Clinical Assessment of Lamina Cribrosa Curvature in Eyes with Primary Open-Angle Glaucoma

Purpose

Quantitative evaluation of lamina cribrosa (LC) posterior bowing in primary open-angle glaucoma (POAG) eyes using swept-source optical coherence tomography.

Methods

Patients with POAG (n = 123 eyes) and healthy individuals of a similar age (n = 92 eyes) were prospectively recruited. Anterior laminar insertion depth (ALID) was defined as the vertical distance between the anterior laminar insertion and a reference plane connecting the Bruch’s membrane openings (BMO). The mean LC depth (mLCD) was approximated by dividing the area enclosed by the anterior LC, the BMO reference plane, and the two vertical lines for ALID measurement by the length between those two vertical lines. The LC curvature index was defined as the difference between the mLCD and the ALID. The factors influencing the LC curvature index were evaluated.

Results

The ALID and mLCD were significantly larger in POAG eyes than in healthy controls (P < 0.05). The LC curvature index was significantly larger in POAG eyes than in healthy controls on both the horizontal (85.8 ± 34.1 vs. 68.2 ± 32.3 μm) and vertical meridians (49.8 ± 38.5 vs. 32.2 ± 31.1 μm, all P < 0.001). Multivariate regression showed significant associations of greater disc area (P < 0.001), vertical C/D ratio (P < 0.001) and mLCD (P < 0.001), smaller rim area (P = 0.001), thinner average RNFLT (P < 0.001), and myopic refraction (P = 0.049) with increased LC curvature index. There was no difference in the LC curvature index between mild (MD > –6 dB) and moderate-to-advanced glaucoma (MD < –6 dB, P = 0.95).

Conclusions

LC posterior bowing was increased in POAG eyes, and was significantly associated with structural optic nerve head (ONH) changes but not with functional glaucoma severity. Quantitative evaluation of LC curvature can facilitate assessment of glaucomatous ONH change.     

On the Use of a Test to Exhaustion Specific to Tennis (TEST) with Ball Hitting by Elite Players

PurposeWe aimed to a) introduce a new Test to Exhaustion Specific to Tennis (TEST) and compare performance (test duration) and physiological responses to those obtained during the 20-m multistage shuttle test (MSST), and b) determine to which extent those variables correlate with performance level (tennis competitive ranking) for both test procedures.MethodsTwenty-seven junior players (8 males, 19 females) members of the national teams of the French Tennis Federation completed MSST and TEST, including elements of the game (ball hitting, intermittent activity, lateral displacement), in a randomized order. Cardiorespiratory responses were compared at submaximal (respiratory compensation point) and maximal loads between the two tests.ResultsAt the respiratory compensation point oxygen uptake (50.1 ± 4.7 vs. 47.5 ± 4.3 mL.min^-1.kg^-1, p = 0.02), but not minute ventilation and heart rate, was higher for TEST compared to MSST. However, load increment and physiological responses at exhaustion did not differ between the two tests. Players’ ranking correlated negatively with oxygen uptake measured at submaximal and maximal loads for both TEST (r = -0.41; p = 0.01 and -0.55; p = 0.004) and MSST (r = -0.38; P = 0.05 and -0.51; p = 0.1).ConclusionUsing TEST provides a tennis-specific assessment of aerobic fitness and may be used to prescribe aerobic exercise in a context more appropriate to the game than MSST. Results also indicate that VO₂ values both at submaximal and maximal load reached during TEST and MSST are moderate predictors of players competitive ranking.     

Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and quantify proteomics data generated by ICAT and SILAC mass spectrometric analyses: application to the proteomics study of membrane proteins from primary human endothelial cells

Proteomics strategies based on nanoflow (nano-) LC-MS/MS allow the identification of hundreds to thousands of proteins in complex mixtures. When combined with protein isotopic labeling, quantitative comparison of the proteome from different samples can be achieved using these approaches. However, bioinformatics analysis of the data remains a bottleneck in large scale quantitative proteomics studies. Here we present a new software named Mascot File Parsing and Quantification (MFPaQ) that easily processes the results of the Mascot search engine and performs protein quantification in the case of isotopic labeling experiments using either the ICAT or SILAC (stable isotope labeling with amino acids in cell culture) method. This new tool provides a convenient interface to retrieve Mascot protein lists; sort them according to Mascot scoring or to user-defined criteria based on the number, the score, and the rank of identified peptides; and to validate the results. Moreover the software extracts quantitative data from raw files obtained by nano-LC-MS/MS, calculates peptide ratios, and generates a non-redundant list of proteins identified in a multisearch experiment with their calculated averaged and normalized ratio. Here we apply this software to the proteomics analysis of membrane proteins from primary human endothelial cells (ECs), a cell type involved in many physiological and pathological processes including chronic inflammatory diseases such as rheumatoid arthritis. We analyzed the EC membrane proteome and set up methods for quantitative analysis of this proteome by ICAT labeling. EC microsomal proteins were fractionated and analyzed by nano-LC-MS/MS, and database searches were performed with Mascot. Data validation and clustering of proteins were performed with MFPaQ, which allowed identification of more than 600 unique proteins. The software was also successfully used in a quantitative differential proteomics analysis of the EC membrane proteome after stimulation with a combination of proinflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lymphotoxin alpha/beta) that resulted in the identification of a full spectrum of EC membrane proteins regulated by inflammation.     

A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells

Chromosomal deletions, as a genetic tool for functional genomics, remain underexploited for vertebrate stem cells mostly because presently available methods are too labor-intensive. To address this, we developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions. When applied to mouse embryonic stem cells (ESCs), this retrovirus-based method yielded deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64% for drug-selected clones. Notably, several of the engineered ESC clones, mostly those with large deletions, showed major alteration in cell fate. In comparison to other methods that have also exploited retroviruses for chromosomal engineering, this modified strategy is more efficient and versatile because it bypasses the need for homologous recombination, and thus can be exploited for rapid and extensive functional screens in embryonic and adult stem cells.     

Characterization and regulation of peptidylglycine α-amidating monooxygenase (PAM) expression in H9c2 cardiac myoblasts

Nitric oxide (NO) is a gaseous, radical molecule that plays a role in various physiological processes in the nervous system such as learning and hippocampal plasticity. It is generated from l-arginine by nitric oxide synthases (NOS), which come in three isoforms depending on the tissue of origin, namely inducible-NOS (iNOS in macrophages), endothelial-NOS (eNOS in endothelial cells) and neural-NOS (nNOS in neural cells). We used epidermal growth factor (EGF)-responsive nestin-positive neural precursor cells originating from the mouse E16 embryonic striatum, and studied the relative expression of NOS isoforms probed with isoform-specific antibody using the avidin-biotin immunohistochemical method. Our data revealed both nNOS and eNOS to be expressed in both neurospheres and desegregated neural precursor cells. However, iNOS signals were virtually undetectable in both cell categories. When the neural precursor cells were carried in the presence of poly-l-ornithine (PLO), there was a strong induction of the expression of iNOS proteins, indicating the possibility that this isoform is amenable to modulation by extracellular cues. These preliminary results suggest both nNOS and eNOS to be important in the physiology of neural precursor cells, and that iNOS might also play a role at certain stages in the life of these cells.     

New challenges in the management of the Brazilian Pantanal and catchment area

The Pantanal wetland is a vast seasonally inundated area of extraordinary landscape and biological diversity and complexity. It is located in the upper portion of the Paraguay River basin in central South America. During the rainy season, increased stream discharge from the surrounding basin produces an annual flood pulse through the Pantanal. Increasing human impact, such as dam construction, deforestation, agricultural related activities, and the Hidrovia project in the Parana-Paraguay waterway, threaten the ecological stability of the Pantanal area. As a result, there is an urgent need to introduce new management practices in the Pantanal Basin. In this paper we present a concept for managing the Pantanal catchment based on the integration of ecological knowledge, institutional organization, and involvement of different stakeholders. We propose approaches for an integrated management of the Pantanal and its catchment based on improving data bases and the empowerment of the stakeholder groups. The latter depends on increasing the level of education and access to information, as well as implementing procedures to improve public involvement and enforcement of environmental regulations.This revised version was published online in March 2005 with corrections to the issue cover date.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.