Report of the International Stem Cell Banking Initiative Workshop Activity: Current Hurdles and Progress in Seed-Stock Banking of Human Pluripotent Stem Cells.

This article summarizes the recent activity of the International Stem Cell Banking Initiative (ISCBI) held at the California Institute for Regenerative Medicine (CIRM) in California (June 26, 2016) and the Korean National Institutes for Health in Korea (October 19-20, 2016). Through the workshops, ISCBI is endeavoring to support a new paradigm for human medicine using pluripotent stem cells (hPSC) for cell therapies. Priority considerations for ISCBI include ensuring the safety and efficacy of a final cell therapy product and quality assured source materials, such as stem cells and primary donor cells. To these ends, ISCBI aims to promote global harmonization on quality and safety control of stem cells for research and the development of starting materials for cell therapies, with regular workshops involving hPSC banking centers, biologists, and regulatory bodies. Here, we provide a brief overview of two such recent activities, with summaries of key issues raised. Stem Cells Translational Medicine 2017;6:1956-1962.

Alzheimer disease research in the 21st century: past and current failures, new perspectives and funding priorities.

Much of Alzheimer disease (AD) research has been traditionally based on the use of animals, which have been extensively applied in an effort to both improve our understanding of the pathophysiological mechanisms of the disease and to test novel therapeutic approaches. However, decades of such research have not effectively translated into substantial therapeutic success for human patients. Here we critically discuss these issues in order to determine how existing human-based methods can be applied to study AD pathology and develop novel therapeutics. These methods, which include patient-derived cells, computational analysis and models, together with large-scale epidemiological studies represent novel and exciting tools to enhance and forward AD research. In particular, these methods are helping advance AD research by contributing multifactorial and multidimensional perspectives, especially considering the crucial role played by lifestyle risk factors in the determination of AD risk. In addition to research techniques, we also consider related pitfalls and flaws in the current research funding system. Conversely, we identify encouraging new trends in research and government policy. In light of these new research directions, we provide recommendations regarding prioritization of research funding. The goal of this document is to stimulate scientific and public discussion on the need to explore new avenues in AD research, considering outcome and ethics as core principles to reliably judge traditional research efforts and eventually undertake new research strategies.

Enhanced long-term depression and impaired reversal learning in phosphodiesterase 4B-knockout (PDE4B-/-) mice.

3'-5'-Cyclic adenosine monophosphate (cAMP) is known to be an important regulator of synaptic plasticity. The effects of cAMP are mediated through downstream effectors such as protein kinase A (PKA), Ca(2+) and cAMP-response element binding protein (CREB). The phosphodiesterase 4 (PDE4) family of enzymes, which is comprised of four genes and at least 25 protein isoforms, mediates the hydrolysis of cAMP, yet little is presently known about the contribution of specific PDE4 isoforms to synaptic plasticity and cognitive behavior. The purpose of the present studies was to determine the contribution of the PDE4B gene in mediating synaptic plasticity and cognitive behavior. Electrophysiological recordings from hippocampal slice preparations of mice deficient in the PDE4B gene (PDE4B(-/-)) showed that knockout animals displayed markedly enhanced basal postsynaptic responses to stimulation and long-term depression as compared to wild-type littermates. Interestingly, no genotypic differences were noted in long-term potentiation experiments following several different induction protocols. On the behavioral level PDE4B(-/-) mice displayed impaired reversal learning in the Morris water maze compared to wild-type littermates, but no differences in acquisition and retention of spatial memory and fear conditioning. Taken together, these results suggest that the PDE4B gene may play a role in synaptic activity and long-term depression and is involved in spatial reversal memory. Our findings support the view that various PDE4 isoforms are non-redundant and have distinct neurological roles.

Crystal structures of IL-2-inducible T cell kinase complexed with inhibitors: insights into rational drug design and activity regulation.

IL-2-inducible T cell kinase plays an essential role in T cell receptor signaling and is considered a drug target for the treatment of Th2-mediated inflammatory diseases. By applying high-throughput protein engineering and crystallization, we have determined the X-ray crystal structures of IL-2-inducible T cell kinase in complex with its selective inhibitor BMS-509744 and the broad-spectrum kinase inhibitors sunitinib and RO5191614. Sunitinib uniquely stabilizes IL-2-inducible T cell kinase in the helix C-in conformation by inducing side chain conformational changes in the ATP-binding site. This preference of sunitinib to bind to an active kinase conformation is reflective of its broad-spectrum kinase activity. BMS-509744 uniquely stabilizes the activation loop in a substrate-blocking inactive conformation, indicating that structural changes described for Src family kinases are also involved in the regulation of IL-2-inducible T cell kinase activity. The observed BMS-509744 binding mode allows rationalization of structure-activity relationships reported for this inhibitor class and facilitates further structure-based drug design. Sequence-based analysis of this binding mode provides guidance for the rational design of inhibitor selectivity.

Enhanced long-term potentiation and impaired learning in phosphodiesterase 4D-knockout (PDE4D) mice.

Elevation of intracellular cyclic adenosine monophosphate (cAMP) concentrations and subsequent regulation of downstream target gene expression through phosphorylation of cAMP-responsive element binding protein (CREB) is hypothesized to underlie the mechanism(s) of long-term memory (LTM) formation. The phosphodiesterase 4 (PDE4) enzyme family is believed to play a key role in LTM by regulating cAMP levels. Thus far, four PDE4 isoforms have been identified (PDE4A, B, C and D); however, the requisite involvement of each of these isoforms in mediating LTM has yet to be elucidated. In the present study, genetic knockout mice were used to investigate the involvement of the PDE4D isoform in both in vitro and in vivo models of learning and memory. Hippocampal synaptic transmission measured electrophysiologically in CA1 slice preparations was similar between wild-type and PDE4D (-/-) mice yet, relative to wild-type controls, knockout mice displayed enhanced early long-term potentiation (LTP) following multiple induction protocols. Interestingly, the PDE4D (-/-) animals exhibited significant behavioral deficits in associative learning using a conditioned fear paradigm as compared with control littermates. The impairment in fear conditioning observed in the PDE4D (-/-) mice could not be attributed to differences in acquisition of the task, alterations in locomotor activity or effects on shock sensitivity. Overall, the in vitro and in vivo alterations in synaptic plasticity observed in the PDE4D (-/-) mice may be explained by adaptive responses occurring throughout development, and suggest that the PDE4D isoform may be an important mediator of LTM formation.

Neuregulin1 (NRG1) signaling through Fyn modulates NMDA receptor phosphorylation: differential synaptic function in NRG1+/- knock-outs compared with wild-type mice.

We previously identified Neuregulin1 (NRG1) as a gene contributing to the risk of developing schizophrenia. Furthermore, we showed that NRG1+/- mutant mice display behavioral abnormalities that are reversed by clozapine, an atypical antipsychotic drug used for the treatment of schizophrenia. We now present evidence that ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), the tyrosine kinase receptor for NRG1 in hippocampal neurons, interacts with two nonreceptor tyrosine kinases, Fyn and Pyk2 (proline-rich tyrosine kinase 2). NRG1 stimulation of cells expressing ErbB4 and Fyn leads to the association of Fyn with ErbB4 and consequent activation. Furthermore, we show that NRG1 signaling, through activation of Fyn and Pyk2 kinases, stimulates phosphorylation of Y1472 on the NR2B subunit of the NMDA receptor (NMDAR), a key regulatory site that modulates channel properties. NR2B Y1472 is hypophosphorylated in NRG1+/- mutant mice, and this defect can be reversed by clozapine at a dose that reverses their behavioral abnormalities. We also demonstrate that short-term synaptic plasticity is altered and theta-burst long-term potentiation is impaired in NRG1+/- mutant mice, and incubation of hippocampal slices from these mice with NRG1 reversed those effects. Attenuated NRG1 signaling through ErbB4 may contribute to the pathophysiology of schizophrenia through dysfunction of NMDAR modulation. Thus, our data support the glutamate hypothesis of schizophrenia.

Selective phosphodiesterase (PDE)-4 inhibitors: a novel approach to treating memory deficit?

Phosphodiesterase-4 (PDE4) belongs to an important family of proteins that regulates the intracellular level of cyclic adenosine monophosphate (cAMP). Several lines of evidence indicate that targeting PDE4 with selective inhibitors may offer novel strategies in the treatment of age-related memory impairment and Alzheimer's disease. The rationale for such an approach stems from preclinical studies indicating that PDE4 inhibitors can counteract deficits in long-term memory caused by pharmacological agents, aging or overexpression of mutant forms of human amyloid precursor proteins. In addition to their pro-cognitive and pro-synaptic plasticity properties, PDE4 inhibitors are potent neuroprotective, neuroregenerative and anti-inflammatory agents. Based on the fact that Alzheimer's disease is a progressive neurodegenerative disorder that is characterised by cognitive impairment, and that neuroinflammation is now recognised as a prominent feature in Alzheimer's pathology, we have concluded that targeting PDE4 with selective inhibitors may offer a novel therapy aimed at slowing progression, prevention and, eventually, therapy of Alzheimer's disease.

Practical synthesis of the new carbapenem antibiotic ertapenem sodium.

[reaction: see text] A practical synthesis for the large-scale production of the new carbapenem antibiotic, [4R,5S,6S]-3-[[(3S,5S)-5-[[(3-Carboxyphenyl)amino]carbonyl]-3-pyrrolidinyl]thio]-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid monosodium salt (ertapenem sodium, 1), has been developed. The synthesis features the novel use of 1,1,3,3-tetramethylguanidine as base for the low-temperature reaction of a thiol, derived from trans-4-hydroxy-L-proline, with the carbapenem nucleus activated as the enol phosphate. Hydrogenolysis of a p-nitrobenzyl ester is effected using a palladium on carbon catalyst to give an overall yield for the two steps of 90%. The use of bicarbonate in the hydrogenolysis was key in providing protection of the pyrrolidine amine as the sodium carbamate improving both the performance of the reaction and the stability of the product. This discovery made processing at manufacturing scale possible. Experimental evidence for the formation of the sodium carbamate is provided. A remarkably expedient process for the simultaneous purification and concentration of the aqueous product stream relies on ion-pairing extraction for the removal of the water-soluble 1,1,3,3-tetramethylguanidine. Crystallization then affords 59-64% overall yield of the monosodium salt form of the product.

High-throughput confirmation of differential display PCR results using reverse Northern blotting.

Nylon filter arrays spotted with differential display PCR (DD-PCR) clones and hybridized with radiolabeled cRNA generated from the source RNA pool (reverse Northern blot) provide a high-throughput means to screen clones for artifacts. Reverse Northern blots also confirm differential gene expression in parallel and require modest quantities of the source RNA pool. We describe a strategy to screen multiple candidates from DD-PCR by high-throughput ligation and transformation, followed by reverse Northern blotting. Purification of re-amplified DD-PCR clones and fabrication of nylon arrays was facilitated by a batch-processing protocol using the widely available Biomek laboratory robot and Bioworks scripts (available from the authors). A strategy to screen out DD-PCR product artifacts of an inappropriate size was also employed. Using these approaches, we identified several mRNAs that are differentially expressed in response to venlafaxine, fluoxetine or desipramine antidepressant treatment in rat C6 glioma cell lines and are candidates for full length clone isolation using 5'-RACE. Such an approach provides a rapid means to eliminate the high percentage of false positive clones from DD-PCR and enables independent confirmation of differential gene expression patterns generated by various experimental conditions.