A Call for Systematic Research on Solute Carriers.

Solute carrier (SLC) membrane transport proteins control essential physiological functions, including nutrient uptake, ion transport, and waste removal. SLCs interact with several important drugs, and a quarter of the more than 400 SLC genes are associated with human diseases. Yet, compared to other gene families of similar stature, SLCs are relatively understudied. The time is right for a systematic attack on SLC structure, specificity, and function, taking into account kinship and expression, as well as the dependencies that arise from the common metabolic space.

In-situ graft-crosslinked gold nanoparticles with high-density surface defects and coated with a polytaurine membrane for the voltammetric determination of dopamine.

Well-dispersed and graft-crosslinked gold nanoparticles (AuNPs) were synthesized by the reduction of tetrachloroaurate with hydrazine at room temperature. The AuNPs possess a high density of surface defects which is due to grafting of n-octanoic acid to polyvinylpyrrolidone. The physical and chemical properties of the resulting AuNPs were characterized by UV-vis, XRD, TEM/HRTEM, SAED, and XPS, respectively. The modified AuNPs were placed on a glassy carbon electrode (GCE) in an electropolymerized taurine layer to obtain a sensitive, selective, stable and rapid electrochemical dopamine sensor. The peak current, typically measured at 0.17 V (vs. SCE), increases linearly in the 1.0 to 120 µM dopamine concentration range, and the limit of detection (at S/N = 3) is 0.16 µM with a sensitivity of 2.94 µA·µM-1·cm-2. The sensor was successfully applied to the determination of dopamine in injections and spiked serum samples. The recoveries from spiked serum samples range from 97.5 to 102.4%, with RSDs ranging between 2.8 and 3.4%. Graphical abstract Schematic representation of a glassy carbon electrode modified with in-situ graft-crosslinked gold nanoparticles combined with an electropolymerized polytaurine membrane. The sensor exhibits excellent features towards dopamine determination.

Structural biology of solute carrier (SLC) membrane transport proteins.

The human solute carriers (SLCs) comprise over 400 different transporters, organized into 65 families ( http://slc.bioparadigms.org/ ) based on their sequence homology and transport function. SLCs are responsible for transporting extraordinarily diverse solutes across biological membranes, including inorganic ions, amino acids, lipids, sugars, neurotransmitters and drugs. Most of these membrane proteins function as coupled symporters (co-transporters) utilizing downhill ion (H+ or Na+) gradients as the driving force for the transport of substrate against its concentration gradient into cells. Other members work as antiporters (exchangers) that typically contain a single substrate-binding site with an alternating access mode of transport, while a few members exhibit channel-like properties. Dysfunction of SLCs is correlated with numerous human diseases and therefore they are potential therapeutic drug targets. In this review, we identified all of the SLC crystal structures that have been determined, most of which are from prokaryotic species. We further sorted all the SLC structures into four main groups with different protein folds and further discuss the well-characterized MFS (major facilitator superfamily) and LeuT (leucine transporter) folds. This review provides a systematic analysis of the structure, molecular basis of substrate recognition and mechanism of action in different SLC family members.

Molecular analysis of human solute carrier SLC26 anion transporter disease-causing mutations using 3-dimensional homology modeling.

The availability of the first crystal structure of a bacterial member (SLC26Dg) of the solute carrier SLC26 family of anion transporters has allowed us to create 3-dimensional models of all 10 human members (SLC26A1-A11, A10 being a pseudogene) of these membrane proteins using the Phyre2 bioinformatic tool. The homology modeling predicted that the SLC26 human proteins, like the SLC26Dg template, all consist of 14 transmembrane segments (TM) arranged in a 7+7 inverted topology with the amino-termini of two half-helices (TM3 and 10) facing each other in the centre of the protein to create the anion-binding site, linked to a C-terminal cytosolic sulfate transporter anti-sigma factor antagonist (STAS) domain. A plethora of human diseases are associated with mutations in the genes encoding human SLC26 transporters, including chondrodysplasias with varying severity in SLC26A2 (~50 mutations, 27 point mutations), congenital chloride-losing diarrhea in SLC26A3 (~70 mutations, 31 point mutations) and Pendred Syndrome or deafness autosomal recessive type 4 in SLC26A4 (~500 mutations, 203 point mutations). We have localized all of these point mutations in the 3-dimensional structures of the respective SLC26A2, A3 and A4 proteins and systematically analyzed their effect on protein structure. While most disease-causing mutations may cause folding defects resulting in impaired trafficking of these membrane glycoproteins from the endoplasmic reticulum to the cell surface - as demonstrated in a number of functional expression studies - the modeling also revealed that a number of pathogenic mutations are localized to the anion-binding site, which may directly affect transport function.

Effect of SLC26 anion transporter disease-causing mutations on the stability of the homologous STAS domain of E. coli DauA (YchM).

The human solute carrier 26 (SLC26) family of anion transporters consists of ten members that are found in various organs in the body including the stomach, intestine, kidney, thyroid and ear where they transport anions including bicarbonate, chloride and sulfate, typically in an exchange mode. Mutations in these genes cause a plethora of diseases such as diastrophic dysplasia affecting sulfate uptake into chondrocytes (SLC26A2), congenital chloride-losing diarrhoea (SLC26A3) affecting chloride secretion in the intestine and Pendred's syndrome (SLC26A4) resulting in hearing loss. To understand how these mutations affect the structures of the SLC26 membrane proteins and their ability to function properly, 12 human disease-causing mutants from SLC26A2, SLC26A3 and SLC26A4 were introduced into the equivalent sites of the sulfate transporter anti-sigma factor antagonist (STAS) domain of a bacterial homologue SLC26 protein DauA (YchM). Biophysical analyses including size-exclusion chromatography, circular dichroism (CD), differential scanning fluorimetry (DSF) and tryptophan fluorescence revealed that most mutations caused protein instability and aggregation. The mutation A463K, equivalent to N558K in human SLC26A4, which is located within α-helix 1 of the DauA STAS domain, stabilized the protein. CD measurements showed that most disease-related mutants had a mildly reduced helix content, but were more sensitive to thermal denaturation. Fluorescence spectroscopy showed that the mutants had more open structures and were more readily denatured by urea, whereas DSF indicated more labile folds. Overall, we conclude that the disease-associated mutations destabilized the STAS domain resulting in an increased propensity to misfold and aggregate.

Highly sensitive graphene-Pt nanocomposites amperometric biosensor and its application in living cell H2O2 detection.

A sensitive hydrogen peroxide (H2O2) sensor was constructed based on graphene-Pt (RGO-Pt) nanocomposites and used to measure the release of H2O2 from living cells. The graphene and Pt nanoparticles (Pt NPs) were modified on glassy carbon electrode (GCE) by the physical adsorption and electrodeposition of K2PtCl6 solution, respectively. Through characterization by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS), it was observed that the electrodeposited Pt NPs were densely covered and well distributed on the entire graphene surface. Electrochemical study demonstrates that the RGO-Pt nanocomposites modified glassy carbon electrode exhibited a high peak current and low overpotential toward the reduction of H2O2. The relevant detection limit of H2O2 is ∼0.2 µM with a wide linear range from 0.5 µM to 3.475 mM, displaying a much higher sensitivity (459 ± 3 mA M(-1) cm(-2), n = 5) than that of Pt nanoparticles or graphene modified electrode. This novel biosensor can measure the H2O2 release from living cells because of its low detection limit, wide linear range, and higher sensitivity.

Rigidity of wedge loop in PACSIN 3 protein is a key factor in dictating diameters of tubules.

BAR (Bin/amphiphysin/Rvs) domain-containing proteins participate in cellular membrane remodeling. The F-BAR proteins normally generate low curvature tubules. However, in the PACSIN subfamily, the F-BAR domain from PACSIN 1 and 2 can induce both high and low curvature tubules. We found that unlike PACSIN 1 and 2, PACSIN 3 could only induce low curvature tubules. To elucidate the key factors that dictate the tubule curvature, crystal structures of all three PACSIN F-BAR domains were determined. A novel type of lateral interaction mediated by a wedge loop is observed between the F-BAR neighboring dimers. Comparisons of the structures of PACSIN 3 with PACSIN 1 and 2 indicate that the wedge loop of PACSIN 3 is more rigid, which influences the lateral interactions between assembled dimers. We further identified the residues that affect the rigidity of the loop by mutagenesis and determined the structures of two PACSIN 3 wedge loop mutants. Our results suggest that the rigidity-mediated conformations of the wedge loop correlate well with the various crystal packing modes and membrane tubulations. Thus, the rigidity of the wedge loop is a key factor in dictating tubule diameters.

Development of matrix certified reference material for accurate determination of docosahexaenoic acid in milk powder.

A novel matrix certified reference material (CRM) of docosahexaenoic acid in milk powder [GBW (E) 100641] was first developed. The CRM candidates was prepared by adding appropriate levels of docosahexaenoic acid to cow's milk, then powder sprayed, lyophilized, mixed, dispensed and sterilized. An optimized acetylchloride-methanol method was proposed and used for the characterization. The CRM characterization was carried out in six laboratories in accordance with ISO Guide 35 requirements. The certified value of CRM was 0.69 mg/g with an uncertainty of 0.08 mg/g (k = 2). The CRM was sufficiently homogeneous between and within bottles and stable up to 6 month at -20℃ and 7 days below 50 â„ƒ. The uncertainty was evaluated by combing the contributions from characterization, homogeneity and stability. Thus, the CRM can be used for quality control and method validation to ensure the accurate and reliable measurements of docosahexaenoic acid in milk for quality monitoring.

Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD.

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV-2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

Inkjet printing of nanoporous gold electrode arrays on cellulose membranes for high-sensitive paper-like electrochemical oxygen sensors using ionic liquid electrolytes.

A simple approach to the mass production of nanoporous gold electrode arrays on cellulose membranes for electrochemical sensing of oxygen using ionic liquid (IL) electrolytes was established. The approach, combining the inkjet printing of gold nanoparticle (GNP) patterns with the self-catalytic growth of these patterns into conducting layers, can fabricate hundreds of self-designed gold arrays on cellulose membranes within several hours using an inexpensive inkjet printer. The resulting paper-based gold electrode arrays (PGEAs) had several unique properties as thin-film sensor platforms, including good conductivity, excellent flexibility, high integration, and low cost. The porous nature of PGEAs also allowed the addition of electrolytes from the back cellulose membrane side and controllably produced large three-phase electrolyte/electrode/gas interfaces at the front electrode side. A novel paper-based solid-state electrochemical oxygen (O(2)) sensor was therefore developed using an IL electrolyte, 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF(6)). The sensor looked like a piece of paper but possessed high sensitivity for O(2) in a linear range from 0.054 to 0.177 v/v %, along with a low detection limit of 0.0075% and a short response time of less than 10 s, foreseeing its promising applications in developing cost-effective and environment-friendly paper-based electrochemical gas sensors.

Cloning, purification, crystallization and preliminary X-ray diffraction analysis of mouse PACSIN 3 protein.

PACSIN-family proteins are cytoplasmic proteins that have vesicle-transport, membrane-dynamics, actin-reorganization and microtubule activities. Here, the N-terminal F-BAR domain of mouse PACSIN 3, which contains 341 amino acids, was successfully cloned, purified and crystallized. The crystal of PACSIN 3 (1-341) diffracted to 2.6 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 54.7, c = 193.7 Å, α = 90, ß = 96.9, γ = 90°. These data should provide further information on PACSIN-family protein structures.

Structure of human stabilin-1 interacting chitinase-like protein (SI-CLP) reveals a saccharide-binding cleft with lower sugar-binding selectivity.

Human secreted protein stabilin-1 interacting chitinase-like protein (SI-CLP) has been identified as a novel member of Glyco_18 domain-containing proteins that is involved in host defense and inflammatory reactions. Efficient secretion of SI-CLP is mediated by its interaction with the endocytic/sorting receptor stabilin-1. SI-CLP is expressed abundantly in macrophages and neutrophils and is up-regulated by Th2 cytokine IL-4 and glucocorticoid, which suggest that SI-CLP could be a marker for adverse effects of glucocorticoid therapy. To gain insight into the biological function of SI-CLP, we determined the crystal structure of SI-CLP at 2.7 Å resolution by x-ray crystallography and found that it featured a typical triose-phosphate isomerase barrel fold with a putative saccharide-binding cleft. Comparison with other chitinase-like proteins showed the cleft to be atypically wide and open. The saccharide-binding capacity of SI-CLP was investigated, and its ligand-binding specificity was found to relate to the length of the oligosaccharides, with preference for chitotetraose. Further investigations reveal that SI-CLP could bind LPS in vitro and neutralize its endotoxin effect on macrophages. Our results demonstrate the saccharide-binding property of SI-CLP by structure and in vitro biochemical analyses and suggest the possible roles of SI-CLP in pathogen sensing and endotoxin neutralization.

Crystallization and preliminary X-ray crystallographic analysis of human PACSIN 1 protein.

PACSIN 1, which is mainly detected in brain tissue, is one of the PACSIN-family proteins involved in endocytosis and recruitment of synaptic vesicles. It binds to dynamin, synaptojanin 1 and N-WASP, and functions in vesicle formation and transport. However, the mechanisms of action of PACSIN 1 in these processes are largely unknown. Here, full-length and five C-terminal truncation constructs of human PACSIN 1 have been successfully expressed and purified in Escherichia coli. PACSIN 1 (1-344) was crystallized and diffracted to a resolution of 3.0 A. The crystal belonged to space group C2, with unit-cell parameters a = 158.65, b = 87.38, c = 91.76 A, alpha = 90.00, beta = 113.61, gamma = 90.00 degrees . There were two molecules in the asymmetric unit and the solvent content was estimated to be about 70.47%.

Crystallization and preliminary X-ray crystallographic studies of human FAIM protein.

Fas apoptosis inhibitory molecule (FAIM), an antagonist of Fas-induced cell death, is highly conserved and is broadly expressed in many tissues. It has been found that FAIM can stimulate neurite outgrowth in PC12 cells and primary neurons. However, the molecular mechanisms of action of FAIM are not understood in detail. Here, full-length human FAIM and two truncation constructs have successfully been cloned, expressed and purified in Escherichia coli. FAIM (1-90) was crystallized and diffracted to a resolution of 2.5 A; the crystal belonged to space group P3(1), with unit-cell parameters a=b=58.02, c=71.11 A, alpha=beta=90, gamma=120 degrees. There were two molecules in the asymmetric unit.

Colonization dynamics of protozoan communities in marine bioassessment surveys using two modified sampling systems.

Colonization dynamics of protozoan communities were investigated at a depth of 1 m in the coastal waters of the Yellow Sea, northern China from May to June 2019, using modified glass slide (mGS) and modified polyurethane foam unit (mPFU) systems. The colonization process and growth curves of protozoa were well fitted to the MacArthur-Wilson and logistic models in both systems, respectively. However, they showed significant differences in both colonization dynamics and biodiversity/functional parameters between the mGS and mPFU systems. The H' (species diversity), the G (colonization rate), and Amax (maximum abundance) were higher, while the value of T90% (the time for reaching 90% equilibrium species number) was lower in the mGS system than those in the mPFU system. Multivariate analyses demonstrated that protozoa showed different models of colonization dynamics in both systems. The results suggest that the mGS system might be more effective than the mPFU system in marine bioassessment surveys.

An approach to assessing ecological quality status due to microalgae bloom using biofilm-dwelling protozoa based on biological trait analysis.

Functional diversity/distinctness measure based trait has been proved to be a robust indicator to summarize the description of community structures and to assess water quality in different types of aquatic environment. In this study, for identifying the shielding effect of microalgae against protozoan grazing, a nine-day survey was conducted by exposing protozoan communities to a series of concentration gradients (100 (control), 104, 105, 106 and 107 cells ml-1) of two microalgae, respectively. Our results showed clear resistance of two test microalgae against protozoan grazing in five treatments. The functional distinctness measures commonly represented a decreasing trend along the gradient of concentrations of both microalgae. Ellipse tests based on the paired functional distinctness indices revealed that community functioning represented an uptrend departure from the expected pattern with the concentrations of both microalgae increase. Therefore, we suggest that the functional distinctness measures might be a reliable approach to detect the ecological effect of microalgae against protozoan grazing.

Seasonal variability in biological trait pattern of biofilm-dwelling protozoa in colonization surveys for marine bioassessment.

Biological trait analysis (BTA) has been proved to be a powerful tool to evaluate marine water quality. The species trait distributions of biofilm-dwelling protozoa were studied in a coastal region of the Yellow Sea, northern China, during a four-season cycle. The BTA demonstrated that: (1) the protozoa showed a significant seasonal variability in biological trait pattern during the colonization process across four seasons; (2) the colonization dynamics in species trait distribution followed different temporal models; (3) the functional dynamics in spring and summer were significantly different from those in autumn and winter (P < 0.05); and (4) functional diversity showed lower values in spring and summer than in autumn and winter. These findings suggest that BTA is subject to a high seasonal variability during colonization surveys when protozoa are used as bioindicators of marine water quality.

Seasonal variation in biological trait distribution of periphytic protozoa in coastal ecosystem: A baseline study for marine bioassessment.

To reveal the seasonal variability in biological trait distribution for monitoring surveys based on periphytic protozoa, a baseline survey was carried out in a coastal region of Yellow Sea, northern China. A total of 40 slide samples were collected in a four season cycle after an exposure time period of 14 days. The results demonstrated that: (1) the community-weighted means (CWM) of algivores with large and medium sizes were high in spring and summer, while bacterivores with small size were high in autumn and winter; (2) there was a significant seasonal variation in the protozoan community functions, especially from spring/summer to autumn and winter; and (3) functional diversity indices generally peaked in spring or summer. Thus, there was a significant seasonal variation in protozoan community functions and this approach may be used to determine an optimal sampling strategy for monitoring programs in marine ecosystems.

Molecularly imprinted electrochemical sensor based on polypyrrole/dopamine@graphene incorporated with surface molecularly imprinted polymers thin film for recognition of olaquindox.

In this paper, an advanced molecularly imprinted electrochemical sensor (MIECS) based on electropolymerized olaquindox (OLA) surface molecularly imprinted polymer thin film on a modified glassy carbon electrode (GCE) was developed for the detection of OLA. It was fabricated by coating dopamine@graphene (DGr) on GCE, then electropolymerizing pyrrole (Py) and molecularly imprinted polymers (MIPs). Graphene (Gr) was introduced for improving conductivity and sensitivity. Dopamine (DA) was used for dispersion and adhesion of Gr. Polypyrrole (PPy) could fix DGr and enhance the current response evidently. The established sensor could selectively recognize OLA but not the analogs of OLA. Some essential parameters controlling the performance of the developed sensor were investigated and optimized. Under optimal conditions, the linear relationship between the current intensity and OLA concentration was obtained from 50 nmol L-1 to 500 nmol L-1 with a limit of detection (LOD) of 7.5 nmol L-1. Analytical results of OLA based on the developed MIECS for fish and feedstuffs showed a good agreement with the results based on high performance liquid chromatography (HPLC).

[Use of Iron-modified Calcite as an Active Capping Material to Control Phosphorus Release from Sediments in Surface Water Bodies].

The use of calcite (CA) as an active capping material has high potential for controlling the release of phosphorus (P) from sediments, but its efficiency still needs to be enhanced. To address this issue, an iron-modified CA (Fe-CA) was prepared, the removal performance of phosphate from aqueous solution by Fe-CA was studied, and the efficiency of the use of Fe-CA as an active capping material to prevent the liberation of P from sediments was investigated. The results showed that Fe-CA exhibited much higher phosphate removal ability than CA. The phosphate removal efficiency of Fe-CA increased with an increase in the Fe-CA dosage. Increasing the initial phosphate concentration gave rise to an increase in the amount of phosphate removed by Fe-CA, and the maximum amount of phosphate removed by Fe-CZ reached 3.09 mg·g-1. Sediment capping with Fe-CA could effectively control the release of soluble reactive P (SRP) from the sediment into the overlying water, leading to a very low concentration of SRP in the overlying water. Additionally, the Fe-CA capping also resulted in the transformation of a small amount of redox-sensitive P (BD-P) and metal-oxide-bound P (NaOH-rP) in sediments to residual P (Res-P), leading to a slight increase in the stability of P in the sediment. The overwhelming majority (90.8%) of P bound by the Fe-CA capping layer existed in the form of NaOH-rP, calcium-bound P (HCl-P), and Res-P, which are relatively very stable. Furthermore, the percentage of bioavailable P (BAP) as a proportion of total extractable P in the P-bound Fe-CA capping layer was very low, and the bound P was re-released with difficulty into the water column for algae growth. Compared to CA capping, the efficiency for the control of sedimentary P release into the overlying water by Fe-CA capping was much higher, and the stability of P bound by the Fe-CA capping layer was also higher. The results of this work indicate that Fe-CA is a very promising active capping material for the interception of the release of P from sediments into the overlying water.