreactive CD4⁺ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca²⁺ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell–astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.     

Coronary thrombi neovascularization in patients with ST-elevation myocardial infarction - clinical and angiographic implications

Introduction

Coronary artery thrombosis in ST-elevation myocardial infarction (STEMI) is a dynamic process often preceded by episodes of silent plaque rupture and subocclusive thrombosis. Thrombus organization is achieved by ingrowth of endothelial and smooth muscle cells. Clinical significance and impact of thrombus neovascularization on primary percutaneous coronary intervention (pPCI) outcome remain unclear. Therefore we investigated composition and neovascularization of thrombi aspirated during pPCI and their association with clinical and angiographic parameters of STEMI patients.

Methods

Aspirated thrombi retrieved from 84 STEMI patients were classified as fresh (< 1 day), lytic (1-5 days) or organized (> 5 days). Thrombus neovascularization was evaluated immunohistochemically using CD34, CD31 and VEGF antibodies. CD34 and CD31 immunopositive (CD34/CD31 +) cells were organized as single, clusters and microvessels. VEGF positivity was graded as low or high, based on thrombus surface immunopositive area.

Results

CD34/CD31 + cells were present in 67% of all aspirated thrombi. Thrombus CD34/CD31 positivity was associated with previous history of angina pectoris (χ² = 6.142, p = 0.013) and lower myocardial blush grade (MBG < 3, χ² = 12.602, p < 0.001). Organization of CD34/CD31 + cells showed inverse association with the extent of VEGF positivity (χ² = 10.607, p = 0.005). Fresh thrombi were associated with shorter ischemic time (U = 237.5, p = 0.002) and MBG 3 (χ² = 6.379, p = 0.012).

Conclusions

Older thrombus age and neovascularization are associated with suboptimal myocardial perfusion in STEMI patients. Thrombus VEGF expression is inversely associated with degree of CD34 + cell organization. Therefore, neovascularization of aspirated thrombi may indicate the duration of thrombosis, coronary microcirculation status and outcome in STEMI patients.     

Fourier components of inhomogeneously broadened water resonances in breast: a new source of MRI contrast.

High spectral and spatial resolution (HiSS) MR data were acquired at 1.5 T using echo-planar spectroscopic imaging from patients with suspicious breast lesions. The water resonances in small voxels are inhomogenously broadened and often have distinct components. Images were calculated with intensity proportional to the Fourier components of the water resonance in each voxel at different offsets from the peak frequency. The results demonstrate that in breast the off-peak Fourier component images of water are qualitatively different from those derived from the peak height of the water resonance. These differences most likely reflect underlying anatomy or physiology. In conventional images, the superposition of the various Fourier components of the water signal may cause loss of detail. The synthesis of water Fourier component images from high spectral and spatial resolution data may provide a new form of contrast, and increase sensitivity to subvoxel physiology and anatomy.     

Spatiotemporal tracking of cells in tissue‐engineered cardiac organoids

Cardiac tissue engineering aims to create myocardial patches for repair of defective or damaged native heart muscle. The inclusion of non‐myocytes in engineered cardiac tissues has been shown to improve the properties of cardiac tissue compared to tissues engineered from enriched populations of myocytes alone. While attempts have been made to mix non‐myocytes (fibroblasts, endothelial cells) with cardiomyocytes, very little is understood about how the tissue properties are affected by varying the respective ratios of the three cell types and how these cells assemble into functional tissues with time. The goal of this study was to investigate the effects of modulating the ratios of the three cell types and to spatially and temporally track cardiac tri‐cultures of cells. Primary neonatal cardiac fibroblasts and D4T endothelial cells were incubated in 5 µM CellTracker^? green dye and CellTracker^? red dye, respectively, while neonatal cardiomyocytes were labelled with 20 µg/mL DAPI. The non‐myocytes were seeded either sequentially (pre‐culture) or simultaneously (tri‐culture) in Matrigel‐coated microchannels and allowed to form organoids, as in our previous studies. We also varied the seeding percentage of cardiomyocytes while keeping the total cell number constant in an attempt to improve the functional properties of the organoids. Organoids were imaged on days 1 and 4. Endothelial cells were seen to aggregate into clusters when simultaneously tri‐cultured with myocytes and fibroblasts, while pre‐cultures contained elongated cells. Functional properties of organoids were improved by increasing the seeding percentage of enriched cardiomyocytes from 40% to 80%. Copyright © 2009 John Wiley & Sons, Ltd.