In vivo electrochemical studies of the effects of cocaine on dopamine nerve terminals in the rat neostriatum

In vivo electrochemical measurements, involving chronoamperometric recordings using monoamine-selective Nafion-coated electrodes, were used to study the effects of locally applied cocaine (50-500 micromolar barrel concentrations) on dopamine (DA) nerve terminals in the neostriatum of the anaesthetized rat. Local application of cocaine did not elicit detectable increases in basal levels of extracellular DA. However, locally applied cocaine significantly augmented the concentration of DA detected following a potassium (K+)-evoked depolarization. Data obtained with a new high-speed chronoamperometric recording technique further support that DA is the predominant species detected electrochemically following potassium-evoked depolarizations both before and after local application of cocaine. Unlike other locally applied uptake inhibitors that we have studied, cocaine failed to augment the time dynamics of released DA. In addition, large doses of the highest concentration of cocaine caused an attenuation of K+-evoked DA release, presumably due to cocaine's local anaesthetic properties. These data suggest that cocaine elevates synaptic levels of DA, but in a manner that is not identical to other potent monoamine uptake inhibitors.     

Primary metabolism in the new human cell line AGE1.HN at various substrate levels: increased metabolic efficiency and α1-antitrypsin production at reduced pyruvate load

Metabolic responses of the new neuronal human cell line AGE1.HN to various substrate levels were analyzed in this study showing that reduced substrate and especially pyruvate load improves metabolic efficiency, leading to improved growth and α₁-antitrypsin (A1AT) production. The adaptation of the metabolism to different pyruvate and glutamine concentrations was analyzed in detail using a full factorial design. The most important finding was an increasingly inefficient use of substrates as well as the reduction of cell proliferation with increasing pyruvate concentrations in the medium. Cultivations with different feeding profiles showed that the highest viable cell density and A1AT concentration (167% of batch) was reached in the culture with the lowest glucose level and without pyruvate feeding. Analysis of metabolic fluxes in the differently fed cultures revealed a more efficient metabolic phenotype in the cultures without pyruvate feeding. The measured in vitro enzyme activities of the selected enzymes involved in pyruvate metabolism were lower in AGE1.HN compared with CHO cells, which might explain the higher sensitivity and different adaptation of AGE1.HN to increased pyruvate concentrations. The results indicate on the one hand that increasing the connectivity between glycolysis and the TCA cycle might improve substrate use and, finally, the production of A1AT. On the other hand, a better balanced substrate uptake promises a reduction of energy spilling which is increased with increasing substrate levels in this cell line. Overall, the results of this study provide important insights into the regulation of primary metabolism and into the adaptation of AGE1.HN to different substrate levels, providing guidance for further optimization of production cell lines and applied process conditions.     

Pregnancy rates, prenatal and postnatal survival of offspring, and litter sizes after reciprocal embryo transfer in DBA/2JHd, C3H/HeNCrl and NMRI mice

Success of embryo transfer is often a limiting factor in transgenic procedures and rederivation efforts, and depends on the genetic background of the donor and recipient strains used. Here we show that embryo transfer to DBA/2J females is possible, and present data on pre- and postnatal success rates after reciprocal embryo transfer using the inbred DBA/2J and C3H/HeN, and outbred NMRI strains. The highest embryo yield was achieved in outbred NMRI females, but embryo yields were similar in DBA/2J and C3H/HeN mice following superovulation despite poor estrus cycle synchronization in DBA/2J females. In-strain transfer of DBA/2J blastocysts (transfer of embryos to recipients from the same strain) resulted in pregnancy rates (57.1%) similar to those obtained following in-strain transfer of C3H/HeN (60.0%) and NMRI mice (83.3%), although the prenatal survival rate of blastocysts was low. Moreover, from the pups born only half survived the postnatal period after transfer of DBA/2J and C3H/HeN blastocysts to DBA/2J recipients. These problems were not observed when transferring NMRI-blastocysts to C3H/HeN and DBA/2J mothers. The number of blastocysts transferred also had a positive effect on the success of embryo transfer. In conclusion, C3H/HeN and DBA/2J females can be used as recipients for embryo transfer procedures for certain donor strains like NMRI, as one major determinant seems to be the genetic background of the embryos transferred. We also recommend to increase the number of DBA/2J blastocysts transferred, and to foster the DBA/2J pups to other DBA/2J mothers postnatally for in-strain transfer of DBA/2J mice.     

Distribution of viral abundance in the reef environment of Key Largo, Florida.

The distribution of viral and microbial abundance in the Key Largo, Fla., reef environment was measured. Viral abundance was measured by transmission electron microscope direct counts and plaque titer on specific bacterial hosts in water and sediment samples from Florida Bay (Blackwater Sound) and along a transect from Key Largo to the outer edge of the reef tract in Key Largo Sanctuary. Water column viral direct counts were highest in Blackwater Sound of Florida Bay (1.2 x 10(7) viruses per ml), decreased to the shelf break (1.7 x 10(6) viruses per ml), and were inversely correlated with salinity (r = -0.97). Viral direct counts in sediment samples ranged from 1.35 x 10(8) to 5.3 x 10(8)/cm(3) of sediment and averaged nearly 2 orders of magnitude greater than counts in the water column. Viral direct counts (both sediment and water column measurements) exceeded plaque titers on marine bacterial hosts (Vibrio natriegens and others) by 7 to 8 orders of magnitude. Water column viral abundance did not correlate with bacterial direct counts or chlorophyll a measurements, and sediment viral parameters did not correlate with water column microbial, viral, or salinity data. Coliphage, which are indicators of fecal pollution, were detected in two water column samples and most sediment samples, yet their concentrations were relatively low (<2 to 15/liter for water column samples, and <2 to 108/cm(3) of sediment). Our findings indicate that viruses are abundant in the Key Largo environment, particularly on the Florida Bay side of Key Largo, and that processes governing their distribution in the water column (i.e., salinity and freshwater input) are independent of those governing their distribution in the sediment environment.     

Greater Ethanol-Induced Locomotor Activation in DBA/2J versus C57BL/6J Mice Is Not Predicted by Presynaptic Striatal Dopamine Dynamics

A large body of research has aimed to determine the neurochemical factors driving differential sensitivity to ethanol between individuals in an attempt to find predictors of ethanol abuse vulnerability. Here we find that the locomotor activating effects of ethanol are markedly greater in DBA/2J compared to C57BL/6J mice, although it is unclear as to what neurochemical differences between strains mediate this behavior. Dopamine elevations in the nucleus accumbens and caudate-putamen regulate locomotor behavior for most drugs, including ethanol; thus, we aimed to determine if differences in these regions predict strain differences in ethanol-induced locomotor activity. Previous studies suggest that ethanol interacts with the dopamine transporter, potentially mediating its locomotor activating effects; however, we found that ethanol had no effects on dopamine uptake in either strain. Ex vivo voltammetry allows for the determination of ethanol effects on presynaptic dopamine terminals, independent of drug-induced changes in firing rates of afferent inputs from either dopamine neurons or other neurotransmitter systems. However, differences in striatal dopamine dynamics did not predict the locomotor-activating effects of ethanol, since the inhibitory effects of ethanol on dopamine release were similar between strains. There were differences in presynaptic dopamine function between strains, with faster dopamine clearance in the caudate-putamen of DBA/2J mice; however, it is unclear how this difference relates to locomotor behavior. Because of the role of the dopamine system in reinforcement and reward learning, differences in dopamine signaling between the strains could have implications for addiction-related behaviors that extend beyond ethanol effects in the striatum.     

Mouse-adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2–infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease.

Studying cross-protection from different coronaviruses is important to inform the research for a universal vaccine. This study uses a mouse-adapted SARS-CoV-2 strain to show that it confers protection from SARS-CoV challenge, suggesting possible immunity from heterologous challenge following natural infection.     

Salvaging Pyrococcus furiosus Protein Targets at SECSG

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.     

Genome-wide meta-analysis identifies regions on 7p21 (AHR) and 15q24 (CYP1A2) as determinants of habitual caffeine consumption

We report the first genome-wide association study of habitual caffeine intake. We included 47,341 individuals of European descent based on five population-based studies within the United States. In a meta-analysis adjusted for age, sex, smoking, and eigenvectors of population variation, two loci achieved genome-wide significance: 7p21 (P = 2.4 × 10(-19)), near AHR, and 15q24 (P = 5.2 × 10(-14)), between CYP1A1 and CYP1A2. Both the AHR and CYP1A2 genes are biologically plausible candidates as CYP1A2 metabolizes caffeine and AHR regulates CYP1A2.     

Maintenance of pluripotency in human embryonic stem cells cultured on a synthetic substrate in conditioned medium

Realizing the potential clinical and industrial applications of human embryonic stem cells (hESCs) is limited by the need for costly, labile, or undefined growth substrates. Here we demonstrate that trypsin passaging of the hESC lines, HUES7 and NOTT1, on oxygen plasma etched tissue culture polystyrene (PE‐TCPS) in conditioned medium is compatible with pluripotency. This synthetic culture surface is stable at room temperature for at least a year and is readily prepared by placing polystyrene substrates in a radio frequency oxygen plasma generator for 5 min. Modification of the polystyrene surface chemistry by plasma etching was confirmed by X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), which identified elemental and molecular changes as a result of the treatment. Pluripotency of hESCs cultured on PE‐TCPS was gauged by consistent proliferation during serial passage, expression of stem cell markers (OCT4, TRA1‐60, and SSEA‐4), stable karyotype and multi‐germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Generation of cost‐effective, easy‐to‐handle synthetic, defined, stable surfaces for hESC culture will expedite stem cell use in biomedical applications. Biotechnol. Bioeng. 2010;105: 130–140. © 2009 Wiley Periodicals, Inc.