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Hepatitis C virus is considered a major public health problem, infecting 2%–3% of the human population. Hepatitis C virus infection causes acute and chronic liver disease, including chronic hepatitis, cirrhosis and hepatocellular carcinoma. In fact, hepatitis C virus infection is the most frequent indication for liver transplantation and a vaccine is not available. Hepatitis C virus displays a narrow host species tropism, naturally infecting only humans, although chimpanzees are also susceptible to experimental infection. To date, there is no evidence for an animal reservoir of viruses closely related to hepatitis C virus which may have crossed the species barrier to cause disease in humans and resulted in the current pandemic. In fact, due to this restricted host range, a robust immunocompetent small animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control and prophylactic vaccine development. Recently, several studies discovered new viruses related to hepatitis C virus, belonging to the hepaci- and pegivirus genera, in small wild mammals (rodents and bats) and domesticated animals which live in close contact with humans (dogs and horses). Genetic and biological characterization of these newly discovered hepatitis C virus-like viruses infecting different mammals will contribute to our understanding of the origins of hepatitis C virus in humans and enhance our ability to study pathogenesis and immune responses using tractable animal models. In this review article, we start with an introduction on the genetic diversity of hepatitis C virus and then focus on the newly discovered viruses closely related to hepatitis C virus. Finally, we discuss possible theories about the origin of this important viral human pathogen.

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

Abstract Background The molecular basis for the genetic risk of ischemic stroke is likely to be multigenic and influenced by environmental factors. Several small case-control studies have suggested associations between ischemic stroke and polymorphisms of genes that code for coagulation cascade proteins and platelet receptors. Our aim is to investigate potential associations between hemostatic gene polymorphisms and ischemic stroke, with particular emphasis on detailed characterization of the phenotype. Methods/Design The Ischemic Stroke Genetic Study is a prospective, multicenter genetic association study in adults with recent first-ever ischemic stroke confirmed with computed tomography or magnetic resonance imaging. Patients are evaluated at academic medical centers in the United States and compared with sex- and age-matched controls. Stroke subtypes are determined by central blinded adjudication using standardized, validated mechanistic and syndromic classification systems. The panel of genes to be tested for polymorphisms includes β-fibrinogen and platelet glycoprotein Ia, Iba, and IIb/IIIa. Immortalized cell lines are created to allow for time- and cost-efficient testing of additional candidate genes in the future. Discussion The study is designed to minimize survival bias and to allow for exploring associations between specific polymorphisms and individual subtypes of ischemic stroke. The data set will also permit the study of genetic determinants of stroke outcome. Having cell lines will permit testing of future candidate risk factor genes.

Sensory neurons are afferent neurons in sensory systems that convert stimuli and transmit information to the central nervous system as electrical signals. Primary afferent neurons that are affected by non-noxious and noxious stimuli are present in the dorsal root ganglia (DRG), and the DRG sensory neurons are used as an in vitro model of the nociceptive response. However, DRG derived from mouse or rat give a low yield of neurons, and they are difficult to culture. To help alleviate this problem, we characterized human induced pluripotent stem cell (hiPSC) derived sensory neurons. They can solve the problems of interspecies differences and supply stability. We investigated expressions of sensory neuron related proteins and genes, and drug responses by Multi-Electrode Array (MEA) to analyze the properties and functions of sensory neurons. They expressed nociceptor, mechanoreceptor and proprioceptor related genes and proteins. They constitute a heterogeneous population of their subclasses. We confirmed that they could respond to both noxious and non-noxious stimuli. We showed that histamine inhibitors reduced histamine-induced neuronal excitability. Furthermore, incubation with a ProTx-II and Nav1.7 inhibitor reduced the spontaneous neural activity in hiPSC-derived sensory neurons. Their responsiveness was different from each drug. We have demonstrated that hiPSC-derived sensory neurons combined with MEA are good candidates for drug discovery studies where DRG in vitro modeling is necessary., (© 2024. The Author(s).)

Emergency fire service (EFS) systems provide rescue operations for emergencies and accidents. If properly designed, they can decrease property loss and mortality. This paper proposes a distributionally robust model (DRM) for optimizing the location of fire stations, the number of fire trucks, and demand assignment for long term planning in an EFS system. This is achieved by minimizing the worst-case expected total cost, including fire station construction cost, purchase cost for fire trucks, transportation cost, and penalty cost for not providing adequate service. The ambiguity in demands and travel durations distributions are captured through moment information and mean absolute deviation. A cutting plane method is used to solve the problem. Due to fact that it is computationally intensive for larger problems, two approximate methods are introduced; one that uses linear decision rules (LDRs), and another that adopts three-point approximations of the distributions. The results show that the heuristic method is especially useful for solving large instances of DRM. Extensive numerical experiments are conducted to analyze the model's performance with respect to different parameters. Finally, data obtained from Hefei (China) demonstrates the practical applicability and value of the model in designing an EFS system in a large metropolitan setting., (© 2022. The Author(s).)

Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here, we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In addition, Cx43 expression is up-regulated in human postmortem tissue and cerebrospinal fluid from ALS patients. Using human induced pluripotent stem cell–derived astrocytes (hiPSC-A) from both familial and sporadic ALS, we establish that Cx43 is up-regulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We also demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes using GAP 19, a mimetic peptide blocker, and tonabersat, a clinically tested small molecule, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Extending the in vitro application of tonabersat with chronic administration to SOD1G93A mice results in MN protection with a reduction in reactive astrocytosis and microgliosis. Taking these data together, our studies identify Cx43 hemichannels as conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.

Salicylate hydroxylase (NahG) has a single redox site in which FAD is reduced by NADH, the O 2 is activated by the reduced flavin, and salicylate undergoes an oxidative decarboxylation by a C(4a)-hydroperoxyflavin intermediate to give catechol. We report experimental results that show the contribution of individual pieces of the FAD cofactor to the observed enzymatic activity for turnover of the whole cofactor. A comparison of the kinetic parameters and products for the NahG-catalyzed reactions of FMN and riboflavin cofactor fragments reveal that the adenosine monophosphate (AMP) and ribitol phosphate pieces of FAD act to anchor the flavin to the enzyme and to direct the partitioning of the C(4a)-hydroperoxyflavin reaction intermediate towards hydroxylation of salicylate. The addition of AMP or ribitol phosphate pieces to solutions of the truncated flavins results in a partial restoration of the enzymatic activity lost upon truncation of FAD, and the pieces direct the reaction of the C(4a)-hydroperoxyflavin intermediate towards hydroxylation of salicylate., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy., Methods: We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy., Results: We generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H 2 O 2 -induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H 2 O 2 -induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment., Conclusions: This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases., (© 2022. The Author(s).)

Since the opening of Earth Observation (EO) archives (USGS/NASA Landsat and EC/ESA Sentinels), large collections of EO data are freely available, offering scientists new possibilities to better understand and quantify environmental changes. Fully exploiting these satellite EO data will require new approaches for their acquisition, management, distribution, and analysis. Given rapid environmental changes and the emergence of big data, innovative solutions are needed to support policy frameworks and related actions toward sustainable development. Here we present the Swiss Data Cube (SDC), unleashing the information power of Big Earth Data for monitoring the environment, providing Analysis Ready Data over the geographic extent of Switzerland since 1984, which is updated on a daily basis. Based on a cloud-computing platform allowing to access, visualize and analyse optical (Sentinel-2; Landsat 5, 7, 8) and radar (Sentinel-1) imagery, the SDC minimizes the time and knowledge required for environmental analyses, by offering consistent calibrated and spatially co-registered satellite observations. SDC derived analysis ready data supports generation of environmental information, allowing to inform a variety of environmental policies with unprecedented timeliness and quality., (© 2021. The Author(s).)

More than 40% of non-small cell lung cancer (NSCLC) patients lack actionable targets and require non-targeted chemotherapeutics. Many become refractory to drugs due to underlying resistance-associated mutations. KEAP1 mutant NSCLCs further activate NRF2 and upregulate its client PTGR1. LP-184, a novel alkylating agent belonging to the acylfulvene class is a prodrug dependent upon PTGR1. We hypothesized that NSCLC with KEAP1 mutations would continue to remain sensitive to LP-184. LP-184 demonstrated highly potent anticancer activity both in primary NSCLC cell lines and in those originating from brain metastases of primary lung cancers. LP-184 activity correlated with PTGR1 transcript levels but was independent of mutations in key oncogenes (KRAS and KEAP1) and tumor suppressors (TP53 and STK11). LP-184 was orders of magnitude more potent in vitro than cisplatin and pemetrexed. Correlative analyses of sensitivity with cell line gene expression patterns indicated that alterations in NRF2, MET, EGFR and BRAF consistently modulated LP-184 sensitivity. These correlations were then extended to TCGA analysis of 517 lung adenocarcinoma patients, out of which 35% showed elevated PTGR1, and 40% of those further displayed statistically significant co-occurrence of KEAP1 mutations. The gene correlates of LP-184 sensitivity allow additional personalization of therapeutic options for future treatment of NSCLC., Competing Interests: CONFLICTS OF INTEREST A.K., J.R.M., U.K., P.S. and K.B. are or have been salaried employees of or consultants to the pharmaceutical company Lantern Pharma Inc. A.K., U.K., P.S., and K.B. hold options to purchase common stock of Lantern Pharma Inc. and are also included as inventors on patent applications filed by Lantern Pharma Inc. J.-P.R. and R.M. are employees of REPROCELL USA, Inc., a commercial contract research organization providing services to Lantern Pharma Inc., (Copyright: © 2021 Kulkarni et al.)

Background: Non-targeted cytotoxics with anticancer activity are often developed through preclinical stages using response criteria observed in cell lines and xenografts. A panel of the NCI-60 cell lines is frequently the first line to define tumor types that are optimally responsive. Open data on the gene expression of the NCI-60 cell lines, provides a unique opportunity to add another dimension to the preclinical development of such drugs by interrogating correlations with gene expression patterns. Machine learning can be used to reduce the complexity of whole genome gene expression patterns to derive manageable signatures of response. Application of machine learning in early phases of preclinical development is likely to allow a better positioning and ultimate clinical success of molecules. LP-184 is a highly potent novel alkylating agent where the preclinical development is being guided by a dedicated machine learning-derived response signature. We show the feasibility and the accuracy of such a signature of response by accurately predicting the response to LP-184 validated using wet lab derived IC50s on a panel of cell lines., Results: We applied our proprietary RADR® platform to an NCI-60 discovery dataset encompassing LP-184 IC50s and publicly available gene expression data. We used multiple feature selection layers followed by the XGBoost regression model and reduced the complexity of 20,000 gene expression values to generate a 16-gene signature leading to the identification of a set of predictive candidate biomarkers which form an LP-184 response gene signature. We further validated this signature and predicted response to an additional panel of cell lines. Considering fold change differences and correlation between actual and predicted LP-184 IC50 values as validation performance measures, we obtained 86% accuracy at four-fold cut-off, and a strong (r = 0.70) and significant (p value 1.36e-06) correlation between actual and predicted LP-184 sensitivity. In agreement with the perceived mechanism of action of LP-184, PTGR1 emerged as the top weighted gene., Conclusion: Integration of a machine learning-derived signature of response with in vitro assessment of LP-184 efficacy facilitated the derivation of manageable yet robust biomarkers which can be used to predict drug sensitivity with high accuracy and clinical value.

The ability to generate human-induced pluripotent stem cell (hiPSC)-derived neural cells displaying region-specific phenotypes is of particular interest for modeling central nervous system biology in vitro. We describe a unique method by which spinal cord hiPSC-derived astrocytes (hiPSC-A) are cultured with spinal cord hiPSC-derived motor neurons (hiPSC-MN) in a multielectrode array (MEA) system to record electrophysiological activity over time. We show that hiPSC-A enhance hiPSC-MN electrophysiological maturation in a time-dependent fashion. The sequence of plating, density, and age in which hiPSC-A are cocultured with MN, but not their respective hiPSC line origin, are factors that influence neuronal electrophysiology. When compared to coculture with mouse primary spinal cord astrocytes, we observe an earlier and more robust electrophysiological maturation in the fully human cultures, suggesting that the human origin is relevant to the recapitulation of astrocyte/motor neuron crosstalk. Finally, we test pharmacological compounds on our MEA platform and observe changes in electrophysiological activity, which confirm hiPSC-MN maturation. These findings are supported by immunocytochemistry and real-time PCR studies in parallel cultures demonstrating human astrocyte mediated changes in the structural maturation and protein expression profiles of the neurons. Interestingly, this relationship is reciprocal and coculture with neurons influences astrocyte maturation as well. Taken together, these data indicate that in a human in vitro spinal cord culture system, astrocytes support hiPSC-MN maturation in a time-dependent and species-specific manner and suggest a closer approximation of in vivo conditions. Stem Cells Translational Medicine 2019;8:1272&1285., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

One of the fundamental limitations in assessing potential efficacy in Central Nervous System (CNS) transplantation of stem cells is the capacity for monitoring cell survival and migration noninvasively and longitudinally. Human glial-restricted progenitor (hGRP) cells (Q-Cells) have been investigated for their utility in providing neuroprotection following transplantation into models of amyotrophic lateral sclerosis (ALS) and have been granted a Food and Drug Administration (FDA) Investigational New Drug (IND) for intraspinal transplantation in ALS patients. Furthermore, clinical development of these cells for therapeutic use will rely on the ability to track the cells using noninvasive imaging methodologies as well as the verification that the transplanted GRPs have disease-relevant activity. As a first step in development, we investigated the use of a perfluorocarbon (PFC) dual-modal ( 19 F magnetic resonance imaging [MRI] and fluorescence) tracer agent to label Q-Cells in culture and following spinal cord transplantation. PFCs have a number of potential benefits that make them appealing for clinical use. They are quantitative, noninvasive, biologically inert, and highly specific. In this study, we developed optimized PFC labeling protocols for Q-Cells and demonstrate that PFCs do not significantly alter the glial identity of Q-Cells. We also show that PFCs do not interfere with the capacity for differentiation into astrocytes either in vitro or following transplantation into the ventral horn of the mouse spinal cord, and can be visualized in vivo by hot spot 19 F MRI. These studies provide a foundation for further preclinical development of PFCs within the context of evaluating Q-Cell transplantation in the brain and spinal cord of future ALS patients using 19 F MRI. Stem Cells Translational Medicine 2019;8:355-365., (© 2019 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

We recently demonstrated that microRNA-218 (miR-218) is greatly enriched in motor neurons and is released extracellularly in amyotrophic lateral sclerosis model rats. To determine if the released, motor neuron-derived miR-218 may have a functional role in amyotrophic lateral sclerosis, we examined the effect of miR-218 on neighbouring astrocytes. Surprisingly, we found that extracellular, motor neuron-derived miR-218 can be taken up by astrocytes and is sufficient to downregulate an important glutamate transporter in astrocytes [excitatory amino acid transporter 2 (EAAT2)]. The effect of miR-218 on astrocytes extends beyond EAAT2 since miR-218 binding sites are enriched in mRNAs translationally downregulated in amyotrophic lateral sclerosis astrocytes. Inhibiting miR-218 with antisense oligonucleotides in amyotrophic lateral sclerosis model mice mitigates the loss of EAAT2 and other miR-218-mediated changes, providing an important in vivo demonstration of the relevance of microRNA-mediated communication between neurons and astrocytes. These data define a novel mechanism in neurodegeneration whereby microRNAs derived from dying neurons can directly modify the glial phenotype and cause astrocyte dysfunction.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type to elucidate underlying disease mechanisms and to identify novel targets that inform on MN health during ALS disease time course. microRNAs (miRNAs) are short, noncoding RNAs that can shape the expression profile of a cell and thus often exhibit cell-type-enriched expression. To determine MN-enriched miRNA expression, we used Cre recombinase-dependent miRNA tagging and affinity purification in mice. By defining the in vivo miRNA expression of MNs, all neurons, astrocytes, and microglia, we then focused on MN-enriched miRNAs via a comparative analysis and found that they may functionally distinguish MNs postnatally from other spinal neurons. Characterizing the levels of the MN-enriched miRNAs in CSF harvested from ALS models of MN disease demonstrated that one miRNA (miR-218) tracked with MN loss and was responsive to an ALS therapy in rodent models. Therefore, we have used cellular expression profiling tools to define the distinct miRNA expression of MNs, which is likely to enrich future studies of MN disease. This approach enabled the development of a novel, drug-responsive marker of MN disease in ALS rodents. SIGNIFICANCE STATEMENT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons (MNs) in the brain and spinal cord are selectively lost. To develop tools to aid in our understanding of the distinct expression profiles of MNs and, ultimately, to monitor MN disease progression, we identified small regulatory microRNAs (miRNAs) that were highly enriched or exclusive in MNs. The signal for one of these MN-enriched miRNAs is detectable in spinal tap biofluid from an ALS rat model, where its levels change as disease progresses, suggesting that it may be a clinically useful marker of disease status. Furthermore, rats treated with ALS therapy have restored expression of this MN RNA marker, making it an MN-specific and drug-responsive marker for ALS rodents., (Copyright © 2017 the authors 0270-6474/17/375574-13$15.00/0.)

43: We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The products of the reactions of 2-(4-methoxyphenyl)ethyl tosylate ( MeO-1-Ts ) and 2-(4-methyphenyl)ethyl tosylate ( Me-1-Ts ) with nucleophilic anions were determined for reactions in 50/50 (v/v) trifluoroethanol/water at 25°C. In many cases the nucleophile selectivity k Nu / k s (M -1 ) for reactions of nucleophile and solvent, calculated from the ratio of product yields, depends upon [Nu - ]. This demonstrates the existence of competing reaction pathways, which show different selectivities for reactions with nucleophiles. A carbon-13 NMR analysis of the products of the reactions of substrate enriched with carbon-13 at the α-carbon, X-1-[α- 13 C]Ts , (X = -OCH 3 , -Me) with nucleophilic anions in 50/50 (v/v) trifluoroethanol/water at 25°C shows the formation of X-1-[β- 13 C]OH , X-1-[β- 13 C]OCH 2 CF 3 and X-1-[β- 13 C]Nu (Nu = Br, Cl, CH 3 CO 2 , Cl 2 CHCO 2 ) from the trapping of symmetrical 4-substituted phenonium ion reaction intermediates X-2 + . The observation of excess label in the α-position, [α- 13 C]/[β- 13 C] > 1.0, for both the water and nucleophile adducts, shows that water and anionic nucleophiles also react by direct substitution at X-1-[α- 13 C]Ts . The ratios of product yields, [α- 13 C]/[β- 13 C] , and observed nucleophile selectivity ( k Nu / k s ) obs were used to dissect the contribution of direct nucleophile addition at Me-1-Ts and trapping of X-2 + to the product yields. The product yields from partitioning of the intermediate gave the nucleophile selectivity k Nu / k s (M -1 ) for X-2 + . Swain-Scott plots of log ( k Nu / k s ) are correlated by slopes of s = 0.78 and s = 0.73 for reactions of MeO-2 + and Me-2 + , respectively. This shows that the sensitivity of bimolecular substitution at X-2 + to changes in nucleophile reactivity is smaller than for nucleophilic substitution at the methyl iodide. Evidence is presented that nucleophile addition to X-2 + proceeds through an "exploded" transition state.

The TIM-barrel fold is described and its propagation throughout the enzyme universe noted. The functions of the individual front loops of the eponymous TIM-barrel of triosephosphate isomerase are presented in a discussion of: (a) electrophilic catalysis, by amino acid side chains from loops 1 and 4, of abstraction of an α-carbonyl hydrogen from substrate dihydroxyacetone phosphate (DHAP) or d-glyceraldehyde 3-phosphate (DGAP). (b) The engineering of loop 3 to give the monomeric variant monoTIM and the structure and catalytic properties of this monomer. (c) The interaction between loops 6, 7 and 8 and the phosphodianion of DHAP or DGAP. (d) The mechanism by which a ligand-gated conformational change, dominated by motion of loops 6 and 7, activates TIM for catalysis of deprotonation of DHAP or DGAP. (e) The conformational plasticity of TIM, and the utilization of substrate binding energy to "mold" the distorted active site loops of TIM mutants into catalytically active enzymes. The features of the TIM-barrel fold that favor effective protein catalysis are discussed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The generation of human induced pluripotent stem cells (hiPSCs) represents an exciting advancement with promise for stem cell transplantation therapies as well as for neurological disease modeling. Based on the emerging roles for astrocytes in neurological disorders, we investigated whether hiPSC-derived astrocyte progenitors could be engrafted to the rodent spinal cord and how the characteristics of these cells changed between in vitro culture and after transplantation to the in vivo spinal cord environment. Our results show that human embryonic stem cell- and hiPSC-derived astrocyte progenitors survive long-term after spinal cord engraftment and differentiate to astrocytes in vivo with few cells from other lineages present. Gene profiling of the transplanted cells demonstrates the astrocyte progenitors continue to mature in vivo and upregulate a variety of astrocyte-specific genes. Given this mature astrocyte gene profile, this work highlights hiPSCs as a tool to investigate disease-related astrocyte biology using in vivo disease modeling with significant implications for human neurological diseases currently lacking animal models.

Background: The RAD-57 pulse CO-oximeter is a lightweight device allowing noninvasive measurement of blood carboxyhemoglobin (S(pCO)). We assessed the diagnostic value of pulse CO-oximetry, comparing S(pCO) values from the RAD-57 to standard laboratory blood carboxyhemoglobin (COHb) measurement in emergency department patients with suspected carbon monoxide (CO) poisoning., Methods: This was a prospective, diagnostic accuracy study according to the Standards for the Reporting of Diagnostic Accuracy Studies criteria in consecutive adult emergency department patients with suspected CO poisoning. S(pCO) was measured with the RAD-57 simultaneously with blood sampling for laboratory blood gas analysis. We made no changes to our standard management of CO poisoning. Blood COHb > 5% for non-smokers, and > 10% for smokers were applied as the reference standard., Results: We included 93 subjects: 37 smokers and 56 non-smokers. CO poisoning was diagnosed in 26 subjects (28%). The S(pCO) values ranged from 1% to 30%, with a median of 4% (IQR 2.7-7.3%). The COHb values ranged from 0% to 34%, with a median of 5% (IQR 2-9%). The mean differences between the COHb and S(pCO) values were -0.2% ± 3.3% (95% limits of agreement of -6.7% and 6.3%) for the whole cohort, -0.7% (limits of agreement -7.7% and 6.2%) for the non-smokers, and 0.6% (limits of agreement -5.0% and 6.2%) for the smokers. The optimal thresholds for detecting CO poisoning were S(pCO) of 9% and 6% for smokers and non-smokers, respectively., Conclusions: S(pCO) measured with the RAD-57 was not a substitute for standard blood COHb measurement. However, noninvasive pulse CO-oximetry could be useful as a first-line screening test, enabling rapid detection and management of CO-poisoned patients in the emergency department.

Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumors in adults. Uncontrolled proliferation and abnormal cell migration are two prominent spatially and temporally disassociated characteristics of GBMs. In this study, we investigated the role of the receptor tyrosine kinase EphB2 in controlling the proliferation/migration dichotomy of GBM. We studied EphB2 gain of function and loss of function in glioblastoma-derived stem-like neurospheres, whose in vivo growth pattern closely replicates human GBM. EphB2 expression stimulated GBM neurosphere cell migration and invasion, and inhibited neurosphere cell proliferation in vitro. In parallel, EphB2 silencing increased tumor cell proliferation and decreased tumor cell migration. EphB2 was found to increase tumor cell invasion in vivo using an internally controlled dual-fluorescent xenograft model. Xenografts derived from EphB2-overexpressing GBM neurospheres also showed decreased cellular proliferation. The non-receptor tyrosine kinase focal adhesion kinase (FAK) was found to be co-associated with and highly activated by EphB2 expression, and FAK activation facilitated focal adhesion formation, cytoskeleton structure change and cell migration in EphB2-expressing GBM neurosphere cells. Taken together, our findings indicate that EphB2 has pro-invasive and anti-proliferative actions in GBM stem-like neurospheres mediated, in part, by interactions between EphB2 receptors and FAK. These novel findings suggest that tumor cell invasion can be therapeutically targeted by inhibiting EphB2 signaling, and that optimal antitumor responses to EphB2 targeting may require concurrent use of anti-proliferative agents.

Tumor-initiating stem cells (also referred to as cancer stem cells, CSCs) are a subpopulation of cancer cells that play unique roles in tumor propagation, therapeutic resistance and tumor recurrence. It is increasingly important to understand how molecular signaling regulates the self-renewal and differentiation of CSCs. Basic helix-loop-helix (bHLH) transcription factors are critical for the differentiation of normal stem cells, yet their roles in neoplastic stem cells are not well understood. In glioblastoma neurosphere cultures that contain cancer stem cells (GBM-CSCs), the bHLH family member inhibitors of DNA binding protein 2 and 4 (Id2 and Id4) were found to be upregulated during the differentiation of GBM-CSCs in response to histone deacetylase inhibitors. In this study, we examined the functions of Id2 and Id4 in GBM neurosphere cells and identified Id proteins as efficient differentiation regulators of GBM-CSCs. Overexpression of Id2 and Id4 promoted the lineage-specific differentiation of GBM neurosphere cells as evidenced by the induction of neuronal/astroglial differentiation markers Tuj1 and GFAP and the inhibition of the oligodendroglial marker GalC. Id protein overexpression also reduced both stem cell marker expression and neurosphere formation potential, a biological marker of cancer cell "stemness." We further showed that Id2 and Id4 regulated GBM neurosphere differentiation through downregulating of another bHLH family member, the oligodendroglial lineage-associated transcription factors (Olig) 1 and 2. Our results provide evidence for distinct functions of Id proteins in neoplastic stem cells, which supports Id proteins and their downstream targets as potential candidates for differentiation therapy in CSCs., (© 2012 Japanese Cancer Association and Hugo W. Moser Research Institute at Kennedy Krieger.)

Social animals learn to perceive their social environment, and their social skills and preferences are thought to emerge from greater exposure to and hence familiarity with some social signals rather than others. Familiarity appears to be tightly linked to multisensory integration. The ability to differentiate and categorize familiar and unfamiliar individuals and to build a multisensory representation of known individuals emerges from successive social interactions, in particular with adult, experienced models. In different species, adults have been shown to shape the social behavior of young by promoting selective attention to multisensory cues. The question of what representation of known conspecifics adult-deprived animals may build therefore arises. Here we show that starlings raised with no experience with adults fail to develop a multisensory representation of familiar and unfamiliar starlings. Electrophysiological recordings of neuronal activity throughout the primary auditory area of these birds, while they were exposed to audio-only or audiovisual familiar and unfamiliar cues, showed that visual stimuli did, as in wild-caught starlings, modulate auditory responses but that, unlike what was observed in wild-caught birds, this modulation was not influenced by familiarity. Thus, adult-deprived starlings seem to fail to discriminate between familiar and unfamiliar individuals. This suggests that adults may shape multisensory representation of known individuals in the brain, possibly by focusing the young's attention on relevant, multisensory cues. Multisensory stimulation by experienced, adult models may thus be ubiquitously important for the development of social skills (and of the neural properties underlying such skills) in a variety of species.

The reaction of 2-methoxyphenylethyl tosylate (MeO-1-Ts) is first-order in [N(3)(-)]. A carbon-13 NMR analysis of the products of the reactions of MeO-1-[α-(13)C]Ts shows the formation of MeO-1-[β-(13)C]OH and MeO-1-[β-(13)C]N(3) from the trapping of a symmetrical 4-methoxyphenonium ion reaction intermediate 2(+). An analysis of the rate and product data provides a value of k(az)/k(s) = 83 M(-1) for partitioning of 2(+) between addition of azide ion and solvent. These data set a limit for the lifetime of 2(+) in aqueous solution., (© 2011 American Chemical Society)

Purpose: We compared the effects of exercise intensity (EI) on the cytokine response to an acute bout of running., Methods: Ten males (mean ± SD VO(2max)= 56.2 ± 8.1 mL·min(-1)·kg(-1)) completed three, counterbalanced, 8-d trials. After three control days, on day 4, participants completed 60 min of running at 55%, 65%, and 75% VO(2max). The cytokines tumor necrosis factor (TNF)-α, interleukin-1β (IL-1β), IL-6, and IL-1 receptor antagonist (ra), and creatine kinase were measured during and for 3 h after exercise and on four follow-up days (FU1-FU4)., Results: RER was higher at 75% V(O2max) compared with both 55% (P < 0.001) and 65% (P < 0.01) VO(2max). IL-1β was undetectable in six participants. There was a small (18%-27%) increase in TNF-α during exercise but no effect of EI. IL-6 concentrations peaked at the end of exercise, with a greater increase at 75% VO(2max), resulting in higher concentrations at the end of exercise and at 30 min after exercise compared with 55% (P < 0.001) and 65% VO(2max) (P < 0.01). IL-1ra concentrations peaked at the end of exercise at 75% VO(2max), resulting in higher (P < 0.05) concentrations at 1-2 h after exercise compared with 55% and 65% VO(2max). Creatine kinase was increased at FU1 and FU2, but there was no effect of EI., Conclusions: Sixty minutes of treadmill running at 75% VO(2max) results in a greater increase in IL-6 but not TNF-α compared with 55% and 65% V(O2max). The higher IL-1ra concentrations at 75% VO(2max) might be related to the higher IL-6 concentrations that precede them.

Deprotonation of carbon and decarboxylation at enzyme active sites proceed through the same carbanion intermediates as for the uncatalyzed reactions in water. The mechanism for the enzymatic reactions can be studied at the same level of detail as for nonenzymatic reactions, using the mechanistic tools developed by physical organic chemists. Triosephosphate isomerase (TIM) catalyzed interconversion of D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate is being studied as a prototype for enzyme catalyzed proton transfer, and orotidine monophosphate decarboxylase (OMPDC) catalyzed decarboxylation of orotidine 5'-monophosphate is being studied as a prototype for enzyme-catalyzed decarboxylation. 1 H NMR spectroscopy is an excellent analytical method to monitor proton transfer to and from carbon catalyzed by these enzymes in D 2 O. Studies of these partial enzyme-catalyzed exchange reactions provide novel insight into the stability of carbanion reaction intermediates, that is not accessible in studies of the full enzymatic reaction. The importance of flexible enzyme loops and the contribution of interactions between these loops and the substrate phosphodianion to the enzymatic rate acceleration are discussed. The similarity in the interactions of OMPDC and TIM with the phosphodianion of bound substrate is emphasized.

Cells can change identity during normal development, in response to tissue damage or defined artificial treatments, or during disease processes such as cancer. Strikingly, not only the reprogramming of tissue cells to an embryonic stem cell-like state, but also the direct conversion from one cell type to another have been described. Direct cell type conversion could represent an alternative strategy for cellular therapies. However, little is known about the actual cellular steps undertaken by a cell as it changes its identity and their possible consequences for the organism. Using an in vivo single-cell system of natural direct reprogramming, in which a C. elegans rectal cell transforms into a motoneuron, we present an in-depth analysis of the cellular transformations involved. We found that the reprogrammed cell transits through intermediate states during direct in vivo reprogramming. We identified and characterised a mutant in the conserved COE transcription factor UNC-3 in which this cellular transformation is blocked. We determined that complete erasure of initial identity first takes place, followed by stepwise, unc-3-dependent, redifferentiation into a motoneuron. Furthermore, unlike in vitro induced reprogramming, reversion to a dedifferentiated identity does not lead to an increase in cellular potential in a natural, in vivo context. Our findings suggest that direct cell type conversion occurs via successive steps, and that dedifferentiation can occur in the absence of cell division. Furthermore, our results suggest that mechanisms are in place in vivo to restrict cell potential during reprogramming, a finding with important implications for regenerative medicine.

It is well known that visual information can affect auditory perception, as in the famous "McGurk effect," but little is known concerning the processes involved. To address this issue, we used the best-developed animal model to study language-related processes in the brain: songbirds. European starlings were exposed to audiovisual compared to auditory-only playback of conspecific songs, while electrophysiological recordings were made in their primary auditory area (Field L). The results show that the audiovisual condition modulated the auditory responses. Enhancement and suppression were both observed, depending on the stimulus familiarity. Seeing a familiar bird led to suppressed auditory responses while seeing an unfamiliar bird led to response enhancement, suggesting that unisensory perception may be enough if the stimulus is familiar while redundancy may be required for unfamiliar items. This is to our knowledge the first evidence that multisensory integration may occur in a low-level, putatively unisensory area of a non-mammalian vertebrate brain, and also that familiarity of the stimuli may influence modulation of auditory responses by vision.

Songbirds are one of the few vertebrate groups (including humans) that evolved the ability to learn vocalizations. During song learning, social interactions with adult models are crucial and young songbirds raised without direct contacts with adults typically produce abnormal songs showing phonological and syntactical deficits. This raises the question of what functional representation of their vocalizations such deprived animals develop. Here we show that young starlings that we raised without any direct contact with adults not only failed to differentiate starlings' typical song classes in their vocalizations but also failed to develop differential neural responses to these songs. These deficits appear to be linked to a failure to acquire songs' functions and may provide a model for abnormal development of communicative skills, including speech.

Schramm and coworkers have punched holes into human purine nucleoside phosphorylase by substitution of glycine for aromatic amino acid residues at a protein lid. The results of studies on the enzymes with holes illuminate hidden chemistry that occurs at the enzyme active site.

The non-enzymatic reaction of 5'-deoxypyridoxal (DPL) with l-alanine in water at 25 degrees C was investigated. DPL reacts with alanine to form an imine, which then undergoes deprotonation at the alpha-amino carbon of alanine to form a resonance delocalized DPL-stabilized carbanion. At early reaction times the only detectable products are pyruvate and the dimeric species formed by addition of the alpha-pyridine stabilized carbanion to DPL. No Claisen-type products of addition of the alpha-amino carbanion to DPL, as was previously reported to form from the reaction between DPL and glycine [K. Toth, T.L. Amyes, J.P. Richard, J.P.G. Malthouse, M.E. Ni Beilliu, J. Am. Chem. Soc. 126 (2004) 10538-10539], are observed. The electrophile reacts instead at the alpha-pyridyl carbon. This dimer is in chemical equilibrium with reactants. At longer reaction times about 50% of DPL is converted to 5'-deoxypyridoxamine, the thermodynamically favored product of formal transamination of DPL.

Direct social contact and social interaction affect speech development in human infants and are required in order to maintain perceptual abilities; however the processes involved are still poorly known. In the present study, we tested the hypothesis that social segregation during development would prevent the proper development of a central auditory area, using a "classical" animal model of vocal development, a songbird. Based on our knowledge of European starling, we raised young female starlings with peers and only adult male tutors. This ensured that female would show neither social bond with nor vocal copying from males. Electrophysiological recordings performed when these females were adult revealed perceptual abnormalities: they presented a larger auditory area, a lower proportion of specialized neurons and a larger proportion of generalist sites than wild-caught females, whereas these characteristics were similar to those observed in socially deprived (physically separated) females. These results confirmed and added to earlier results for males, suggesting that the degree of perceptual deficiency reflects the degree of social separation. To our knowledge, this report constitutes the first evidence that social segregation can, as much as physical separation, alter the development of a central auditory area.

Categorization is essential to all cognitive processes, but identifying the neural substrates underlying categorization processes is a real challenge. Among animals that have been shown to be able of categorization, songbirds are particularly interesting because they provide researchers with clear examples of categories of acoustic signals allowing different levels of recognition, and they possess a system of specialized brain structures found only in birds that learn to sing: the song system. Moreover, an avian brain nucleus that is analogous to the mammalian secondary auditory cortex (the caudo-medial nidopallium, or NCM) has recently emerged as a plausible site for sensory representation of birdsong, and appears as a well positioned brain region for categorization of songs. Hence, we tested responses in this non-primary, associative area to clear and distinct classes of songs with different functions and social values, and for a possible correspondence between these responses and the functional aspects of songs, in a highly social songbird species: the European starling. Our results clearly show differential neuronal responses to the ethologically defined classes of songs, both in the number of neurons responding, and in the response magnitude of these neurons. Most importantly, these differential responses corresponded to the functional classes of songs, with increasing activation from non-specific to species-specific and from species-specific to individual-specific sounds. These data therefore suggest a potential neural substrate for sorting natural communication signals into categories, and for individual vocal recognition of same-species members. Given the many parallels that exist between birdsong and speech, these results may contribute to a better understanding of the neural bases of speech.

The values of K(i) for methylphosphate dianion (MP(2-)) inhibition of the cleavage of 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) catalyzed by 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane (Zn(2)(1)(H(2)O)) approach a small limiting value of 6 microM at pH

The interaction between ammonia and the benzene radical cation has been investigated by gas-phase studies of mass selected ion clusters {C(6)H(6)-(NH(3))(n=0-8)}(+) via tandem quadrupole mass spectrometry and through calculations. Experiments show a special stability for the cluster ion that contains four ammonias: {C(6)H(6)(NH(3))(4)}(+). Calculations provide evidence that the first ammonia forms a weak dative bond to the cyclohexadienyl radical cation, {C(6)H(6)-NH(3)}(+), where there is a transfer of electrons from ammonia to benzene. Additional solvating ammonia molecules form stabilizing hydrogen bonds to the ring-bound ammonia {C(6)H(6)-NH(3)}(+).(NH(3))(n), which cause cooperative changes in the structure of the cluster complex. Free ammonia is a weak hydrogen bond donor, but electron transfer from NH(3) to the benzene ring that strengthens the dative bond will increase the hydrogen acidity and the strength of the cluster hydrogen bonds to the added ammonia. A progressive "tightening" of this dative bond is observed upon addition of the first, second, and third ammonia to give a cluster stabilized by three N-(+)H x N hydrogen bonds. This shows that the energetic cost of tightening the dative bond is recovered with dividends in the formation of stable cluster hydrogen bonds.

The 5'-deoxypyridoxal stabilized glycine carbanion has been generated in water at neutral and mildly basic pH. At pH < 7, this carbanion reacts mainly with the carbonyl carbon of 1 to form a stable Claisen-type adduct. At pH > or = 8, this carbanion reacts with the iminium carbon of the pyridoxal-glycine iminium ion to form the second Claisen-type adduct 3 as the major reaction product.

A massive fish kill affecting exclusively common carp (Cyprinus carpio carpio) in the St. Lawrence River, Québec, Canada, during the summer of 2001 was investigated by use of laboratory diagnostic methods and by an attempt to experimentally induce the disease. The ultimate causes of mortality were opportunistic bacterial infections with Aeromonas hydrophila and Flavobacterium sp. secondary to immunosuppression induced by physiologic (i.e., spawning) and environmental (i.e., high temperatures and low water levels) stressors, and possibly enhanced by an infection causing lymphocytic encephalitis observed in 9 of 18 (50%) fish examined. Experimental induction of disease was attempted in captured wild carp by administration of crude and filtered (particulate <0.22 microm) inocula prepared from a homogenate of tissues from carp affected by the natural outbreak. Although significant clinical disease or mortality was not induced by experimental challenge, lymphocytic encephalitis similar to the one observed in naturally affected carp was induced in four of seven (57%) fish administered crude inoculum and four of seven (57%) fish administered filtered inoculum. None of the control fish inoculated with sterile phosphate-buffered saline (n = 6) were affected by encephalitis. The cause of the encephalitis observed in carp from the natural outbreak and in experimentally inoculated fish could not be determined by use of virus isolation and transmission electron microscopy.

Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATPand temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.

Carbanion ylides are strongly stabilized by electrostatic interactions between opposing charges at neighboring atoms and this stabilizing electrostatic interaction increases with decreasing dielectric constant of the medium through which the charges interact. Consequently, there is a large increase in the thermodynamic driving force, with decreasing dielectric constant of the reaction medium, for deprotonation of cationic carbon acids and decarboxylation to form related ylides. This favors catalysis of the formation of unstable ylides at enzyme active sites of low dielectric constant. A brief survey of enzymes that catalyze deprotonation of cationic carbon acids and related decarboxylation reactions shows catalysis generally occurs for substrates that are bound in a deep pocket on the protein, with an apparent dielectric constant that is much lower than for the solvent water. In several cases, proton transfer is to a catalytic residue that is relatively weakly solvated in water. We suggest that there is a strong advantage for evolution of protein catalysts that utilize weakly solvated basic side chains which are relatively easily buried in nonpolar active sites that are favorable for zwitterion formation.

This study tests the effect on the laying rhythm of a light cycle reaching directly the encephala via a diode in Japanese quail maintained in constant darkness. In DD, all the birds expressed their free-running laying rhythm (period close to 25 h). When the diode is switched on 14 h per 24 h cycle, females showed the same organization as in LD with the same laying time. Thus, a photoperiodic cycle, where light was only perceived through the skull of the female quail, could synchronize its laying rhythm. This device with a LED is an interesting alternative solution to eye-patching or blinding of birds.

Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.

Hemispheric specialisation for speech in humans has been well documented. The lateralisation for song production observed in songbirds is reminiscent of this hemispheric dominance. In order to investigate whether song perception is also lateralised, we made multiunit recordings of the neuronal activity in the field L of starlings during the presentation of species-specific and artificial non-specific sounds. We observed a systematic stronger activation in one hemisphere than in the other one during the playback of species-specific sounds, with inter-subject variability in the predominant hemisphere for song perception. Such an asymmetry was not observed for artificial non-specific sounds. Thus, our results suggest that, at least at the individual level, the two hemispheres of the starlings' brain perceive and process conspecific signals differently.

An E461G mutation of beta-galactosidase results in the disappearance of the high pL (L = H, D) downward break in the rate profiles for k(cat)/K(m) for wild-type enzyme-catalyzed hydrolysis of 4-nitrophenyl beta-D-galactopyranoside (Gal-OPNP) and a decrease from (k(cat))(HOH)/(k(cat))(DOD) = 1.7 to (k(cat))(HOH)/(k(cat))(DOD) = 1.2 in the solvent deuterium isotope effect. These observations provide evidence that the propionic acid side chain of Glu 461 is protonated at catalytically active free beta-galactosidase and they are consistent with a role for this residue in Brønsted acid catalysis at the leaving group. The earlier observation that this same E461G mutation results in the loss of a downward break at high pH in the rate profile for k(s) for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to water cannot be simply explained by a mechanism in which the single side chain of Glu 461 functions to provide general acid catalysis in the rate limiting step for formation of the beta-D-galactopyranosyl intermediate and general base catalysis of breakdown of this intermediate. Evidence is presented that there may be different catalytic mechanisms, with different roles for the side chain for Glu-461, for nucleophilic addition of water and of small alkyl alcohols to the beta-D-galactopyranosyl reaction intermediate., (Copyright 2001 Academic Press.)