The supplementation of the sialic acid biosynthetic pathway with exogenously supplied N-acetylmannosamine (ManNAc) analogs has many potential biomedical and biotechnological applications. In this work, we explore the structure-activity relationship of ManNAc analogs on cell viability and metabolic flux into the sialic acid biosynthetic pathway to gain a better understanding of the fundamental biology underlying "glycosylation engineering" technology. A panel of ManNAc analogs bearing various modifications on the hydroxyl groups as well as substitutions at the N-acyl position was investigated. Increasing the carbon chain length of ester derivatives attached to the hydroxyl groups increased the metabolic efficiency of sialic acid production, whereas similar modification to the N-acyl group decreased efficiency. In both cases, increases in chain length decreased cell viability; DNA ladder formation, Annexin V-FITC two-dimensional flow cytometry assays, caspase-3 activation, and down-regulation of sialoglycoconjugate-processing enzymes established that the observed growth inhibition and toxicity resulted from apoptosis. Two of the panel of 12 analogs tested, specifically Ac 4 ManNLev and Ac 4 ManNHomoLev, were highly toxic. Interestingly, both of these analogs maintained a ketone functionality in the same position relative to the core monosaccharide structure, and both also inhibited flux through the sialic acid pathway (the remainder of the less toxic analogs either increased or had no measurable impact on flux). These results provide fundamental insights into the role of sialic acid metabolism in apoptosis by demonstrating that ManNAc analogs can modulate apoptosis both indirectly via hydroxylgroup effects and directly through N-acyl-group effects.The term "sialic acid engineering" refers to a technique where non-natural N-acetylmannosamine (ManNAc) 1 analogs intercept the sialic acid biosynthetic pathway and are incorporated into cellular sialoglycoconjugates in the place of sialic acid residues (Fig. 1) (1, 2). The impetus behind this strategy is to mimic nature, which uses Ͼ50 different forms of sialic acid to modulate the structure and function of sialic acid-bearing glycoproteins and lipids (3). By using synthetic N-acyl-modified ManNAc analogs, the surfaces of living cells can be endowed with novel properties not found in nature (4) that, depending on the exact analog used to perform this "submolecular microsurgery" (5), have the potential to elicit a variety of changes in the behavior of the host cell. Theoretically, the ability to modify the cell surface and recombinant sialoglycoconjugates with molecular precision has the potential to regulate any biological process governed by sialic acid, such as cell growth and differentiation, communication among different cells, recognition of soluble factors, and attachment to, or disengagement from, the extracellular matrix (6). In practice, sialic acid engineering methods have already been demonstrated to regulate cellular responses ranging from adhesion to prolif...

"Sialic acid engineering" refers to the strategy where cell surface carbohydrates are modified by the biosynthetic incorporation of metabolic intermediates, such as non-natural N-acetylmannosamine (ManNAc) analogues, into cellular glycoconjugates. While this technology has promising research, biomedical, and biotechnological applications due to its ability to endow the cell surface with novel physical and chemical properties, its adoption on a large scale is hindered by the inefficient metabolic utilization of ManNAc analogues. We address this limitation by proposing the use of acetylated ManNAc analogues for sialic acid engineering applications. In this paper, the metabolic flux of these "second-generation" compounds into a cell, and, subsequently, into the target sialic acid biosynthetic pathway is characterized in detail. We show that acetylated ManNAc analogues are metabolized up to 900-fold more efficiently than their natural counterparts. The acetylated compounds, however, decrease cell viability under certain culture conditions. To determine if these toxic side effects can be avoided, we developed an assay to measure the cellular uptake of acetylated ManNAc from the culture medium and its subsequent flux into sialic acid biosynthetic pathway. This assay shows that the majority ( > 80%) of acetylated ManNAc is stored in a cellular "reservoir" capable of safely sequestering this analogue. These results provide conditions that, from a practical perspective, enable the acetylated analogues to be used safely and efficaciously and therefore offer a general strategy to facilitate metabolic substrate-based carbohydrate engineering efforts. In addition, these results provide fundamental new insights into the metabolic processing of non-natural monosaccharides.

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Genetically encoded voltage indicators (GEVIs) continue to evolve, resulting in many different probes with varying strengths and weaknesses. Developers of new GEVIs tend to highlight their positive features. A recent article from an independent laboratory has compared the signal/noise ratios of a number of GEVIs. Such a comparison can be helpful to investigators eager to try to image the voltage of excitable cells. In this perspective, we will present examples of how the biophysical features of GEVIs affect the imaging of excitable cells in an effort to assist researchers when considering probes for their specific needs.

Metabolic substrate-based sialic acid engineering techniques, where exogenously supplied N-acetylmannosamine (ManNAc) analogues are utilized by the sialic acid biosynthetic pathway, allow the cell surface to be endowed with novel physical and chemical properties and show promise for increasing the quality of recombinant glycoproteins. The in vitro toxicity of many ManNAc analogues, however, hinders the large-scale adoption of this technology. In this study, we used a selection strategy where cells were subjected to progressively higher levels of ManNAc analogues to establish novel cell lines that showed decreased sensitivity to analogue-induced in vitro toxicity. The decreased sensitivity to sugar analogue-induced apoptosis, demonstrated by the Annexin V-FITC detection method and DNA fragmentation assays, corresponded to increased sialic acid production in the resistant cell lines. The ManNAc analogue-resistant cell lines exhibited cross-resistance to apoptosis induced by staurosporine and an apoptosis-activating Fas antibody. We propose that the selection strategy employed to develop these novel cell lines, which serve as superior hosts for substrate-based sialic acid engineering applications, will generally apply to the development of host cell lines for biotechnology applications.

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The CA1 area in the mammalian hippocampus is essential for spatial learning. Pyramidal cells are the hippocampus output neurons and their activities are regulated by inhibition exerted by a diversified population of interneurons. Lateral inhibition has been suggested as the mechanism enabling the reconfiguration of pyramidal cell assembly activity observed during spatial learning tasks in rodents. However, lateral inhibition in the CA1 lacks the overwhelming evidence reported in other hippocampal areas such as the CA3 and the dentate gyrus. The use of genetically encoded voltage indicators and fast optical recordings permits the construction of cell-type specific response maps of neuronal activity. Here, we labelled mouse CA1 pyramidal neurons with the genetically encoded voltage indicator ArcLight and optically recorded their response to Schaffer Collaterals stimulation in vitro. By undertaking a manifold learning approach, we report a hyperpolarization-dominated area focused in the perisomatic region of pyramidal cells receiving late excitatory synaptic input.Functional network organization metrics revealed that information transfer was higher in this area. The localized hyperpolarization disappeared when GABA A receptors were pharmacologically blocked. This is the first report where the spatiotemporal pattern of lateral inhibition is visualized in the CA1 by expressing a genetically encoded voltage indicator selectively in principal neurons. Our analysis suggests a fundamental role of lateral inhibition in CA1 information processing.

Cerebellar granule cells (GCs) relay mossy fiber (MF) inputs to Purkinje cell dendrites via their axons, the parallel fibers (PFs), which are individually located at a given sublayer of the molecular layer (ML). Although a certain degree of heterogeneity among GCs has been recently reported, variability of GC responses to MF inputs has never been associated with their most notable structural variability, location of their projecting PFs in the ML. Here, we utilize an adeno-associated virus (AAV)-mediated labeling technique that enables us to categorize GCs according to the location of their PFs, and compare the Ca2+ responses to MF stimulations between three groups of GCs, consisting of either GCs having PFs at the deep (D-GCs), middle (M-GCs), or superficial (S-GCs) sublayer. Our structural analysis revealed that there was no correlation between position of GC soma in the GC layer and location of its PF in the ML, confirming that our AAV-mediated labeling was important to test the projection-dependent variability of the Ca2+ responses in GCs. We then found that the Ca2+ responses of D-GCs differed from those of M-GCs. Pharmacological experiments implied that the different Ca2+ responses were mainly attributable to varied distributions of GABAA receptors (GABAARs) at the synaptic and extrasynaptic regions of GC dendrites. In addition to GABAAR distributions, amounts of extrasynaptic NMDA receptors appear to be also varied, because Ca2+ responses were different between D-GCs and M-GCs when glutamate spillover was enhanced. Whereas the Ca2+ responses of S-GCs were mostly equivalent to those of D-GCs and M-GCs, the blockade of GABA uptake resulted in larger Ca2+ responses in S-GCs compared with D-GCs and M-GCs, implying existence of mechanisms leading to more excitability in S-GCs with increased GABA release. Thus, this study reveals MF stimulation-mediated non-uniform Ca2+ responses in the cerebellar GCs associated with the location of their PFs in the ML, and raises a possibility that combination of inherent functional variability of GCs and their specific axonal projection contributes to the information processing through the GCs.