Mass Spectrometry-Based Disulfide Mapping of Lysyl Oxidase-like 2

Lysyl oxidase-like 2 (LOXL2) catalyzes the oxidative deamination of peptidyl lysines and hydroxylysines to promote extracellular matrix remodeling. Aberrant activity of LOXL2 has been associated with organ fibrosis and tumor metastasis. The lysine tyrosylquinone (LTQ) cofactor is derived from Lys653 and Tyr689 in the amine oxidase domain via post-translational modification. Based on the similarity in hydrodynamic radius and radius of gyration, we recently proposed that the overall structures of the mature LOXL2 (containing LTQ) and the precursor LOXL2 (no LTQ) are very similar. In this study, we conducted a mass spectrometry-based disulfide mapping analysis of recombinant LOXL2 in three forms: a full-length LOXL2 (fl-LOXL2) containing a nearly stoichiometric amount of LTQ, Δ1-2SRCR-LOXL2 (SRCR1 and SRCR2 are truncated) in the precursor form, and Δ1-3SRCR-LOXL2 (SRCR1, SRCR2, SRCR3 are truncated) in a mixture of the precursor and the mature forms. We detected a set of five disulfide bonds that is conserved in both the precursor and the mature recombinant LOXL2s. In addition, we detected a set of four alternative disulfide bonds in low abundance that is not associated with the mature LOXL2. These results suggest that the major set of five disulfide bonds is retained post-LTQ formation.     

Drugs,Guts, Brains,but Not Rock and Roll: The Need to Consider the Role of Gut Microbiota in Contemporary Mental Health and Wellness of Emerging Adults

Emerging adulthood (ages 18–25) is a critical period for neurobiological development and the maturation of the hypothalamic–pituitary–adrenal axis. Recent findings also suggest that a natural perturbation of the gut microbiota (GM), combined with other factors, may create a unique vulnerability during this period of life. The GM of emerging adults is thought to be simpler, less diverse, and more unstable than either younger or older people. We postulate that this plasticity in the GM suggests a role in the rising mental health issues seen in westernized societies today via the gut–brain–microbiota axis. Studies have paid particular attention to the diversity of the microbiota, the specific function and abundance of bacteria, and the production of metabolites. In this narrative review, we focus specifically on diet, physical activity/exercise, substance use, and sleep in the context of the emerging adult. We propose that this is a crucial period for establishing a stable and more resilient microbiome for optimal health into adulthood. Recommendations will be made about future research into possible behavioral adjustments that may be beneficial to endorse during this critical period to reduce the probability of a “dysbiotic” GM and the emergence and severity of mental health concerns.     

Encapsulation of Nanoparticles Using Nitrilotriacetic Acid End‐Functionalized Polystyrenes and Their Application for the Separation of Proteins

The use of nitrilotriacetic acid end‐functionalized polystyrenes (NTA‐PS) as a multifunctional nanocarrier for the aqueous dispersion of CdSe, γ‐Fe₂O₃ and gold nanoparticles (NPs) is described. When the amphiphilic end‐ functionalized polystyrenes and NPs are dissolved together in tetrahydrofuran, the addition of water causes the spontaneous formation of micellar aggregates, resulting in the successful encapsulation and aqueous dispersion of NPs. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), photoluminescence (PL) spectroscopy, and vibrating sample magnetometer (VSM) are used to characterize the structure and properties of the NPs‐containing micellar aggregates (nanocarrier). After complexation of Ni²⁺ with NTA on the surface of the nanocarrier containing γ‐Fe₂O₃, specific binding between Ni‐NTA complex and histidine‐tagged (His‐tagged) proteins enables selective separation of His‐tagged proteins using a magnet.     

Functionalization of Tailored Porous Carbon Monolith for Decontamination of Radioactive Substances

As the control over radioactive species becomes critical for the contemporary human life, the development of functional materials for decontamination of radioactive substances has also become important. In this work, a three-dimensional (3D) porous carbon monolith functionalized with Prussian blue particles was prepared through removal of colloidal silica particles from exfoliated graphene/silica composite precursors. The colloidal silica particles with a narrow size distribution were used to act a role of hard template and provide a sufficient surface area that could accommodate potentially hazardous radioactive substances by adsorption. The unique surface and pore structure of the functionalized porous carbon monolith was examined using electron microscopy and energy-dispersive X-ray analysis (EDS). The effective incorporation of PB nanoparticles was confirmed using diverse instrumentations such as X-ray diffraction (XRD), Fourier-transform infrared (FT-IR), and X-ray photoelectron spectroscopy (XPS). A nitrogen adsorption/desorption study showed that surface area and pore volume increased significantly compared with the starting precursor. Adsorption tests were performed with ¹³³Cs ions to examine adsorption isotherms using both Langmuir and Freundlich isotherms. In addition, adsorption kinetics were also investigated and parameters were calculated. The functionalized porous carbon monolith showed a relatively higher adsorption capacity than that of pristine porous carbon monolith and the bulk PB to most radioactive ions such as ¹³³Cs, ⁸⁵Rb, ¹³⁸Ba, ⁸⁸Sr, ¹⁴⁰Ce, and ²⁰⁵Tl. This material can be used for decontamination in expanded application fields.     

DOT1L Methyltransferase Regulates Calcium Influx in Erythroid Progenitor Cells in Response to Erythropoietin

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca²⁺) influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca²⁺ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca²⁺ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (Dot1l^KO) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1l^KO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca²⁺ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca²⁺ influx by TRPC2, Dot1l^KO HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca²⁺ influx. Such heightened Ca²⁺ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.     

Two additional cases of metformin‐associated encephalopathy in patients with end‐stage renal disease undergoing hemodialysis

We report on two additional cases of metformin‐associated encephalopathy in patients with end‐stage renal disease (ESRD) undergoing hemodialysis. Two patients were seen at our hospital with abnormal neurological signs and symptoms. Magnetic resonance imaging (MRI) revealed the same pattern of high signal intensity in both basal ganglia in T2‐weighted images in the two patients. The two patients had started taking metformin 5 and 6 weeks earlier at the same dose of 1000 mg per day. Metformin was immediately stopped, and regular hemodialysis was conducted. Their signs and symptoms resolved completely after these measures. The high signal intensity in both ganglia in T2‐weighted MRI also disappeared. We should suspect metformin‐induced encephalopathy and withdraw the drug when presented with diabetic patients with chronic kidney disease and neurological signs and symptoms of unknown cause.     

Functional characterization of purified pear protease and its proteolytic activities with casein and myofibrillar proteins

This study was performed to characterize pear protease proteolytic activity and investigate the use of pear protease as a meat tenderizer. Pear protease was purified and stabilized by 5% dextrin during lyophilization (dry) or concentration (liquid). Pear protease was further characterized with respect to pH, thermodynamics, and enzyme kinetics. Pear protease was stable at a pH range of 5-8 with an optimum pH of 6.5. From Arrhenius plots, liquid protease showed higher temperature dependency (23.49 kJ/mol) than dry protease (18.62 kJ/mol) due to its higher activation energy. The kcat/Km, catalytic efficiency of enzyme, was similar with 2.9 and 2.7 µM/min with dry and liquid proteases. Pear protease was evaluated for its proteolytic activities with casein and beef myofibrillar proteins by individually and combination with fig and kiwifruit proteases. These result indicated that pear and kiwifruit proteases could be complementary to be a desirable product for meat tenderization.     

Identification of cellulolytic bacteria associated with tunic softness syndrome in the sea squirt, Halocynthia roretzi

The edible ascidian, sea squirt Halocynthia roretzi (Drasche) is marine invertebrate that is a valuable source of foods and bioactive compounds. A severe disease of the sea squirt characterized by degeneration of tunic fibers formed of bundled cellulose microfibrils has occurred. We hypothesized that bacteria lyse the cellulose fibril structure, cellulase activity may be a causative agent of the disease. Among the bacteria isolated from diseased sea squirt, Pseudoalteromonas sp. NO3 had cellulase activity based on a Congo red overlay assay and starch-reducing activity. Sea squirts exhibited 40–100% cumulative mortality after injection with Pseudoalteromonas sp. NO3 using doses of 2×10⁶?2×10⁸ colony forming unit (CFU)/individual. Dead sea squirts possess thinner and ruptured tunics, which were similar to natural outbreaks. These results suggest that Pseudoalteromonas sp. NO3 possessing cellulase activity is one of the causes of tunic softness syndrome in sea squirt.     

Development of an Automated Method for Folin‐Ciocalteu Total Phenolic Assay in Artichoke Extracts

Abstract: We developed a system to run the Folin‐Ciocalteu (F‐C) total phenolic assay, in artichoke extract samples, which is fully automatic, consistent, and fast. The system uses 2 high performance liquid chromatography (HPLC) pumps, an autosampler, a column heater, a UV/Vis detector, and a data collection system. To test the system, a pump delivered 10‐fold diluted F‐C reagent solution at a rate of 0.7 mL/min, and 0.4 g/mL sodium carbonate at a rate of 2.1 mL/min. The autosampler injected 10 μL per 1.2 min, which was mixed with the F‐C reagent and heated to 65 °C while it passed through the column heater. The heated reactant was mixed with sodium carbonate and color intensity was measured by the detector at 600 nm. The data collection system recorded the color intensity, and peak area of each sample was calculated as the concentration of the total phenolic content, expressed in μg/mL as either chlorogenic acid or gallic acid. This new method had superb repeatability (0.7% CV) and a high correlation with both the manual method (r²= 0.93) and the HPLC method (r²= 0.78). Ascorbic acid and quercetin showed variable antioxidant activity, but sugars did not. This method can be efficiently applied to research that needs to test many numbers of antioxidant capacity samples with speed and accuracy.