Structural elucidation of how ARF small GTPases induce membrane tubulation for vesicle fission.

The ADP-Ribosylation Factor (ARF) small GTPases have been found to act in vesicle fission through a direct ability to tubulate membrane. Here, we have used cryo-electron microscopy (EM) to solve the structure of an ARF6 protein lattice assembled on tubulated membrane to 3.9 Å resolution. ARF6 forms tetramers that polymerize into helical arrays to form this lattice. We identify, and confirm functionally, protein contacts critical for this lattice formation. The solved structure also suggests how the ARF amphipathic helix is positioned in the lattice for membrane insertion, and how a GTPase-activating protein (GAP) docks onto the lattice to catalyze ARF-GTP hydrolysis in completing membrane fission. As ARF1 and ARF6 are structurally conserved, we have also modeled ARF1 onto the ARF6 lattice, which has allowed us to pursue the reconstitution of Coat Protein I (COPI) vesicles to confirm more definitively that the ARF lattice acts in vesicle fission. Our findings are notable for having achieved the first detailed glimpse of how a small GTPase bends membrane and having provided a molecular understanding of how an ARF protein acts in vesicle fission.

Kinetics of low radioactive wastewater imbibition and radionuclides sorption in partially saturated ternary-binder mortar.

This study seeks to assess the imbibition kinetics of low radioactive wastewater (from the DayaBay nuclear power plant) into a partially saturated ternary-binder mortar, as well as the sorption kinetics of 60Co and 137Cs from the water. Mortar samples with the initial saturation degrees of 0, 0.4, 0.6, 0.8 and 1.0 were prepared for the wastewater treatment. Pore structure of the mortar was characterized using water vapor sorption isotherm and mercury intrusion porosimetry tests interpreted by the Guggenheim-Anderson-de Boer isothermal equilibrium, and volume- and energy-based fractal models. Results show that the mortar has consistent fractal pore structure between the models, and the liquid imbibitions follow the fractal imbibition kinetics, in which the parameters are non-linearly impacted by the initial saturation degrees. The sorption rate and retention capacity of 137Cs are much lower than those of 60Co, and both follow the Brouers-Sotolongo fractional kinetics. The findings uncover the complex liquid imbibition and radionuclides sorption kinetics in cement-based porous materials, and the in-situ data would contribute to the material designs and sorption controls for large scale in-situ treatments of wastewater from nuclear power plant.

Structural insights into membrane remodeling by SNX1.

The sorting nexin (SNX) family of proteins deform the membrane to generate transport carriers in endosomal pathways. Here, we elucidate how a prototypic member, SNX1, acts in this process. Performing cryoelectron microscopy, we find that SNX1 assembles into a protein lattice that consists of helical rows of SNX1 dimers wrapped around tubular membranes in a crosslinked fashion. We also visualize the details of this structure, which provides a molecular understanding of how various parts of SNX1 contribute to its ability to deform the membrane. Moreover, we have compared the SNX1 structure with a previously elucidated structure of an endosomal coat complex formed by retromer coupled to a SNX, which reveals how the molecular organization of the SNX in this coat complex is affected by retromer. The comparison also suggests insight into intermediary stages of assembly that results in the formation of the retromer-SNX coat complex on the membrane.

The Unusual Homodimer of a Heme-Copper Terminal Oxidase Allows Itself to Utilize Two Electron Donors.

The heme-copper oxidase superfamily comprises cytochrome c and ubiquinol oxidases. These enzymes catalyze the transfer of electrons from different electron donors onto molecular oxygen. A B-family cytochrome c oxidase from the hyperthermophilic bacterium Aquifex aeolicus was discovered previously to be able to use both cytochrome c and naphthoquinol as electron donors. Its molecular mechanism as well as the evolutionary significance are yet unknown. Here we solved its 3.4 Šresolution electron cryo-microscopic structure and discovered a novel dimeric structure mediated by subunit I (CoxA2) that would be essential for naphthoquinol binding and oxidation. The unique structural features in both proton and oxygen pathways suggest an evolutionary adaptation of this oxidase to its hyperthermophilic environment. Our results add a new conceptual understanding of structural variation of cytochrome c oxidases in different species.

A bibliometric analysis of infectious diseases in patients with liver transplantation in the last decade.

BACKGROUND: A bibliometric analysis was performed to reveal the current status of investigations in infectious diseases in patients with liver transplantation (LT) and to prioritize future research needs. METHODS: The present study comprehensively retrieved publications relevant to infectious diseases in LT recipients published between 2010 and 2020. The search was conducted on the Web of Science (WoS) database. A bibliometric analysis was conducted through machine learning and visualization tools, including VOSviewer, Bibliographic Item Co-Occurrence Matrix Builder, and Graphical Clustering Toolkit. Research hotspots and trends in the field were assessed, while the contributions and collaborations of countries, institutions, and authors were documented. RESULTS: A total of 691 publications were analyzed. Research output sharply increased in 2015, with a fast drop afterward. "Liver transplantation" was the most frequent keyword, with strong links to "hepatitis C virus" and "infection". Study areas included risk factors of infectious diseases in LT recipients, pathogens causing post-transplantation infections, antibacterial therapy and prophylaxis for peritransplant infection complications, living donor LT, and pediatric LT. The efficacy and safety of direct-acting antivirals (DAAs) for hepatitis C virus (HCV) infection among liver transplant recipients has attracted recent research interest. Didier Samuel was the most productive author, while Xavier Forns was the top-cited author. Shanghai Jiao Tong University was the most productive contributor, and Gilead Sciences was the most cited organization. Moreover, the USA was the greatest contributor. Gastroenterology was the most cited journal, while Liver Transplantation was the most prolific journal. CONCLUSIONS: This bibliometric analysis will better understand the research status of infectious complications in LT recipients and forecast future research trends. Priority should be given to identifying risk factors for peritransplantation infections and effective treatments against infectious complications in the coming years.

High mortality associated with gram-negative bacterial bloodstream infection in liver transplant recipients undergoing immunosuppression reduction.

BACKGROUND: Immunosuppression is an important factor in the incidence of infections in transplant recipient. Few studies are available on the management of immunosuppression (IS) treatment in the liver transplant (LT) recipients complicated with infection. The aim of this study is to describe our experience in the management of IS treatment during bacterial bloodstream infection (BSI) in LT recipients and assess the effect of temporary IS withdrawal on 30 d mortality of recipients presenting with severe infection. AIM: To assess the effect of temporary IS withdrawal on 30 d mortality of LT recipients presenting with severe infection. METHODS: A retrospective study was conducted with patients diagnosed with BSI after LT in the Department of Liver Surgery, Renji Hospital from January 1, 2016 through December 31, 2017. All recipients diagnosed with BSI after LT were included. Univariate and multivariate Cox regression analysis of risk factors for 30 d mortality was conducted in the LT recipients with Gram-negative bacterial (GNB) infection. RESULTS: Seventy-four episodes of BSI were identified in 70 LT recipients, including 45 episodes of Gram-positive bacterial (GPB) infections in 42 patients and 29 episodes of GNB infections in 28 patients. Overall, IS reduction (at least 50% dose reduction or cessation of one or more immunosuppressive agent) was made in 28 (41.2%) cases, specifically, in 5 (11.9%) cases with GPB infections and 23 (82.1%) cases with GNB infections. The 180 d all-cause mortality rate was 18.5% (13/70). The mortality rate in GNB group (39.3%, 11/28) was significantly higher than that in GPB group (4.8%, 2/42) (P = 0.001). All the deaths in GNB group were attributed to worsening infection secondary to IS withdrawal, but the deaths in GPB group were all due to graft-versus-host disease. GNB group was associated with significantly higher incidence of intra-abdominal infection, IS reduction, and complete IS withdrawal than GPB group (P < 0.05). Cox regression showed that rejection (adjusted hazard ratio 7.021, P = 0.001) and complete IS withdrawal (adjusted hazard ratio 12.65, P = 0.019) were independent risk factors for 30 d mortality in patients with GNB infections after LT. CONCLUSION: IS reduction is more frequently associated with GNB infection than GPB infection in LT recipients. Complete IS withdrawal should be cautious due to increased risk of mortality in LT recipients complicated with BSI.

Cryo-EM structures of S-OPA1 reveal its interactions with membrane and changes upon nucleotide binding.

Mammalian mitochondrial inner membrane fusion is mediated by optic atrophy 1 (OPA1). Under physiological conditions, OPA1 undergoes proteolytic processing to form a membrane-anchored long isoform (L-OPA1) and a soluble short isoform (S-OPA1). A combination of L-OPA1 and S-OPA1 is essential for efficient membrane fusion; however, the relevant mechanism is not well understood. In this study, we investigate the cryo-electron microscopic structures of S-OPA1-coated liposomes in nucleotide-free and GTPγS-bound states. S-OPA1 exhibits a general dynamin-like structure and can assemble onto membranes in a helical array with a dimer building block. We reveal that hydrophobic residues in its extended membrane-binding domain are critical for its tubulation activity. The binding of GTPγS triggers a conformational change and results in a rearrangement of the helical lattice and tube expansion similar to that of S-Mgm1. These observations indicate that S-OPA1 adopts a dynamin-like power stroke membrane remodeling mechanism during mitochondrial inner membrane fusion.

Effectiveness of tigecycline in the treatment of infections caused by carbapenem-resistant gram-negative bacteria in pediatric liver transplant recipients: A retrospective study.

INTRODUCTION: Tigecycline (TGC) is effective for the infections caused by carbapenem-resistant gram-negative bacteria (CRGNB) in adults, but it is not investigated systematically in children because of concern about adverse effects. This study aimed to analyze the effectiveness of TGC in treating CRGNB infections in children after receiving liver transplant. METHODS: The subjects in this retrospective study were pediatric liver transplant recipients treated with TGC for at least 3 days to fight microbiologically verified CRGNB infection after initial antibiotic failure during the period from January 2014 to May 2018. Clinical and microbiological outcomes were reviewed to evaluate the efficacy and safety of TGC. RESULTS: Of the 1177 pediatric liver transplant recipients, 13 patients were eligible for inclusion in this analysis. All the patients received TGC at dose of 2 mg/kg every 12 hours for a duration of 10.1 ± 5.1 days on average to treat CRGNB infections, including complicated intra-abdominal infection, ventilator-associated pneumonia, and bloodstream infection. The isolates included Klebsiella pneumoniae (69.2%, 9/13) and Acinetobacter baumannii (30.8%, 4/13). Clinical efficacy was achieved in 84.6% (11/13) and pathogen eradicated in 69.2% (9/13) of the patients. The overall mortality rate was 15.4% (2/13). No TGC-related serious adverse event was reported. CONCLUSION: Tigecycline can be considered in combination antimicrobial regimen for treating CRGNB-related infections in pediatric liver transplant recipients.

A 3.3†Å-Resolution Structure of Hyperthermophilic Respiratory Complex†III Reveals the Mechanism of Its Thermal Stability.

Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Šresolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N-terminus of cyt. c1 was found to greatly enhance the interaction between cyt. b and cyt. c1 , and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment.

Minimal change disease induced by tiopronin: a rare case report and a review of the literature.

Tiopronin (TP), a glycine derivative with a free thiol, is extensively used for the treatment of cystinuria. Moreover, TP is usually prescribed as hepatoprotective medicine in China. In the present case, a 36-year-old female who presented with foamy urine and general edema was admitted to the hospital. She had been taking TP for six months to treat drug-induced liver injury due to anti-tuberculosis drugs including isoniazid, rifampicin and pyrazinamide. The urine tests at admission revealed nephritic-range proteinuria with a daily urinary protein level of 8,024 mg. Meanwhile, albumin and cholesterol levels were abnormal. The light microscopy was negative and electron microscopy showed foot process effacement. Thus, minimal change disease (MCD) was diagnosed, and TP was consequently discontinued. Finally, the patient accomplished complete remission within five weeks after the cessation of TP without undergoing glucocorticoid therapy. TP was speculated to play an antigenic role in this adverse effect. To date, there are only two similar cases documented in the literature. Herein, we first report a case of a Chinese patient who generated MCD after prolonged TP administration. Clinicians should be wary of the occurrence of MCD due to TP when administering long-term therapy of TP. A weekly urinalysis may be useful for early identification of TP-induced MCD.

ACAP1 assembles into an unusual protein lattice for membrane deformation through multiple stages.

Studies on the Bin-Amphiphysin-Rvs (BAR) domain have advanced a fundamental understanding of how proteins deform membrane. We previously showed that a BAR domain in tandem with a Pleckstrin Homology (PH domain) underlies the assembly of ACAP1 (Arfgap with Coil-coil, Ankryin repeat, and PH domain I) into an unusual lattice structure that also uncovers a new paradigm for how a BAR protein deforms membrane. Here, we initially pursued computation-based refinement of the ACAP1 lattice to identify its critical protein contacts. Simulation studies then revealed how ACAP1, which dimerizes into a symmetrical structure in solution, is recruited asymmetrically to the membrane through dynamic behavior. We also pursued electron microscopy (EM)-based structural studies, which shed further insight into the dynamic nature of the ACAP1 lattice assembly. As ACAP1 is an unconventional BAR protein, our findings broaden the understanding of the mechanistic spectrum by which proteins assemble into higher-ordered structures to achieve membrane deformation.

Correction: New interfaces on MiD51 for Drp1 recruitment and regulation.

[This corrects the article DOI: 10.1371/journal.pone.0211459.].

New interfaces on MiD51 for Drp1 recruitment and regulation.

Mitochondrial fission is facilitated by dynamin-related protein Drp1 and a variety of its receptors. However, the molecular mechanism of how Drp1 is recruited to the mitochondrial surface by receptors MiD49 and MiD51 remains elusive. Here, we showed that the interaction between Drp1 and MiD51 is regulated by GTP binding and depends on the polymerization of Drp1. We identified two regions on MiD51 that directly bind to Drp1, and found that dimerization of MiD51, relevant to residue C452, is required for mitochondrial dynamics regulation. Our Results have suggested a multi-faceted regulatory mechanism for the interaction between Drp1 and MiD51 that illustrates the potentially complicated and tight regulation of mitochondrial fission.

Structural Insight into the Specific DNA Template Binding to DnaG primase in Bacteria.

Bacterial primase initiates the repeated synthesis of short RNA primers that are extended by DNA polymerase to synthesize Okazaki fragments on the lagging strand at replication forks. It remains unclear how the enzyme recognizes specific initiation sites. In this study, the DnaG primase from Bacillus subtilis (BsuDnaG) was characterized and the crystal structure of the RNA polymerase domain (RPD) was determined. Structural comparisons revealed that the tethered zinc binding domain plays an important role in the interactions between primase and specific template sequence. Structural and biochemical data defined the ssDNA template binding surface as an L shape, and a model for the template ssDNA binding to primase is proposed. The flexibility of the DnaG primases from B. subtilis and G. stearothermophilus were compared, and the results implied that the intrinsic flexibility of the primase may facilitate the interactions between primase and various partners in the replisome. These results shed light on the mechanism by which DnaG recognizes the specific initiation site.

Structural characterization of coatomer in its cytosolic state.

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli by using BrkA autotransporter.

BACKGROUND: Bacterial surface display technique enables the exogenous proteins or polypeptides displayed on the bacterial surface, while maintaining their relatively independent spatial structures and biological activities. The technique makes recombinant bacteria possess the expectant functions, subsequently, directly used for many applications. Many proteins could be used to achieve bacterial surface display, among them, autotransporter, a member of the type V secretion system of gram-negative bacteria, has been extensively studied because of its modular structure and apparent simplicity. However, autotransporter has not been widely used at present due to lack of a convenient genetic vector system. With our recently characterized autotransporter BrkA (Bordetella serum-resistance killing protein A) from Bordetella pertussis, we are aiming to develop a new autotransporter-based surface display system for potential wide application. RESULTS: Here, we construct a bacterial surface display system named as BrkAutoDisplay, based on the structure of autotransporter BrkA. BrkAutoDisplay is a convenient system to host exogenous genes. In our test, this system is good to efficiently display various proteins on the outer membrane surface of Escherichia coli, including green fluorescent protein (GFP), various enzymes and single chain antibody. Moreover, the displayed GFP possesses green fluorescence, the enzymes CotA, EstPc and PalA exhibit catalytic activity 0.12, 6.88 and 0.32 mU (per 5.2 × 10(8) living bacteria cells) respectively, and the single chain antibody fragment (scFv) can bind with its antigen strongly. Finally, we showed that C41(DE3) is a good strain of E. coli for the successful functionality of BrkAutoDisplay. CONCLUSIONS: We designed a new bacterial display system called as BrkAutoDisplay and displayed various exogenous proteins on E. coli surface. Our results indicate that BrkAutoDisplay system is worthy of further study for industrial applications.

A PH domain in ACAP1 possesses key features of the BAR domain in promoting membrane curvature.

The BAR (Bin-Amphiphysin-Rvs) domain undergoes dimerization to produce a curved protein structure, which superimposes onto membrane through electrostatic interactions to sense and impart membrane curvature. In some cases, a BAR domain also possesses an amphipathic helix that inserts into the membrane to induce curvature. ACAP1 (Arfgap with Coil coil, Ankyrin repeat, and PH domain protein 1) contains a BAR domain. Here, we show that this BAR domain can neither bind membrane nor impart curvature, but instead requires a neighboring PH (Pleckstrin Homology) domain to achieve these functions. Specific residues within the PH domain are responsible for both membrane binding and curvature generation. The BAR domain adjacent to the PH domain instead interacts with the BAR domains of neighboring ACAP1 proteins to enable clustering at the membrane. Thus, we have uncovered the molecular basis for an unexpected and unconventional collaboration between PH and BAR domains in membrane bending.

Molecular insights into the membrane-associated phosphatidylinositol 4-kinase IIα.

Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structure impairs our understanding of the mechanism regulating kinase activity. Here we present the crystal structure of human PI4KIIα in ADP-bound form. The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks. Two structural insertions, a palmitoylation insertion and an RK-rich insertion, endow PI4KIIα with the 'integral' membrane-binding feature. Molecular dynamics simulations, biochemical and mutagenesis studies reveal that the palmitoylation insertion, containing an amphipathic helix, contributes to the PI-binding pocket and anchors PI4KIIα to the membrane, suggesting that fluctuation of the palmitoylation insertion affects PI4KIIα's activity. We conclude from our results that PI4KIIα's activity is regulated indirectly through changes in the membrane environment.

Revealing various coupling of electron transfer and proton pumping in mitochondrial respiratory chain.

Cellular respiration is the process that releases energy from food and supplies energy for life processes. The mitochondrial respiratory chain is the final and most important step for cellular respiration and is located on the inner membrane of mitochondrion and comprises four large trans-membrane protein complexes (respiratory chain Complexes I, II, III and IV) as well as ubiquinone between Complexes I/II and III and cytochrome c between Complexes III and IV. The function of mitochondrial respiratory chain is biological oxidation by transferring electrons from NADH and succinate to oxygen and then generating proton gradient across the inner membrane. Such proton gradient is utilized by ATP synthase (ATPase, also called as Complex V) to produce energy molecules ATP. Structural studies of mitochondrial respiratory membrane protein complexes are important to understand the mechanism of electron transfer and the redox-coupled proton translocation across the inner membrane. Here, according to the time line, we reviewed the great achievements on structural studies of mitochondrial respiratory complexes in the past twenty years as well as the recent research progresses on the structures of mitochondrial respiratory supra-complexes.

Flexible interwoven termini determine the thermal stability of thermosomes.

Group II chaperonins, which assemble as double-ring complexes, assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner. The molecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated. Here, we selected the group II chaperonins (cpn-α and cpn-ß), also called thermosomes, from Acidianus tengchongensis and investigated their assembly and thermal stability. We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities. Cpn-ß is more thermally stable than cpn-α, while the thermal stability of the hetero thermosome cpn-αß is intermediate. Cryo-electron microscopy reconstructions of cpn-α and cpn-ß revealed the interwoven densities of their non-conserved flexible N/C-termini around the equatorial planes. The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interactions in the assembly and thermal stability of the thermosomes.