John Steele

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.

SorCS1 and SorL1/SorLA/LR11 belong to the sortilin family of vacuolar protein sorting-10 (Vps10) domain-containing proteins. Both are genetically associated with Alzheimer's disease (AD), and SORL1 expression is decreased in the brains of patients suffering from AD. SORCS1 is also genetically associated with types 1 and 2 diabetes mellitus (T1DM, T2DM). We have undertaken a study of the possible role(s) for SorCS1 in metabolism of the Alzheimer's amyloid-β peptide (Aβ) and the Aβ precursor protein (APP), to test the hypothesis that Sorcs1 deficiency might be a common genetic risk factor underlying the predisposition to AD that is associated with T2DM. Overexpression of SorCS1cβ-myc in cultured cells caused a reduction ( p = 0.002) in Aβ generation. Conversely, endogenous murine Aβ 40 and Aβ 42 levels were increased (Aβ 40 , p = 0.044; Aβ 42 , p = 0.007) in the brains of female Sorcs1 hypomorphic mice, possibly paralleling the sexual dimorphism that is characteristic of the genetic associations of SORCS1 with AD and DM. Since SorL1 directly interacts with Vps35 to modulate APP metabolism, we investigated the possibility that SorCS1cβ-myc interacts with APP, SorL1, and/or Vps35. We readily recovered SorCS1:APP, SorCS1:SorL1, and SorCS1:Vps35 complexes from nontransgenic mouse brain. Notably, total Vps35 protein levels were decreased by 49% ( p = 0.009) and total SorL1 protein levels were decreased by 29% ( p = 0.003) in the brains of female Sorcs1 hypomorphic mice. From these data, we propose that dysfunction of SorCS1 may contribute to both the APP/Aβ disturbance underlying AD and the insulin/glucose disturbance underlying DM.

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aβ42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Alzheimer disease (AD) is a form of neurodegeneration that develops over the course of multiple decades and as a result of the accumulation of the pathogenic amyloid-β (Aβ) peptide, also known as A4. In late-stage AD, failure of autophagic clearance results in neuronal cell bodies that are almost entirely consumed by autophagic vacuoles (AVs). Previously, we have shown that the potential AD drug latrepirdine (aka Dimebon(®)), a Russian antihistamine that has shown mixed results in phase II clinical trials in AD, regulates metabolism of the amyloid-β/A4 precursor protein (APP). In two Molecular Psychiatry papers in 2012, we sought to determine the mechanism through which latrepirdine regulates APP metabolism and to determine, using an Alzheimer mouse model, whether latrepirdine provides protection from the toxicity associated with the accumulation of Aβ. In cultured cells, we provided evidence that latrepirdine stimulates MTOR- and ATG5-dependent autophagy, leading to the reduction of intracellular levels of APP metabolites, including Aβ. Consistent with this finding, we found that chronic latrepirdine administration resulted in increased levels of the biomarkers thought to correlate with autophagy activation in the brains of TgCRND8 (APP K670M, N671L, V717F) or wild-type mice, and that treatment was associated with abrogation of behavioral deficit, reduction in Aβ neuropathology, and prevention of autophagic failure among TgCRND8 mice.

Human structural congenital malformations are the leading cause of infant mortality in the United States. Estimates from the United States Center for Disease Control and Prevention (CDC) determine that close to 3% of all United States newborns present with birth defects; the worldwide estimate approaches 6% of infants presenting with congenital anomalies. The scientific community has recognized for decades that the majority of birth defects have undetermined etiologies, although we propose that environmental agents interacting with inherited susceptibility genes are the major contributing factors. Neural tube defects (NTDs) are among the most prevalent human birth defects and as such, these malformations will be the primary focus of this review. NTDs result from failures in embryonic central nervous system development and are classified by their anatomical locations. Defects in the posterior portion of the neural tube are referred to as meningomyeloceles (spina bifida), while the more anterior defects are differentiated as anencephaly, encephalocele, or iniencephaly. Craniorachischisis involves a failure of the neural folds to elevate and thus disrupt the entire length of the neural tube. Worldwide NTDs have a prevalence of approximately 18.6 per 10,000 live births. It is widely believed that genetic factors are responsible for some 70% of NTDs, while the intrauterine environment tips the balance toward neurulation failure in at risk individuals. Despite aggressive educational campaigns to inform the public about folic acid supplementation and the benefits of providing mandatory folic acid food fortification in the United States, NTDs still affect up to 2,300 United States births annually and some 166,000 spina bifida patients currently live in the United States, more than half of whom are now adults. Within the context of this review, we will consider the role of maternal nutritional status (deficiency states involving B vitamins and one carbon analytes) and the potential modifiers of NTD risk beyond folic acid. There are several well-established human teratogens that contribute to the population burden of NTDs, including: industrial waste and pollutants [e.g., arsenic, pesticides, and polycyclic aromatic hydrocarbons (PAHs)], pharmaceuticals (e.g., anti-epileptic medications), and maternal hyperthermia during the first trimester. Animal models for these teratogens are described with attention focused on valproic acid (VPA; Depakote). Genetic interrogation of model systems involving VPA will be used as a model approach to discerning susceptibility factors that define the gene-environment interactions contributing to the etiology of NTDs.

The synthesis of a prototype trisubstituted piperazinedione combinatorial library of 1,000 compounds has been achieved from three precursor sets — two sets of ten α-amino acids and one set of ten aldehydes. A sodium triacetoxyborohydride-mediated reductive alkylation was crucial to the success of the multi-step synthesis on resin. This protocol represents a new method to augment compound files rapidly with novel heterocyclic entities for high-speed screening.

This review aims to give an overview of current good practice in the prosecution of Lead Generation. It will assess experiences across the field as judged from the contents of the limited number of peer-review disclosures to date. It will also rely heavily on the experiences of the authors from many campaigns within this organisation. Its focus will be on the assembly of an appropriate compound collection for application in High Throughput Screening (HTS), the prosecution of HTS, the profiling of HTS output and, lastly the Hit-to-Lead optimisation of selected HTS output. Excluded from the scope are detailed aspects of library design [1], parallel synthesis [2], virtual library applications [3], virtual screening [4] and fragment screening [5] approaches, all of which have been the subject of recent reviews.

Recent evidence suggests that high molecular weight soluble oligomeric Abeta (oAbeta) assemblies (also known as Abeta-derived diffusible ligands, or ADDLs) may represent a primary neurotoxic basis for cognitive failure in Alzheimer disease (AD). To date, most in vivo studies of oAbeta/ADDLs have involved injection of assemblies purified from the cerebrospinal fluid of human subjects with AD or from the conditioned media of Abeta-secreting cells into experimental animals. We sought to study the bioactivities of endogenously formed oAbeta/ADDLs generated in situ from the physiological processing of human amyloid precursor protein (APP) and presenitin1 (PS1) transgenes.We produced and histologically characterized single transgenic mice overexpressing APP(E693Q) or APP(E693Q) X PS1DeltaE9 bigenic mice. APP(E693Q) mice were studied in the Morris water maze (MWM) task at 6 and 12 months of age. Following the second MWM evaluation, mice were sacrificed, and brains were assayed for Abetatotal, Abeta40, Abeta42, and oAbeta/ADDLs by enzyme-linked immunosorbent assay (ELISA) and were also histologically examined. Based on results from the oAbeta/ADDL ELISA, we assigned individual APP(E693Q) mice to either an undetectable oAbeta/ADDLs group or a readily detectable oAbeta/ADDLs group. A days to criterion (DTC) analysis was used to determine delays in acquisition of the MWM task.Both single transgenic and bigenic mice developed intraneuronal accumulation of APP/Abeta, although only APP(E693Q) X PS1Delta9 bigenic mice developed amyloid plaques. The APP(E693Q) mice did not develop amyloid plaques at any age studied, up to 30 months. APP(E693Q) mice were tested for spatial learning and memory, and only 12-month-old APP(E693Q) mice with readily detectable oAbeta/ADDLs displayed a significant delay in acquisition of the MWM task when compared to nontransgenic littermates.These data suggest that cerebral oAbeta/ADDL assemblies generated in brain in situ from human APP transgenes may be associated with cognitive impairment. We propose that a DTC analysis may be a sensitive method for assessing the cognitive impact in mice of endogenously generated oligomeric human Abeta assemblies. ANN NEUROL 2010.

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aβ42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

Latrepirdine (Dimebon(TM)) was originally marketed as a non-selective antihistamine in Russia. It was repurposed as an effective treatment for patients suffering from Alzheimer's disease (AD) and Huntington's disease (HD) following preliminary reports showing its neuroprotective functions and ability to enhance cognition in AD and HD models. However, latrepirdine failed to show efficacy in phase III trials in AD and HD patients following encouraging phase II trials. The failure of latrepirdine in the clinical trials has highlighted the importance of understanding the precise mechanism underlying its cognitive benefits in neurodegenerative diseases before clinical evaluation. Latrepirdine has shown to affect a number of cellular functions including multireceptor activity, mitochondrial function, calcium influx and intracellular catabolic pathways; however, it is unclear how these properties contribute to its clinical benefits. Here, we review the studies investigating latrepirdine in cellular and animal models to provide a complete evaluation of its mechanisms of action in the central nervous system. In addition, we review recent studies that demonstrate neuroprotective functions for latrepirdine-related class of molecules including the β-carbolines and aminopropyl carbazoles in AD, Parkinson's disease and amyotrophic lateral sclerosis models. Assessment of their neuroprotective effects and underlying biological functions presents obvious value for developing structural analogues of latrepirdine for dementia treatment.

Abstract Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer’s disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.

A novel class of 2-(4-heterocyclylphenyl)-1,4-dihydropyridines (2-38) possessing antagonist activity against platelet activating factor (PAF) was prepared by the Hantzsch synthesis from a variety of ethyl 4'-heterocyclic-substituted benzoylacetates, aryl or heteroaryl aldehydes, and substituted 3-aminocrotonamides or 3-aminocrotonate esters. Structure-activity relationships were evaluated where PAF antagonist activity was measured in vitro by determining the concentration of compound (IC50) required to inhibit the PAF-induced aggregation of rabbit washed platelets, and in vivo by determining the oral dose (ED50) which protected mice from a lethal injection of PAF. The nature of the substituent at the dihydropyridine 2-position was found to be important for both in vitro and in vivo activity, whereas there was greater flexibility for structural variation at the 4- and 5-positions. The most potent compound was 4-(2-chlorophenyl)-1,4-dihydro-3-(ethoxycarbonyl)-6-methyl-2-[4-(2- methylimidazo[4,5-c]pyrid-1-yl)phenyl]-5-[N-(2- pyridyl)carbamoyl]pyridine (17, UK-74,505), IC50 = 4.3 nM, ED50 = 0.26 mg/kg po, which was found to be approximately 33 times more potent in vitro (rabbit platelet aggregation) and about 8 times more potent in vivo (murine lethality) than WEB2086. Compound 17 also exhibited a long duration of action in the dog (inhibition of PAF-induced whole blood aggregation ex vivo was maintained for greater than 24 h following a single oral dose of 75 micrograms/kg) and was highly selective as a PAF antagonist, showing only weak affinity (IC50 = 6600 nM) for the [3H]nitrendipine binding site. As a result of its high oral potency, selectivity, and duration of action, UK-74,505 has been selected for clinical evaluation.

Proteins secreted by strains of Mycobacterium tuberculosis during short-term, zinc-sufficient batch culture were identified in order to define antigens likely to be relevant to the pathogenesis of human disease. [35S]Methionine-labelled proteins in supernatants of 4-7 d cultures were separated by PAGE under both denaturing and non-denaturing conditions, and the position of labelled material was determined. Secreted protein patterns of M. tuberculosis were quite similar to those of Bacillus Calmette-Guérin (BCG) but differed by the absence of the 46 kDa dimeric protein specific to BCG and by the presence in large amounts of a 23 kDa protein which, when denatured, gave 13 kDa subunits. This 13 kDa subunit protein constituted up to 20% of secreted proteins in classical strains of M. tuberculosis of phage type B but was not detected in phage type I strains from South India. This may be relevant to the different pathogenicity of these strains. Western blot analysis showed that antigens defined in supernatants of short-term (3 d) cultures of M. tuberculosis constituted a small subset of those seen in supernatants of organisms cultured for longer periods. One of the secreted proteins has the interesting property of binding to fibronectin. The available monoclonal antibodies and antisera have been used to identify lines on immunoblots corresponding to the secreted/released antigens of M. tuberculosis. The present findings suggest that there are major secreted antigens to which antibodies do not yet appear to have been produced experimentally.

Latrepirdine (Dimebon), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer's disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model, Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of amyloid-β (Aβ)42 aggregates. Latrepirdine was shown to upregulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro green fluorescent protein (GFP) tagged Aβ yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aβ42. GFP-Aβ42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aβ42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aβ42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aβ42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aβ42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aβ42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aβ42.

Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer’s pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger γ-secretase-inhibitor-sensitive biogenesis of Aβ42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer’s amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-β (Aβ) oligomers but never develop Aβ plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch Aβ-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain Aβ monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer’s disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

Recent studies have implicated specific assembly subtypes of β-amyloid (Aβ) peptide, specifically soluble oligomers (soAβ) as disease-relevant structures that may underlie memory loss in Alzheimer disease. Removing existing soluble and insoluble Aβ assemblies is thought to be essential for any attempt at stabilizing brain function and slowing cognitive decline in Alzheimer disease. IV immunoglobulin (IVIg) therapies have been shown to contain naturally occurring polyclonal antibodies that recognize conformational neoepitopes of soluble or insoluble Aβ assemblies including soAβ. These naturally occurring polyclonal antibodies have been suggested to underlie the apparent clinical benefits of IVIg. However, direct evidence linking anti-Aβ antibodies to the clinical bioactivity of IVIg has been lacking.Five-month-old female Dutch APP E693Q mice were treated for 3 months with neat IVIg or with IVIg that had been affinity-depleted over immobilized Aβ conformers in 1 of 2 assembly states. Memory was assessed in a battery of tests followed by quantification of brain soAβ levels using standard anti-soAβ antibodies.We provide evidence that NU4-type soAβ (NU4-soAβ) assemblies accumulate in the brains of Dutch APP E693Q mice and are associated with defects in memory, even in the absence of insoluble Aβ plaques. Memory benefits were associated with depletion from APP E693Q mouse brain of NU4-soAβ and A11-soAβ but not OC-type fibrillar Aβ oligomers.We propose that targeting of specific soAβ assembly subtypes may be an important consideration in the therapeutic and/or prophylactic benefit of anti-Aβ antibody drugs.

The concepts of 'drug-like' and 'lead-like' chemical properties are having a major influence on the selection of compounds for high-throughput screening, and in the design of lead generation libraries. Medicinal chemists are recycling 'privileged' drug-like structures, whilst aiming to seek optimal physical properties for oral delivery. This approach biases the chemical profiles of compound screening collections towards known structures. Novel library synthesis, creating new chemical classes to address intellectual property, toxicity issues, and less chemically tractable targets, though considered risky, is warranted.:

The suitability of neutralized perfluorosulphonic acid (Nafion®) as a surface for the attachment and growth of human cells was investigated in tissue culture. Nafion was equivalent to tissue culture polystyrene (TCP), and markedly better than polytetrafluoroethylene (Teflon®), for the attachment and growth of human umbilical artery endothelial (HUAE) cells. The attachment and growth of HUAE cells on Fn-coated Nafion was equivalent to that on Fn-coated TCP. The contribution to the attachment and spreading of HUAE cells that is due to adsorption of serum fibronectin (Fn) or vitronectin (Vn) on to the Nafion or TCP was directly tested by selective removal of Fn or Vn from the serum before addition to the culture medium. HUAE cells seeded on to Nafion or TCP in medium depleted of Vn failed to attach and spread on to these surfaces, as measured after 4 or 24 h of culture. HUAE cells seeded in medium depleted of Fn, but containing Vn, attached and spread on to Nafion, albeit to a decreased extent as compared to that in intact serum when measured after 4 h of culture, and there was no effect of depletion of Fn when measured after 24 h of culture. HUAE cells seeded on to TCP in medium depleted of Fn became attached and spread during 4 h of culture. Our results show that Nafion is a suitable polymeric surface for the attachment and growth of human cells, including endothelial cells. For HUAE cells, adsorption on to the surface of an adhesive glycoprotein, such as Vn or Fn, is an essential step for attachment and spreading of the cells onto the Nafion surface.

TiCl4 catalyzes the essentially quantitative coupling of chiral acetals 1 with 1-t-butoxy-1-t-butyldimethylsilyloxyethene 2 to generate β-alkoxycarboxylates in which the new asymmetric center is formed with excellent diastereoselection. β-hydroxycarboxylic acids of high ee result from removal of the chiral auxiliary. The procedure has been applied to the synthesis of R-(+)-α-lipoic acid 10.

Background Interscalene brachial plexus block (ISBPB) provides excellent analgesia after rotator cuff surgery but is associated with diaphragm dysfunction. In this study, ISBPB with 20 mL of 0.125% or 0.25% bupivacaine were compared to assess the effect on diaphragm function, oxygen saturation, pain control, opioid requirements, and patient satisfaction. Materials and methods In this prospective, randomized, double-blind study, 30 adults undergoing outpatient arthroscopic rotator cuff repair were enrolled to receive ultrasound-guided interscalene brachial plexus catheter placement with 20 mL of 0.125% (n = 15) or 0.25% bupivacaine (n = 15). Diaphragm function and oxygen saturation were assessed before ISBPB placement and on discharge from the postanesthesia care unit. Postoperative pain scores, opioid requirements, and patient satisfaction were compared. Results Diaphragm function and oxygen saturation were superior in the low concentration group. Absent or paradoxic motion of the diaphragm was present in 78% of the 0.25% group compared with 21% of patients in the 0.125% group (P = .008). Oxygen saturation decreased 4.3% in the 0.25% group compared with a decrease of 2.6% in the 0.125% group (P = .04). Pain scores averaged 1 of 10 in the 0.25% group and 0 of 10 in the 0.125% group (P = .02). Opioid requirements and patient satisfaction were not different between the two groups. Conclusions In this randomized, double-blind comparison of ISBPB performed with 20 mL of 0.125% or 0.25% bupivacaine, diaphragm function and oxygen saturation were superior in patients treated with more dilute bupivacaine. Furthermore, there were no clinically significant differences in pain scores, and no statistically significant differences in opioid requirements and patient satisfaction. Interscalene brachial plexus block (ISBPB) provides excellent analgesia after rotator cuff surgery but is associated with diaphragm dysfunction. In this study, ISBPB with 20 mL of 0.125% or 0.25% bupivacaine were compared to assess the effect on diaphragm function, oxygen saturation, pain control, opioid requirements, and patient satisfaction. In this prospective, randomized, double-blind study, 30 adults undergoing outpatient arthroscopic rotator cuff repair were enrolled to receive ultrasound-guided interscalene brachial plexus catheter placement with 20 mL of 0.125% (n = 15) or 0.25% bupivacaine (n = 15). Diaphragm function and oxygen saturation were assessed before ISBPB placement and on discharge from the postanesthesia care unit. Postoperative pain scores, opioid requirements, and patient satisfaction were compared. Diaphragm function and oxygen saturation were superior in the low concentration group. Absent or paradoxic motion of the diaphragm was present in 78% of the 0.25% group compared with 21% of patients in the 0.125% group (P = .008). Oxygen saturation decreased 4.3% in the 0.25% group compared with a decrease of 2.6% in the 0.125% group (P = .04). Pain scores averaged 1 of 10 in the 0.25% group and 0 of 10 in the 0.125% group (P = .02). Opioid requirements and patient satisfaction were not different between the two groups. In this randomized, double-blind comparison of ISBPB performed with 20 mL of 0.125% or 0.25% bupivacaine, diaphragm function and oxygen saturation were superior in patients treated with more dilute bupivacaine. Furthermore, there were no clinically significant differences in pain scores, and no statistically significant differences in opioid requirements and patient satisfaction.

Endosomal sorting of the Alzheimer amyloid precursor protein (APP) plays a key role in the biogenesis of the amyloid-β (Aβ) peptide. Genetic lesions underlying Alzheimer's disease (AD) can act by interfering with this physiological process. Specifically, proteins involved in trafficking between endosomal compartments and the trans -Golgi network (TGN) [including the retromer complex (Vps35, Vps26) and its putative receptors (sortilin, SorL1, SorCS1)] have been implicated in the molecular pathology of late-onset AD. Previously, we demonstrated a role for SorCS1 in APP metabolism and Aβ production and, while we implicated a role for the retromer in this regulation, the underlying mechanism remained poorly understood. Here, we provide evidence for a motif within the SorCS1c cytoplasmic tail that, when manipulated, results in perturbed sorting of APP and/or its fragments to endosomal compartments, decreased retrograde TGN trafficking, and increased Aβ production in H4 neuroglioma cells. These perturbations apparently do not involve turnover of the cell surface APP pool, but rather they involve intracellular APP and/or its fragments, downstream of APP endocytosis.

Alzheimer's disease (AD) is a complex and slowly progressing dementing disorder that results in neuronal and synaptic loss, deposition in brain of aberrantly folded proteins, and impairment of spatial and episodic memory. Most studies of mouse models of AD have employed analyses of cognitive status and assessment of amyloid burden, gliosis, and molecular pathology during disease progression. Here we sought to understand the behavioral, cellular, ultrastructural, and molecular changes that occur at a pathological stage equivalent to the early stages of human AD. We studied the TgCRND8 mouse, a model of aggressive AD amyloidosis, at an early stage of plaque pathology (3 months of age) in comparison to their wildtype littermates and assessed changes in cognition, neuron and spine structure, and expression of synaptic glutamate receptor proteins. We found that, at this age, TgCRND8 mice display substantial plaque deposition in the neocortex and hippocampus and impairment on cued and contextual memory tasks. Of particular interest, we also observed a significant decrease in the number of neurons in the hippocampus. Furthermore, analysis of CA1 neurons revealed significant changes in apical and basal dendritic spine types, as well as altered expression of GluN1 and GluA2 receptors. This change in molecular architecture within the hippocampus may reflect a rising representation of inherently less stable thin spine populations, which can cause cognitive decline. These changes, taken together with toxic insults from amyloid-β protein, may underlie the observed neuronal loss.

Maternal folate (vitamin B9) status is the largest known modifier of neural tube defect risk, so we evaluated folate-related mechanisms of action for dolutegravir (DTG) developmental toxicity.Folate receptor 1 (FOLR1) was examined as a target for DTG developmental toxicity using protein and cellular interaction studies and an animal model.FOLR1 competitive binding studies were used to test DTG for FOLR1 antagonism. Human placenta cell line studies were used to test interactions with DTG, folate, and cations. Zebrafish were selected as an animal model to examine DTG-induced developmental toxicity and rescue strategies.FOLR1 binding studies indicate DTG is a noncompetitive FOLR1 antagonist at therapeutic concentrations. In-vitro testing indicates calcium (2 mmol/l) increases FOLR1-folate interactions and alters DTG-FOLR1-folate interactions and cytotoxicity. DTG does not inhibit downstream folate metabolism by dihydrofolate reductase. Early embryonic exposure to DTG is developmentally toxic in zebrafish, and supplemental folic acid can mitigate DTG developmental toxicity.Folates and FOLR1 are established modifiers of risk for neural tube defects, and binding data indicates DTG is a partial antagonist of FOLR1. Supplemental folate can ameliorate increased developmental toxicity due to DTG in zebrafish. The results from these studies are expected to inform and guide future animal models and clinical studies of DTG-based antiretroviral therapy in women of childbearing age.

Neural tube defects (NTDs) are a broad class of congenital birth defects that result from the failure of neural tube closure during neurulation. Folic acid supplementation has been shown to prevent the occurrence of NTDs by as much as 70% in some human populations, and folate deficiency in a pregnant woman is associated with increased risk for having an NTD affected infant. Thus, folate transport-related genes and genes involved in the subsequent folate-mediated one-carbon metabolic pathway have long been considered primary candidates to study the genetic etiology of human NTDs. Herein, we review the genes involved in folate transport and one-carbon metabolism thus far identified as contributing variants that influence human NTD risk, and place these findings in the context of our evolving understanding of the complex genetic architecture underlying these defects.

Neural tube defects (NTDs) are a classic example of preventable birth defects for which there is a proven-effective intervention, folic acid (FA); however, further methods of prevention remain unrealized. In the decades following implementation of FA nutritional fortification programs throughout at least 87 nations, it has become apparent that not all NTDs can be prevented by FA. In the United States, FA fortification only reduced NTD rates by 28-35% (Williams et al., 2015). As such, it is imperative that further work is performed to understand the risk factors associated with NTDs and their underlying mechanisms so that alternative prevention strategies can be developed. However, this is complicated by the sheer number of genes associated with neural tube development, the heterogeneity of observable phenotypes in human cases, the rareness of the disease, and the myriad of environmental factors associated with NTD risk. Given the complex genetic architecture underlying NTD pathology and the way in which that architecture interacts dynamically with environmental factors, further prevention initiatives will undoubtedly require precision medicine strategies that utilize the power of human genomics and modern tools for assessing genetic risk factors. Herein, we review recent advances in genomic strategies for discovering genetic variants associated with these defects, and new ways in which biological models, such as mice and cell culture-derived organoids, are leveraged to assess mechanistic functionality, the way these variants interact with other genetic or environmental factors, and their ultimate contribution to human NTD risk.

New Mo(II) complexes BnEt(3)N(+)[Mo(CO)(4)ClBr(2)](-) (A) and Mo(CO)(5)(OTf)(2) (B) and their W(II) congeners D and E have been developed as catalysts for the title reactions. Unlike other Lewis acids, the latter catalysts exhibit cis-stereoselectivity in the cyclization of citronellal (1 --> 3 with A and 1 --> 5 with B). Isotopic labeling allowed formulation of the reaction mechanism, according to which these complexes act as bulky Lewis acids, eta(1)-coordinated to the carbonyl oxygen. The stereochemistry appears to be controlled by the protruding ligand L(p), which dictates the boatlike transition state III. The kinetically formed cis-alkenol 3 can be equilibrated by [Mo(CO)(4)Br(2)](2) (C) or ZnCl(2) to its trans-epimer 2 via a retro-ene reaction.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRate enhancement of biomimetic polyene cyclizations by a cation-stabilizing auxiliaryWilliam S. Johnson, Stephen D. Lindell, and John SteeleCite this: J. Am. Chem. Soc. 1987, 109, 19, 5852–5853Publication Date (Print):September 1, 1987Publication History Published online1 May 2002Published inissue 1 September 1987https://doi.org/10.1021/ja00253a048RIGHTS & PERMISSIONSArticle Views527Altmetric-Citations51LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (270 KB) Get e-Alerts Get e-Alerts

Abstract Background Recent reports suggest that latrepirdine (Dimebon™, dimebolin), a retired Russian antihistamine, improves cognitive function in aged rodents and in patients with mild to moderate Alzheimer's disease (AD). However, the mechanism(s) underlying this benefit remain elusive. AD is characterized by extracellular accumulation of the amyloid-β (Aβ) peptide in the brain, and Aβ-lowering drugs are currently among the most popular anti-amyloid agents under development for the treatment of AD. In the current study, we assessed the effect of acute dosing of latrepirdine on levels of extracellular Aβ using in vitro and in vivo experimental systems. Results We evaluated extracellular levels of Aβ in three experimental systems, under basal conditions and after treatment with latrepirdine. Mouse N2a neuroblastoma cells overexpressing Swedish APP were incubated for 6 hr in the presence of either vehicle or vehicle + latrepirdine (500pM-5 μM). Synaptoneurosomes were isolated from TgCRND8 mutant APP-overexpressing transgenic mice and incubated for 0 to 10 min in the absence or presence of latrepirdine (1 μM or 10 μM). Drug-naïve Tg2576 Swedish mutant APP overexpressing transgenic mice received a single intraperitoneal injection of either vehicle or vehicle + latrepirdine (3.5 mg/kg). Picomolar to nanomolar concentrations of acutely administered latrepirdine increased the extracellular concentration of Aβ in the conditioned media from Swedish mutant APP-overexpressing N2a cells by up to 64% (p = 0.01), while a clinically relevant acute dose of latrepirdine administered i.p. led to an increase in the interstitial fluid of freely moving APP transgenic mice by up to 40% (p = 0.01). Reconstitution of membrane protein trafficking and processing is frequently inefficient, and, consistent with this interpretation, latrepirdine treatment of isolated TgCRND8 synaptoneurosomes involved higher concentrations of drug (1-10 μM) and led to more modest increases in extracellular Aβ x-42 levels (+10%; p = 0.001); of note, however, was the observation that extracellular Aβ x-40 levels did not change. Conclusions Here, we report the surprising association of acute latrepirdine dosing with elevated levels of extracellular Aβ as measured in three independent neuron-related or neuron-derived systems, including the hippocampus of freely moving Tg2576 mice. Given the reported association of chronic latrepirdine treatment with improvement in cognitive function, the effects of chronic latrepirdine treatment on extracellular Aβ levels must now be determined.

Blast-induced traumatic brain injury (TBI) has been a major cause of morbidity and mortality in the conflicts in Iraq and Afghanistan. How the primary blast wave affects the brain is not well understood. In particular, it is unclear whether blast injures the brain through mechanisms similar to those found in non-blast closed impact injuries (nbTBI). The β-amyloid (Aβ) peptide associated with the development of Alzheimer's disease is elevated acutely following TBI in humans as well as in experimental animal models of nbTBI. We examined levels of brain Aβ following experimental blast injury using enzyme-linked immunosorbent assays for Aβ 40 and 42. In both rat and mouse models of blast injury, rather than being increased, endogenous rodent brain Aβ levels were decreased acutely following injury. Levels of the amyloid precursor protein (APP) were increased following blast exposure although there was no evidence of axonal pathology based on APP immunohistochemical staining. Unlike the findings in nbTBI animal models, levels of the β-secretase, β-site APP cleaving enzyme 1, and the γ-secretase component presenilin-1 were unchanged following blast exposure. These studies have implications for understanding the nature of blast injury to the brain. They also suggest that strategies aimed at lowering Aβ production may not be effective for treating acute blast injury to the brain.

Currently, the field is awaiting the results of several pivotal Phase III clinical Alzheimer's disease (AD) trials that target amyloid-β (Aβ). In light of the recent biomarker studies that indicate Aβ levels are at their most dynamic 5-10 years before the onset of clinical symptoms, it is becoming uncertain whether direct approaches to target Aβ will achieve desired clinical efficacy. AD is a complex neurodegenerative disease caused by dysregulation of numerous neurobiological networks and cellular functions, resulting in synaptic loss, neuronal loss, and ultimately impaired memory. While it is clear that Aβ plays a key role in the pathogenesis of AD, it may be a challenging and inefficient target for mid-to-late stage AD intervention. Throughout the course of AD, multiple pathways become perturbed, presenting a multitude of possible therapeutic avenues for design of AD intervention and prophylactic therapies. In this chapter, we sought to first provide an overview of Aβ-directed strategies that are currently in development, and the pivotal Aβ-targeted trials that are currently underway. Next, we delve into the biology and therapeutic designs associated with other key areas of research in the field including tau, protein trafficking and degradation pathways, ApoE, synaptic function, neurotrophic/neuroprotective strategies, and inflammation and energy utilization. For each area we have provided a comprehensive and balanced overview of the therapeutic strategies currently in preclinical and clinical development, which will shape the future therapeutic landscape of AD.

Thrombin-activatable fibrinolysis inhibitor (TAFI) has emerged as a key link between the coagulation and fibrinolysis cascades and represents a promising new target for the treatment of thrombosis. A novel series of imidazolepropionic acids has been designed that exhibit high potency against activated TAFI (TAFIa) and excellent selectivity over plasma carboxypeptidase N (CPN). Structure activity relationships suggest that the imidazole moiety plays a key role in binding to the catalytic zinc of TAFIa, and this has been supported by crystallographic studies using porcine pancreatic carboxypeptidase B as a surrogate for TAFIa. The SAR program led to the identification of 21 (TAFIa Ki = 10 nM, selectivity TAFIa/CPN > 1000) as a candidate for clinical development. Compound 21 exhibited antithrombotic efficacy in a rabbit model of venous thrombosis, yet had no effect on surgical bleeding in the rabbit. In addition, 21 exhibited an excellent preclinical and clinical pharmacokinetic profile, characterized by paracellular absorption, low clearance, and a low volume of distribution, fully consistent with its physicochemical properties of low molecular weight (MW = 239) and high hydrophilicity (log D = -2.8). These data indicate 21 (UK-396,082) has potential as a novel TAFIa inhibitor for the treatment of thrombosis and other fibrin-dependent diseases in humans.

Abstract Background The accumulation of amyloid beta (Aβ) oligomers or fibrils is thought to be one of the main causes of synaptic and neuron loss, believed to underlie cognitive dysfunction in Alzheimer’s disease (AD). Neuron loss has rarely been documented in amyloid precursor protein (APP) transgenic mouse models. We investigated whether two APP mouse models characterized by different folding states of amyloid showed different neuronal densities using an accurate method of cell counting. Findings We examined total cell and neuronal populations in Swedish/Indiana APP mutant mice (TgCRND8) with severe Aβ pathology that includes fibrils, plaques, and oligomers, and Dutch APP mutant mice with only Aβ oligomer pathology. Using the isotropic fractionator, we found no differences from control mice in regional total cell populations in either TgCRND8 or Dutch mice. However, there were 31.8% fewer hippocampal neurons in TgCRND8 compared to controls, while no such changes were observed in Dutch mice. Conclusions We show that the isotropic fractionator is a convenient method for estimating neuronal content in milligram quantities of brain tissue and represents a useful tool to assess cell loss efficiently in transgenic models with different types of neuropathology. Our data support the hypothesis that TgCRND8 mice with a spectrum of Aβ plaque, fibril, and oligomer pathology exhibit neuronal loss whereas Dutch mice with only oligomers, showed no evidence for neuronal loss. This suggests that the combination of plaques, fibrils, and oligomers causes more damage to mouse hippocampal neurons than Aβ oligomers alone.

Neural tube closure is a critical early step in central nervous system development that requires precise control of metabolism to ensure proper cellular proliferation and differentiation. Dysregulation of glucose metabolism during pregnancy has been associated with neural tube closure defects (NTDs) in humans suggesting that the developing neuroepithelium is particularly sensitive to metabolic changes. However, it remains unclear how metabolic pathways are regulated during neurulation. Here, we used single-cell mRNA-sequencing to analyze expression of genes involved in metabolism of carbon, fats, vitamins, and antioxidants during neurulation in mice and identify a coupling of glycolysis and cellular proliferation to ensure proper neural tube closure. Using loss of miR-302 as a genetic model of cranial NTD, we identify misregulated metabolic pathways and find a significant upregulation of glycolysis genes in embryos with NTD. These findings were validated using mass spectrometry-based metabolite profiling, which identified increased glycolytic and decreased lipid metabolites, consistent with a rewiring of central carbon traffic following loss of miR-302. Predicted miR-302 targets Pfkp, Pfkfb3, and Hk1 are significantly upregulated upon NTD resulting in increased glycolytic flux, a shortened cell cycle, and increased proliferation. Our findings establish a critical role for miR-302 in coordinating the metabolic landscape of neural tube closure.

In the presence of triethylsilane and trifluoroacetic acid, the reaction between indoles and aldehydes in dichloromethane at 0°C, results in good yields of C-3 reductively alkylated products. The transformation is most effective for the preparation of 3-(arylmethyl)indoles 6 from aromatic aldehydes.

A combinatorial library of 30,752 compounds has been synthesised from a set of 32 natural and unnatural amino acids. The library was designed to include the known endothelin antagonist, FR-139,317, as a positive control, and this and a number of close analogues were shown to be the most potent compounds. Thus, combinatorial libraries may be used both to discover leads and rapidly explore SAR. A combinatorial library of 30,752 compounds has been synthesised from a set of 32 natural and unnatural amino acids. The library was designed to include the known endothelin antagonist, FR-139,317, as a positive control, and this and a number of close analogues were shown to be the most potent compounds. Thus, combinatorial libraries may be used both to discover leads and rapidly explore SAR.

Certain mutant Alzheimer's amyloid-β (Aβ) peptides (that is, Dutch mutant APP(E693Q)) form complexes with gangliosides (GAβ). These mutant Aβ peptides may also undergo accelerated aggregation and accumulation upon exposure to GM2 and GM3. We hypothesized that increasing β-hexosaminidase (β-hex) activity would lead to a reduction in GM2 levels, which in turn, would cause a reduction in Aβ aggregation and accumulation. The small molecule OT1001 is a β-hex-targeted pharmacological chaperone with good bioavailability, blood-brain barrier penetration, high selectivity for β-hex and low cytotoxicity. Dutch APP(E693Q) transgenic mice accumulate oligomeric Aβ as they age, as well as Aβ oligomer-dose-dependent anxiety and impaired novel object recognition (NOR). Treatment of Dutch APP(E693Q) mice with OT1001 caused a dose-dependent increase in brain β-hex levels up to threefold over those observed at baseline. OT1001 treatment was associated with reduced anxiety, improved learning behavior in the NOR task and dramatically reduced GAβ accumulation in the subiculum and perirhinal cortex, both of which are brain regions required for normal NOR. Pharmacological chaperones that increase β-hex activity may be useful in reducing accumulation of certain mutant species of Aβ and in preventing the associated behavioral pathology.

Following the discovery of moderately potent antagonist activity platelet-activating factor (PAF) in 2-methyl-1-phenylimidazo[4,5-c]pyridine (2) (IC50 = 840 nM), 19 derivatives (3-21) were prepared which incorporated various lipophilic groups attached to the phenyl 4-position. Structure-activity relationships were evaluated where PAF antagonist activity was measured in vitro by determining the concentration of compound (IC50) required to inhibit the PAF-induced aggregation of rabbit washed platelets and in vivo by determining the oral dose (ED50) which protected mice from a lethal injection of PAF. [1,5]Benzodiazepines, e.g., 14 (2,3-dihydro-1-methyl-4-[4-(2-methylimidazo[4,5-c] pyrid-1-yl)phenyl]-1H-[1,5]benzodiazepin-2-one) (IC50 = 4.9 nM, Ed50 = 0.03 mg/kg po), were found to possess equivalent or superior potency to the 1,4-dihydropyridine PAF antagonist UK-74,505 (1,4-(2-chlorophenyl)-1,4-dihydro-3-(ethoxycarbonyl)-6-methyl-2- [4-(2-methylimidazo[4,5-c]pyrid-1-yl)phenyl]-5-[N-(2-pyridyl) carbamoyl]pyridine) in vitro and in vivo. Furthermore, a potent benzazepine, 21 (7,8-dichloro-1-methyl-4-[4-(methylimidazo[4,5-c]pyrid-1-yl) phenyl]-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one) (IC50 = 0.5 nM, ED50 = 0.03 mg/kg po), was discovered. These investigations prompted the synthesis and evaluation of additional diazepine derivatives, which are described in the following paper. The relationship between the key PAF antagonist pharmacophores of 2-methyl-1-phenylimidazo[4,5-c]pyridine, a triazolothienodiazepine (WEB2170), and a pyrrolothiazolidine (RP-52,770) is discussed.

A method for the gas chromatographic analysis of ten phenolic glycosides commonly found in Salix species is described. These compounds can be successfully separated using cyclohexanedimethanol succinate on Chromosorb G, with the exception of fragilin and salidroside. The latter two compounds can be separated using the other columns described. Infrared and ultraviolet data of some fractions collected are also given and the methods are applied to a crystalline fraction of a crude glycoside extract from a species of Salix.

N-(5-Bromo-3-methoxypyrazin-2-yl)-5-chlorothiophene-2-sulfonamide 1 was identified as a hit in a CCR4 receptor antagonist high-throughput screen (HTS) of a subset of the AstraZeneca compound bank. As a hit with a lead-like profile, it was an excellent starting point for a CCR4 receptor antagonist program and enabled the rapid progression through the Lead Identification and Lead Optimization phases resulting in the discovery of two bioavailable CCR4 receptor antagonist candidate drugs.

DNA damage response (DDR) genes orchestrating the network of DNA repair, cell cycle control, are essential for the rapid proliferation of neural progenitor cells. To date, the potential association between specific DDR genes and the risk of human neural tube defects (NTDs) has not been investigated. Using whole-genome sequencing and targeted sequencing, we identified significant enrichment of rare deleterious RAD9B variants in spina bifida cases compared to controls (8/409 vs. 0/298; p = .0241). Among the eight identified variants, the two frameshift mutants and p.Gln146Glu affected RAD9B nuclear localization. The two frameshift mutants also decreased the protein level of RAD9B. p.Ser354Gly, as well as the two frameshifts, affected the cell proliferation rate. Finally, p.Ser354Gly, p.Ser10Gly, p.Ile112Met, p.Gln146Glu, and the two frameshift variants showed a decreased ability for activating JNK phosphorylation. RAD9B knockdowns in human embryonic stem cells profoundly affected early differentiation through impairing PAX6 and OCT4 expression. RAD9B deficiency impeded in vitro formation of neural organoids, a 3D cell culture model for human neural development. Furthermore, the RNA-seq data revealed that loss of RAD9B dysregulates cell adhesion genes during organoid formation. These results represent the first demonstration of a DDR gene as an NTD risk factor in humans.

Background Cerebral folate deficiency (CFD) syndrome is characterised by a low concentration of 5-methyltetrahydrofolate in cerebrospinal fluid, while folate levels in plasma and red blood cells are in the low normal range. Mutations in several folate pathway genes, including FOLR1 (folate receptor alpha , FRα) , DHFR (dihydrofolate reductase) and PCFT (proton coupled folate transporter ) have been previously identified in patients with CFD. Methods In an effort to identify causal mutations for CFD, we performed whole exome sequencing analysis on eight CFD trios and identified eight de novo mutations in seven trios. Results Notably, we found a de novo stop gain mutation in the capicua (CIC) gene. Using 48 sporadic CFD samples as a validation cohort, we identified three additional rare variants in CIC that are putatively deleterious mutations. Functional analysis indicates that CIC binds to an octameric sequence in the promoter regions of folate transport genes: FOLR1 , PCFT and reduced folate carrier (Slc19A1; RFC1 ). The CIC nonsense variant (p.R353X) downregulated FOLR1 expression in HeLa cells as well as in the induced pluripotent stem cell (iPSCs) derived from the original CFD proband. Folate binding assay demonstrated that the p.R353X variant decreased cellular binding of folic acid in cells. Conclusion This study indicates that CIC loss of function variants can contribute to the genetic aetiology of CFD through regulating FOLR1 expression. Our study described the first mutations in a non-folate pathway gene that can contribute to the aetiology of CFD.

Neural tube defects (NTDs) are a group of common and severe congenital malformations. The PI3K-AKT signalling pathway plays a crucial role in the neural tube development. There is limited evidence concerning any possible association between aberrant methylation in PI3K-AKT signalling pathway genes and NTDs. Therefore, we aimed to investigate potential associations between aberrant methylation of PI3K-AKT pathway genes and NTDs. Methylation studies of PI3K-AKT pathway genes utilizing microarray genome-methylation data derived from neural tissues of ten NTD cases and eight non-malformed controls were performed. Targeted DNA methylation analysis was subsequently performed in an independent cohort of 73 NTD cases and 32 controls to validate the methylation levels of identified genes. siRNAs were used to pull-down the target genes in human embryonic stem cells (hESCs) to examine the effects of the aberrant expression of target genes on neural cells. As a result, 321 differentially hypermethylated CpG sites in the promoter regions of 30 PI3K-AKT pathway genes were identified in the microarray data. In target methylation analysis, CHRM1, FGF19, and ITGA7 were confirmed to be significantly hypermethylated in NTD cases and were associated with increased risk for NTDs. The down-regulation of FGF19, CHRM1, and ITGA7 impaired the formation of rosette-like cell aggregates. The down-regulation of those three genes affected the expression of PAX6, SOX2 and MAP2, implying their influence on the differentiation of neural cells. This study for the first time reported that hypermethylation of PI3K-AKT pathway genes such as CHRM1, FGF19, and ITGA7 is associated with human NTDs.

Boron concentration profiles in rapid thermally annealed Si and strained Si1−xGex in situ doped, epitaxial layers were measured using secondary-ion-mass spectroscopy. Comparison of the Si1−xGex samples to the Si samples after rapid thermal annealing revealed a retarded B diffusivity inside the strained Si1−xGex layers. A simple empirical expression for the B retardation, which depended linearly on the Ge concentration, was developed and incorporated into a diffusion model for dopants in heterostructures. This model accurately simulated the measured B concentration profiles over a wide range of Ge fractions (0%–10%), B peak concentrations (2×1018–3×1019cm−3), and rapid thermal annealing conditions (900–1025 °C for 20–30 s).

Abstract Choosing the right compounds to synthesise from large virtual combinatorial libraries is a current challenge for the pharmaceutical industry. Herein we describe a highly optimised method that aligns lead‐like properties with compound diversity. The methods are illustrated by considering a two‐dimensional library based on the interesting spirocyclic bis ‐azetidine template. magnified image A practical and pragmatic method is demonstrated that aligns lead‐like properties with compound diversity for the picking of compounds to synthesise from large virtual libraries. Methods are highlighted for decreasing synthetic attrition through the prior filtration of reagents sets grouped by reaction type. Also disclosed are protocols that use a combination of predicted physicochemical parameters and potential toxicological liabilities to enable the synthesis of lead‐like compounds with a low potential risk of exhibiting toxicity or undesirable physicochemical properties. Lastly, a compound‐picking process for a 2D compound matrix is demonstrated that maximises the diversity coverage whilst minimising synthetic effort. Thus a very highly optimised process is shown that delivers premium sample quality where lead‐likeness and novelty are aligned to afford the best possible enhancement for the corporate compound collection.

The separation of phenolic glycosides on thin layers of silica gel G and polyamide−cellulose is given, and effects of humidity control discussed. This technique is advocated for the detection of phenolic glycosides in the genus Salix and the results obtained from the bark of S. gracilis Anderss. var. textoris Fern. (S. petiolaris Sm.) are recorded. Tremuloidin, which previously has been found in both the bark and leaves of Populus species, but only in the leaves of Salix species 2, was detected in the bark of S. gracilis var. textoris.

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Valproic acid (VPA, valproate, Depakote) is a commonly used anti-seizure medication (ASM) in the treatment of epilepsy and a variety of other neurological disorders. While VPA and other ASMs are efficacious for management of seizures, they also increase the risk for adverse pregnancy outcomes, including neural tube defects (NTDs). Thus, the utility of these drugs during pregnancy and in women of childbearing potential presents a continuing public health challenge. Elucidating the underlying genetic or metabolic risk factors for VPA-affected pregnancies may lead to development of non-teratogenic ASMs, novel prevention strategies, or more targeted methods for managing epileptic pregnancies. To address this challenge, we performed unbiased, whole embryo metabolomic screening of E8.5 mouse embryos from two inbred strains with differential susceptibility to VPA-induced NTDs. We identified metabolites of differential abundance between the two strains, both in response to VPA exposure and in the vehicle controls. Notable enriched pathways included lipid metabolism, carnitine metabolism, and several amino acid pathways, especially cysteine and methionine metabolism. There also was increased abundance of ω-oxidation products of VPA in the more NTD-sensitive strain, suggesting differential metabolism of the drug. Finally, we found significantly reduced levels of hypotaurine in the susceptible strain regardless of VPA status. Based on this information, we hypothesized that maternal supplementation with L-carnitine (400 mg/kg), coenzyme A (200 mg/kg), or hypotaurine (350 mg/kg) would reduce VPA-induced NTDs in the sensitive strain and found that administration of hypotaurine prior to VPA exposure significantly reduced the occurrence of NTDs by close to one-third compared to controls. L-carnitine and coenzyme A reduced resorption rates but did not significantly reduce NTD risk in the sensitive strain. These results suggest that genetic variants or environmental exposures influencing embryonic hypotaurine status may be factors in determining risk for adverse pregnancy outcomes when managing the health care needs of pregnant women exposed to VPA or other ASMs.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy, and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Antagonism of the chemokine receptor CXCR2 has been proposed as a strategy for the treatment of inflammatory diseases such as arthritis, chronic obstructive pulmonary disease and asthma. Earlier series of bicyclic CXCR2 antagonists discovered at AstraZeneca were shown to have low solubility and poor oral bioavailability. In this Letter we describe the design, synthesis and characterisation of a new series of monocyclic CXCR2 antagonists with improved solubility and good pharmacokinetic profiles.

The effects of Ge in the epitaxial-base on the reliability of Si/Ge/sub x/Si/sub 1-x//Si heterojunction bipolar transistors were investigated. The ten-year time-to-failure under emitter-base junction reverse-bias stress was measured at the designed operation voltage by the current-acceleration method and compared to that of Si bipolar junction transistors with no Ge (x=0). The investigation shows that the Ge incorporated by the reduced pressure chemical vapor deposition epitaxial technology to give the ramp-type Ge profile has no adverse effects on the transistor reliability.

High-performance Si and SiGe epitaxial base bipolar transistors have been fabricated using a commercially available, reduced pressure, epitaxial reactor. The SiGe devices exhibit exceptional Early voltages in the range of 400-500 V, and an f/sub T/ of 31 GHz with a BV/sub CEO/ of 7.6 V and BV/sub CBO/ of 16 V. These results demonstrate that SiGe has potential as a commercially viable technology for analog, digital, and mixed-signal applications.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Annals of NeurologyVolume 69, Issue 2 p. 221-225 Editorial Apomorphine and Alzheimer Aβ: Roles for regulated α cleavage, autophagy, and antioxidation?†‡§ John W. Steele BA, John W. Steele BA Departments of Neurology and Psychiatry, Alzheimer's Disease Research Center, and Programs in Neuroscience and in Pharmacology and Systems Biology, Mount Sinai School of Medicine and James J Peters Veterans Affairs Medical Center New York, NYSearch for more papers by this authorSam Gandy MD, PhD, Sam Gandy MD, PhD Departments of Neurology and Psychiatry and Alzheimer's Disease Research Center, Mount Sinai School of Medicine and James J Peters Veterans Affairs Medical Center New York, NYSearch for more papers by this author John W. Steele BA, John W. Steele BA Departments of Neurology and Psychiatry, Alzheimer's Disease Research Center, and Programs in Neuroscience and in Pharmacology and Systems Biology, Mount Sinai School of Medicine and James J Peters Veterans Affairs Medical Center New York, NYSearch for more papers by this authorSam Gandy MD, PhD, Sam Gandy MD, PhD Departments of Neurology and Psychiatry and Alzheimer's Disease Research Center, Mount Sinai School of Medicine and James J Peters Veterans Affairs Medical Center New York, NYSearch for more papers by this author First published: 10 January 2011 https://doi.org/10.1002/ana.22359Citations: 8 † We thank B. Wustman for helpful suggestions and discussions. ‡ Potential Conflicts of Interest § S.G.: research grant support, Amicus Pharmaceuticals; consultancy, Diagenic, Amicus Pharmaceuticals, JAI, Pfizer-Janssen Alzheimer's Immunotherapy Alliance; paid presentations, NCME; safety monitoring board, Johnson & Johnson. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume69, Issue2February 2011Pages 221-225 RelatedInformation

Macroautophagy (autophagy) is a conserved cellular pathway that regulates the degradation of long-lived proteins, protein aggregates, and cellular organelles. Autophagy is essential for maintaining neuronal homeostasis; however, neuronal autophagic efficiency decreases with age. Therefore, aging is one of the greatest risk factors for development of Alzheimer's disease (AD), a slowly progressing form of neurodegeneration that develops over the course of 10-20 years prior to the onset of overt clinical symptoms. AD is defined neuropathologically by the presence of extracellular aggregates of the amyloidogenic protein amyloid-β (Aβ) and intracellular accumulation of the microtubule-associated protein tau. At end-stage Alzheimer's disease, abnormal autophagic pathology has been reported in human brain and in multiple mouse models of AD, suggesting that an intimate association may exist between neuronal autophagy stasis and Alzheimer's-related pathology. Here, we highlight recent evidence that the autophagic pathway plays a role in both the generation and clearance of the pathogenic Aβ protein and its precursors. The primary focus of this review is to examine the compelling research that highlights the autophagic pathway as a therapeutic target for AD and to discuss the therapeutic space around autophagy-regulating programs for AD. Finally, we propose that programs targeting autophagy regulation for AD ought to consider prophylactic or early stage intervention trials based on evidence against druggability of this pathway in late-stage disease.

In this study, we demonstrate the use of Molecular topology (MT) in an Alzheimer's disease (AD) drug discovery program. MT uses and expands upon the principles governing the molecular connectivity theory of numerically characterizing molecular structures, in the present case, active anti-AD drugs/agents, using topological descriptors to build models. Topological characterization has been shown to embody sufficient molecular information to provide strong correlation to therapeutic efficacy.We used MT to include multiple bioactive properties that allows for the identification of multi-functional single agent compounds, in this case, the dual functions of β-amyloid (Aβ) -lowering and anti-oligomerization. Using this technology, we identified and designed novel compounds in chemical classes unrelated to current anti-AD agents that exert dual Aβ lowering and anti-Aβ oligomerization activities in animal models of AD. AD is a multifaceted disease with different pathological features.Our study, for the first time, demonstrated that MT can provide novel strategy for discovering drugs with Aβ lowering and anti-aggregation dual activities for AD.

We will describe the first modular integration of a SiGe:C heterojunction bipolar transistor (SiGe:C HBTs) into a conventional 0.25 /spl mu/m, epi-free CMOS platform. The high temperature stability of base doping profiles in SiGe:C HBTs and an optimized collector linkage have allowed the modular integration of an npn device with f/sub T//f/sub max/ of 55/90 GHz into two variants of a conventional epi-free 0.25 /spl mu/m CMOS platform. In both cases, the original CMOS steps and the electrical parameters of the CMOS devices remain essentially unchanged. Yield and electrical characteristics of the integrated SiGe:C HBT are shown to be the same as those from a bipolar-only process.

Background/Purpose In 2004, a heritable occurrence of spina bifida was reported in sheep on a farm in the United States. We maintained and characterized the spina bifida phenotype in this flock to assess its potential as an alternative surgical model. Methods A breeding strategy was developed in which the sheep were crossed to maintain or increase the occurrence of spina bifida. Measurements and observations were recorded regarding lesion size, birthweight, ambulatory capacity, or urological function, and necropsies were performed on spina bifida afflicted lambs in conjunction with magnetic resonance imaging to determine the character of the spina bifida defects and assess the presence of Chiari-like malformations or hydrocephalus. Results The defects were observed to be more prevalent in ram lambs, and the rate of spina bifida per litter could be increased through backcrossing or by selection of a productive ewe breed. The lambs displayed a range of ambulatory and urological deficits which could be used to evaluate new fetal repair methodologies. Finally, affected lambs were shown to demonstrate severe Chiari malformations and hydrocephalus. Conclusions We have determined that use of these sheep as a natural source for spina bifida fetuses is feasible and could supplement the deficits of current sheep models for myelomeningocele repair. Level of evidence Level IV.

To present an 8-year (1985-1992) cumulative analysis of the 12,743 calls received by the Medication Information Line for the Elderly (MILE), a consumer-oriented drug information service.Data on all calls received by MILE were documented using a standard format. Certain calls were selected by the pharmacist for follow-up based on the nature and predicted severity of the inquiry.MILE is located within the Faculty of Pharmacy of the University of Manitoba and staffed by experienced, practicing pharmacists. Calls were received from the elderly and their care providers. Calls were initially taken on a local Winnipeg line, although in 1987 a toll-free number was made available to rural Manitoba residents.The majority of the calls received by MILE were from women aged 65 years or older who accessed MILE on their own initiative. Ninety-one percent of the calls were rated as not serious. Only 6% of calls were from outside Winnipeg. Although 94% of the older consumers reported having a regular pharmacist, they commonly never thought of using their pharmacist for inquiries. The drugs cited most often by consumers, nurses, physicians, and dentists were the nonsteroidal antiinflammatory agents, cardiac drugs, diuretics, antihypertensives, benzodiazepines, and antidepressants. The type of inquiries most frequently involved adverse drug reactions, drug interactions, and therapeutic use. The drugs cited and inquiries made by pharmacist callers were more diverse than those of consumers and other healthcare professionals.Many older drug consumers have medication information needs that are not being met. Since a large proportion of the callers appeared to be self-motivated women, MILE may not be accessing all older consumers in need of information. This analysis also suggests that many older consumers are not aware that the pharmacist is available and capable of providing drug information.

A previously described gas-liquid chromatographic screening test for the detection of phenolic glycosides has been applied to the determination of the phenolic glycoside chromatograms of nineteen samples of Populus species. The results have been used as a chemotaxonomic check on existing morphological classification of the samples into the Leuce, Aegeiros and Tacamahaca sections of the genus.

A method is described for the gas chromatographic analysis of a number of phenols and related compounds that are commonly found in members of the Salicaccae and in other plants. The phenols listed can be resolved by using one or more columns. The procedure was successfully applied to various plant smples and is extremely useful for identification of aglycones from phenolic glycosides. Application of the procedure to Populus samples was not uniformly successful.

November bark of Salix petiolaris Sm. was extracted with acetone and ethanol and the extracts were subjected to chromatogaphy on polyamide columns. The various column fractions were monitored by thin-layer chromatographic and gas-liquid chromatographic procedures. From these results and by isolation of crystalline material, the bark was shown to contain salicin, picein, vimalin, salicyloylsalicin, salireposide, grandidentatin, populin, tremulacin and/or tremuloidin, salicyloylsalicin-2-O-benzoate, (+)-catechin and β-sitosterol.

Abstract Through the advantages conferred in the speed of synthesis, combinatorial chemistry is making a significant impact on the process of drug discovery. The mix‐and‐split paradigm has been an effective method for the production of compound mixtures, although there is now a need for new, fast library approaches to generate well‐characterised single compounds. Having already demonstrated the successful preparation and application of library mixtures, we have now developed a novel combinatorial method for the production of single compounds.

Salix tyrrellii is an endemic taxon described from the active sand dunes south of Lake Athabasca, Saskatchewan. It is related to S. planifolia from which it differs in having slender erect branches which are often slightly tortuose, a pro- longed juvenile growth form, smaller and thicker leaves with stomata on both sur- faces and low concentrations of phenolic glycosides in bark tissues. Its amphisto- matous leaves link this taxon to some arctic populations of S. planifolia in the N.W.T. and Manitoba which may be ancestral to it. The growth form of S. tyrrellii is probably an adaptation to active sand dune habitats. Phenolic glycosides may play a role in its adaptation to active sand dunes. Because of its close relationship to S. planifolia, the new combination S. planifolia ssp. tyrreliji (Raup) Argus is proposed. An extensive system of active sand dunes lies along the south shore of Lake Athabasca in northwestern Saskatchewan and adjacent Alberta. Large dune fields covering 300 sq km and 30 sq km are located near the mouths of the William and MacFarlane Rivers, respectively. The region of actively blowing sand extends over 90 km along the lake shore and up to 20 km inland (Map 1). The dune fields consist of loose sand, some of which is blown into dunes up to 35 m tall, overlying gravel pavements and sandy glacio-fluvial materials. Smaller dunes scattered throughout the region are parabola dunes that have developed from blowouts in the sandy Pinus banksiana and Picea mariana forests that stabilize much of the sand area. The active sand habitat supports a distinctive flora made up of an unusually large number of endemic taxa together with disjunct arctic species and the more common boreal and northern Great Plains species.

Abstract A gas-liquid chromatography procedure was used to examine the effect of different extraction methods on the apparent free phenolic glycoside content of the bark of Salix petiolaris Sm. Previously reported decomposition of phenolic glycosides in plant extracts treated with lead subacetate was confirmed. Decomposition and interconversion of certain pure glycosides in commonly used extraction solvents were demonstrated and the effect of solvents on extracts of the above bark is described.

Data generated in Niemann Pick type C1 (NPC1) human embryonic and human induced pluripotent stem cell derived neurons complement on-going studies in animal models and provide the first example, in disease-relevant human cells, of processes that underlie preferential neuronal defects in a NPC1. Our work and that of other investigators in human neurons derived from stem cells highlight the importance of performing rigorous mechanistic studies in relevant cell types to guide drug discovery and therapeutic development, alongside of existing animal models. Through the use of human stem cell-derived models of disease, we can identify and discover or repurpose drugs that revert early events that lead to neuronal failure in NPC1. Together with the study of disease pathogenesis and efficacy of therapies in animal models, these strategies will fulfill the promise of stem cell technology in the development of new treatments for human diseases. This article is part of a Special Issue entitled SI: Exploiting human neurons.

In the presence of 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), methyl benzylpenicillinate 1,1-dioxide (3a) is converted into (2S,3R)-1-(1-methoxycarbonyl-2-methylprop-1-enyl)-4-oxo-3-phenylacetamidoazetidine-2-sulphinic acid (5a). Corresponding reactions are observed with the p-nitrobenzyl and 2-iodoethyl esters of benzylpenicillinic acid 1,1-dioxide (3b and c), with methyl phenoxymethylpenicillinate 1,1-dioxide (3d), with methyl benzyloxypenicillinate 1,1-dioxide (3i), with methyl 6β-phthalimidopenicillanate 1,1-dioxide (3h), with methyl 6α-chloropenicillanate 1,1-dioxide (4b), and with p-nitrobenzyl penicillanate 1,1-dioxide (4c). Unidentified non-β-lactam products are produced when methyl penicillinate 1,1-dioxide (3e), methyl methylpenicillinate 1,1-dioxide (3f), and benzyl 6β-triphenylmethylaminopenicillanate 1,1-dioxide (3g) are treated with DBN.

Abstract A detailed description is given of a gas-liquid chromatographic screening test for the detection of phenolic glycosides in 10-mg samples of plant material. The test was applied to a large number of P. deltoides Marsh.bark samples in order to examine and compare glycoside content patterns in the “western” (var. occidentalis ) and “eastern” (var. deltoides ) types.

The design of a series of dual thromboxane synthase inhibitor/thromboxane receptor antagonists based on a 3-[2-[(arylsulfonyl)amino]ethyl]benzenepropanoic acid thromboxane receptor antagonist template is described. Introduction of a 5-(1H-imidazol-1-ylmethyl), a 5-(3-pyridinylmethyl), or a 5-(3-pyridinyloxy) substituent leads to dual agents with thromboxane synthase inhibitory activity comparable with that of dazmegrel (7). In addition, 3-pyridinylalkyl substituents also make a significant contribution to thromboxane receptor binding. Oral administration of compound 74 (5 mg/kg) to conscious dogs produces long-lasting thromboxane synthase inhibition and thromboxane receptor blockade as measured by inhibition of U46619-induced platelet aggregation ex vivo.

The ACPI (Advanced Configuration and Power Interface) specification was developed to provide a standardized approach to configuring the hardware, systems, and software necessary for power and thermal management within the PC. Temperature sensors and system monitors are a core part of any reliable thermal management system. This paper discusses ways to use temperature sensing in the PC for optimum conformance to the ACPI specification. While the discussion focuses on PC applications, many of the concepts presented here also apply to electronic systems in general. As we will see, efficient, cost-effective solutions are possible with the right component choices. Components are available that can off-load much of the ACPI requirement from the operating system, thereby enabling the computer to achieve its full performance potential.

The various recorded methods of synthesizing 10-methylphenothiazine were reviewed, and an improved process is suggested which gives consistent yields of 50–60% purified product. A series of bis-Schiff's bases was prepared by condensation of 3-formyl-10-methylphenothiazine with a number of diamines, and reduction of this series yielded a corresponding series of bis-secondary amines. The secondary amines did not show an NH absorption band in the infrared spectra unless considerably higher than normal concentrations were used, but nuclear magnetic resonance studies in deuteriochloroform confirmed the existence of the ―CH 2 NH― group. A model compound of the same type (namely, 3-phenyliminomethylene-10-methyl-phenothiazine) was synthesized, and it was found to exhibit exactly the same effects in the infrared spectra and in the nuclear magnetic resonance spectra in deuteriochloroform. In dimethyl sulfoxide-d 6 , the model compound showed clear evidence of coupling between the NH and the adjacent methylene group, an effect which is rarely seen.Quaternization of the secondary amines was unsuccessful in all but a few cases.

The metabolism and excretion of styrene oxide-glutathione conjugates were studied in the intact rat and by isolated perfusion techniques in rat lung, liver and kidney. The intact animals converted the styrene oxide-glutathione conjugates almost entirely to styrene oxide-n-acetylcysteine derivatives mercapturic acids), with much smaller amounts of cysteine, cysteinylglycine and (unchanged) glutathione conjugates. Only small amounts of biliary excretion (approximately 5%) and less than 1% of fecal excretion of radioactivity were found. The isolated perfused kidney retains the capacity to transform virtually all of the glutathione conjugates at the dose level studied (25-30 mumol/kidney) and the principal component in urine from the isolated kidney was the cysteine conjugate. Isolated perfused lung experiments demonstrated that this organ has a minor role in styrene oxide-thioether conjugate. Isolated perfused lung experiments demonstrated that this organ has a minor role in styrene oxide-thioether conjugate metabolism. The isolated perfused liver experiments showed that phenobarbital pretreatment has little effect on biliary excretion but barbital pretreatment has little effect on biliary excretion but that it causes profound qualitative and quantitative effects on metabolism of styrene oxide-glutathione conjugates in the perfusion medium. The high level of styrene oxide-cysteine conjugates in the perfusion medium of an isolated perfused liver, especially after phenobarbital treatment, is consistent with the theory that gamma-glutamyltransferase activity is extracellular. The high-pressure liquid chromatography system of analysis described offers the ability to simultaneously separate and quantitate the major thioether conjugates of styrene oxide and other electrophilic epoxides.

Following the Colombia accident, the Extravehicular Mobility Units (EMU) onboard ISS were unused for several months. Upon startup, the units experienced a failure in the coolant system. This failure resulted in the loss of Extravehicular Activity (EVA) capability from the US segment of ISS. With limited on-orbit evidence, a team of chemists, engineers, metallurgists, and microbiologists were able to identify the cause of the failure and develop recovery hardware and procedures. As a result of this work, the ISS crew regained the capability to perform EVAs from the US segment of the ISS.

The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1(∆exon8)). We previously reported that PS1 L271V increased amyloid beta (Aβ) 42/40 ratios, while PS1(∆exon8) reduced Aβ42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1(∆exon8) did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1(∆exon8) is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1(∆exon8) in vivo by crossing PS1(∆exon8) transgenics with either PS1-null or Dutch APP(E693Q) mice. As a control, we crossed APP(E693Q) with mice expressing a deletion in an adjacent exon (PS1(∆exon9)). PS1(∆exon8) did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation. These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions.

Benzyltriethylammonium chloropentacarbonylmolybdate (1) and chloropentacarbonyltungstate (2) complexes react with CF3SO3Ag in DME via a combination of chloride exchange and a redox process, as revealed by cyclic voltammetry and ESR spectrometry. The resulting intermediate M(I) species disproportionate to M(0) and M(II) so that 3 equiv of TfOAg are required for the quantitative conversion into the M(II) complex. Analogous PPN complexes 3 and 4 only undergo the redox process (in DME); in this instance, the chloride exchange is precluded, presumably due to strong pairing of the complex anion with the counterion.

Methyl 6β-(dihydro-4H-1,3,5-dioxazin-5-yl)penicillanate is converted into methyl (6S,9S)-8,8-dimethyl-2-oxo-5-oxa-7-thia-1,4-diazabicyclo[4.3.0]non-3-ene-9-carboxylate by the action of potassium permanganate in aqueous acetic acid.

Neural tube defects (NTDs) are a group of severe congenital malformations caused by a failure of neural tube closure during early embryonic development. Although extensively investigated, the genetic etiology of NTDs remains poorly understood. FKBP8 is critical for proper mammalian neural tube closure. Fkbp8-/- mouse embryos showed posterior NTDs consistent with a diagnosis of spina bifida (SB). To date, no publication has reported any association between FKBP8 and human NTDs. Using Sanger sequencing on genomic DNA samples from 472 SB and 565 control samples, we identified five rare (MAF ≤ 0.001) deleterious variants in SB patients, while no rare deleterious variant was identified in the controls (P = 0.0191). p.Glu140* affected FKBP8 localization to the mitochondria and created a truncated form of the FKBP8 protein, thus impairing its interaction with BCL2 and ultimately leading to an increase in cellular apoptosis. p.Ser3Leu, p.Lys315Asn and p.Ala292Ser variants decreased FKBP8 protein level. p.Lys315Asn further increased the cellular apoptosis. RNA sequencing on anterior and posterior tissues isolated from Fkbp8-/- and wildtype mice at E9.5 and E10.5 showed that Fkbp8-/- embryos have an abnormal expression profile within tissues harvested at posterior sites, thus leading to a posterior NTD. Moreover, we found that Fkbp8 knockout mouse embryos have abnormal expression of Wnt3a and Nkx2.9 during the early stage of neural tube development, perhaps also contributing to caudal specific NTDs. These findings provide evidence that functional variants of FKBP8 are risk factors for SB, which may involve a novel mechanism by which Fkbp8 mutations specifically cause SB in mice.

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo . The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo , but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo .

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Abstract The design of a series of dual thromboxane synthase inhibitor/thromboxane receptor antagonists based on an indole thromboxane synthase inhibitor template is described. The indole-5-propanoic acid derivatives 17, 22 and 23 were found to be potent dual agents in vitro.

In many states, students with learning disabilities are required to take algebra in high school. These students are usually served in inclusive settings (the general education classroom), where they often struggle with algebra because the content is so abstract. Although limited research and literature are available on algebra instruction for students with special needs, recognized strategies that promote students' learning can help make the algebra experience more enjoyable and successful for them and for the other students in the class, as well. This article highlights characteristics of students with learning disabilities, reviews current literature on algebra and students with learning disabilities, summarizes some of the recommendations, and describes how they can be put into practice.

The first page of this article is displayed as the abstract.

The design of a series of thromboxane receptor antagonists based on 3-(2-[{(4-chlorophenyl)sulfonyl}amino]ethyl)benzenepropanoic acid (1) is described. Addition of an arylmethyl group at the 5-position of 1 gave exceptionally potent agents in vitro and in vivo, with 13a (UK-147,535) giving complete blockade of the TxA2 receptor for greater than 12 hours in dogs, following an oral dose of 0.1 mg/kg.

<div class="htmlview paragraph">The ISS (International Space Station) ITCS (Internal Thermal Control System) includes two internal coolant loops that utilize an aqueous based coolant for heat transfer. A silver salt biocide had previously been utilized as an additive in the coolant formulation to control the growth and proliferation of microorganisms within the coolant loops. Ground-based and in-flight testing demonstrated that the silver salt was rapidly depleted, and did not act as an effective long-term biocide. Efforts to select an optimal alternate biocide for the ITCS coolant application have been underway and are now in the final stages.</div> <div class="htmlview paragraph">An extensive evaluation of biocides was conducted to down-select to several candidates for test trials and was reported on previously. Criteria for that down-select included: the need for safe, non-intrusive implementation and operation in a functioning system; the ability to control existing planktonic and biofilm residing microorganisms; a negligible impact on system wetted materials of construction; and a negligible reactivity with existing coolant additives.</div> <div class="htmlview paragraph">Candidate testing to provide data for the selection of an optimal alternate biocide is now in the final stages. That testing has included rapid biocide effectiveness screening using Biolog MT2 plates to determine minimum inhibitory concentration (amount that will inhibit visible growth of microorganisms), time kill studies to determine the exposure time required to completely eliminate organism growth, materials compatibility exposure evaluations, and coolant compatibility studies. This paper reports the current status of the effort to select an alternate biocide for the ISS ITCS coolant. The results of various test results to select the optimal candidate are presented.</div>

The n.m.r. and mass spectral data are used to show that chloroacetylation of 1-azaphenothiazine gave 1-hydroxy-2-chloroacetyl-6-thia-10b-aza-2a-azoniaaceanthrylene hydroxide inner salt and not the expected 10-chloroacetyl-1-azaphenothiazine.

An EMU water processing kit (Airlock Coolant Loop Recovery -- A/L CLR) was developed as a corrective action to Extravehicular Mobility Unit (EMU) coolant flow disruptions experienced on the International Space Station (ISS) in May of 2004 and thereafter. A conservative duty cycle and set of use parameters for A/L CLR use and component life were initially developed and implemented based on prior analysis results and analytical modeling. Several initiatives were undertaken to optimize the duty cycle and use parameters of the hardware. Examination of post-flight samples and EMU Coolant Loop hardware provided invaluable information on the performance of the A/L CLR and has allowed for an optimization of the process. The intent of this paper is to detail the evolution of the A/L CLR hardware, efforts to optimize the duty cycle and use parameters, and the final recommendations for implementation in the post-Shuttle retirement era.

The Oxygen Generation System (OGS) Hydrogen Dome Assembly Orbital Replacement Unit (ORU) serial number 00001 suffered a cell stack high-voltage shutdown on July 5, 2010. The Hydrogen Dome Assembly ORU was removed and replaced with the on-board spare ORU serial number 00002 to maintain OGS operation. The Hydrogen Dome Assembly ORU was returned from ISS on STS-133/ULF-5 in March 2011 with test, teardown and evaluation (TT&E) and failure analysis to follow.

Animal models of genetic forms of AD have not fully recapitulated the human disease. Furthermore, over 95% of subjects with common sporadic forms of Alzheimer's disease (AD) lack identifiable mutations. Developing an induced pluripotent stem cells (iPSCs) model from AD patient fibroblasts provides the best pathway for studying brain cells from these patients and identifying potentially pathogenic subcellular phenotypes. We have created iPS cells from patients carrying presenilin-1 (PSEN1) mutations, which are responsible for the most common form of autosomal dominant, 100% penetrant, inherited AD. A total of twelve affected and unaffected control patients from the FAD1 (A246E) and FAD4 (M146L) families were used to generate iPS lines. These in turn have been successfully differentiated into human forebrain neurons for use in mechanistic studies of Aβ production, cell death, and other AD-relevant biochemical changes. Human fibroblasts were reprogrammed via retroviral addition of the four Yamanakatranscription factors - Oct4, KLF4, Sox2, and c-Myc. iPS lines were analyzed by a variety of methods to characterize pluripotency and retroviral transgene silencing. We have differentiated control and AD iPS cells into forebrain neurons utilizing both embryoid body (EB) and monolayer methods, including generation of cholinergic basal forebrain neurons that are vulnerable in AD. Initial studies indicate that human iPS neuronal cells can respond to exogenous oligomerized abeta in a similar manner to hippocampal rodent neurons by induction of pro-apoptotic proteins such as Bim. Our preliminary studies suggest substantial biochemical and phenotypic differences between control and AD neuronal cells, including an increased ratio of secreted Aβ42/Aβ40, and enhanced cell death in response to apoptotic stimuli. We have successfully generated an iPSC model of early-onset AD that reflects multiples aspects of the disease biochemically and phenotypically at the cellular level. The recapitulation of brain molecular phenotypes affirms the promise of this approach and validates our efforts at extending this strategy to the discovery of subcellular phenotypes underlying common sporadic AD. Furthermore, by developing a human in vitro model of cholinergic basal forebrain neurons, we have created a platform that can be used for high throughput screening campaigns to identify novel therapeutics.

Animal models of genetic forms of AD have not fully recapitulated the human disease. Furthermore, over 95% of subjects with common sporadic forms of Alzheimer's disease (AD) lack identifiable mutations. Developing an induced pluripotent stem cells (iPSCs) model from AD patient fibroblasts provides the best pathway for studying brain cells from these patients and identifying potentially pathogenic subcellular phenotypes. We have created iPS cells from patients carrying presenilin-1 (PSEN1) mutations, which are responsible for the most common form of autosomal dominant, 100% penetrant, inherited AD. A total of twelve affected and unaffected control patients from the FAD1 (A246E) and FAD4 (M146L) families were used to generate iPS lines. These in turn have been successfully differentiated into human forebrain neurons for use in mechanistic studies of Aβ production, cell death, and other AD-relevant biochemical changes. Human fibroblasts were reprogrammed via retroviral addition of the four Yamanaka transcription factors - Oct4, KLF4, Sox2, and c-Myc. iPS lines were analyzed by a variety of methods to characterize pluripotency and retroviral transgene silencing. We have differentiated control and AD iPS cells into forebrain neurons utilizing both embryoid body (EB) and monolayer methods, including generation of cholinergic basal forebrain neurons that are vulnerable in AD. Initial studies indicate that human iPS neuronal cells can respond to exogenous oligomerized abeta in a similar manner to hippocampal rodent neurons by induction of pro-apoptotic proteins such as Bim. Our preliminary studies suggest substantial biochemical and phenotypic differences between control and AD neuronal cells, including an increased ratio of secreted Aβ42/Aβ40, and enhanced cell death in response to apoptotic stimuli. We have successfully generated an iPSC model of early-onset AD that reflects multiples aspects of the disease biochemically and phenotypically at the cellular level. The recapitulation of brain molecular phenotypes affirms the promise of this approach and validates our efforts at extending this strategy to the discovery of subcellular phenotypes underlying common sporadic AD. Furthermore, by developing a human in vitro model of cholinergic basal forebrain neurons, we have created a platform that can be used for high throughput screening campaigns to identify novel therapeutics.

Neural tube closure is a complex process driven by mechanical forces, but this process can be disturbed leading to development defects. So, to understand the interplay between forces and tissue stiffness during neurulation, we developed a multimodal Brillouin microscopy and optical coherence system (OCT). OCT provides structural guidance while mapping the biomechanical properties of embryonic neural tube using Brillouin microscopy. 3D-OCT, 2D-OCT, and 2D-Brillouin images of Mthfd1l and Fuz knockout mouse embryos at gestation days 9.5 and 10.5 were acquired. Our results show overall decrease in the stiffness of homozygotic knockout neural tube tissues compared to the wildtype.

Abstract Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy, and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower compared to that of wild-type embryos, while exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in both nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness for normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

In the presence of 1,5-diazabicyclo[4.3.0]non-5-ene (DBN)(0.5 mol equiv.) and deuterium oxide, methyl benzylpenicillinate 1,1-dioxide (1a) was converted into its 3,6-dideuteriated derivative (3a), its 3,6-dideuteriated 6-epimer (3b), and (2R,3S)-1-(1-methoxycarbonyl-2-methylprop-1-enyl)-4-oxo-3-phenylacetamido[3-2H]azetidine-2-sulphinic acid (2b). Under similar conditions, methyl penicillanate 1,1-dioxide (1c) afforded its 3-deuteriated derivative (3c) which was then transformed into (2R)-(1-methoxycarbonyl-2-methylprop-1-enyl)-4-oxoazetidine-2-sulphinic acid (2c). The aforementioned results indicate that the β-elimination reactions occur by E1cB pathways, in which C-3 anionic intermediates, i.e.(4a) and (4b), are formed reversibly.