Calorimetric investigation of copper(II) binding to Aβ peptides: thermodynamics of coordination plasticity

Metal ions have been shown to play a critical role in β-amyloid (Aβ) neurotoxicity, thus prompting an intense investigation into the formation of metal–Aβ complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal–protein interactions, including those in metal–Aβ complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu²⁺ binding to Aβ16, N-acetyl-Aβ16, Aβ28, N-acetyl-Aβ28, and Aβ28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aβ16 was found to be 1.1 (±0.13) × 10⁹ and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aβ28 variants lacking a potential Cu²⁺-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu²⁺ binding to Aβ and other intrinsically disordered peptides.     

Beneficial effects of a polar fraction of garlic (Allium sativum Linn) oil in rats fed with two different high fat diets

Feeding a diet containing 20% of sesame oil (SO) or coconut oil (CNO) along with 2% cholesterol to rats for two months showed differences in their serum and tissue lipid profile and certain enzyme activities. Hyperlipidemia and related oxidative effects were more pronounced in coconut oil fed rats than those fed sesame oil. Feeding a combination of the oils (10% CNO +10% SO) lowered significantly the hyperlipidemia and certain other deleterious effects of CNO. Feeding a polar fraction of garlic oil (PFGO) prepared in the same way as for ajoene and administered at a dosage of 100 mg/kg along with each of the above oil containing diets counteracted significantly the hyperlipidemic, oxidant and also most of the other deleterious effects of the oils like raised lipid levels in serum and tissues, raised serum levels of AST and tissue levels of HMGCoA reductase and the lowered serum and tissue levels of glutathione reductase. The results support the claims that ajoene, the major polar compound of garlic oil, has very good biological action, which warrants further study.     

Expression of a GALACTINOL SYNTHASE gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.     

Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine

Traditional 2D cell cultures do not accurately recapitulate tumor heterogeneity, and insufficient human cell lines are available. Patient-derived xenograft (PDX) models more closely mimic clinical tumor heterogeneity, but are not useful for high-throughput drug screening. Recently, patient-derived organoid cultures have emerged as a novel technique to fill this critical need. Organoids maintain tumor tissue heterogeneity and drug-resistance responses, and thus are useful for high-throughput drug screening. Among various biological tissues used to produce organoid cultures, circulating tumor cells (CTCs) are promising, due to relative ease of ascertainment. CTC-derived organoids could help to acquire relevant genetic and epigenetic information about tumors in real time, and screen and test promising drugs. This could reduce the need for tissue biopsies, which are painful and may be difficult depending on the tumor location. In this review, we have focused on advances in CTC isolation and organoid culture methods, and their potential applications in disease modeling and precision medicine.     

A high-throughput approach to developing and optimizing mixed-mode membrane chromatography for protein purification

Membrane chromatography has been established as a viable alternative to packed-bed column chromatography for the purification of therapeutic proteins. Purification via membrane chromatography offers key advantages, including higher productivity and reduced buffer usage. Unlike column chromatography purification, the utilization of high-throughput screening in order to reduce development times and material requirements has been a challenge for membrane chromatography. This research focused on the development of a new, high-throughput screening technique for use in screening membrane chromatography conditions for monoclonal antibody purification. The developed screen utilizes a 96-well plate format, thereby allowing for the screening of multiple different membrane conditions at once. For this study, four mixed-mode cation exchange membranes and one cation exchange membrane were evaluated on the plate. The screen is performed in a similar manner to that of a resin slurry plate screen, however, instead of a single loading step, the antibody feed was loaded in 50 mg/ml increments up to a maximum loading of 450 mg/ml. Performing a similar, incremental loading on a resin plate would be impractical, as mixing times are substantially longer due to pore diffusion limitations. However, due to the significantly faster rate of mass transfer for membranes relative to resin, mixing times could be reduced by up to a factor of sixty on the membrane plate. Additional optimization showed that higher hydrophobicity can potentially lead to slower kinetics and mixing times that may need to be adjusted accordingly. The end result is a screen that has been proven to provide results comparable to those obtained on larger-scale membrane purification runs while also enabling exploration of a much greater operating space and significantly reducing the feed materials required.     

Environmental impact and bioremediation of seleniferous soils and sediments

Selenium concentrations in the soil environment are directly linked to its transfer in the food chain, eventually causing either deficiency or toxicity associated with several physiological dysfunctions in animals and humans. Selenium bioavailability depends on its speciation in the soil environment, which is mainly influenced by the prevailing pH, redox potential, and organic matter content of the soil. The selenium cycle in the environment is primarily mediated through chemical and biological selenium transformations. Interactions of selenium with microorganisms and plants in the soil environment have been studied in order to understand the underlying interplay of selenium conversions and to develop environmental technologies for efficient bioremediation of seleniferous soils. In situ approaches such as phytoremediation, soil amendment with organic matter and biovolatilization are promising for remediation of seleniferous soils. Ex situ remediation of contaminated soils by soil washing with benign leaching agents is widely considered for removing heavy metal pollutants. However, it has not been applied until now for remediation of seleniferous soils. Washing of seleniferous soils with benign leaching agents and further treatment of Se-bearing leachates in bioreactors through microbial reduction will be advantageous as it is aimed at removal as well as recovery of selenium for potential re-use for agricultural and industrial applications. This review summarizes the impact of selenium deficiency and toxicity on ecosystems in selenium deficient and seleniferous regions across the globe, and recent research in the field of bioremediation of seleniferous soils.     

Synthesis of alkaline protease by catabolite repression- resistant Thermoactinomyces sp. E79 mutant

The production of alkaline protease from Thermoactinomyces sp. E79 was repressed by 0.2% (w/v) glucose in the medium. Catabolite repression-resistant mutant M1 was obtained by combined treatment of UV light and N-methyl- N-nitro-N-nitrosoguanidine. The glucose uptake studies accomplished by [14C]glucose showed that the mutant has lost its ability for glucose uptake. The protease production by mutant M1 in the enzyme production medium was 62 U/mg, which was twice that of the wild-type strain.