The structure of MgtE in the absence of magnesium provides new insights into channel gating.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Structural insights into eRF3 and stop codon recognition by eRF1.

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.

A genetic approach for analyzing the co-operative function of the tRNA mimicry complex, eRF1/eRF3, in translation termination on the ribosome.

During termination of translation in eukaryotes, a GTP-binding protein, eRF3, functions within a complex with the tRNA-mimicking protein, eRF1, to decode stop codons. It remains unclear how the tRNA-mimicking protein co-operates with the GTPase and with the functional sites on the ribosome. In order to elucidate the molecular characteristics of tRNA-mimicking proteins involved in stop codon decoding, we have devised a heterologous genetic system in Saccharomyces cerevisiae. We found that eRF3 from Pneumocystis carinii (Pc-eRF3) did not complement depletion of S. cerevisiae eRF3. The strength of Pc-eRF3 binding to Sc-eRF1 depends on the GTP-binding domain, suggesting that defects of the GTPase switch in the heterologous complex causes the observed lethality. We isolated mutants of Pc-eRF3 and Sc-eRF1 that restore cell growth in the presence of Pc-eRF3 as the sole source of eRF3. Mapping of these mutations onto the latest 3D-complex structure revealed that they were located in the binding-interface region between eRF1 and eRF3, as well as in the ribosomal functional sites. Intriguingly, a novel functional site was revealed adjacent to the decoding site of eRF1, on the tip domain that mimics the tRNA anticodon loop. This novel domain likely participates in codon recognition, coupled with the GTPase function.

Anesthesia Management Using Remimazolam for Lower Limb Amputation in a Patient With Septic Shock Due to Necrotizing Fasciitis.

We report a case of a patient with necrotizing fasciitis and septic shock caused by streptococcal toxic shock syndrome, who was anesthetized and managed with remimazolam. The patient, a woman in her 40s, was admitted to the ICU with a diagnosis of necrotizing fasciitis of the right lower extremity and septic shock and was scheduled for above-the-knee amputation under general anesthesia. She was anesthetized with remimazolam for sedation and fentanyl and remifentanil for analgesia. Intraoperatively, we were able to maintain hemodynamic stability with similar or only slightly higher doses of circulatory agonists during admission. In the present case, remimazolam, an ultrashort-acting benzodiazepine, was safely used to provide anesthesia to a patient in septic shock due to necrotizing fasciitis, who was receiving high doses of vasopressor agents for cardiovascular support, as it was necessary to select an anesthetic drug that would cause minimal circulatory depression.

Omnipotent role of archaeal elongation factor 1 alpha (EF1α in translational elongation and termination, and quality control of protein synthesis.

The molecular mechanisms of translation termination and mRNA surveillance in archaea remain unclear. In eukaryotes, eRF3 and HBS1, which are homologous to the tRNA carrier GTPase EF1α, respectively bind eRF1 and Pelota to decipher stop codons or to facilitate mRNA surveillance. However, genome-wide searches of archaea have failed to detect any orthologs to both GTPases. Here, we report the crystal structure of aRF1 from an archaeon, Aeropyrum pernix, and present strong evidence that the authentic archaeal EF1α acts as a carrier GTPase for aRF1 and for aPelota. The binding interface residues between aRF1 and aEF1α predicted from aRF1·aEF1α·GTP ternary structure model were confirmed by in vivo functional assays. The aRF1/eRF1 structural domain with GGQ motif, which corresponds to the CCA arm of tRNA, contacts with all three structural domains of aEF1α showing striking tRNA mimicry of aRF1/eRF1 and its GTPase-mediated catalysis for stop codon decoding. The multiple binding capacity of archaeal EF1α explains the absence of GTPase orthologs for eRF3 and HBS1 in archaea species and suggests that universal molecular mechanisms underlie translational elongation and termination, and mRNA surveillance pathways.

The CGA codon decoding through tRNAArg (ICG) supply governed by Tad2/Tad3 in Saccharomyces cerevisiae.

The CGA codon is a rare codon in Saccharomyces cerevisiae and is known to be inefficiently decoded by wobble pairing with Arg-tRNA(ICG). The tRNAArg (ICG) is post-transcriptionally edited from tRNAArg (ACG) by the anticodon first adenosine deamination enzyme Tad2/Tad3 complex. Experimental consecutive CGA codons cause ribosome stalling to result in the reduction of the encoding protein product. In this study, the additional supply of tRNAArg (ACG) genes that produce decoding Arg-tRNA(ICG) promoted the product level from the CGA12-luc reporter, revealing that the product reduction is essentially due to inefficient decoding and deficiency in the tRNA supply. The mature tRNAArg (ICG) and the precursor tRNAArg (ACG) ratios examined for cellular tRNA fraction revealed that the tRNAArg (ICG) ratio is maintained at less than 30% and is responsive to the Tad2/Tad3 expression level.

Systematic genetic identification of functional domains on collided di-ribosomes responsible for rescue pathways upon translation arrest in Saccharomyces cerevisiae.

Translation elongation becomes arrested when various obstacles arise, such as a series of inefficient rare codons or stable RNA secondary structures, thus causing ribosomal stalling along the mRNA. Certain wasteful and persistent stalling states are resolved by ribosome rescue pathways. For instance, collisions between stalled and subsequent ribosomes are thought to induce ubiquitination of ribosomal S20 protein by the E3 ubiquitin ligase Hel2, which triggers subsequent rescue reactions. Although structural studies have revealed specific contact sites between collided ribosomes, the ribosomal regions crucial for the rescue reaction remain uncharacterized. In this study, we performed a systematic genetic analysis to identify the molecular regions required for ribosome rescue in Saccharomyces cerevisiae. A series of dominant negative mutations capable of abolishing the rescue reaction were isolated in ribosomal proteins S20 and Asc1. Moreover, mutations in both proteins clustered on the surface of ribosomes between the collided ribosome interfaces, aligned in such a way that they seemingly faced each other. Further analysis via the application of the split-TRP1 protein assay revealed that the mutation of either protein distinctively affected the functional interaction between Hel2 and Asc1, suggesting the development of differential functionality at the interface between collided ribosomes. Our results provide novel and complementary insights into the detailed molecular mechanisms of ribosomal rescue pathways.

A rapid and convenient enzyme digestion method for the analysis of N-glycans using exoglycosidase-impregnated polyacrylamide gels fabricated in an automatic pipette tip.

Efficient enzymatic digestion methods are critical for the characterization and identification of glycans. Glycan hydrolysis enzymes are widely utilized for the identification of glycoprotein or glycolipid glycans. The commonly utilized in solution glycan hydrolysis methods require several hours of incubation with enzymes for complete removal of their target monosaccharides. To develop an efficient and simple method for the rapid release of monosaccharides from glycoprotein glycans, we fabricated exoglycosidase-impregnated acrylamide gels in an automatic pipette tip. Our automated enzymatic reactors are based on the simple photochemical copolymerization of monomers comprising acrylamide and methylene-bis-acrylamide-containing enzymes with an azobis compound functioning as the photocatalytic initiator. After filling the tip of the automatic pipette with these acrylamide solutions, polymerization of the acrylamide gel solution was performed by irradiation with a LED. The immobilized enzymes maintained their activities in the pipette tips and their action was completed by fully automatic pipetting for 10 to 30 min. We utilized 8-aminopyrene-1, 3, 6-trisulfonic acid (APTS)-labeled glycans as a substrate and measured by capillary electrophoresis (CE) before and after enzymatic digestion. We demonstrated that this method exhibited quantitative enzymatic and specific cleavage of monosaccharides from glycoprotein glycans.

Human ABCE1 exhibits temperature-dependent heterologous co-functionality in S. cerevisiae.

ABCE1 protein (Rli1 in Saccharomyces cerevisiae) is a unique ribosome recycling factor that is composed of an N-terminal FeS cluster domain and two ATPase domains. Here, we report that heterologous expression of human ABCE1 in S. cerevisiae is unable to complement conditional knockout of ABCE1 (Rli1), at a typical experimental temperature of 30 °C. However, low but significant growth was observed at high temperature, 37 °C. Considering the close interaction of ABCE1 with translation factors and ribosomal components, the observed temperature-dependent complementation may be attributed to heterologous co-functionality of ABCE1 with S. cerevisiae factor(s), and might reflect functional upregulation of human ABCE1 at its functional temperature.

Improving Skin cancer Management with ARTificial Intelligence (SMARTI): protocol for a preintervention/postintervention trial of an artificial intelligence system used as a diagnostic aid for skin cancer management in a specialist dermatology setting.

INTRODUCTION: Convolutional neural networks (CNNs) can diagnose skin cancers with impressive accuracy in experimental settings, however, their performance in the real-world clinical setting, including comparison to teledermatology services, has not been validated in prospective clinical studies. METHODS AND ANALYSIS: Participants will be recruited from dermatology clinics at the Alfred Hospital and Skin Health Institute, Melbourne. Skin lesions will be imaged using a proprietary dermoscopic camera. The artificial intelligence (AI) algorithm, a CNN developed by MoleMap Ltd and Monash eResearch, classifies lesions as benign, malignant or uncertain. This is a preintervention/postintervention study. In the preintervention period, treating doctors are blinded to AI lesion assessment. In the postintervention period, treating doctors review the AI lesion assessment in real time, and have the opportunity to then change their diagnosis and management. Any skin lesions of concern and at least two benign lesions will be selected for imaging. Each participant's lesions will be examined by a registrar, the treating consultant dermatologist and later by a teledermatologist. At the conclusion of the preintervention period, the safety of the AI algorithm will be evaluated in a primary analysis by measuring its sensitivity, specificity and agreement with histopathology where available, or the treating consultant dermatologists' classification. At trial completion, AI classifications will be compared with those of the teledermatologist, registrar, treating dermatologist and histopathology. The impact of the AI algorithm on diagnostic and management decisions will be evaluated by: (1) comparing the initial management decision of the registrar with their AI-assisted decision and (2) comparing the benign to malignant ratio (for lesions biopsied) between the preintervention and postintervention periods. ETHICS AND DISSEMINATION: Human Research Ethics Committee (HREC) approval received from the Alfred Hospital Ethics Committee on 14 February 2019 (HREC/48865/Alfred-2018). Findings from this study will be disseminated through peer-reviewed publications, non-peer reviewed media and conferences. TRIAL REGISTRATION NUMBER: NCT04040114.

Misdecoding of rare CGA codon by translation termination factors, eRF1/eRF3, suggests novel class of ribosome rescue pathway in S. cerevisiae.

The CGA arginine codon is a rare codon in Saccharomyces cerevisiae. Thus, full-length mature protein synthesis from reporter genes with internal CGA codon repeats are markedly reduced, and the reporters, instead, produce short-sized polypeptides via an unknown mechanism. Considering the product size and similar properties between CGA sense and UGA stop codons, we hypothesized that eukaryote polypeptide-chain release factor complex eRF1/eRF3 catalyses polypeptide release at CGA repeats. Herein, we performed a series of analyses and report that the CGA codon can be, to a certain extent, decoded as a stop codon in yeast. This also raises an intriguing possibility that translation termination factors eRF1/eRF3 rescue ribosomes stalled at CGA codons, releasing premature polypeptides, and competing with canonical tRNAICG to the CGA codon. Our results suggest an alternative ribosomal rescue pathway in eukaryotes. The present results suggest that misdecoding of low efficient codons may play a novel role in global translation regulation in S. cerevisiae.

Crystal structure of plant vacuolar iron transporter VIT1.

The iron ion is an essential cofactor in several vital enzymatic reactions, such as DNA replication, oxygen transport, and respiratory and photosynthetic electron transfer chains, but its excess accumulation induces oxidative stress in cells. Vacuolar iron transporter 1 (VIT1) is important for iron homeostasis in plants, by transporting cytoplasmic ferrous ions into vacuoles. Modification of the VIT1 gene leads to increased iron content in crops, which could be used for the treatment of human iron deficiency diseases. Furthermore, a VIT1 from the malaria-causing parasite Plasmodium is considered as a potential drug target for malaria. Here we report the crystal structure of VIT1 from rose gum Eucalyptus grandis, which probably functions as a H+-dependent antiporter for Fe2+ and other transition metal ions. VIT1 adopts a novel protein fold forming a dimer of five membrane-spanning domains, with an ion-translocating pathway constituted by the conserved methionine and carboxylate residues at the dimer interface. The second transmembrane helix protrudes from the lipid membrane by about 40 Å and connects to a three-helical bundle, triangular cytoplasmic domain, which binds to the substrate metal ions and stabilizes their soluble form, thus playing an essential role in their transport. These mechanistic insights will provide useful information for the further design of genetically modified crops and the development of anti-malaria drugs.

Tight interaction of eEF2 in the presence of Stm1 on ribosome.

The stress-related protein Stm1 interacts with ribosomes, and is implicated in repressing translation. Stm1 was previously studied both in vivo and in vitro by cell-free translation systems using crude yeast lysates, but its precise functional mechanism remains obscure. Using an in vitro reconstituted translation system, we now show that Stm1 severely inhibits translation through its N-terminal region, aa 1 to 107, and this inhibition is antagonized by eEF3. We found that Stm1 stabilizes eEF2 on the 80 S ribosome in the GTP-bound form, independently of eEF2's diphthamide modification, a conserved post-translational modification at the tip of domain IV. Systematic analyses of N- or C-terminal truncated mutants revealed that the core region of Stm1, aa 47 to 143, is crucial for its ribosome binding and eEF2 stabilization. Stm1 does not inhibit the 80 S-dependent GTPase activity of eEF2, at least during the first round of GTP-hydrolysis. The mechanism and the role of the stable association of eEF2 with the ribosome in the presence of Stm1 are discussed in relation to the translation repression by Stm1.

Mutations in the G-domain of Ski7 cause specific dysfunction in non-stop decay.

Ski7 functions as a cofactor in both normal mRNA turnover and non-stop mRNA decay (NSD) mRNA surveillance in budding yeast. The N-terminal region of Ski7 (Ski7N) interacts with the ski-complex and the exosome. The C-terminal region of Ski7 (Ski7C) binds guanine nucleotides and shares overall sequence and structural homology with the proteins of the translational GTPase superfamily, especially the tRNA/tRNA-mimic carrier protein subfamilies such as EF1α, eRF3, and Hbs1. Previous reports showed that Ski7N polypeptide functions adequately in vivo, while Ski7C, if any, only slightly. Furthermore, Ski7C does not exhibit GTP-hydrolysing activities under normal conditions. Therefore, the physiological and functional significance of the conserved Ski7C is unclear. Here, we report strong genetic evidence suggesting differential roles for Ski7N and Ski7C in normal and specific mRNA turnover pathways by creating/isolating mutations in both Ski7N and Ski7C conserved motifs using indicator yeast strains. We concluded that Ski7C participates in mRNA surveillance as a regulatory module competitively with the Hbs1/Dom34 complex. Our results provide insights into the molecular regulatory mechanisms underlying mRNA surveillance.

Remifentanil-induced alterations in neutrophil numbers after surgery.

BACKGROUND: Neutrophils are the first line of defense against invasive microorganisms during and after surgery. There is a possibility that different opioid analgesics used during surgery have different effects on the leucocyte count. We retrospectively analyzed the numbers of leucocytes, neutrophils, and lymphocytes just after surgery in patients who received remifentanil-based anesthesia and those who received fentanyl-based anesthesia.In female patients who underwent modified mastectomy or simple mastectomy with resection of a lymph node(s) or with biopsy of a sentinel lymph node(s) between January 2010 and December 2013 (n = 83), propensity score pairwise matching was performed according to the patient's age and procedure, and forty patients (Remifentanil group and Fentanyl group; n = 20 each) were analyzed. FINDINGS: Postoperative numbers of leucocytes and neutrophils were significantly lower in patients who received remifentanil-based anesthesia than in those who received fentanyl-based anesthesia (p = 0.03, p = 0.014; leucocytes and neutrophils, respectively). The increases in the numbers of leucocytes and neutrophils were significantly lower in the patients in the remifentanil group (p = 0.009, p = 0.0046; increase in leucocytes and neutrophils, respectively). CONCLUSIONS: In conclusion, remifentanil-based anesthesia attenuates postoperative leucocyte and neutrophil counts. It is unknown whether this phenomenon indicates the possibility of immunosuppression. Further studies are required.

Crystal structures of the TRIC trimeric intracellular cation channel orthologues.

Ca2+ release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca2+ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.

Outward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportin.

In vertebrates, the iron exporter ferroportin releases Fe(2+) from cells into plasma, thereby maintaining iron homeostasis. The transport activity of ferroportin is suppressed by the peptide hormone hepcidin, which exhibits upregulated expression in chronic inflammation, causing iron-restrictive anaemia. However, due to the lack of structural information about ferroportin, the mechanisms of its iron transport and hepcidin-mediated regulation remain largely elusive. Here we report the crystal structures of a putative bacterial homologue of ferroportin, BbFPN, in both the outward- and inward-facing states. Despite undetectable sequence similarity, BbFPN adopts the major facilitator superfamily fold. A comparison of the two structures reveals that BbFPN undergoes an intra-domain conformational rearrangement during the transport cycle. We identify a substrate metal-binding site, based on structural and mutational analyses. Furthermore, the BbFPN structures suggest that a predicted hepcidin-binding site of ferroportin is located within its central cavity. Thus, BbFPN may be a valuable structural model for iron homeostasis regulation by ferroportin.