Surfactant protein D contributes to ocular defense against Pseudomonas aeruginosa in a murine model of dry eye disease.

Dry eye disease can cause ocular surface inflammation that disrupts the corneal epithelial barrier. While dry eye patients are known to have an increased risk of corneal infection, it is not known whether there is a direct causal relationship between these two conditions. Here, we tested the hypothesis that experimentally-induced dry eye (EDE) increases susceptibility to corneal infection using a mouse model. In doing so, we also examined the role of surfactant protein D (SP-D), which we have previously shown is involved in corneal defense against infection. Scopolamine injections and fan-driven air were used to cause EDE in C57BL/6 or Black Swiss mice (wild-type and SP-D gene-knockout). Controls received PBS injections and were housed normally. After 5 or 10 days, otherwise uninjured corneas were inoculated with 10(9) cfu of Pseudomonas aeruginosa strain PAO1. Anesthesia was maintained for 3 h post-inoculation. Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation. SP-D was measured by Western immunoblot, and corneal pathology assessed from 6 h to 4 days. EDE mice showed reduced tear volumes after 5 and 10 days (each by ∼75%, p<0.001) and showed fluorescein staining (i.e. epithelial disruption). Surprisingly, there was no significant difference in corneal pathology between EDE mice and controls (∼10-14% incidence). Before bacterial inoculation, EDE mice showed elevated SP-D in ocular washes. After inoculation, fewer bacteria were recovered from ocular washes of EDE mice (<2% of controls, p = 0.0004). Furthermore, SP-D knockout mice showed a significant increase in P. aeruginosa corneal colonization under EDE conditions. Taken together, these data suggest that SP-D contributes to corneal defense against P. aeruginosa colonization and infection in EDE despite the loss of barrier function to fluorescein

MicroRNA-762 is upregulated in human corneal epithelial cells in response to tear fluid and Pseudomonas aeruginosa antigens and negatively regulates the expression of host defense genes encoding RNase7 and ST2.

Mucosal surfaces regulate defenses against infection and excessive inflammation. We previously showed that human tears upregulated epithelial expression of genes encoding RNase7 and ST2, which inhibited Pseudomonas aeruginosa invasion of human corneal epithelial cells. Here, microRNA microarrays were used to show that a combination of tear fluid exposure (16 h) then P. aeruginosa antigens (3 h) upregulated miR-762 and miR-1207, and down-regulated miR-92 and let-7b (all > 2-fold) in human corneal epithelial cells compared to P. aeruginosa antigens alone. RT-PCR confirmed miR-762 upregulation ∼ 3-fold in tear-antigen exposed cells. Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization. However, P. aeruginosa invasion was increased > 3-fold by a miR-762 mimic which reduced RNase7 and ST2 gene expression. Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir. Combination of tear fluid and miR-762 antagomir increased RNase7 and ST2 gene expression. These data show that mucosal fluids, such as tears, can modulate epithelial microRNA expression to regulate innate defense genes, and that miR-762 negatively regulates RNase7, ST2 and Rab5a genes. Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria. These data also suggest a complex relationship between tear induction of miR-762, its modulation of innate defense genes, and P. aeruginosa internalization

3D quantitative imaging of unprocessed live tissue reveals epithelial defense against bacterial adhesion and subsequent traversal requires MyD88.

While a plethora of in vivo models exist for studying infectious disease and its resolution, few enable factors involved in the maintenance of health to be studied in situ. This is due in part to a paucity of tools for studying subtleties of bacterial-host interactions at a cellular level within live organs or tissues, requiring investigators to rely on overt outcomes (e.g. pathology) in their research. Here, a suite of imaging technologies were combined to enable 3D and temporal subcellular localization and quantification of bacterial distribution within the murine cornea without the need for tissue processing or dissection. These methods were then used to demonstrate the importance of MyD88, a central adaptor protein for Toll-Like Receptor (TLR) mediated signaling, in protecting a multilayered epithelium against both adhesion and traversal by the opportunistic bacterial pathogen Pseudomonas aeruginosa ex vivo and in vivo

Sternal pain after rigid fixation: a pilot study of randomization rigid vs conventional wire closure.

Objective: Rigid sternal fixation may provide better sternal closure than conventional sternal wire closure. We performed a prospective randomized study to investigate if rigid closure reduces postoperative sternal pain. Methods: Patients undergoing CABG ± valve surgery between July 2011 and January 2012 were prospectively randomized into conventional wire closure (group C) or rigid fixation using sternal plates (group R). Pain scores were determined at 6 AM using a numeric rating scale (0 no pain, 5 moderate pain, 10 worst possible pain). Narcotic pain medication requirement from day 1 to 5 was collected and converted into intravenous morphine equivalent. Results: Among the total of 26 patients, 11 patients were in Group R (10 male and 1 female, age 67 ± 8.0) and 15 patients were in Group C (13 male and 2 female, age 66 ± 9.9). Preoperative risk factors and procedure were identical between the two groups. Pain scores were not significantly different between 2 groups. Narcotic requirement was smaller in group R (15.7 mg intravenous morphine equivalent in group R in day 1vs 18.4 mg intravenous morphine equivalent in day 1 in group C in day 1, 13.1 mg vs 12.5 mg in day 2, 9.4 mg vs 10.5 mg in day 3, 6.9 mg vs 7.7 mg in day 4, and 6.2 mg vs 6.9 mg in day 5) than group C. Total iv narcotic given over 5 days was 24 ± 41 mg in group R and 34 mg ± 54 mg in group C (p=0.60). Conclusion: Randomized data rom this ongoing study showed a trend of fewer narcotic requirement especially intravenous narcotics in group R than in group C. Implications: Rigid fixation may potentially improve immediate sternal pain after open heart surgery. Less narcotic requirement potentially facilitate early return to the daily activity

Phosphorylation and calcium antagonistically tune myosin-binding protein C\u27s structure and function

During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C\u27s N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain\u27s extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C\u27s inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C\u27s calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C\u27s phosphorylation state

A Comparison of the Intrinsic Shapes of Two Different Types of Dwarf Galaxies: Blues Compact Dwarfs and Dwarf Ellipticals

We measure the apparent shapes for a sample of 62 blue compact dwarf galaxies (BCDs), and compare them with the apparent shapes for a sample of 80 dwarf elliptical galaxies (dEs). The BCDs are flatter, on average, than the dEs, but the difference is only marginally significant. We then use both non-parametric and parametric techniques to determine possible distributions of intrinsic shapes for the BCDs. The hypothesis that BCDs are oblate spheroids can be ruled out with a high confidence level ($> 99$), but the hypothesis that they are prolate spheroids cannot be excluded. The apparent shapes of BCDs are totally consistent with the hypothesis that they are triaxial ellipsoids. If the intrinsic axis ratios, $\beta$ and $\gamma$, are distributed according to a Gaussian with means $\beta_0$ and $\gamma_0$ and standard deviation $\sigma$, we find the best-fitting distribution for BCDs has $(\beta_0,\gamma_0,\sigma)= (0.66,0.55,0.16)$, while that for dEs has $(\beta_0,\gamma_0,\sigma)= (0.85,0.64,0.24)$. Our results are consistent with the hypothesis that BCDs have a close evolutionary relation with dEs.Comment: total 23 pages, 9 figures, and 1 Table, submitted to ApJ on Sep 19 1997. Email addresses: [email protected], [email protected], [email protected], [email protected], [email protected]

Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling

The beating heart exhibits remarkable contractile fidelity over a lifetime, which reflects the tight coupling of electrical, chemical, and mechanical elements within the sarcomere, the elementary contractile unit. On a beat-to-beat basis, calcium is released from the ends of the sarcomere and must diffuse toward the sarcomere center to fully activate the myosin- and actin-based contractile proteins. The resultant spatial and temporal gradient in free calcium across the sarcomere should lead to nonuniform and inefficient activation of contraction. We show that myosin-binding protein C (MyBP-C), through its positioning on the myosin thick filaments, corrects this nonuniformity in calcium activation by exquisitely sensitizing the contractile apparatus to calcium in a manner that precisely counterbalances the calcium gradient. Thus, the presence and correct localization of MyBP-C within the sarcomere is critically important for normal cardiac function, and any disturbance of MyBP-C localization or function will contribute to the consequent cardiac pathologies

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism

The conceptualisation and impact of a post-graduate course in lesson and learning study

This paper tells a story about the design, development and impact of a post-graduate Masters-level module aimed at (1) enabling groups of teachers within schools to develop innovative approaches to teaching and learning on the basis of their own Lesson Studies and (2) creating a school network of excellence for Lesson Study in the area as a context for building a cumulative evidence-base, which focuses on identifying and resolving enduring problems of teaching and learning in schools. The first part of the paper outlines the curriculum for the module and sets the innovative conceptual framework that underpins its design . This framework is innovative because it connects and unifies a number of distinct pedagogical perspectives. It links the methodology of Japanese Lesson Study with Stenhouse’s idea of ‘the teacher as a researcher’ and his ‘process model’ of curriculum development as an alternative to the globally dominant ‘objectives model’. Then in turn, the framework incorporates Marton and Booth’s pedagogical theory of ‘variation’. The paper argues that linking and fusing Lesson Study methodology with this wider context of pedagogical ideas unambiguously renders teacher research as learnin