Jonathan Franks

Carbon nanotubes degraded by neutrophil myeloperoxidase induce less pulmonary inflammation

We have shown previously that single-walled carbon nanotubes can be catalytically biodegraded over several weeks by the plant-derived enzyme, horseradish peroxidase. However, whether peroxidase intermediates generated inside human cells or biofluids are involved in the biodegradation of carbon nanotubes has not been explored. Here, we show that hypochlorite and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase catalyse the biodegradation of single-walled carbon nanotubes in vitro, in neutrophils and to a lesser degree in macrophages. Molecular modelling suggests that interactions of basic amino acids of the enzyme with the carboxyls on the carbon nanotubes position the nanotubes near the catalytic site. Importantly, the biodegraded nanotubes do not generate an inflammatory response when aspirated into the lungs of mice. Our findings suggest that the extent to which carbon nanotubes are biodegraded may be a major determinant of the scale and severity of the associated inflammatory responses in exposed individuals.

Histone deacetylase 6–mediated selective autophagy regulates COPD-associated cilia dysfunction

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) that are associated with epithelial cell dysfunction, cilia shortening, and mucociliary clearance disruption. Exposure to CS reduced cilia length and induced autophagy in vivo and in differentiated mouse tracheal epithelial cells (MTECs). Autophagy-impaired (Becn1+/- or Map1lc3B-/-) mice and MTECs resisted CS-induced cilia shortening. Furthermore, CS increased the autophagic turnover of ciliary proteins, indicating that autophagy may regulate cilia homeostasis. We identified cytosolic deacetylase HDAC6 as a critical regulator of autophagy-mediated cilia shortening during CS exposure. Mice bearing an X chromosome deletion of Hdac6 (Hdac6-/Y) and MTECs from these mice had reduced autophagy and were protected from CS-induced cilia shortening. Autophagy-impaired Becn1-/-, Map1lc3B-/-, and Hdac6-/Y mice or mice injected with an HDAC6 inhibitor were protected from CS-induced mucociliary clearance (MCC) disruption. MCC was preserved in mice given the chemical chaperone 4-phenylbutyric acid, but was disrupted in mice lacking the transcription factor NRF2, suggesting that oxidative stress and altered proteostasis contribute to the disruption of MCC. Analysis of human COPD specimens revealed epigenetic deregulation of HDAC6 by hypomethylation and increased protein expression in the airways. We conclude that an autophagy-dependent pathway regulates cilia length during CS exposure and has potential as a therapeutic target for COPD.

Liver-Specific β-Catenin Knockout Mice Have Bile Canalicular Abnormalities, Bile Secretory Defect, and Intrahepatic Cholestasis

Beta‐catenin plays important roles in liver physiology and hepatocarcinogenesis. While studying the role of β‐catenin in diet‐induced steatohepatitis, we recently found that liver‐specific β‐catenin knockout (KO) mice exhibit intrahepatic cholestasis. This study was undertaken to further characterize the role of β‐catenin in biliary physiology. KO mice and wild‐type (WT) littermates were fed standard chow or a diet supplemented with 0.5% cholic acid for 2 weeks. Chow‐fed KO mice had higher serum and hepatic total bile acid levels and lower bile flow rate than WT mice. Expression levels of bile acid biosynthetic genes were lower and levels of major bile acid exporters were similar, which therefore could not explain the KO phenotype. Despite loss of the tight junction protein claudin‐2, KO mice had preserved functional integrity of tight junctions. KO mice had bile canalicular morphologic abnormalities as evidenced by staining for F‐actin and zona occludens 1. Electron microscopy revealed dilated and tortuous bile canaliculi in KO livers along with decreased canalicular and sinusoidal microvilli. KO mice on a cholic acid diet had higher hepatic and serum bile acid levels, bile ductular reaction, increased pericellular fibrosis, and dilated, misshapen bile canaliculi. Compensatory changes in expression levels of several bile acid transporters and regulatory genes were found in KO livers. Conclusion: Liver‐specific loss of β‐catenin leads to defective bile canalicular morphology, bile secretory defect, and intrahepatic cholestasis. Thus, our results establish a critical role for β‐catenin in biliary physiology. (HEPATOLOGY 2010)

Invasion of Erythrocytes by Francisella tularensis

Francisella tularensis is the causative agent of tularemia and is classified as a category A biodefense agent by the Centers for Disease Control and Prevention because of its highly infectious nature. F. tularensis infects leukocytes and exhibits an extracellular phase in the blood of the host. It is unknown, however, whether F. tularensis can infect erythrocytes; thus, we examined this possibility in vivo and in vitro. In the murine model of pulmonary type A tularemia, we showed the presence of intraerythrocytic bacteria by double-immunofluorescence microscopy and ex vivo gentamicin protection of the purified erythrocyte fraction. In vitro, F. tularensis invaded human erythrocytes, as shown in the gentamicin protection assays, double-immunofluorescence microscopy, flow cytometry, scanning electron microscopy, and transmission electron microscopy with immunogold labeling of the bacteria. Additional in vitro tests indicated that serum complement-dependent and complement-independent mechanisms contribute to erythrocyte invasion. Our results reveal a novel intraerythrocytic phase during F. tularensis infection.

Ex vivo carbon monoxide delivery inhibits intimal hyperplasia in arterialized vein grafts

AIMS Veins are still the best conduits available for arterial bypass surgery. When these arterialized vein grafts fail, it is often due to the development of intimal hyperplasia (IH). We investigated the feasibility and efficacy of the ex vivo pre-treatment of vein grafts with soluble carbon monoxide (CO) in the inhibition of IH. METHODS AND RESULTS The inferior vena cava was excised from donor rats and placed as an interposition graft into the abdominal aorta of syngeneic rats. Prior to implantation, vein grafts were stored in cold Lactated Ringer (LR) solution with or without CO saturation (bubbling of 100% CO) for 2 h. Three and 6 weeks following grafting, vein grafts treated with cold LR for 2 h developed IH, whereas grafts implanted immediately after harvest demonstrated significantly less IH. Treatment in CO-saturated LR significantly inhibited IH and reduced vascular endothelial cell (VEC) apoptosis. Electron microscopy revealed improved VEC integrity with less platelet/white blood cell aggregation in CO-treated grafts. The effects of CO in preventing IH were associated with activation of hypoxia inducible factor-1α (HIF-1α) and an increase in vascular endothelial growth factor (VEGF) expression at 3-6 h after grafting. Treatment with a HIF-1α inhibitor completely abrogated the induction of VEGF by CO and reversed the protective effects of CO on prevention of IH. CONCLUSION Ex vivo treatment of vein grafts in CO-saturated LR preserved VEC integrity perioperatively and significantly reduced neointima formation. These effects appear to be mediated through the activation of the HIF1α/VEGF pathway.

RTD-1mimic containing γPNA scaffold exhibits broad-spectrum antibacterial activities.

Macrocyclic peptides with multiple disulfide cross-linkages, such as those produced by plants and those found in nonhuman primates, as components of the innate immunity, hold great promise for molecular therapy because of their broad biological activities and high chemical, thermal, and enzymatic stability. However, for some, because of their intricate spatial arrangement and elaborate interstrand cross-linkages, they are difficult to prepare de novo in large quantities and high purity, due to the nonselective nature of disulfide-bond formation. We show that the disulfide bridges of RTD-1, a member of the θ-defensin subfamily, could be replaced with noncovalent Watson-Crick hydrogen bonds without significantly affecting its biological activities. The work provides a general strategy for engineering conformationally rigid, cyclic peptides without the need for disulfide-bond reinforcement.

Ultrathin DSAEK Tissue Prepared With a Low-Pulse Energy, High-Frequency Femtosecond Laser

Purpose: To evaluate the endothelial cell survival and stromal bed quality when creating deep stromal cuts with a low–pulse energy, high-frequency femtosecond laser to produce “ultrathin” tissue for Descemet stripping automated endothelial keratoplasty. Methods: Seventeen corneas were used for this study. Five corneas were cut with the laser at a depth of 420 to 500 &mgr;m to produce a tissue thickness of approximately ⩽70 &mgr;m. Five corneas served as an uncut comparison group. Vital dye staining and computer digitized planimetry analysis were performed on these corneas. The 7 remaining corneas were cut for scanning electron microscopy evaluation. Results: The mean central posterior stromal thickness of cut corneas was 60.6 &mgr;m (range, 43–72 &mgr;m). Endothelial cell damage in cut and comparison corneas was 3.92% ± 2.22% (range, 1.71%–6.51%) and 4.15% ± 2.64% (range, 1.21%–7.01%), respectively (P = 0.887). Low-magnification (×12) scanning electron microscopy revealed a somewhat irregular-appearing surface with concentric rings peripherally. Qualitative grading of higher magnification (×50) central images resulted in an average score of 2.56 (between smooth and rough). Conclusions: Ultrathin tissue for Descemet stripping automated endothelial keratoplasty can be safely prepared with minimal endothelial cell damage using a low–pulse energy, high-frequency femtosecond laser; however, the resulting stromal surface quality may not be optimal with this technique.

Sickle Cell Trait Increases Red Blood Cell Storage Hemolysis and Post-Transfusion Clearance in Mice.

Background Transfusion of blood at the limits of approved storage time is associated with lower red blood cell (RBC) post-transfusion recovery and hemolysis, which increases plasma cell-free hemoglobin and iron, proposed to induce endothelial dysfunction and impair host defense. There is noted variability among donors in the intrinsic rate of storage changes and RBC post-transfusion recovery, yet genetic determinants that modulate this process are unclear. Methods We explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hbatm1PazHbbtm1TowTg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS). Findings Human and transgenic HbAS RBCs demonstrate accelerated storage time-dependent hemolysis and reduced post-transfusion recovery in mice. The rapid post-transfusion clearance of stored HbAS RBC is unrelated to macrophage-mediated uptake or intravascular hemolysis, but by enhanced sequestration in the spleen, kidney and liver. HbAS RBCs are intrinsically different from HbAA RBCs, with reduced membrane deformability as cells age in cold storage, leading to accelerated clearance of transfused HbAS RBCs by entrapment in organ microcirculation. Interpretation The common genetic variant HbAS enhances RBC storage dysfunction and raises provocative questions about the use of HbAS RBCs at the limits of approved storage.

Correlative Fluorescence and Electron Microscopy in 3D

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in 2D to the locations previously highlighted by fluorescence markers, or subsequent high resolution acquisition of tomographic information using a TEM [1]. While fluorescence microscopy can provide insight into location, interactions and transport of chemical species in biological specimens by utilizing fluorescent markers for imaging, the ultimate resolution of the technique is still limited to ~200 nm range using standard approaches and between 20 and 120nm using super-resolution methods. The electron microscopy techniques (SEM and TEM) can provide the next step in resolution (in sub 1 nm regime) to help understand the interactions at the cellular level. The TEM, however, suffers from a relatively small field of view – just 1-2 mm.

The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.