We have demonstrated an easy, economic, one-step synthetic route to water-soluble fluorescent carbon dots derived from the thermal upcycling of urine. These “pee-dots” (PDs), which primarily comprise hydrophile-decorated amorphous carbon, exhibit bright, stable, excitation wavelength dependent fluorescence in aqueous solution and are shown to be useful nanoscale labels in cell imaging applications. Cytotoxicity studies demonstrate that these PDs are benign toward model cell lines, even at concentrations as high as 500 μg mL⁻¹. Notably, this approach converts an otherwise useless, negatively-valued byproduct of human life into a value-added nanoscale product while simultaneously pasteurizing the waste stream. The reported PDs proved to be effective nanoprobes for the fluorescence-based detection of heavy metal ions of environmental concern, particularly Cu²⁺ and Hg²⁺ ions which were found to be strong quenchers of their fluorescence. Interestingly, the optical properties and nanoscale dimensions of the PDs are a direct reflection of the diet (e.g., vitamin C or asparagus (sulfur) fortified) followed by the urine donor.

The role of neural cell adhesion molecule (NCAM) in the development and maturation of the neuromuscular junction (NMJ) was explored by characterizing structurally and functionally NMJs from postnatal day 11 (P11) to P30 +/+, +/−, and −/− NCAM null mutant mice. Differences in NCAM levels resulted in alterations in the size and shape of NMJs, with −/− NMJs being smaller. Additionally both the withdrawal of polyneuronal innervation and the selective accumulation of synaptic vesicle protein in the presynaptic terminal were delayed. These observations suggest that the bidirectional signaling responsible for these events is impaired at −/− NMJs. Functionally, miniature end plate potential size, end plate potential size, and quantal content did not differ from that of wild type under either normal or low release conditions. However at normal release conditions, −/− NMJs, unlike +/+ NMJs, lacked paired-pulse facilitation. The most striking abnormality was the inability of NCAM null junctions to maintain transmitter output with repetitive stimuli. Combined electrophysiological and FM1-43-labeling studies suggest that NCAM null junctions are unable either to dock or to mobilize a sufficient number of vesicles at high but physiological rates of transmitter release. Taken together our observations show that many aspects of transmission are normal and, thus, that many presynaptic and postsynaptic molecules have assembled properly in the absence of NCAM. However, the fact that NCAM was required for specific aspects of transmission, including paired-pulse facilitation and reliable transmission with repetitive stimuli, suggests that NCAM either is directly involved in these processes or is required for the proper organization and/or function of other molecules underlying these processes.

Mice that lack all three major isoforms of neural cell adhesion molecule (NCAM) (180 and 140 kDa transmembrane, and 120 kDa glycosylphosphatidylinositol linked) were previously shown to exhibit major alterations in the maturation of their neuromuscular junctions (NMJs). Specifically, even by postnatal day 30, they failed to downregulate from along their axons and terminals an immature, brefeldin A-sensitive, synaptic vesicle-cycling mechanism that used L-type Ca²⁺channels. In addition, these NCAM null NMJs were unable to maintain effective transmitter output with high-frequency repetitive stimulation, exhibiting both severe initial depression and subsequent cyclical periods of total transmission failures that were of presynaptic origin. As reported here, mice that lack only the 180 kDa isoform of NCAM downregulated the immature vesicle-cycling mechanism on schedule, implicating either the 140 or 120 kDa NCAM isoforms in this important maturational event. However, 180 NCAM-deficient mice still exhibited many functional transmission defects. Although 180 NCAM null NMJs did not show the severe initial depression of NCAM null NMJs, they still had cyclical periods of complete transmission failure. In addition, several presynaptic molecules were expressed at lower levels or were more diffusely localized. Thus, the 180 kDa isoform of NCAM appears to play an important role in the molecular organization of the presynaptic terminal and in ensuring effective transmitter output with repetitive stimulation. Our results also suggest that PKC and MLCK (myosin light chain kinase) may be downstream effectors of NCAM in these processes. Together, these results indicate that different isoforms of NCAM mediate distinct and important events in presynaptic maturation.

Intracellular nicotinamide phosphoribosyltransferase (iNAMPT) is the rate-limiting enzyme of the mammalian NAD+ biosynthesis salvage pathway. Using inducible and conditional knockout (cKO) mice, we show that Nampt gene deletion in adult projection neurons leads to a progressive loss of body weight, hypothermia, motor neuron (MN) degeneration, motor function deficits, paralysis, and death. Nampt deletion causes mitochondrial dysfunction, muscle fiber type conversion, and atrophy, as well as defective synaptic function at neuromuscular junctions (NMJs). When treated with nicotinamide mononucleotide (NMN), Nampt cKO mice exhibit reduced motor function deficits and prolonged lifespan. iNAMPT protein levels are significantly reduced in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, indicating the involvement of NAMPT in ALS pathology. Our findings reveal that neuronal NAMPT plays an essential role in mitochondrial bioenergetics, motor function, and survival. Our study suggests that the NAMPT-mediated NAD+ biosynthesis pathway is a potential therapeutic target for degenerative MN diseases.