A method to quantify regional axonal transport blockade at the optic nerve head after short term intraocular pressure elevation in mice.

Axonal transport blockade is an initial step in retinal ganglion cell (RGC) degeneration in glaucoma and targeting maintenance of normal axonal transport could confer neuroprotection. We present an objective, quantitative method for assessing axonal transport blockade in mouse glaucoma models. Intraocular pressure (IOP) was elevated unilaterally in CD1 mice for 3 days using intracameral microbead injection. Longitudinal sections of optic nerve head (ONH) were immunofluorescently labeled for myelin basic protein (MBP) and amyloid precursor protein (APP), which is transported predominantly orthograde by neurons. The beginning of the myelin transition zone, visualized with the MBP label, was more posterior with elevated IOP, 288.8 ± 40.9 µm, compared to normotensive control eyes, 228.7 ± 32.7 µm (p = 0.030, N = 6 pairs). Glaucomatous regional APP accumulations in retina, prelaminar ONH, unmyelinated ONH, and myelinated optic nerve were identified by objective qualification of pixels with fluorescent intensity greater than the 97.5th percentile value of control eyes (suprathreshold pixels). This method segregated images with APP blockade from those with normal transport of APP. The fraction of suprathreshold pixels was significantly higher following IOP elevation than in normotensive controls in the unmyelinated ONH and myelinated nerve regions (paired analyses, p = 0.02 and 0.003, respectively, N = 12), but not in retina or prelaminar ONH (p = 0.91 and 0.08, respectively). The mean intensity of suprathreshold pixels was also significantly greater in glaucoma than in normotensive controls in prelaminar ONH, unmyelinated ONH and myelinated optic nerve (p = 0.01, 0.01, 0.002, respectively). Using this method, subconjunctival glyceraldehyde, which is known to worsen long-term RGC loss with IOP elevation, also produced greater APP blockade, but not statistically significant compared to glaucoma alone. Systemic losartan, which aids RGC axonal survival in glaucoma, reduced APP blockade, but not statistically significant compared to glaucoma alone. The method provides a short-term assessment of axonal injury for use in initial tests of neuroprotective therapies that may beneficially affect RGC transport in animal models of glaucoma.

Next-Generation Probiotics Targeting Clostridium difficile through Precursor-Directed Antimicrobial Biosynthesis.

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.

Novel reuterin-related compounds suppress odour by periodontopathic bacteria.

OBJECTIVE: Halitosis is caused by volatile sulphur compounds including methyl mercaptan (CH3 SH) in the oral cavity and is a serious problem that limits interpersonal social communication. The aim of study was to evaluate the effects of reuterin-related compounds (RRCs) on halitosis-related periodontopathic bacteria in vitro. MATERIALS AND METHODS: RRC-01, RRC-02 and RRC-03 (32 and 64 µg ml-1 ) in culture media containing Fusobacterium nucleatum JCM8523 and Porphyromonas gingivalis ATCC33277 were used. The effects of RRCs on CH3 SH production and detectable odour by F. nucleatum and P. gingivalis were examined by CH3 SH production assay and organoleptic test, respectively. The number of bacterial cells was also measured using an ATP assay. In P. gingivalis treated with RRCs, the expression of mgl gene, which is responsible for CH3 SH production, was examined by qRT-PCR. RESULTS: CH3 SH production and the score of detectable odour from F. nucleatum and P. gingivalis culture media containing RRCs were significantly lower than that without RRCs (P < 0.05). The expression of mgl gene in P. gingivalis was significantly downregulated by RRC-01 (P < 0.01), but not by RRC-02 or RRC-03. CONCLUSIONS: RRCs are potent oral care products for preventing halitosis via reducing CH3 SH production.

An in vitro intact globe expansion method for evaluation of cross-linking treatments.

PURPOSE: To measure the tissue mechanical response to elevated intraocular pressure (IOP) using intact globe expansion of rabbit eyes. This method examined rabbit kit (2-3 weeks old) eyes as a model for weakened tissue and evaluated riboflavin/UVA and glyceraldehyde cross-linking treatments. METHODS: The ocular shape of enucleated eyes was photographed during a 24-hour period while a controlled IOP was imposed (either low IOP = 22 mm Hg or high IOP = 85 mm Hg). Untreated controls consisted of kit eyes tested at both low- and high IOP and adult eyes tested at high IOP. Treated kit eyes (dextran controls, riboflavin/UVA treatment of the cornea, and glyceraldehyde treatment of the entire globe) were tested at high IOP. RESULTS: Low IOP elicited negligible creep of the sclera and very gradual creep of the cornea. In contrast, high IOP induced up to an 8% strain in the sclera and a 15% strain in the cornea of rabbit kit eyes. The expansion of adult eyes was less than one third that of kit eyes at the same, high IOP. Riboflavin/UVA treatment of corneas reduced expansion compared with that in both dextran-treated and untreated control corneas. Glyceraldehyde treatment prevented expansion of the cornea and sclera. CONCLUSIONS: The intact globe expansion method (GEM) imposes a loading geometry comparable to in vivo conditions and can quantify changes in mechanical stability as a function of testing conditions (e.g., IOP, tissue maturation, and therapeutic cross-linking) with small sample sizes and small variability. Rabbit kit eyes provide a model of weak tissue suitable for screening treatments that strengthen the cornea and sclera.

Enhanced survival of sickle erythrocytes upon treatment with glyceraldehyde.

Glyceraldehyde has been demonstrated to be an antisickling agent in vitro. In the present investigation, chromium-51 red cell studies were used to investigate the life span in vivo of sickle erythrocytes after treatment with glyceraldehyde in vitro. The mean survival (T1/2) of control cells was 5.8 +/- 1.6 days, whereas cells treated with 10 mmol/L or 20 mmol/L glyceraldehyde survived 9.0 +/- 1.4 (P less than .05) and 11.3 +/- 0.8 (P less than .002) days, respectively. The extent of modification by glyceraldehyde was 0.4 to 1.0 lysine residue per hemoglobin tetramer. These studies demonstrate not only a prolongation of the life span of sickle erythrocytes by treatment with glyceraldehyde but also the absence of any deleterious effects that would be revealed by this study.

Comparative evaluation of fifteen anti-sickling agents.

Fifteen compounds reported to be inhibitors of gelation or sickling were studied by standard methods. These tests included (1) the determination of the solubility of deoxyhemoglobin S or Csat, (2) evaluation of sickling in whole SS blood at various pO2s, (3) measurement of the oxygen affinity of hemoglobin and blood, and (4) examination of red cell indices and morphology. Among the 4 noncovalent agents tested, butylurea was the most potent inhibitor of gelation and sickling in vitro; however, relatively high concentrations were required compared to the covalent agents. In the latter group, bis-(3,5 dibromosalicyl)-fumarate, nitrogen mustard, and dimethyladipimidate were especially effective inhibitors of gelation and/or sickling. All of these compounds require further development before they can be considered for clinical use.