Neural Correlates of Sexual Cue Reactivity in Individuals with and without Compulsive Sexual Behaviours

Although compulsive sexual behaviour (CSB) has been conceptualized as a “behavioural” addiction and common or overlapping neural circuits may govern the processing of natural and drug rewards, little is known regarding the responses to sexually explicit materials in individuals with and without CSB. Here, the processing of cues of varying sexual content was assessed in individuals with and without CSB, focusing on neural regions identified in prior studies of drug-cue reactivity. 19 CSB subjects and 19 healthy volunteers were assessed using functional MRI comparing sexually explicit videos with non-sexual exciting videos. Ratings of sexual desire and liking were obtained. Relative to healthy volunteers, CSB subjects had greater desire but similar liking scores in response to the sexually explicit videos. Exposure to sexually explicit cues in CSB compared to non-CSB subjects was associated with activation of the dorsal anterior cingulate, ventral striatum and amygdala. Functional connectivity of the dorsal anterior cingulate-ventral striatum-amygdala network was associated with subjective sexual desire (but not liking) to a greater degree in CSB relative to non-CSB subjects. The dissociation between desire or wanting and liking is consistent with theories of incentive motivation underlying CSB as in drug addictions. Neural differences in the processing of sexual-cue reactivity were identified in CSB subjects in regions previously implicated in drug-cue reactivity studies. The greater engagement of corticostriatal limbic circuitry in CSB following exposure to sexual cues suggests neural mechanisms underlying CSB and potential biological targets for interventions.     

An investigation of enumeration and DNA partitioning in Bacillus subtilis L-form bacteria

R.N. WATERHOUSE, E.J. ALLAN, F. AMIJEE, V.J. UNDRILL AND L.A. GLOVER. 1994. Cell numbers of two morphogenic forms of Bacillus subtilis (the cell-walled parental and the derived stable cell wall-deficient L-form) have been compared by two methods: DNA hybridization (i.e. deduced genome numbers) and viable cell counts (i.e. number of colony-forming units (cfu)). The DNA hybridization method was shown to be a reliable and reproducible method for estimating genome numbers. Comparison of different L-form populations showed that the two methods of enumeration gave different values, with the deduced genome numbers much higher (by several orders of magnitude) than cell numbers deduced from viable cell counts. In contrast, when a culture of the cell-walled form was enumerated, the discrepancy between the two methods was low (by a factor of about 6) The combination of a high number of L-form genomes detected by DNA hybridization and a relatively low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA replication and cell division processes in L-form bacteria. This suggestion was further substantiated by assessing the stability of plasmid pPL608 in a transformed B. subtilis L-form cell line, where even in the presence of continued kanamycin selection, 25% of the population lost kanamycin resistance. The results are discussed with particular reference to cell division in cell wall-deficient, stable L-form bacteria.     

Stabilization of globular proteins via introduction of temperature-activated elastin-based switches

To investigate whether swapping native turns of a globular protein with an elastin-based turn sequence (VPGVG) can increase its thermostability, we have performed molecular dynamics simulations of wild-type chymotrypsin inhibitor 2 (CI2) and variants containing elastin-based turns at 10 degrees C and 40 degrees C. Wild-type CI2 is more stable at 10 degrees C, while both of the variant forms are more stable at 40 degrees C. Detailed analyses indicate that the elastin-based turns do indeed contribute to the inverse temperature behavior of the modified proteins. Therefore, swapping a wild-type turn sequence with an elastin-based turn provides a novel way to both improve stability of target proteins at body temperature and to possibly introduce a temperature-sensitive switch.     

TIM-4 expressed on APCs induces T cell expansion and survival

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c(+) and CD11b(+) cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.     

Measurements of mitochondrial K+ fluxes in whole rat hearts using 87Rb-NMR

The rubidium efflux from hypothermic rat hearts perfused by theLangendorff method at 20°C was studied. At thistemperature ⁸⁷Rb-NMR efflux experiments showed theexistence of two ⁸⁷Rb pools: cytoplasmic and mitochondrial.Rat heart mitochondria showed a very slow exchange of mitochondrialRb⁺ for cytoplasmic K⁺. After washout ofcytosolic Rb⁺, mitochondria kept a stable Rb⁺level for >30 min. Rb⁺ efflux from mitochondria wasstimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmalpermeabilization and concomitant cellular energy depletion by saponin(0.01 mg/ml for 4 min) in the presence of a perfusate mimickingintracellular conditions, or by ATP-sensitive K (K_ATP)channel openers. DNP, a mitochondrial uncoupler, caused the onset ofmitochondrial Rb⁺ exchange; however, the washout was notcomplete (80 vs. 56% in control). Energy deprivation by saponin, whichpermeabilizes the sarcolemma, resulted in a rapid and completeRb⁺ efflux. The mitochondrial Rb⁺ efflux rateconstant (k) decreased in the presence of glibenclamide, aK_ATP channel inhibitor (5 µM;k = 0.204 ± 0.065 min¹; n = 8),or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM,respectively; k = 0.134 ± 0.021 min¹;n = 4) in the saponin experiments (saponin only;k = 0.321 ± 0.079 min¹; n = 3),indicating the inhibition of mitochondrial K_ATP channels. Thus hypothermia in combination with ⁸⁷Rb-NMR allowed theprobing of the mitochondrial K⁺ pool in whole heartswithout mitochondrial isolation.

     

Reverse-Polynomial Dilution Calibration Methodology Extends Lower Limit of Quantification and Reduces Relative Residual Error in Targeted Peptide Measurements in Blood Plasma

Matrix effect is the alteration of an analyte''s concentration-signal response caused by co-existing ion components. With electrospray ionization (ESI), matrix effects are believed to be a function of the relative concentrations, ionization efficiency, and solvation energies of the analytes within the electrospray ionization droplet. For biological matrices such as plasma, the interactions between droplet components is immensely complex and the effect on analyte signal response not well elucidated. This study comprised of three sequential quantitative analyses: we investigated whether there is a generalizable correlation between the range of unique ions in a sample matrix (complexity); the amount of matrix components (concentration); and matrix effect, by comparing an E. coli digest matrix (∼2600 protein proteome) with phospholipid depleted human blood plasma, and unfractionated, nondepleted human plasma matrices (∼10⁷ proteome) for six human plasma peptide multiple reaction monitoring assays. Our data set demonstrated analyte-specific interactions with matrix complexity and concentration properties resulting in significant ion suppression for all peptides (p < 0.01), with nonuniform effects on the ion signals of the analytes and their stable-isotope analogs. These matrix effects were then assessed for translation into relative residual error and precision effects in a low concentration (∼0–250 ng/ml) range across no-matrix, complex matrix, and highly complex matrix, when a standard addition stable isotope dilution calibration method was used. Relative residual error (%) and precision (CV%) by stable isotope dilution were within <20%; however, error in phospholipid-depleted and nondepleted plasma matrices were significantly higher compared with no-matrix (p = 0.006). Finally a novel reverse-polynomial dilution calibration method with and without phospholipid-depletion was compared with stable isotope dilution for relative residual error and precision. Reverse-polynomial dilution techniques extend the Lower Limit of Quantification and reduce error (p = 0.005) in low-concentration plasma peptide assays and is broadly applicable for verification phase Tier 2 multiplexed multiple reaction monitoring assay development within the FDA-National Cancer Institute (NCI) biomarker development pipeline.Plasma is the overriding human medium sampled for established and novel protein biomarkers (1, 2). As of 2011, 1929 high-confidence proteins have been cataloged by the Human Plasma Proteome Project, with estimates that there are up to 10⁷ unique protein sequences in plasma that span a concentration range across 10 orders of magnitude (1, 3). 99% of the protein mass in plasma is made up of 22 proteins including Albumin, Fibrinogen, and a range of immunoglobulins, leaving more than 1900 known small proteins and essentially the entirety of the projected plasma proteome in the remaining 1% (4). It is these low-mass, low abundance proteins such as the Interleukins, C-Reactive Protein, and Carcinoma Antigen 125 (CA125), that are indicative of many important physiological and pathological processes, and proteomic scientists and clinicians have thus focused their efforts in qualitatively and quantitatively defining this fraction for novel biomarkers (4–6).The development of plasma biomarkers is a large-scale undertaking that spans discovery, verification, and validation phases in a multistage pipeline: Thousands of “discovered” differentiated proteins are evaluated for probability of effect, from which 10–100s of proteins are then selected for targeted quantification in verification phase to evaluate sensitivity and specificity for its intended indication (2, 7). Finally a panel of the strongest marker candidates is progressed to validation phase, and FDA-level validated quantitative assays are used to test the clinical utility of the biomarker panel. Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS)¹ is the most robust analytical method available for proteomic scientists in this pipeline, able to separate complex mixtures and specifically and sensitively identify and quantify its components (2, 7–10), The ability to ionize and evaporate the contents of a liquid sample (coupling LC to MS/MS) is the basis that allows this to happen (9). Electrospray Ionization (ESI) is the most widely used ionization apparatus in LC-MS/MS bioanalysis because of its ionization efficiency and stability and low chemical specificity (9, 10). Although these properties make ESI very robust, the complexity of biological matrices poses a significant challenge for LC-ESI-MS/MS-based quantitation; despite chromatography and nanospray technology, the ESI droplet of a plasma peptide-digest sample (given its immense range of unique protein/peptide sequences and concentrations) can contain an unknown multitude of co-eluting components that “compete” to dissolve from the droplet and reach gas phase, suppressing and varying the signal intensity responses for a given analyte concentration (9–13). These ionization competing elements can also go on to produce isobaric signals in the third quadrupole that interfere with an analyte''s transition signals (14). Termed “matrix effects,” these phenomena of complex sample matrices can significantly impede quantitative accuracy (15). For high-throughput clinical assays, matrix effects are controlled for by preparing calibration standards in the same biological matrix to mimic the conditions of the samples intended for study as per FDA bioanalytical method validation guidelines (16). The catch to this technique is that the signal from the endogenous analyte in the background matrix hinders accuracy when the nominal concentration is close to or below the endogenous signal (14, 17). There is a need for broadly applicable methods of controlling matrix effects and increasing accuracy in low concentration MRM peptide assays for nondepleted, unfractionated plasma that can be adopted for the highly multiplexed, high throughput, “Tier 2” MS assays required in verification phase of the biomarker development pipeline (2, 8). Several simple methods have independently demonstrated the ability to increase accuracy in various hyphenated-MS assays in complex matrices: “Reverse” curves utilize the stable-isotope analog not as an internal standard but as a surrogate calibration analyte to circumvent interference from the endogenous analyte signal and extend assay Lower Limit(s) of Quantification (LLOQ), and nonlinear calibration techniques have proven to more accurately reflect the concentration-MS detector response at the low and high end of concentration gradients (8, 14, 18–21). Specifically in the case of biological matrices, phospholipids are particularly deleterious ion suppressing elements because of their easily ionizable, polar, and hydrophobic moieties that can have complex interactions with co-eluting analytes as well as the chromatography stationary and mobile phases required for most other analytes (22–25). Combination solid-phase extraction (SPE) and phospholipid removal techniques have proved to effectively minimize ion suppression effects in ESI-MS assays (22–25).In this study, we investigated whether there is a generalizable linear correlation between the number of unique ions (complexity) in a biological sample matrix, the amount of ionizable matrix content (concentration), and matrix effects, for six human plasma peptides comparing serial dilutions of an Escherichia Coli (E. coli) peptide-digest against phospholipid-depleted and nondepleted unfractionated human plasma peptide-digest (highly complex) matrices. We examined the influence of matrix effects on relative residual error in a low-concentration (∼0–250 ng/ml) plasma peptide range, and compared the utility of a reverse-polynomial dilution (RPD) calibration method versus standard addition stable-isotope dilution (SID) in phospholipid-depleted and nondepleted unfractionated human plasma. A peptide-centric matrix effect is reported and the effect of the endogenous analyte signal on relative residual error in low-concentration (∼0–250 ng/ml) plasma peptide assays is established. A RPD calibration technique that extends LLOQ and reduces relative residual error in low-concentration plasma peptide MRM assays is presented.     

Increasing temperature accelerates protein unfolding without changing the pathway of unfolding

We have traditionally relied on extremely elevated temperatures (498K, 225 degrees C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298K to 498K. The simulation lengths vary from 94ns to 20ns, for a total simulation time of 344ns, or 0.34 micros. At 298K, the protein is very stable over the full 50ns simulation. At 348K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25ns, explores partially unfolded conformations for 20ns, and then refolds over the last 35ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.     

RAPID ASSEMBLY OF A WOUND PLUG: STAGE ONE OF A TWO‐STAGE WOUND REPAIR MECHANISM IN THE GIANT UNICELLULAR CHLOROPHYTE DASYCLADUS VERMICULARIS (CHLOROPHYCEAE)1

Upon injury, selected coenocytic algae are capable of forming temporary wound plugs to prevent detrimental cytoplasmic loss. Wound plugs of Dasycladus vermicularis ([Scropoli] Krasser) were harvested 5 min post‐injury and dried. The plug material contained 94% water and can be considered a hydrogel. The gel plug extended several millimeters from the cut end and filled the space inside the cell wall, which resulted from cytoplasmic retraction. Total organic carbon included 55% sugars, 5%–15% protein, and 0.18% lipids. The major sugars were glucose, galactose, mannose, and galacturonic acid. Fluorescein isothiocyanate‐lectins specific for these sugars were localized around the plug matrix. Sulfur content calculated as sulfate corresponded to 17% of the carbohydrate by weight, and sulfated material was detected in plugs by Alcian Blue staining. Formation of the initial plug occurred within 1 min of injury and was not significantly perturbed by the addition of ionic, antioxidant, or chelating agents to the seawater medium. However, addition of exogenous d (+)‐galactose and d (+)‐glucose prevented formation of the nascent gel plug. Wound plugs that were allowed to form from 10 min up until 24 h post‐injury were isolated and incubated with selected biochemical probes to identify the biochemical processes involved in plug formation. The operative strategy in Dasycladus to prevent “cytoplasmic hemorrhage” required availability of sequestered carbohydrate and lectin precursor components throughout the thallus for plug assembly. Once the initial assembly had commenced, additional biochemical interactions were initiated (as a function of time) to promote structural integrity.     

A prion protein epitope selective for the pathologically misfolded conformation

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.