Recombinant human natural autoantibodies against GPIIb/IIIa inhibit binding of autoantibodies from patients with AITP

Autoimmune thrombocytopenic purpura (AITP) is caused by autoantibodies predominantly against platelet membrane glycoproteins (GP) IIb/IIIa and GPIb/IX. Naturally occurring autoantibodies have been described against a variety of autoantigens; it has been suggested that perturbation of their regulation may be associated with autoimmune diseases. Using a combinatorial Fab phagemid library from an individual immunized with human RhD+ red blood cells, we evaluated the presence of natural anti-GPIIb/IIIa autoantibodies as well as their relation to AITP-associated anti-GPIIb/IIIa autoantibodies. Selection on native GPIIb/IIIa and characterization of positive clones by inhibition studies against murine monoclonal anti-GPIIb/IIIa antibodies and by DNA analysis revealed the presence of two distinct recombinant anti-GPIIb/IIIa autoantibodies, which partially inhibited binding of affinity-purified platelet-associated autoantibodies from 8/12 AITP patients. Our results demonstrated that GPIIb/IIIa-specific Fab directed against conformational epitopes within the GPIIb/IIIa complex may be cloned from the genome of an individual immunized with RhD+ red blood cells, who was not affected by AITP. The partial inhibition of binding of platelet-associated autoantibodies from AITP patients to GPIIb/IIIa by the recombinant anti-GPIIb/IIIa phage clones suggests recognition of closely related antigenic epitopes. These phage clones may represent down-regulated, potentially pathological autoantibodies and could be used as new tools for investigation of AITP.     

Andrographolide Derivatives Target the KEAP1/NRF2 Axis and Possess Potent Anti-SARS-CoV-2 Activity

Naturally occurring compounds represent a vast pool of pharmacologically active entities. One of such compounds is andrographolide, which is endowed with many beneficial properties, including the activity against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). To initiate a drug repurposing or hit optimization campaign, it is imperative to unravel the primary mechanism(s) of the antiviral action of andrographolide. Here, we showed by means of a reporter gene assay that andrographolide exerts its anti-SARS-CoV-2 effects by inhibiting the interaction between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2-related factor 2 (NRF2) causing NRF2 upregulation. Moreover, we demonstrated that subtle structural modifications of andrographolide could lead to derivatives with stronger on-target activities and improved physicochemical properties. Our results indicate that further optimization of this structural class is warranted to develop novel COVID-19 therapies.     

Identification of transmembrane domain residues determinant in the structure-function relationship of the human platelet-activating factor receptor by site-directed mutagenesis

Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.     

Novel cyclic analogs of angiotensin II with cyclization between positions 5 and 7: conformational and biological implications

To study the conformational features of molecular recognition of angiotensin II (Asp1-Arg2-Val3-Tyr4-Val/IIe5-His6-Pro7-Phe8, AII), the synthesis and biological testing of several cyclic analogs of AII cyclized between positions 5 and 7 have been performed. The synthesized analogs were Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Pen7)-Phe8 (3), Sar1-Arg2-Val3-Tyr4-cyclo(Asp5-His6-Apt7)-Phe8 (4), Sar1-Arg2-Val3-Tyr4-cyclo(Glu5-His6-Apt7)-Phe8 (5), Sar1-Arg2-Val3-Tyr4-cyclo-(Cys5-His6-Mpt7)-Phe8 (6), Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Mpc7)-Phe8 (7), Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpt7)-Phe8 (8), and Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpc7)-Phe8 (9), where Apt stands for 4-amino-trans-proline, and Mpt and Mpc for 4-mercapto-trans- and -cis-prolines, respectively. Compound (9) showed good affinity at AT-1 receptors, namely a KD = 20 nM. In functional assays, it showed the characteristics of a weak partial agonist with a relative affinity of 0.26% of that for AII and an intrinsic efficacy, alpha E, of 0.42. Molecular modeling suggested a possible explanation for this finding: the relatively strong binding and the weak partial agonistic activity of compound 9 are due to interaction with AT-1 receptor of only two functionally important groups, namely, the side chains of the His6 and Phe8 residues.     

Regularity of rotational travelling water waves

Several recent results on the regularity of streamlines beneath a rotational travelling wave, along with the wave profile itself, will be discussed. The survey includes the classical water wave problem in both finite and infinite depth, capillary waves and solitary waves as well. A common assumption in all models to be discussed is the absence of stagnation points.     

Microstructural Evolution and Mechanical Behaviour of Metastable Cu-Zr-Co Alloys

Three different Cu-Zr-Co alloys, namely Cu40Zr37.5Co22.5, Cu42.5Zr45Co12.5 and Cu49Zr49Co2, were obtained by rapid cooling. The microstructure and phase formation of as-cast rods with diameters of 2 mm are compared with those of the respective ingots. An increasing Co content stabilises the B2 CuZr phase and leads to the precipitation of a ternary Cu-Zr-Co phase. The variation of the cooling rate affects the size of the B2 dendrites as well as the volume fraction and the morphology of the interdendritic phases. The mechanical properties were determined in compression and all alloys show a certain plastic deformability despite the presence of several binary and ternary intermetallic phases. The deformation mechanisms are discussed on the basis of the microstructures and the constituent phases.     

Dynamic permeation method to determine partition coefficients of highly hydrophobic chemicals between poly(dimethylsiloxane) and water

Measurement of partition coefficients between poly(dimethylsiloxane) (PDMS) and water (KPDMSw) becomes more and more difficult as the hydrophobicity of the compound increases. Experimental challenges include long extraction times, sorption to various surfaces and materials, and incomplete dissolution of the compound in the aqueous phase. In order to avoid these artifacts and to shorten experimental time, a dynamic permeation method was developed. According to steady-state diffusion theory, KPDMSw is inversely proportional to the permeation rate through the aqueous boundary layer (ABL) from the donor PDMS to the acceptor PDMS. A simple ABL permeation reactor can thus be applied to determine KPDMSw values of hydrophobic chemicals within a few days. The obtained values were in good agreement with those obtained using a conventional shaking method and the partition controlled delivery system. A good linear correlation was obtained between the logarithm of the 1-octanol/water partition coefficient (log Kow) from the literature and log KPDMSw over 6 orders of magnitude.     

The case for solar/hydrogen energy

Since sustainable, technologically-converted solar energy is the likely basis for our post-fossil-energy future, there is a basic need for solar-produced fuels. It is noteworthy that heat and electricity, solely, are being developed as solar-energy delivery means, while historically civilizations depend on fuels. Hydrogen, a clean, efficiently-used fuel, can be readily derived from water using any of a number of both proved and prospective solar-energy conversion technologies—both direct and indirect (hydropower, wind, etc.). Solar/hydrogen (and oxygen) can also extend depleting fossil-energy resources while ameliorating environmental degradation. The Hydrogen Energy System concept is overviewed as background.A recent ‘Solar/Hydrogen Systems Assessment’ delineated early-availability systems based on photovoltaic, thermal/heat-engine, wind and hydropower solar conversion, and associated water electrolysis to yield product hydrogen and oxygen as ‘hydrogen energy’. Involved technologies being highly modular, good economics of equipment manufacture and deployment are inherent, as is early availability and as-needed rates of construction (in contrast, e.g., with nuclear-plant experience). Proved technological means exist for transporting, storing and distributing hydrogen energy to end-users.Most significant, both small-scale (local, dispersed) and large-scale (central, remote) solar/hydrogen generation facilities can be established in balance with prevailing societal-selection dictates. Involving a readily storable, transportable ‘energy currency’, then-existing hydrogen-energy systems can be inter-tied as desired, providing load-management-related economic advantages to both the energy-user and the ‘energy utility’ of that era. Future solar/hydrogen-electric residences might, as is illustrated, buy and sell hydrogen and electricity in a ‘grid-cooperative’ arrangement.The salient operative question concerns the efficacy of ‘conventional wisdom’ in the energy free-market decision-making process. Will early-enough, adequate level-of-effort programmes be implemented to ensure non-disruptive meeting of tomorrow's demand worldwide? In an aura of business-as-usual, solar/hydrogen's timely contribution to ‘picking up the load’ from exhaustible fossil fuels in the face of still-escalating world energy demand is judged most problematic. Consequently, an unprecedented cooperative world effort for the research, development, demonstration and deployment of solar hydrogen energy delivery capabilities is suggested.     

Acrylamide in almond products

Acrylamide was determined in 86 different almond products, such as roasted almonds, almond-containing bakery products, raw almonds, and marzipan. The highest acrylamide concentrations were found in dark roasted almonds, while only moderate acrylamide contents were determined in bakery products. Roasting experiments under different process conditions showed that acrylamide increases with time and that temperature has a much stronger effect on acrylamide formation than time. During roasting reducing sugars are consumed faster and to a larger extent than free asparagine, suggesting that the content of reducing sugars may be a critical factor for acrylamide formation in roasted almonds. Acrylamide was found to decrease in roasted almonds during storage at room temperature.