Structure‐based design of combinatorial mutagenesis libraries

The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high‐throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general‐purpose method, called “Structure‐based Optimization of Combinatorial Mutagenesis ” (SOCoM ), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library‐averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β‐lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure‐based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large‐scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure‐based assessments, such as the energy gap between alternative conformational or bound states.     

Selective facilitation of LTP in the ventral hippocampus by calcium stores

The effects of low concentrations of caffeine and ryanodine on field excitatory postsynaptic potentials (EPSPs) and long-term potentiation (LTP) were studied in CA1 region of slices of dorsal and ventral hippocampus (DH and VH, respectively). There was a striking difference between the two regions in the magnitude of effect of both drugs, as well as the ability to interact with a tetanic stimulation to produce LTP. Low concentration of caffeine (1 mM) produced a postsynaptic increase in the slope of population EPSPs in VH, and facilitated LTP in this region. Low concentration of ryanodine (0.2 μM) was able to convert short-term potentiation (STP) to LTP in VH only. These effects are postsynaptic and they reflect a higher concentration of ryanodine receptors (RyRs) in the VH compared to the DH.     

alpha,beta-Dehydrophenylalanine choline esters, a new class of reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase

Our goal was to design, synthesize, and evaluate new cholinesterase inhibitors. Fourteen dehydroamino acids esterified to choline and to its ternary analog were synthesized by a new method that gave a yield of 84-93%. The potency of the amino acid ester derivatives was tested by measuring K(i) values for inhibition of human red cell acetylcholinesterase and human plasma butyrylcholinesterase. The most potent compound was a choline ester of dehydrophenylalanine where the amine group of the amino acid was derivatized with a benzoyl group containing a methoxy in the 2-position, CH(3)O(C(6)H(4))CONHC(CHC(6)H(5))COOCH(2)CH(2)N(+)(CH(3))(3). This compound was a strong inhibitor of both human acetylcholinesterase and human butyrylcholinesterase, with K(i) values of 10 microM and 0.08 microM, respectively. These K(i) values are comparable to that of Rivastigmine. Docking of the most potent compound into the active site of human butyrylcholinesterase showed that the lowest energy model had two benzene rings oriented towards Trp 82 and Tyr 332 whereas the positively charged nitrogen group was stabilized by Trp 231. This orientation placed the ester group 3.89 A from the active site Ser 198, a distance too far for covalent bonding, explaining why the esters are inhibitors rather than substrates. This class of anticholinesterase agents has the potential for therapeutic utility in the treatment of disorders of the cholinergic system.     

Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis

One prime feature of alcoholic liver disease (ALD) is iron accumulation in hepatic macrophages/Kupffer cells (KC) associated with enhanced NF-kappaB activation. Our recent work demonstrates a peroxynitrite-mediated transient rise in intracellular labile iron (ILI) as novel signaling for endotoxin-induced IKK and NF-kappaB activation in rodent KC. The present study investigated the mechanism of KC iron accumulation and its effects on ILI response in experimental ALD. We also tested ILI response in human blood monocytes. Chronic alcohol feeding in rats results in increased expression of transferrin (Tf) receptor-1 and hemochromatosis gene (HFE), enhanced iron uptake, an increase in nonheme iron content, and accentuated ILI response for NF-kappaB activation in KC. Ex vivo treatment of these KC with an iron chelator abrogates the increment of iron content, ILI response, and NF-kappaB activation. The ILI response is evident in macrophages derived from human blood monocytes by PMA treatment but not in vehicle-treated monocytes, and this differentiation-associated phenomenon is essential for maximal TNF-alpha release. PMA-induced macrophages load iron dextran and enhance ILI response and TNF-alpha release. These effects are reproduced in KC selectively loaded in vivo with iron dextran in mice and more importantly aggravate experimental ALD. Our results suggest enhanced iron uptake as a mechanism of KC iron loading in ALD and demonstrate the ILI response as a function acquired by differentiated macrophages in humans and as a priming mechanism for ALD.     

Regulatable gutless adenovirus vectors sustain inducible transgene expression in the brain in the presence of an immune response against adenoviruses

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding beta-galactosidase (beta-gal) driven by a TetOn system containing the rtTAS(s)M2 transactivator and the tTS(Kid) repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-beta-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-beta-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-beta-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-beta-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.     

Biphasic effects of atropine on sensory-evoked hippocampal rhythmical slow activity in urethane-anaesthetized rats

This study investigated the response of hippocampal RSA, recorded from electrodes in CA1 and the contralateral dentate gyrus of urethane-anaesthetized rats, to atropine sulphate administered at 15 min intervals in a cumulative dose-response schedule (1, 3, 10, 50 and 50 mg x kg(-1) i.p.). The power of CA1 and dentate gyrus RSA in the 3-7 Hz band was increased after administering the first 3 doses of atropine (1, 3 and 10 mg x kg(-1) cumulatively) in rats held in the stereotaxic frame or removed from the frame and given electrical sensory stimulation to the base of the tail. This increase in RSA was dependent on sensory input, since it was not seen in animals outside the frame unless sensory stimulation was given, and it was abolished by increasing the dose of atropine (an additional 50 and 50 mg x kg(-1) cumulatively). Methylatropine (6 mg x kg(-1) i.p.) did not increase RSA power. The biphasic effect of atropine on sensory-evoked hippocampal RSA activity may be explained by differential effects at pre- and post-synaptic sites e.g. in the septo-hippocampal system or on pathways processing sensory information.     

Theta driving both inhibits and potentiates the effects of nicotine on dentate gyrus responses

The medial septal area of conscious rats was stimulated through previously implanted electrodes at a frequency of 7.7 Hz for 20 min each day for 7 days to evoke rhythmic slow activity in CA1 at a similar frequency to spontaneous theta. Two weeks later in the anaesthetized rats the effects of a single subcutaneous injection of nicotine (0.4 mg x kg(-1)) on fEPSPs, evoked in the dentate gyrus by separate stimulation of the MPP and LPP, were studied and compared with those obtained in controls. Nicotine increased the firing of locus coeruleus neurones and the slope of the fEPSPs evoked by LPP stimulation, but not by MPP stimulation. Prior theta driving considerably increased the effect of nicotine on the responses evoked by stimulation of the MPP and abolished the nicotine-induced potentiation of the responses evoked by stimulation of the LPP. The results are attributed to theta driving increasing the amount of noradrenaline released by nicotine and to noradrenaline producing a beta-adrenoceptor long-lasting potentiation at the medial perforant path synapse and a long-lasting depression at the lateral perforant path synapse.     

An untargeted metabolomics method for archived newborn dried blood spots in epidemiologic studies

Introduction

For pediatric diseases like childhood leukemia, a short latency period points to in-utero exposures as potentially important risk factors. Untargeted metabolomics of small molecules in archived newborn dried blood spots (DBS) offers an avenue for discovering early-life exposures that contribute to disease risks.

Objectives

The purpose of this study was to develop a quantitative method for untargeted analysis of archived newborn DBS for use in an epidemiological study (California Childhood Leukemia Study, CCLS).

Methods

Using experimental DBS from the blood of an adult volunteer, we optimized extraction of small molecules and integrated measurement of potassium as a proxy for blood hematocrit. We then applied this extraction method to 4.7-mm punches from 106 control DBS samples from the CCLS. Sample extracts were analyzed with liquid chromatography—high resolution mass spectrometry (LC-HRMS) and an untargeted workflow was used to screen for metabolites that discriminate population characteristics such as sex, ethnicity, and birth weight.

Results

Thousands of small molecules were measured in extracts of archived DBS. Normalizing for potassium levels removed variability related to varying hematocrit across DBS punches. Of the roughly 1000 prevalent small molecules that were tested, multivariate linear regression detected significant associations with ethnicity (three metabolites) and birth weight (15 metabolites) after adjusting for multiple testing.

Conclusions

This untargeted workflow can be used for analysis of small molecules in archived DBS to discover novel biomarkers, to provide insights into the initiation and progression of diseases, and to provide guidance for disease prevention.

     

Retina of Vertebrates: An Internal Cell Reserve for Regeneration

The recent data were summarized concerning the presence in the retina of fish, amphibians and birds of additional sources of growth and regeneration, alternative to the already known sources, such as growth zone of eye, pigment epithelium, and cells–precursors of rods, and which are localized in the inner nuclear layer of retina. These sources are represented by as yet not finally identified oval small cells and cells of Müller glia. Both types of cells are capable of proliferating and producing precursors for various differentiated cells, including photoreceptors or their additional precursors. The current immunochemistry data are provided, which were obtained using markers of proliferation, proneural phenotype, and specific cell differentiation in the growing retina and in the retina after various damages. The regulatory mechanisms and methods of the stimulation of proliferation of the cells, which are sources of increase in the number and restoration of photoreceptors, interneurons, and glial cells of vertebrate retina, are discussed.