17 ß-estradiol mineralization in human waste products and soil in the presence and the absence of antimicrobials.

Natural steroidal estrogens, such as 17 ß-estradiol (E2), as well as antimicrobials such as doxycycline and norfloxacin, are excreted by humans and hence detected in sewage sludge and biosolid. The disposal of human waste products on agricultural land results in estrogens and antibiotics being detected as mixtures in soils. The objective of this study was to examine microbial respiration and E2 mineralization in sewage sludge, biosolid, and soil in the presence and the absence of doxycycline and norfloxacin. The antimicrobials were applied to the media either alone or in combination at total rates of 4 and 40 mg kg-1, with the 4 mg kg-1 rate being an environmentally relevant concentration. The calculated time that half of the applied E2 was mineralized ranged from 294 to 418 days in sewage sludge, from 721 to 869 days in soil, and from 2,258 to 14,146 days in biosolid. E2 mineralization followed first-order and the presence of antimicrobials had no significant effect on mineralization half-lives, except for some antimicrobial applications to the human waste products. At 189 day, total E2 mineralization was significantly greater in sewage sludge (38 ±0.7%) > soil (23 ±0.7%) > biosolid (3 ±0.7%), while total respiration was significantly greater in biosolid (1,258 mg CO2) > sewage sludge (253 mg CO2) ≥ soil (131 mg CO2). Strong sorption of E2 to the organic fraction in biosolid may have resulted in reduced E2 mineralization despite the high microbial activity in this media. Total E2 mineralization at 189 day was not significantly influenced by the presence of doxycycline and/or norfloxacin in the media. Antimicrobial additions also did not significantly influence total respiration in media, except that total CO2 respiration at 189 day was significantly greater for biosolid with 40 mg kg-1 doxycycline added, relative to biosolid without antimicrobials. We conclude that it is unlikely for doxycycline and norfloxacin, or their mixtures, to have a significant effect on E2 mineralization in human waste products and soil. However, the potential for E2 to be persistent in biosolids, with and without the presence of antimicrobials, is posing a challenge for biosolid disposal to agricultural lands.

Draft Genome Sequence of Clavibacter michiganensis subsp. nebraskensis Strain DOAB 397, Isolated from an Infected Field Corn Plant in Manitoba, Canada.

In 2014, the pathogen Clavibacter michiganensis subsp. nebraskensis was isolated from symptomatic corn leaves in Manitoba, Canada. We report the draft genome sequence of C. michiganensis subsp. nebraskensis DOAB 397, consisting of 3.059 Mb with 73.0% G+C content, 2,922 predicted protein-coding sequences, 45 tRNAs, 3 rRNAs, and 37 pseudogenes.

17 ß-estradiol and 17 α-ethinylestradiol mineralization in sewage sludge and biosolids.

Natural steroid estrogens (e.g., 17 ß-estradiol, E2), synthetic steroid estrogens (e.g., 17 α-ethinylestradiol, EE2) and pharmaceutical antibiotics (e.g., ciprofloxacin) are chemicals detected in biosolids and sewage sludges because they partition into the solids fraction during the wastewater treatment process. This research utilized a three-way factorial design (six media × two estrogens × three antibiotic treatments) to quantify cumulative E2 and EE2 mineralization over 133 d (MAX) in a range of sewage sludge and biosolid samples in the presence (4 and 40 mg kg(-1)) and absence of ciprofloxacin. The same three-way factorial design was utilized to quantify the impact of the six media, E2 or EE2, and ciprofloxacin on cumulative soil respiration over 133 d (RESP). Minimal ciprofloxacin mineralization was observed (<0.05% over 133 d), but despite its persistence, ciprofloxacin had no significant effect on MAX of E2 or EE2, and, in general, no significant effect on RESP. MAX ranged from 38.38% to 48.44% for E2 but from only 0.72% to 24.27% for EE2 although RESP was relatively similar, ranging from 101.00 to 866.54 mg CO2 in the presence of E2 and from 69.55 to 893.95 mg CO2 in the presence of EE2. The sorption-limited bioavailability of EE2, which is inherently resistant to biodegradation due to chemical structure, as MAX and Freundlich sorption coefficients (Kf) were negatively correlated. As such, the Kf values of EE2 were largest in composted biosolids in which EE2 was particularly resistant to microbial degradation as the MAX of EE2 was <3%. In contrast, the MAX of E2 showed a positive association with the Kf values of E2 because some steps in the E2 transformation process have been found to occur in the sorbed phase. The MAX of E2 was significantly greater in the biosolid and composted biosolid media than in any other media, whereas the MAX of E2 decreased in the following order: secondary sewage sludge > primary sewage sludge > biosolid = composted biosolid. This suggests that sewage sludges in municipal lagoons and pre-treatment holding lagoons are a more favorable media for mineralization of EE2, whereas biosolids in post-treatment storage lagoons are a more favorable media for the mineralization of E2. The presence of ciprofloxacin will have no impact on the potential E2 or EE2 mineralization rates in these cases.

Deciphering proteins and their functions in the regenerating retina.

Neurons of the mammalian CNS, including retinal ganglion cells, lack, in contrast to the PNS, the ability to regenerate axons spontaneously after injury. Regeneration of the CNS is extremely complex and involves various molecular factors and cells. Therewith the regenerative process remains an enormous scientific and clinical challenge. This article provides an overview of proteins that play a crucial role in axon regeneration of retinal ganglion cells and their underlying signaling pathways. In this context, we elucidate the role of 2D gel electrophoresis and highlight some additional proteins, altered upon regeneration by using this highly sensitive method.

Axonal regeneration in the organotypically cultured monkey retina: biological aspects, dependence on substrates and age-related proteomic profiling.

Injury to the mature primate and subprimate optic nerve results in irreversible impairment and loss of vision, because the retinal ganglion cells (RGCs) fail to regenerate their cut axons within the optic nerve interior. This study was performed to examine whether aging monkey RGCs retain the ability to regenerate their axons in organ culture and whether axonal regeneration is associated with specific proteomic profile. Retinal stripes obtained from marmoset eyes (C. jacchus) were cultured between the day of birth and adult stages on different substrates like laminin-1, laminin-2, collagen, matrigel and poly-D-lysine. No neurotrophic factors were added to the medium. Axonal growth was monitored with microscopy and immunohistochemistry. Onset and rate of growth was examined with time-lapse videography. Vigorous regeneration of axons occurred from identifiable morphological types of RGCs throughout all stages of life, although the numbers of axons decreased with age. Axonal growth occurred virtually only on laminin-1. Growth correlated with re-expression of the laminin-1 receptor alpha6-integrin and sustained staining for GAP-43 as shown by immunohistochemistry and immunoblotting. At proteomic level, there is a maturation-dependent change in the protein immunostaining within the retina. When retinal slices of the same age were compared, regeneration-specific protein staining included calmodulin, fatty acid binding protein, alpha-crystallin, IFN-gamma, cyclin-dependent kinase inhibitor (p21), beta-hemoglobin, 60s-ribosomal protein, GAP-DH and ADP-ribosylation factor (ARF). To our knowledge these data are the first from subhuman animals to suggest that axonal regeneration of injured RGCs is correlated to expression of identifiable proteins within the retina.

Potential role of Pax-2 in retinal axon navigation through the chick optic nerve stalk and optic chiasm.

The degree of fiber decussation at the optic chiasm differs between species, ranging from complete crossing in lower vertebrates to highly complex patterns of intermingling of the fibers from the two eyes seen in mammals and birds. Understanding the genetic control of fiber guidance through the chiasm is therefore important to unravel the developmental mechanisms within the visual system. Here we first report on early stages of chiasm formation, with pioneering axons from the left eye consistently arriving earlier than their counterparts from the right eye. This initial left-right asymmetry is transient and no functional significance is assigned to it yet. Secondly, we examined formation of the chiasm in relation with the expression of the transcription factor Pax-2 along the ventral eye cup and optic nerve stalk. Finally, in order to examine causal involvement of Pax-2 in chiasm formation, the gene was overexpressed along the neuraxis and in the eye cup at embryonic stages preceding the exit of axons from the eye, and hence arrival of axons at the chiasm. When studied with neuroanatomical tracing, Pax-2 overexpression resulted in visibly anomalous decussation of axons at the chiasm. A likely consequence of this perturbation was erroneous arrival of axons at the tectum, as observed by anterograde staining from the retina. These data suggest that balanced expression of Pax-2 results in the correct formation of the chick chiasm at early stages by imposing accurate pathfinding within the optic stalk and the midline.

Regeneration of ganglion cell axons into a peripheral nerve graft alters retinal expression of glial markers and decreases vulnerability to re-axotomy.

PURPOSE: To compare the effect of cutting the optic nerve versus replacing the cut optic nerve with a peripheral nerve (PN) graft on retinal glial markers, and to determine whether the PN graft can stabilize regenerating retinal ganglion cells (RGCs), thus preventing their death following re-axotomy. METHODS: Retinas harvested after ganglion cell regeneration into a sciatic nerve graft were compared to untreated control retinas and retinas obtained following optic nerve axotomy. Glial-specific proteins such as glial fibrillary acidic protein (GFAP), Bcl-2 and complement-3 receptor (Ox-42) were examined using immunohistochemistry. Ganglion cells that survived the second axotomy were quantified on retinal flat mounts by retrograde labeling from the graft. RESULTS: GFAP expression in astrocytes and Muller cells was elevated in axotomized retinas when compared to controls, and an additional up-regulation in Muller cells was found in retinas following ganglion cell regeneration. Increased GFAP expression in retinas containing regenerated neurons was accompanied by increased Bcl-2 expression with latter being confined to Muller cells. Moreover, re-axotomy of the regenerated axons within the graft did not result in significant retrograde degeneration of RGCs within 28 days. CONCLUSIONS: The data suggest that the graft stabilizes the regenerating RGCs to an extent reminiscent of peripheral neurons, a process that may involve the interaction between neuronal and glial elements.

Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: comparative proteomic studies on rat retina and mouse brain.

A long-chain derivative of 1,3-dioxolane sodium propyloxy sulfate, with similar denaturing and electrophoretic properties as SDS, and facilitated protein identification following polyacrylamide gel electrophoresis (PAGE) for Coomassie-stained protein bands, has been tested. Comparative acid-labile surfactant/sodium dodecyl sulfate two-dimensional (ALS/SDS 2-D)-PAGE experiments of lower abundant proteins from the proteomes of regenerating rat retina and mouse brain show that peptide recovery for mass spectrometry (MS) mapping is significantly enhanced using ALS leading to more successful database searches. ALS may influence some procedures in proteomic analysis such as the determination of protein content and methods need to be adjusted to that effect. The promising results of the use of ALS in bioanalytics call for detailed physicochemical investigations of surfactant properties.