Does Optic Nerve Head Surface Topography Change Prior to Loss of Retinal Nerve Fiber Layer Thickness: A Test of the Site of Injury Hypothesis in Experimental Glaucoma

Purpose

To test the hypothesis that optic nerve head (ONH) deformation manifesting as changes in its mean surface height precedes thinning of the peripapillary retinal nerve fiber layer (RNFL) in experimental glaucoma (EG).

Methods

68 rhesus macaque monkeys each had three or more baseline imaging sessions under manometric intraocular pressure (IOP) control to obtain average RNFL thickness (RNFLT) and the ONH surface topography parameter mean position of the disc (MPD). Laser photocoagulation was then applied to the trabecular meshwork of one eye to induce chronic, mild-to-moderate IOP elevation and bi-weekly imaging continued. Event analysis was applied to determine for each parameter when an ‘endpoint’ occurred (signficant change from baseline) for eight different endpoint criteria. Specificity was assessed in the group of 68 fellow control eyes. Classical signal detection theory and survival analysis were used to compare MPD with RNFLT.

Results

Regardless of the endpoint criterion, endpoints were always more frequent for MPD than for RNFLT. The discriminability index (d’) was 2.7 ± 0.2 for MPD and 1.9 ± 0.2 for RNFLT (p<0.0001). Endpoints were reached by MPD an average of 1-2 months earlier than by RNFLT (p<0.01). At the onset of the first specific, detectable MPD change in EG eyes, there was still no significant change in RNFLT on average (p=0.29) and only 25% of individual eyes exhibited signficant reduction. In contrast, at onset of signficant RNFLT change, MPD had already changed an average of 101 µm from baseline (p<0.0001) and 71% of the individual eyes had exhibited significant change. The magnitude of MPD change was more than could be explained on the basis of axon loss alone.

Conclusions

This study demonstrates that the average surface height of the ONH changes prior to any detectable loss of average peripapillary RNFL thickness in non-human primate eyes with experimental glaucoma.     

Gene overexpression/suppression analysis of candidate virulence factors of Candida albicans

We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enable simultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involved insertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adherence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis. In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibility to neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenously disseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitro phenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumulation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins. These data indicate that the same gene can increase or decrease candidal virulence in distinct models of infection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally, these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidal virulence factors.     

Does drainage pay? Quantifying agricultural profitability associated with wetland drainage practices and canola production in Alberta

Conversion of wetlands in cultivated agricultural landscapes is one of the primary drivers of wetland loss in Alberta, Canada, despite a provincial wetland policy that prioritizes wetland avoidance. While other sectors of the agricultural industry have established initiatives to maintain wetlands, a common narrative within the conventional cropping sector is that wetland retention leads to lost acreage and overlap of crop inputs, and that there are financial benefits associated with wetland drainage. The objective of this research was to explicitly quantify crop productivity within drained wetland basins, in an effort to better understand the extent to which producers financially benefit from drainage practices. Working collaboratively with canola producers in central Alberta over the 2019 growing season, wetland basins within four quarter sections were mapped using an Unmanned Aerial Vehicle, and wetland basins with clear evidence of surface drainage were identified. Agricultural input and yield data provided by producers was then used to quantify profitability within each drained basin. Average profit for drained basins for each producer ranged between ? $145/acre and $76/acre, with an average of $55/acre across all operations. This is compared to an average profit of $203/acre for non-wetland areas across all operations. The results suggest that the financial benefits of drainage are highly variable, and for many drained basins, producers may experience financial losses that may be overlooked when profits are examined only at the field- or operation-level. While this study included a small number of operations, and was limited to one type of crop over a single growing season, the results still provide important insight into the extent to which producers benefit financially from the practice of wetland drainage.

     

Hydrological and biological processes modulate carbon,nitrogen and phosphorus flux from the St. Lawrence River to its estuary (Quebec,Canada)

Changes in atmospheric deposition, stream water chemistry, and solute fluxes were assessed across 15 small forested catchments. Dramatic changes in atmospheric deposition have occurred over the last three decades, including a 70% reduction in sulphur (S) deposition. These changes in atmospheric inputs have been associated with expected changes in levels of acidity, sulphate and base cations in streams. Soil retention of S appeared to partially explain rates of chemical recovery. In addition to these changes in acid–base chemistry we also observed unexpected changes in nitrogen (N) biogeochemistry and nutrient stoichiometry of stream water, including decreased stream N concentrations. Among all catchments the average flux of dissolved inorganic nitrogen (DIN) was best predicted by average runoff, soil chemistry (forest floor C/N) and levels of acid deposition (both S and N). The rate of change in stream DIN flux, however, was much more closely correlated with reductions in rates of S deposition rather than those of DIN. Unlike DIN fluxes, the average concentrations as well as the rates of decline in streamwater nitrate (NO₃) concentration over time were tightly linked to stream dissolved organic carbon/dissolved organic nitrogen ratios DOC/DON and DON/TP rather than catchment characteristics. Declines in phosphorus adsorption with increasing soil pH appear to contribute to the relationship between C, N, and P in our study catchments. Our observations suggest that catchment P availability and its alteration due to environmental changes (e.g. acidification) might have profound effects on N cycling and catchment N retention that have been largely unrecognized.     

The role of the plastic surgeon in the management of surgical infection.

At our Medical Center, our reconstructive service has actively sought referrals of acute and chronic infections by declaring an interest in undertaking the integrated management and reconstruction of these cases. The practices of the two senior surgeons were reviewed for three academic years (1992 to 1995). Cases of surgical infection were analyzed as to site, ablative procedures, and reconstructive procedures. In total, 139 patients with 147 infections were identified. Sites of infection included head and neck (9.5 percent), trunk and pelvis (39.5 percent), upper extremity (22 percent), and lower extremity (29 percent). One-hundred thirty-one ablative procedures were done on this group, as were 126 reconstructive procedures, including 17 fasciocutaneous flaps, 26 pedicled muscle flaps, and 28 microsurgical flaps. With a mean follow-up of 14 months, 92 percent of these patients had resolution of infection. The 8 percent failure group included recurrences, amputation, and death. This series demonstrates that a plastic surgery service can attract a diverse population of surgical infections and manage them successfully with ablation and a wide variety of reconstructive procedures. The coordination of ablation and reconstruction may be optimally performed by the plastic surgeon.     

Role of VEGF in small bowel adaptation after resection: the adaptive response is angiogenesis dependent

Previous work in our group has demonstrated that mouse salivary gland has the highest concentration of salivary-derived VEGF protein compared with other organs and is essential for normal palatal mucosal wound healing. We hypothesize that salivary VEGF plays an important role in maintaining the integrity of the gastrointestinal mucosa following small bowel resection (SBR). Thirty-five 8- to 10-wk-old C57BL/6 female mice were divided into seven treatment groups: 1) sham (transaction and anastomosis, n = 5); 2) SBR (n = 8); 3) sialoadenectomy and small bowel resection (SAL+SBR, n = 8); 4) sialoadenectomy and small bowel resection with EGF supplementation (SAL+SBR+EGF, n = 9); 5) sialoadenectomy and small bowel resection with VEGF supplementation (SAL+SBR+VEGF, n = 9); 6) sialoadenectomy and small bowel resection supplemented with EGF and VEGF (SAL+ SBR+VEGF+EGF, n = 6); 7) selective inhibition of VEGF in the submandibular gland by Ad-VEGF-Trap following small bowel resection (Ad-VEGF-Trap+SBR, n = 7). Adaptation was after 3 days by ileal villus height and crypt depth. The microvascular response was evaluated by CD31 immunostaining and for villus-vessel area ratio by FITC-labeled von Willebrand factor immunostaining. The adaptive response after SBR was significantly attenuated in the SAL group in terms of villus height (250.4 +/- 8.816 vs. 310 +/- 19.35, P = 0.01) and crypt depth (100.021 +/- 4.025 vs. 120.541 +/- 2.82, P = 0.01). This response was partially corrected by orogastric VEGF or EGF alone. The adaptive response was completely restored when both were administered together, suggesting that salivary VEGF and EGF both contribute to intestinal adaptation. VEGF increases the vascular density (6.4 +/- 0.29 vs. 6.1 +/- 0.29 vs. 5.96 +/- 0.20) and villus-vessel area ratio (0.713 +/- 0.01 vs. 0.73 +/- 0.01) in the adapting bowel. Supplementation of both EGF and VEGF fully rescues adaptation, suggesting that the adaptive response may be dependent on VEGF-driven angiogenesis. These results support a previously unrecognized role for VEGF in the small bowel adaptive response.     

Pleiotropic functions of TNF-alpha determine distinct IKKbeta-dependent hepatocellular fates in response to LPS

TNF-alpha influences morbidity and mortality during the course of endotoxemia. However, the complex pleiotropic functions of TNF-alpha remain poorly understood. We evaluated how hepatic induction of NF-kappaB and TNF-alpha influence survival and hepatocellular death in a lethal murine model of endotoxic shock. Using dominant-negative viral vectors to inhibit the IKK complex, we demonstrate through this study that the liver is a major source of TNF-alpha during the course of lethal endotoxemia and that IKKbeta (but not IKKalpha) is predominantly responsible for activating NF-kappaB and TNF-alpha in the liver after LPS administration. Using TNF-alpha knockout mice and hepatic-specific inhibition of IKKbeta, we demonstrate that the status of TNF-alpha and NF-kappaB balances necrotic and apoptotic fates of hepatocytes in the setting of endotoxemia. In the presence of TNF-alpha, inhibiting hepatic IKKbeta resulted in increased survival, reduced serum proinflammatory cytokines, and reduced hepatocyte necrosis in response to a lethal dose of endotoxin. In contrast, inhibiting hepatic IKKbeta in TNF-alpha knockout mice resulted in decreased survival and increased caspase 3-mediated hepatocyte apoptosis after endotoxin challenge, despite a reduced proinflammatory cytokine response. In the presence of TNF-alpha, NF-kappaB-dependent hepatocellular necrosis predominated, while in the absence of TNF-alpha, NF-kappaB primarily influenced apoptotic fate of hepatocytes. Changes in JNK phosphorylation after LPS challenge were also dynamically affected by both IKKbeta and TNF-alpha; however, this pathway could not solely explain the differential outcomes in hepatocellular fates. In conclusion, our studies demonstrate that induction of NF-kappaB and TNF-alpha balances protective (antiapoptotic) and detrimental (proinflammatory) pathways to determine hepatocellular fates during endotoxemia.     

Insights from atomic-resolution X-ray structures of chemically synthesized HIV-1 protease in complex with inhibitors

The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.