Recognition of the gluconeogenic enzyme,Pck1, via the Gid4 E3 ligase: An in silico perspective

Gluconeogenesis, the reverse process of glycolysis, is a favorable mechanism at conditions of glucose deprivation. Pck1 is a rate‐limiting gluconeogenic enzyme, where its deficiency or mutation contributes to serious clinical situations as neonatal hypoglycemia and liver failure. A recent report confirms that Pck1 is a target for proteasomal degradation through its proline residue at the penultimate position, recognized by Gid4 E3 ligase, but with a lack of informative structural details. In this study, we delineate the localized sequence motif, degron, that specifically interact with Gid4 ligase and unravel the binding mode of Pck1 to the Gid4 ligase by using molecular docking and molecular dynamics. The peptide/protein docking HPEPDOCK web server along with molecular dynamic simulations are applied to demonstrate the binding mode and interactions of a Pck1 wild type (SPSK) and mutant (K4V) with the recently solved structure of Gid4 ligase. Results unveil a distinct binding mode of the mutated peptide compared with the wild type despite having comparable binding affinities to Gid4. Moreover, the four‐residue peptide is found insufficient for Gid4 binding, while the seven‐residue peptide suffices for binding to Gid4. The amino acids S134, K135, and N137 in the loop L1 (between β1 and β2) of the Gid4 are essential for the stabilization of the seven‐residue peptide in the binding site of the ligase. The presence of Val⁴ instead of Lys⁴ smashes the H‐bonds that are formed between Lys⁴ and Gid4 in the wild type peptide, making the peptide prone to bind with the other side of the binding pocket (L4 loop of Gid4). The dynamics of Gid4 L3 loop is affected dramatically once K4V mutant Pck1 peptide is introduced. This opens the door to explore the mutation effects on the binding mode and smooth the path to target protein degradation by design competitive and non‐competitive inhibitors.     

Organometallic estrogens: synthesis, interaction with lamb uterine estrogen receptor, and detection by infrared spectroscopy

As an integral part of the development of a new technique using organometallic markers for the detection of hormone receptors by FT-IR spectroscopy, a series of estradiol derivatives labeled with Cr(CO)3 or Cr(CO)2CS fragments on the A ring has been synthesized. The stereochemistry of one of these steroids, alpha-[3-(dimethyl-tert-butylsiloxy)-17 beta-estradiol]dicarbonyl(thiocarbonyl)chromium(0), has been established by X-ray diffraction. The organochromium-labeled steroids are stable in aqueous methanol solution, and their relative binding affinities to estrogen receptor have been determined; these values vary from 0.4 to 28%. The complex exhibiting the strongest affinity, [3-O-(3-hydroxypropyl)-17 beta-estradiol]-chromium tricarbonyl complex, has been prepared in a tritiated form with a high specific activity (4.1 Ci/mmol). This tritiated hormone binds reversibly to the estradiol receptor in lamb uterine cytosol with an affinity (Kd = 0.85 nM) and number of binding sites (n = 770 fmol/mg of protein) close to the values observed for estradiol itself. The level of nonspecific binding is low, and the hormone is not bound significantly to other nontarget tissues. The observation that the binding affinity of the steroid depends on which side of the steroidal A ring the organometallic label is bound demonstrates the nonequivalence of the two sides of the A ring with respect to the receptor site. The FT-IR spectra of the organochromium markers in the v(CO) region can be used for the detection of the estradiol receptor in lamb uterine cytosol.     

Proliferative capacity of epitope-specific CD8 T-cell responses is inversely related to viral load in chronic human immunodeficiency virus type 1 infection

The relationship between the function of human immunodeficiency virus (HIV)-specific CD8 T-cell responses and viral load has not been defined. In this study, we used a panel of major histocompatibility complex class I tetramers to examine responses to frequently targeted CD8 T-cell epitopes in a large cohort of antiretroviral-therapy-na?ve HIV type 1 clade C virus-infected persons in KwaZulu Natal, South Africa. In terms of effector functions of proliferation, cytokine production, and degranulation, only proliferation showed a significant correlation with viral load. This robust inverse relationship provides an important functional correlate of viral control relevant to both vaccine design and evaluation.     

Nicotine potentiates the neurogenic contractile response of rabbit bladder tissue via nicotinic acetylcholine receptors: nitric oxide and prostaglandins have no role in this process

Nicotine, a nicotinic acetylcholine receptors (nAChRs) agonist, has a role in modulation of the neurotransmitter release following nerve stimulation in both the central and peripheral nervous systems. The aim of this study was to determine whether electrical field stimulation (EFS)-evoked contractions are altered in rabbit bladder in the presence of nicotine and, if an alteration occurs, to investigate the effects of nitric oxide and prostaglandins on nicotine-induced alternation in isolated rabbit bladder. EFS-evoked contractile responses from rabbit bladder obtained were recorded with isometric force displacement transducers. Nicotine was added to preparations at various concentrations. The effects of hexamethonium, cadmium (Cd(2+)), indomethacin and N-nitro-L-arginine methyl ester (L-NAME) were tested on the EFS-evoked contractions in the presence of nicotine. Nicotine led to a dose-dependent increase in the amplitude of the EFS-evoked contractile responses. Cd(2+) and hexamethonium inhibited the nicotine-induced increase in EFS-evoked responses, whereas indomethacin and L-NAME had no effect. In conclusion, nicotine increased the EFS-evoked contractile responses possibly by facilitating release of neurotransmitters from nerve terminals by a mechanism dependent on the influx of Ca(2+) from voltage-gated Ca(2+) channels (VGCCs) via activation of nAChRs in isolated rabbit bladder. Nitric oxide and prostaglandins do not have a physiological role in the regulation of neurotransmitter release.     

Linear solvation energy relationship modeling and kinetic studies on reactive extraction of succinic acid by tridodecylamine dissolved in MIBK

Equilibrium and kinetic studies for the extraction of succinic acid from aqueous solution with tridodecylamine diluted in MIBK are reported. All measurements were carried out at 298.15 K. The extent to which the organic phase may be loaded with succinic acid is expressed as a loading ratio, Z. The equilibrium data were also interpreted by a proposed mechanism of three reactions of complexation by which (1:1) and (2:1) acid-amine complexes are formed. Kinetics of extraction of succinic acid by tridodecylamine in MIBK has also been determined. Kinetic studies for the extraction of succinic acid from aqueous solution with tridodecylamine diluted in MIBK were carried out using a stirred cell for kinetic studies. The results of the liquid-liquid equilibrium measurements were correlated by a linear solvation energy relationship (LSER) model, which takes into account physical interactions. From the regression coefficients, information on the solvent-solute interaction is obtained and solvation models are proposed.     

In vitro and in silico assessment of the developability of a designed monoclonal antibody library

Despite major advances in antibody discovery technologies, the successful development of monoclonal antibodies (mAbs) into effective therapeutic and diagnostic agents can often be impeded by developability liabilities, such as poor expression, low solubility, high viscosity and aggregation. Therefore, strategies to predict at the early phases of antibody development the risk of late-stage failure of antibody candidates are highly valuable. In this work, we employ the in silico solubility predictor CamSol to design a library of 17 variants of a humanized mAb predicted to span a broad range of solubility values, and we examine their developability potential with a battery of commonly used in vitro and in silico assays. Our results demonstrate the ability of CamSol to rationally enhance mAb developability, and provide a quantitative comparison of in vitro developability measurements with each other and with more resource-intensive solubility measurements, as well as with in silico predictors that offer a potentially faster and cheaper alternative. We observed a strong correlation between predicted and experimentally determined solubility values, as well as with measurements obtained using a panel of in vitro developability assays that probe non-specific interactions. These results indicate that computational methods have the potential to reduce or eliminate the need of carrying out laborious in vitro quality controls for large numbers of lead candidates. Overall, our study provides support to the emerging view that the implementation of in silico tools in antibody discovery campaigns can ensure rapid and early selection of antibodies with optimal developability potential.     

Green leaf volatiles: biosynthesis,biological functions and their applications in biotechnology

Plants have evolved numerous constitutive and inducible defence mechanisms to cope with biotic and abiotic stresses. These stresses induce the expression of various genes to activate defence‐related pathways that result in the release of defence chemicals. One of these defence mechanisms is the oxylipin pathway, which produces jasmonates, divinylethers and green leaf volatiles (GLVs) through the peroxidation of polyunsaturated fatty acids (PUFAs). GLVs have recently emerged as key players in plant defence, plant–plant interactions and plant–insect interactions. Some GLVs inhibit the growth and propagation of plant pathogens, including bacteria, viruses and fungi. In certain cases, GLVs released from plants under herbivore attack can serve as aerial messengers to neighbouring plants and to attract parasitic or parasitoid enemies of the herbivores. The plants that perceive these volatile signals are primed and can then adapt in preparation for the upcoming challenges. Due to their ‘green note’ odour, GLVs impart aromas and flavours to many natural foods, such as vegetables and fruits, and therefore, they can be exploited in industrial biotechnology. The aim of this study was to review the progress and recent developments in research on the oxylipin pathway, with a specific focus on the biosynthesis and biological functions of GLVs and their applications in industrial biotechnology.     

From global proteome profiling to single targeted molecules of follicular fluid and oocyte: contribution to embryo development and IVF outcome

The development of in vitro fertilization (IVF) techniques for infertility management has led to the investigation of the proteome of follicular fluid and oocyte. In addition, different markers contributing to oocyte maturation and embryo development potential have been reported in the literature. Different techniques were utilized to analyze whole proteome or single protein markers in follicular fluid and oocytes, particularly in animal models. Data from several studies have generated large amounts of information, however, an ideal profile to predict the best oocytes and embryos suitable for implantation are still to be uncovered. The identification of such profiles and markers from follicular fluid, oocytes and endometrium should help scientists and clinicians develop better strategies to improving clinical outcome of IVF cycles.