Structural Basis for the Function of the C-Terminal Proton Release Pathway in the Calcium Pump

The calcium pump (sarco/endoplasmic reticulum Ca²⁺-ATPase, SERCA) plays a major role in calcium homeostasis in muscle cells by clearing cytosolic Ca²⁺ during muscle relaxation. Active Ca²⁺ transport by SERCA involves the structural transition from a low-Ca²⁺ affinity E2 state toward a high-Ca²⁺ affinity E1 state of the pump. This structural transition is accompanied by the countertransport of protons to stabilize the negative charge and maintain the structural integrity of the transport sites and partially compensate for the positive charges of the two Ca²⁺ ions passing through the membrane. X-ray crystallography studies have suggested that a hydrated pore located at the C-terminal domain of SERCA serves as a conduit for proton countertransport, but the existence and function of this pathway have not yet been fully characterized. We used atomistic simulations to demonstrate that in the protonated E2 state and the absence of initially bound water molecules, the C-terminal pore becomes hydrated in the nanosecond timescale. Hydration of the C-terminal pore is accompanied by the formation of water wires that connect the transport sites with the cytosol. Water wires are known as ubiquitous proton-transport devices in biological systems, thus supporting the notion that the C-terminal domain serves as a conduit for proton release. Additional simulations showed that the release of a single proton from the transport sites induces bending of transmembrane helix M5 and the interaction between residues Arg762 and Ser915. These structural changes create a physical barrier against full hydration of the pore and prevent the formation of hydrogen-bonded water wires once proton transport has occurred through this pore. Together, these findings support the notion that the C-terminal proton release pathway is a functional element of SERCA and also provide a mechanistic model for its operation in the catalytic cycle of the pump.     

Computational Investigation Identified Potential Chemical Scaffolds for Heparanase as Anticancer Therapeutics

Heparanase (Hpse) is an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains. Its upregulated expression is implicated in tumor growth, metastasis and angiogenesis, thus making it an attractive target in cancer therapeutics. Currently, a few small molecule inhibitors have been reported to inhibit Hpse, with promising oral administration and pharmacokinetic (PK) properties. In the present study, a ligand-based pharmacophore model was generated from a dataset of well-known active small molecule Hpse inhibitors which were observed to display favorable PK properties. The compounds from the InterBioScreen database of natural (69,034) and synthetic (195,469) molecules were first filtered for their drug-likeness and the pharmacophore model was used to screen the drug-like database. The compounds acquired from screening were subjected to molecular docking with Heparanase, where two molecules used in pharmacophore generation were used as reference. From the docking analysis, 33 compounds displayed higher docking scores than the reference and favorable interactions with the catalytic residues. Complex interactions were further evaluated by molecular dynamics simulations to assess their stability over a period of 50 ns. Furthermore, the binding free energies of the 33 compounds revealed 2 natural and 2 synthetic compounds, with better binding affinities than reference molecules, and were, therefore, deemed as hits. The hit compounds presented from this in silico investigation could act as potent Heparanase inhibitors and further serve as lead scaffolds to develop compounds targeting Heparanase upregulation in cancer.     

The Age-Sensitive Efficacy of Calorie Restriction on Mitochondrial Biogenesis and mtDNA Damage in Rat Liver

Calorie restriction (CR) is the most efficacious treatment to delay the onset of age-related changes such as mitochondrial dysfunction. However, the sensitivity of mitochondrial markers to CR and the age-related boundaries of CR efficacy are not fully elucidated. We used liver samples from ad libitum-fed (AL) rats divided in: 18-month-old (AL-18), 28-month-old (AL-28), and 32-month-old (AL-32) groups, and from CR-treated (CR) 28-month-old (CR-28) and 32-month-old (CR-32) counterparts to assay the effect of CR on several mitochondrial markers. The age-related decreases in citrate synthase activity, in TFAM, MFN2, and DRP1 protein amounts and in the mtDNA content in the AL-28 group were prevented in CR-28 counterparts. Accordingly, CR reduced oxidative mtDNA damage assessed through the incidence of oxidized purines at specific mtDNA regions in CR-28 animals. These findings support the anti-aging effect of CR up to 28 months. Conversely, the protein amounts of LonP1, Cyt c, OGG1, and APE1 and the 4.8 Kb mtDNA deletion content were not affected in CR-28 rats. The absence of significant differences between the AL-32 values and the CR-32 counterparts suggests an age-related boundary of CR efficacy at this age. However, this only partially curtails the CR benefits in counteracting the generalized aging decline and the related mitochondrial involvement.     

Prediction of Chameleonic Efficiency

Chameleonic properties, i. e., the capacity of a molecule to hide polarity in non-polar environments and expose it in water, help achieving sufficient permeability and solubility for drug molecules with high MW. We present models of experimental measures of polarity for a set of 24 FDA approved drugs (MW 405-1113) and one PROTAC (MW 1034). Conformational ensembles in aqueous and non-polar environments were generated using molecular dynamics. A linear regression model that predicts chromatographic apparent polarity (EPSA) with a mean unsigned error of 10 Ų was derived based on separate terms for donor, acceptor, and total molecular SASA. A good correlation (R²=0.92) with an experimental measure of hydrogen bond donor potential, Δlog P_oct-tol, was found for the mean hydrogen bond donor SASA of the conformational ensemble scaled with Abraham's A hydrogen bond acidity. Two quantitative measures of chameleonic behaviour, the chameleonic efficiency indices, are introduced. We envision that the methods presented herein will be useful to triage designed molecules and prioritize those with the best chance of achieving acceptable permeability and solubility.     

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.     

Nano fibrillated cellulose-based foam by Pickering emulsion: Preparation,characterizations, and application as dye adsorbent

An ecofriendly and biodegradable porous structure was prepared from drying aqueous foams based on nano fibrillated cellulose (NFC), extracted from softwood pulp by subcritical water/CO₂ treatment (SC-NFC). The primary aim of this work was to use the modified SC-NFC as stabilizer for a water-based Pickering emulsion which upon drying, yielded porous cellulosic materials, a good dye adsorbent. In order to exploit the carboxymethylated SC-NFC (CMSC-NFC, with a degree of substitution of 0.35 and a charge density of 649 μeqv/g) as a stabilizer for water-based Pickering emulsion in subsequent step, an optimized quantity of octyl amine (30 mg/g of SC-NFC) was added to make them partially hydrophobic. A series of dry foam structures were prepared by varying the concentrations of treated CMSC-NFCs and 4 wt% was found to be the optimum concentration to yield foam with high porosity (99%) and low density (0.038 g/cc) along with high compression strength (0.24 MPa), superior to the conventionally extracted NFC. The foams were applied to capture as high as 98% of methylene blue dyes, making them a potential green candidate for treating industrial effluent. In addition, the dye adsorption kinetics and isotherms were found to be well suited with second order kinetics and Langmuir isotherm models.     

Targeted editing of intronic-splicing silencer enhancement of SMN2 Exon 7 inclusion by CRISPR/Case 9

Spinal muscular atrophy (SMA) is an autosomal recessive hereditary neuromuscular disease. Exon 7 and 8 of survival of motor neuron 1 (SMN1) gene or only exon 7 homology deletion leads to the failure to produce a full-length SMN gene. The copy number of SMN2 gene with high homology of SMN1 affects the degree of disease and was the target gene for targeting therapy, in which splicing silencer in intron 7 was the key to suppress the inclusion of exon 7. In this study, we projected to use CRISPR/Case 9 for the targeted editing of intronic-splicing silencer (ISS) sequence to promote the inclusion of SMN2 exon 7 and increase the production of SMN2 full-length (FL) gene expression. It happens that there was a protospacer adjacent motif (PAM) at one end of the ISS sequence according to the design of sgRNA. The recombinant vector of sgRNA HSMN2 CRISPR/Case 9 was constructed and transfected into HEK293 cells. Sequencing results showed that the ISS sequence could be edited accurately and targeting in the predicted direction, in which deleting small fragments, inserting small amounts and mutation. Quantitative analysis of RT-PCR products by restriction enzyme of DdeI digestion showed that the FL of SMN2 increased by 8% (P < 0.05). In the primary cultured chondrocytes of SMA mice, in which sgRNA HSMN2 CRISPR/Case9 recombinant vector transfection could increase the SMN2 FL gene by 23% (P < 0.05) and significantly improve SMN protein levels (P < 0.05). CRISPR/Case 9 is an effective tool for gene editing and therapy of hereditary diseases, but it is rarely reported in the treatment of SMA diseases. This study shows that CRISPR/Case 9 was first used for the precision target of ISS sequence editing, which can effectively promote the production of SMN2 FL gene expressions, in which there was an important clinical reference value.     

Tropfenbewegung und Stofftransport in technischen Flüssig/flüssig-Systemen. Teil 2: Auswirkung von Grenzflächeneffekten und Verunreinigungen

The design of a liquid/liquid contact apparatus necessitates the knowledge of fluid dynamics and mass transfer in the dispersion. Prediction of process parameters is challenging due to swarm and interfacial effects. Single drop investigations are suitable to enable a sophisticated dimensioning based on few simple experiments combined with published theories and correlations. In the first part of this article, the fundamentals of fluid dynamics and mass transfer in absence of interfacial effects were explained. Here, an overview of relevant interfacial phenomena and the influence of different substances typically occurring in technical applications as impurities or accompanying components is given.     

Computational Fluid Dynamics versus Experiment: An Investigation on Liquid Weeping of Nye Trays

The weeping phenomenon was investigated using some experimental tests and a numerical model. The tests were performed within a 1.22‐m‐diameter pilot‐scale column including two chimney trays and two Nye test trays with an air‐water system. The rates of weeping were measured in the Nye trays with two heights of the weir and a hole area of 5 %. Moreover, the weeping rates in the outlet and inlet halves of the Nye tray and the total weeping rate were calculated. In the next step, an Eulerian‐Eulerian computational fluid dynamics (CFD) technique was used. The results show good agreement between the attained CFD findings and the experimental data.     

A Highly Sensitive,Reversible, and Bidirectional Humidity Actuator by Calcium Carbonate Ionic Oligomers Incorporated Poly(Vinylidene Fluoride)

Sensitivity and multi-directional motivation are major two factors for developing optimized humidity-response materials, which are promising for sensing, energy production, etc. Organic functional groups are commonly used as the water sensitive units through hydrogen bond interactions with water molecules in actuators. The multi-coordination ability of inorganic ions implies that the inorganic ionic compounds are potentially superior water sensitive units. However, the particle forms of inorganic ionic compounds produced by classical nucleation limit the number of exposed ions to interact with water. Recent progress on the inorganic ionic oligomers has broken through the limitation of classical nucleation, and realized the molecular-scaled incorporation of inorganic ionic compounds into an organic matrix. Here, the incorporation of hydrophilic calcium carbonate ionic oligomers into hydrophobic poly(vinylidene fluoride) (PVDF) is demonstrated. The ultra-small calcium carbonate oligomers within a PVDF film endow it with an ultra-sensitive, reversible, and bidirectional response. The motivation ability is superior to other bidirectional humidity-actuators at present, which realizes self-motivation on an ice surface, converting the chemical potential energy of the humidity gradient from ice to kinetic energy.