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The treatment of industrial wastewater containing complex foulants demands high efficiency yet cost-effective separation process. The composite hollow fiber nanofiltration membranes suffer from tedious fabrication procedures, impeding the large volume fabrication and application. Herein, we reveal a very simplified designing strategy by polyelectrolyte modification of the co-extruded polyimide/polyethersulphone dual-layer hollow fibers with hierarchical structure and chemistry. The active layer's thickness of the dual-layer hollow fiber substrate is only 800 nm, the thinnest in literature. Compared to integral active single layer hollow fiber, the ultrathin active layer not only leads to over 4 times increase in mechanical flexibility and tenfold reduction in material cost but also enables fully chemical transition from imide to cross-linked polyamide, minimizing the intrinsic hydrolysis tendency of active material. The optimized membranes with a well-defined pore radius of 0.23 nm have 93–95% rejection to Mg2+ and Ca2+ and PWP of 9.1 LMH/bar. Moreover, the composite membranes exhibit backwash ability and stability in the heated feed solutions, which meets the demand for direct treatment of industrial effluents. This work paves the way for the large volume fabrication and application of hollow fiber nanofiltration membranes for treating municipal water, industrial wastewater, and other applications.

Highly permeable two-dimensional membranes are desirable for efficient molecular sieving and resource recovery through confined-space mass transfer via nanosheets restacking. However, their application requires addressing the swelling issue of 2D nanosheets in solvents, weak interfacial interaction between the adjacent nanosheets, and performance declination caused by over-restacking. This study reports an interfacial adhesion enhanced MXene membrane via pillaring carbon nanotubes (CNTs) combined with an upgraded post-treatment method consisting of fan-drying and air-drying. The combined drying method induces the nanosheets stacking to a partial-dehydration membrane structure, granting the neat MXene membrane with stable anti-swelling property. The strong π-π interactions and van der Waals forces of CNTs impel the close-fitting of MXene nanosheets for enhanced anti-swelling property and interfacial bonding force. The CNTs with confined-space mass transfer effect and high mechanical strength elevate the pillared MXene/CNTs-CTAB membrane performance for 5 times (the pure water permeance was enhanced from 20.09 L·m-2·h-1·bar-1 to 100.89 L·m-2·h-1·bar-1), endowing the membrane with high anti-pressure property. The membrane with precise molecular sieving property exhibits great potential in resource recovery and energy-saving.

Delamination of the dual-layer membrane fabricated from immiscible polymers is recognized as the main obstacle to advance the technology. The current work discloses an innovative approach to address the delamination issue by incorporating specific functional groups on the polymer chains. Delamination-free P84®/Ultem® dual-layer hollow fiber nanofiltration membranes were obtained by incorporating 4-Fluoro-2-(trifluoromethyl) benzylamine (FTB) in P84® polyimide. The progressively incorporated FTB on P84® decreases solubility parameter difference and establishes specific intermolecular interactions between the polymer pair which contributes to the interpenetration of molecules at the interfacial region of the dual layers. The interpenetrated network structure at the interfacial region renders the robust integrity of the dual-layer membranes. The one-step fabricated membranes had an over 99% rejection of tetracycline (MW: 444.4 Da) in methanol with pure methanol permeance of 3.7 LMH/bar. The elaborated mechanism may potentially enlarge the scope of selecting immiscible polymers for fabricating dual-layer membranes by molecular designing.

The objective of this study was to develop a novel and effective method for the evaluation of the postmortem interval using infrared microspectroscopy. Postmortem rat liver samples were investigated at 10, 20, and 30°C. Time-dependent changes in the images were observed from variations in the spatial distribution in the liver tissue that were correlated with the postmortem interval. This relationship, which has not been reported previously, offers a novel approach to supplement conventional forensic methods.

Motivation: The identification of orthologous gene pairs is generally based on sequence similarity. Gene pairs that are mutually ‘best hits’ between the genomes being compared are asserted to be orthologs. Although this method identifies most orthologous gene pairs with high confidence, it will miss a fraction of them, especially genes in duplicated gene families. In addition, the approach depends heavily on the completeness and quality of gene annotation. When the gene sequences are not correctly represented the approach is unlikely to find the correct ortholog. To overcome these limitations, we have developed an approach to identify orthologous gene pairs using shared chromosomal synteny and the annotation of protein function. Results: Assembled mouse and human genomes were used to identify the regions of conserved synteny between these genomes. ‘Syntenic anchors’ are conserved non-repetitive locations between mouse and human genomes. Using these anchors, we identified blocks of sequences that contain consistently ordered anchors between the two genomes (syntenic blocks). The synteny information has been used to help us identify orthologous gene pairs between mouse and human genomes. The approach combines the mutual selection of the best tBlastX hits between human and mouse transcripts, and inferring gene orthologous relationships based on sharing syntenic anchors, collocating in the same syntenic blocks and sharing the same annotated protein function. Using this approach, we were able to find 19 357 orthologous gene pairs between human and mouse genomes, a 20% increase in the number of orthologs identified by conventional approaches. Contact: richard.mural@celera.com

The amino acid sequence of the light chain of Acanthamoebamyosin IC deduced from the cDNA sequence comprises 149 aminoacids with a calculated molecular weight of 16739. All but the 3N-terminal residues were also determined by amino acid sequencingof the purified protein, which also showed the N-terminus to beblocked. Phylogenetic analysis shows Acanthamoeba myosin IC lightchain to be more similar to the calmodulin subfamily of EF-handcalcium-modulated proteins than to the myosin II essential lightchain or regulatory light chain subfamilies. In pairwisecomparisons, the myosin IC light chain sequence is most similarto sequences of calmodulins (∼50% identical) and a squidcalcium-binding protein (∼43% identical); the sequence is∼37% identical to the calcium-binding essential light chainof Physarum myosin II and ∼30% identical to the essentiallight chain of Acanthamoeba myosin II, and the essential lightchain and regulatory light chain of Dictyostelium myosin II. Thesequence predicts four helix-loop-helix domains with possiblecalcium-binding sites in domains I and III, suggesting thatcalcium may affect the activity of this unconventional myosin.This is the first report of the sequence of an unconventionalmyosin light chain other than calmodulin

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by ( i ) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, ( ii ) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, ( iii ) its K m for ATP, and ( iv ) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same K m for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.

The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (AMIs)—AMIA, AMIB, and AMIC—are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin’s actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-Å resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (time-averaged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. The tail domain of vertebrate intestinal brush-border myosin I has been observed to swing through 31° on binding of ADP. However, it was predicted on grounds of differing kinetics that any such effects with AMIC should be small [Jontes, J. D., Ostap, E. M., Pollard, T. D. & Milligan, R. A. (1998) J. Cell Biol. 141, 155–162]. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.

Acanthamoeba class I myosins are unconventional, single-headed myosins that express actin-activated Mg 2+ -ATPase and in vitro motility activities only when a single serine or threonine in the heavy chain is phosphorylated by myosin I heavy chain kinase (MIHCK). Some other, but not most, class I myosins have the same consensus phosphorylation site sequence, and the two known class VI myosins have a phosphorylatable residue in the homologous position, where most myosins have an aspartate or glutamate residue. Recently, we found that the catalytic domain of Acanthamoeba MIHCK has extensive sequence similarity to the p21-activated kinase (PAK)/STE20 family of kinases from mammals and yeast, which are activated by small GTP-binding proteins. The physiological substrates of the PAK/STE20 kinases are not well characterized. In this paper we show that PAK1 has similar substrate specificity as MIHCK when assayed against synthetic substrates and that PAK1 phosphorylates the heavy chain (1 mol of P i per mol) and activates Acanthamoeba myosin I as MIHCK does. These results, together with the known involvement of Acanthamoeba myosin I, yeast myosin I, STE20, PAK, and small GTP-binding proteins in membrane- and cytoskeleton-associated morphogenetic transformations and activities, suggest that myosins may be physiological substrates for the PAK/STE20 family and thus mediators of these events.

The actin-activated Mg(2+)-ATPase and in vitro motility activities of the three Acanthamoeba myosin I isozymes depend upon phosphorylation of their single heavy chains by myosin I heavy chain kinase. Previously, the kinase had been shown to be activated by autophosphorylation, which is enhanced by acidic phospholipids, or simply by binding to purified plasma membranes in the absence of significant autophosphorylation. In this paper, we show that the rate of phosphorylation of myosin I by unphosphorylated kinase is approximately 20-fold faster when both the myosin I and the kinase are bound to acidic phospholipid vesicles than when both are soluble. This activation is not due to an increase in the local concentrations of vesicle-bound kinase and myosin I. Thus, acidic phospholipids, like membranes, can activate myosin I heavy chain kinase in the absence of significant autophosphorylation, i.e. membrane proteins are not required. Kinetic studies show that both binding of kinase to phospholipid vesicles and autophosphorylation of kinase in the absence of phospholipid increase the Vmax relative to soluble, unphosphorylated kinase with either an increase in the apparent Km (when myosin I is the substrate) or no significant change in Km (when a synthetic peptide is the substrate). Kinetic data showed that autophosphorylation of phospholipid-bound kinase is both intermolecular and intervesicular, and that phosphorylation of phospholipid-bound myosin I by phospholipid-bound kinase is also intervesicular even when the kinase and myosin are bound to the same vesicles. The relevance of these results to the activation of myosin I heavy chain kinase and phosphorylation of myosin I isozymes in situ are discussed.

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of (3)H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-(3)H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K(off)) of the bound RuBP was 4.8 x 10(-4) per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO(2), and Mg(2+), and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg(2+) in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg(2+), ATP (but not the nonhydrolyzable analog, adenosine-5'-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-(3)H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.

The interactions of ATP and ADP with rubisco activase purified from spinach were investigated by measuring enhanced fluorescence due to ANS-binding to the protein. Evidence of conformational changes was observed from the differences in the interaction of ANS with rubisco activase in the presence of excess ATP and ADP. Fluorescent changes associated with the titration of a rubisco activase-ANS mixture with ATP and ADP indicated that the binding of ADP to rubisco activase was much tighter than that of ATP. The concentration of Mg2+ and pH had significant effects on the affinities of rubisco activase for ATP and ADP, with higher pH and Mg2+ concentration facilitating the binding of ATP to rubisco activase in the presence of ADP. The physiological implications of the binding characteristics of ATP and ADP with rubisco activase on the light-dark regulation of rubisco are discussed.