The mineralocorticoid hormone aldosterone regulates sodium and potassium homeostasis but also adversely modulates the maladaptive process of cardiac adverse remodeling post-myocardial infarction. Through activation of its mineralocorticoid receptor (MR), a classic steroid hormone receptor/transcription factor, aldosterone promotes inflammation and fibrosis of the heart, the vasculature, and the kidneys. This is why MR antagonists reduce morbidity and mortality of heart disease patients and are part of the mainstay pharmacotherapy of advanced human heart failure. A plethora of animal studies using cell type–specific targeting of the MR gene have established the importance of MR signaling and function in cardiac myocytes, vascular endothelial and smooth muscle cells, renal cells, and macrophages. In terms of its signaling properties, the MR is distinct from nuclear receptors in that it has, in reality, two physiological hormonal agonists: not only aldosterone but also cortisol. In fact, in several tissues, including in the myocardium, cortisol is the primary hormone activating the MR. There is a considerable amount of evidence indicating that the effects of the MR in each tissue expressing it depend on tissue- and ligand-specific engagement of molecular co-regulators that either activate or suppress its transcriptional activity. Identification of these co-regulators for every ligand that interacts with the MR in the heart (and in other tissues) is of utmost importance therapeutically, since it can not only help elucidate fully the pathophysiological ramifications of the cardiac MR`s actions but also help design and develop novel better MR antagonist drugs for heart disease therapy. Among the various proteins the MR interacts with are molecules involved in cardiac G protein-coupled receptor (GPCR) signaling. This results in a significant amount of crosstalk between GPCRs and the MR, which can affect the latter`s activity dramatically in the heart and in other cardiovascular tissues. This review summarizes the current experimental evidence for this GPCR-MR crosstalk in the heart and discusses its pathophysiological implications for cardiac adverse remodeling as well as for heart disease therapy. Novel findings revealing non-conventional roles of GPCR signaling molecules, specifically of GPCR-kinase (GRK)-5, in cardiac MR regulation are also highlighted.

We propose that quarks and gluon flux tubes emerge from networks of standing vacuum waves. Each unbound nucleon, in its ground state, may be electromagnetically modeled as massless quantized charge on two pairs of orbiting arcs. Each charge arc is associated with a superposition of radially-oriented vacuum fundamental harmonics. These quantum superpositions of radial waves are coupled to nucleon mass-energy. The charge arcs orbit on the two surfaces of a spindle torus with polar charge-exclusion zones. These ground-state models of unbound nucleons may be interpreted as pairs of virtual Möbius bands. The optimal triangular Möbius band may explain proton uniqueness. These unbound proton and neutron models are shown to be precisely connected via a parameter dependent on neutron mass and the sum of the up and down bare quark masses. Due to this precise connection, and the relatively high experimental precision of proton magnetic moment, neutron magnetic moment is calculated about two orders of magnitude more precisely than the most accurate experiments to date. This quantum network-based approach to modeling unbound low-energy nucleons calculates several other measurable parameters.

BackgroundThe use of antimicrobials to treat food animals may result in antimicrobial residues in foodstuffs of animal origin. The European Medicines Association (EMA) and World Health Organization (WHO) define safe antimicrobial concentrations in food based on acceptable daily intakes (ADIs). It is unknown if ADI doses of antimicrobials in food could influence the antimicrobial susceptibility of human-associated bacteria. ObjectivesThis aim of this study was to evaluate if the consumption of ADI doses of erythromycin could select for erythromycin resistance in a Galleria mellonella model of Streptococcus pneumoniae infection. Methods A chronic model of S. pneumoniae infection in G. mellonella larvae was used for the experiment. Inoculation of larvae with S. pneumoniae was followed by injections of EMA (0.0875 μg/ml) and WHO (0.012 μg/ml) ADI doses of erythromycin proceeded by isolation of S. pneumoniae colonies on selective agar plates. Minimum inhibitory concentrations (MICs) of resistant colonies were measured, and whole genome sequencing (WGS) was performed followed by variant calling to determine the genetic modifications.   Results Exposure to single doses of both EMA and WHO ADI doses of erythromycin resulted in the emergence of erythromycin resistance in S. pneumoniae. Emergent resistance to erythromycin was associated with a mutation in rplA. Conclusion In our in vivo model, even single dose of erythromycin that are classified as acceptable by the WHO and EMA induced significant increases in erythromycin MICs in S. pneumoniae. These results suggest the need to include the induction of antimicrobial resistance (AMR) as a significant criterion for determining ADIs.

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.

Using assembly theory, we investigate the assembly pathways of fixed-length binary strings formed by joining the individual bits present in the assembly pool and the strings that entered the pool as a result of previous joining operations. We show that the string assembly index is bounded from below and above, and we conjecture about the form of the upper bound. We show that a string having the smallest assembly index can be assembled by an elegant trivial program of length equal to this index, if the length of the string is expressible as a product of Fibonacci numbers. We conjecture that there is no binary program that has a length shorter than the length of the string having the largest assembly index that could assemble this string. We conjecture that a black hole surface is defined by a balanced distinct string that satisfies the upper bound of a distinct string assembly index. The results confirm that four Planck areas provide a minimum information capacity that provides a minimum thermodynamic (Bekenstein-Hawking) entropy. Knowing that the problem of determining the assembly index is at least NP-complete, we conjecture that the problem of determining the assembly index of a given binary string is NP-complete, while the problem of creating the string so that it would have a predetermined maximum assembly index is NP-hard. Therefore, once the new information is assembled by a dissipative structure or by a human, increasing the information entropy according to the second law of infodynamics, it is subject to the second law of thermodynamics, and nature seeks to optimize its assembly pathway.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well-known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum. Cytoplasm obtained from different E. coli strains was examined as an example of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

We work within a Winterberg framework where space, i.e., the vacuum, consists of a two component superfluid/super-solid made up of a vast assembly (sea) of positive and negative mass Planck particles, called planckions. These material particles interact indirectly, and have very strong restoring forces keeping them a finite distance apart from each other within their respective species. Because of their mass compensating effect, the vacuum appears massless, charge-less, without pressure, net energy density or entropy. In addition, we consider two variable models, where, , is Newton’s constant, and, , increases with an increase in cosmological time. We argue that there are at least two competing models for the quantum vacuum within such a framework. The first follows a strict extension of Winterberg’s model. This leads to nonsensible results, if increases, going back in cosmological time, as the length scale inherent in such a model will not scale properly. The second model introduces a different length scale, which does scale properly, but keeps the mass of the Planck particle as, the Planck mass. Moreover we establish a connection between ordinary matter, dark matter, and dark energy, where all three mass densities within the Friedman equation must be interpreted as residual vacuum energies, which only surface, once aggregate matter has formed, at relatively low temperatures. The symmetry of the vacuum will be shown to be broken, because of the different scaling laws, beginning with the formation of elementary particles. Much like waves on an ocean where positive and negative planckion mass densities effectively cancel each other out and form a zero vacuum energy density/ zero vacuum pressure surface, these positive mass densities are very small perturbations (anomalies) about the mean. This greatly alleviates, i.e., minimizes the cosmological constant problem, a long standing problem associated with the vacuum.