Electrical synapses between mushroom body neurons are critical for consolidated memory retrieval in Drosophila.

Electrical synapses between neurons, also known as gap junctions, are direct cell membrane channels between adjacent neurons. Gap junctions play a role in the synchronization of neuronal network activity; however, their involvement in cognition has not been well characterized. Three-hour olfactory associative memory in Drosophila has two components: consolidated anesthesia-resistant memory (ARM) and labile anesthesia-sensitive memory (ASM). Here, we show that knockdown of the gap junction gene innexin5 (inx5) in mushroom body (MB) neurons disrupted ARM, while leaving ASM intact. Whole-mount brain immunohistochemistry indicated that INX5 protein was preferentially expressed in the somas, calyxes, and lobes regions of the MB neurons. Adult-stage-specific knockdown of inx5 in αß neurons disrupted ARM, suggesting a specific requirement of INX5 in αß neurons for ARM formation. Hyperpolarization of αß neurons during memory retrieval by expressing an engineered halorhodopsin (eNpHR) also disrupted ARM. Administration of the gap junction blocker carbenoxolone (CBX) reduced the proportion of odor responsive αß neurons to the training odor 3 hours after training. Finally, the α-branch-specific 3-hour ARM-specific memory trace was also diminished with CBX treatment and in inx5 knockdown flies. Altogether, our results suggest INX5 gap junction channels in αß neurons for ARM retrieval and also provide a more detailed neuronal mechanism for consolidated memory in Drosophila.

Additive Expression of Consolidated Memory through Drosophila Mushroom Body Subsets.

Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αß, α'ß', and γ neurons. Previously we demonstrated that two parallel pathways mediated ARM consolidation: the serotonergic dorsal paired medial (DPM)-αß neurons and the octopaminergic anterior paired lateral (APL)-α'ß' neurons. This finding prompted us to ask how this composite ARM is retrieved. Here, we showed that blocking the output of αß neurons and that of α'ß' neurons each impaired ARM retrieval, and blocking both simultaneously had an additive effect. Knockdown of radish and octß2R in αß and α'ß' neurons, respectively, impaired ARM. A combinatorial assay of radish mutant background rsh1 and neurotransmission blockade confirmed that ARM retrieved from α'ß' neuron output is independent of radish. We identified MBON-ß2ß'2a and MBON-ß'2mp as the MB output neurons downstream of αß and α'ß' neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, we showed that α'ß' neurons could be functionally subdivided into α'ß'm neurons required for ARM retrieval, and α'ß'ap neurons required for ARM consolidation. Our work demonstrated that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval.

Mushroom body glycolysis is required for olfactory memory in Drosophila.

Glucose catabolism, also known as glycolysis, is important for energy generation and involves a sequence of enzymatic reactions that convert a glucose molecule into two pyruvate molecules. The glycolysis process generates adenosine triphosphate as a byproduct. In this study, we investigated whether glycolysis plays a role in maintaining neuronal functions in the Drosophila mushroom bodies (MBs), which are generally accepted to be an olfactory learning and memory center. Our data showed that individual knockdown of glycolytic enzymes in the MBs, including hexokinase (HexA), phosphofructokinase (Pfk), or pyruvate kinase (PyK), disrupts olfactory memory. Whole-mount brain immunostaining indicated that pyruvate kinase is strongly expressed in the MB αß, α'ß', and γ neuron subsets. We conclude that HexA, Pfk, and PyK are required in each MB neuron subset for olfactory memory formation. Our data therefore indicates that glucose catabolism in the MBs is important for olfactory memory formation in Drosophila.

Klebsiella pneumoniae yggG gene product: a zinc-dependent metalloprotease.

Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.

Performance evaluation of knowledge management among hospital employees.

PURPOSE: The purpose of this study is to investigate the cognition of knowledge management (KM) among hospital employees and the relationship between KM and the KM enabler activities (financial, customer, internal business processes, learning and growth) in a regional hospital in Taiwan. DESIGN/METHODOLOGY/APPROACH: Both qualitative and quantitative research were used in this study. The instrument was conducted using in-depth interviews of three policy-makers as participants. The quantitative data were collected from a regional hospital in the Northern part of Taiwan with a 77 percent effective response rate (n=154). FINDINGS: The findings in this paper indicate that the cognition and demand for KM in subordinates is close to the expectations of policy-makers. The policy-makers expect subordinates working in the hospital to be brave in taking on new responsibilities and complying with hospital operation norms. KM is emphasized as a powerful and positive asset. Moreover, understanding KM predicts good performance in an organization. RESEARCH LIMITATIONS/IMPLICATIONS: The findings in this paper can be generalized to other regional hospitals. The findings may be applied to a wider population. PRACTICAL IMPLICATIONS: This study can provide insights into the perceptions and cognitions of workers in a hospital about KM and the activities of KM enablers. The responses and perceptions observed in the interviews in this study, as well as the quantitative research results could be useful to other hospitals and individuals who engage KM as a new management trend. ORIGINALITY/VALUE: This study suggested KM guidelines for policy-makers who are experienced managers.

Laser Capture Microdissection of Single Neurons with Morphological Visualization Using Fluorescent Proteins Fused to Transmembrane Proteins.

Gene expression analysis in individual neuronal types helps in understanding brain function. Genetic methods expressing fluorescent proteins are widely used to label specific neuronal populations. However, because cell type specificity of genetic labeling is often limited, it is advantageous to combine genetic labeling with additional methods to select specific cell/neuronal types. Laser capture microdissection is one of such techniques with which one can select a specific cell/neuronal population based on morphological observation. However, a major issue is the disappearance of fluorescence signals during the tissue processing that is required for high-quality sample preparation. Here, we developed a simple, novel method in which fluorescence signals are preserved. We use genetic labeling with fluorescence proteins fused to transmembrane proteins, which shows highly stable fluorescence retention and allows for the selection of fluorescent neurons/cells based on morphology. Using this method in mice, we laser-captured neuronal somata and successfully isolated RNA. We determined that ∼100 cells are sufficient to obtain a sample required for downstream applications such as quantitative PCR. Capability to specifically microdissect targeted neurons was demonstrated by an ∼10-fold increase in mRNA for fluorescent proteins in visually identified neurons expressing the fluorescent proteins compared with neighboring cells not expressing it. We applied this method to validate virus-mediated single-cell knockout, which showed up to 92% reduction in knocked-out gene RNA compared with wild-type neurons. This method using fluorescent proteins fused to transmembrane proteins provides a new, simple solution to perform gene expression analysis in sparsely labeled neuronal/cellular populations, which is especially advantageous when genetic labeling has limited specificity.

Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition.

Choline kinase beta (CKß) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKß is important for normal mitochondrial function and muscle development as the lack of the ckß gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CKß phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKß by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKß phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKß was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKß residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKß phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKß to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKß, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.