A short guide on blue fluorescent proteins: limits and perspectives.

The advent of the so-called colorful biology era is in line with the discovery of fluorescent proteins (FPs), which can be widely used to detect the intracellular locations of macromolecules or to determine the abundance of metabolites in organelles. The application of multiple FPs that emit different spectra and colors could be implemented to precisely evaluate cellular events. FPs were initially established with the emergence of the green fluorescent protein (GFP) from jellyfish. Red fluorescent proteins (RFPs) from marine anemones and several corals adopt fluorescent chromophores that are similar to GFP. Chromophores of GFP and GFP-like FPs are formed through the oxidative rearrangement of three chromophore-forming residues, thereby limiting their application to only oxidative environments. Alternatively, some proteins can be fluorescent upon their interaction with cellular prosthetic cofactors and, thus, work in aerobic and anaerobic conditions. The modification of an NADPH-dependent blue fluorescent protein (BFP) also expanded its application to the quantization of NADPH in the cellular environment. However, cofactor-dependent BFPs have an intrinsic weakness of poor photostability with a high fluorescent background. This review explores GFP-derived and NADPH-dependent BFPs with a focus on NADPH-dependent BFPs, which might be technically feasible in the near future upon coupling with two-photon fluorescence microscopy or nucleic acid-mimickers. KEY POINTS: • Oxidation-dependent GFP-like BFPs and redox-free NADPH-dependent BFPs • GFPs of weak photostability and intensity with a high fluorescent background • Real-time imaging using mBFP under two-photon fluorescence microscopy.

Engineering of two thiamine diphosphate-dependent enzymes for the regioselective condensation of C1-formaldehyde into C4-erythrulose.

A number of carboligases, which catalyze condensation of C1- and/or C2-aldehydes into multi-carbon products, have been reported. However, their catalytic activities and/or regioselectivities remained rather low. Thereby, this study has focused on engineering of C1 and C2 carboligases for the regioselective condensation of C1-formaldehyde into C4-erythrulose via C2-glycolaldehyde. The crystal structure of the glyoxylate carboligase from Escherichia coli (EcGCL) was elucidated in complex with glycolaldehyde. A structure-guided rationale generated several mutants, one of whose catalytic activity reached 15.6 M-1·s-1, almost 10 times greater than the wild-type enzyme. Another variant (i.e., EcGCL_R484M/N283Q/L478M/M488L/R284K) has shown significantly increased stability to the glycolaldehyde toxicity, enabling production of glycolaldehyde to 31 mM from 75 mM formaldehyde (conversion: 83 %). Besides, the E1 subunit of α-ketoglutarate dehydrogenase complex from Vibrio vulnificus (VvSucA) was engineered as a regiospecific C2 carboligase for condensation of glycolaldehyde into erythrulose. The combination of EcGCL_R484M/N283Q/L478M/M488L/R284K and VvSucA_K228L led to the cascade production of erythrulose to 8 mM from 90 mM formaldehyde via glycolaldehyde without byproduct formation. This study will contribute to valorization of C1 gases into industrially relevant multi-carbon products in an environment-friendly way.

One-Pot Biosynthesis of 2-Keto-4-hydroxybutyrate from Cheap C1 Compounds Using Rationally Designed Pyruvate Aldolase and Methanol Dehydrogenase.

One-carbon chemicals (C 1s) are potential building blocks as they are cheap, sustainable, and abiotic components. Methanol-derived formaldehyde can be another versatile building block for the production of 2-keto-4-hydroxyacid derivatives that can be used for amino acids, hydroxy carboxylic acids, and chiral aldehydes. To produce 2-keto-4-hydroxybutyrate from C 1s in an environment-friendly way, we characterized an aldolase from Pseudomonas aeruginosa PAO1 (PaADL), which showed much higher catalytic activity in condensing formaldehyde and pyruvate than the reported aldolases. By applying a structure-based rational approach, we found a variant (PaADLV121A/L241A) that exhibited better catalytic activities than the wild-type enzyme. Next, we constructed a one-pot cascade biocatalyst system by combining PaADL and a methanol dehydrogenase (MDH) and, for the first time, effectively produced 2-keto-4-hydroxybutyrate as the main product from pyruvate and methanol via an enzymatic reaction. This simple process applied here will help design a green process for the production of 2-keto-4-hydroxyacid derivatives.

Structural characterization of the type I-B CRISPR Cas7 from Thermobaculum terrenum.

Clustered regularly interspaced short palindromic repeats (CRISPR) in many prokaryotes functions as an adaptive immune system against mobile genetic elements. A heterologous ribonucleoprotein silencing complex composed of CRISPR-associated (Cas) proteins and a CRISPR RNA (crRNA) neutralizes the incoming mobile genetic elements. The type I and III silencing complexes commonly include a protein-helical backbone of several copies of identical subunits, for example, Cas7 in the type I silencing complex. In this study, we structurally characterized type I-B Cas7 (Csh2 from Thermobaculum terrenum; TterCsh2). The revealed crystal structure of TterCsh2 shows a typical glove-like architecture of Cas7, which consists of a palm, a thumb, and a finger domain. Csh2 proteins have 5 conserved sequence motifs that are arranged to form a presumable crRNA-binding site in the TterCsh2 structure. This crRNA binding site of TterCsh2 is structurally and potentially comparable to those observed in helix-forming Cas7 structures in other sub-types. Analysis of the reported Cas7 structures and their sequences suggests that Cas7s can be divided into at least two sub-classes. These data will broaden our understanding on the Cascade complex of CRISPR/Cas systems.

Structural features of a minimal intact methyltransferase of a type I restriction-modification system.

Type I restriction-modification enzymes are oligomeric proteins composed of methylation (M), DNA sequence-recognition (S), and restriction (R) subunits. The different bipartite DNA sequences of 2-4 consecutive bases are recognized by two discerned target recognition domains (TRDs) located at the two-helix bundle of the two conserved regions (CRs). Two M-subunits and a single S-subunit form an oligomeric protein that functions as a methyltransferase (M2S1 MTase). Here, we present the crystal structure of the intact MTase from Vibrio vulnificus YJ016 in complex with the DNA-mimicking Ocr protein and the S-adenosyl-L-homocysteine (SAH). This MTase includes the M-domain with a helix tail (M-tail helix) and the S1/2-domain of a TRD and a CR α-helix. The Ocr binds to the cleft of the TRD surface and SAH is located in the pocket within the M-domain. The solution- and negative-staining electron microscopy-based reconstructed (M1S1/2)2 structure reveals a symmetric (S1/2)2 assembly using two CR-helices and two M-tail helices as a pivot, which is plausible for recognizing two DNA regions of same sequence. The conformational flexibility of the minimal M1S1/2 MTase dimer indicates a particular state resembling the structure of M2S1 MTases.

Crystal structures of UDP-N-acetylmuramic acid L-alanine ligase (MurC) from Mycobacterium bovis with and without UDP-N-acetylglucosamine.

Peptidoglycan comprises repeating units of N-acetylmuramic acid, N-acetylglucosamine and short cross-linking peptides. After the conversion of UDP-N-acetylglucosamine (UNAG) to UDP-N-acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by UDP-N-acetylmuramic acid L-alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, the crystal structure of Mycobacterium bovis MurC (MbMurC) is reported, which exhibits a three-domain architecture for the binding of UNAM, ATP and an amino acid as substrates, with a nickel ion at the domain interface. The ATP-binding loop adopts a conformation that is not seen in other MurCs. In the UNAG-bound structure of MbMurC, the substrate mimic interacts with the UDP-binding domain of MbMurC, which does not invoke rearrangement of the three domains. Interestingly, the glycine-rich loop of the UDP-binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety of the ligand, but not with the pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC.

Resuscitative Endovascular Balloon Occlusion of the Aorta for an Iliac Artery Aneurysm: Case Report.

Isolated iliac artery aneurysm (IAA) is rare, but can be fatal. Emergency surgery is performed in cases of hemorrhagic shock due to a suddenly ruptured IAA, which may have a high mortality rate because of massive non-compressible torso hemorrhage (NCTH). Recently, resuscitative endovascular balloon occlusion of the aorta (REBOA) has been accepted as an alternative to aortic cross-clamping via open thoracotomy to achieve hemostasis in trauma patients with profound shock due to NCTH and is considered an emerging bridging therapy for damage control. However, there is limited information on the use of REBOA in non-trauma patients with shock. Herein, we describe a patient with impending cardiac arrest due to isolated ruptured IAA, in whom perioperative bleeding was successfully controlled by REBOA.

Expandability of Cephalic Veins after Brachial Plexus Block in Arteriovenous Fistula Formation for Hemodialysis.

BACKGROUND: Arteriovenous fistula (AVF) for hemodialysis is essential for patients with end-stage renal disease. However, it is difficult to maintain AVF reliably. It is vitally important to select proper blood vessels for AVF formation. In a previous study, a minimum diameter of 3 mm for the autologous vein was proposed. However, patients who did not meet the minimum vascular diameter before anesthesia, but fulfilled other criteria, showed satisfactory venous dilatation after brachial plexus block (BPB). This study investigated the extent of vein expansion by BPB and the surgical outcomes of dilated veins after BPB. METHODS: Sixty-one patients who underwent AVF formation using an autologous vein between August 2018 and December 2019 were included in the study. The clinical characteristics of the patient groups, hemodynamic parameters including the diameter of blood vessels before and after BPB, and complications were investigated. Based on the venous diameter measured by sonography before anesthesia, patients were divided into group A (26 patients) and group B (35 patients), with venous diameters <3 mm and ≥3 mm, respectively. RESULTS: The venous diameter expanded after anesthesia by 41% overall, by 62% in group A, and by 25% in group B. This difference between groups A and B was statistically significant (p=0.001). No other variables showed statistically significant differences. CONCLUSION: Sufficient venous dilatation was observed after BPB. Therefore, if the vein is sufficiently dilated after BPB, even in patients with a pre-anesthesia venous diameter <3 mm, surgery may still be performed with an expected desirable outcome.

Clinical Outcomes of Arteriovenous Graft in End-Stage Renal Disease Patients with an Unsuitable Cephalic Vein for Hemodialysis Access.

BACKGROUND: As the population of patients with end-stage renal disease has grown older, the proportion of patients with poorly preserved vasculature has concomitantly increased. Thus, arteriovenous grafts (AVG) have been used more frequently to access blood vessels for hemodialysis. Despite this increasing demand, studies of AVG are limited. In this study, we examined the surgical outcomes of upper-limb AVG creation. METHODS: Among the arteriovenous fistula formation procedures performed between January 2014 and March 2019 at Dankook University Hospital, 42 cases involved AVG creation. We compared patients in whom the axillary vein was used (group A; brachioaxillary AVG [B-Ax AVG]; n=20) with those in whom upper limb veins were used (group B; brachiobasilic AVG or brachioantecubital AVG; n=22). RESULTS: The 1-year primary patency rate was higher in group A than in group B (57.9% vs. 41.7%; p=0.262). The incidence of postoperative complications was not significantly different between groups. CONCLUSION: AVG using the axillary vein showed no major differences in safety or functionality compared to AVG using other veins. Therefore, accounting for age, underlying disease, and expected patient lifespan, B-Ax AVG can be considered an acceptable surgical method.

Structure-Guided Generation of a Redox-Independent Blue Fluorescent Protein from mBFP.

Fluorescent proteins, such as the green fluorescent protein, are used for detection of cellular components and events. However, green fluorescent protein and its derivatives have limited usage under anaerobic conditions and require a long maturation time. On the other hand, the NADPH-dependent blue fluorescent protein (BFP) without oxidative modification of residues is instantly functional in both aerobic and anaerobic systems. BFP proteins belong to a short-chain dehydrogenase/reductase (SDR) protein family, and their fluorescent property changes with reaction time in the presence of a substrate. With the aim of developing a better fluorescent reporter independent of redox state, we elucidated the crystal structure of a tetrameric mBFP from soil metagenomes with and without NADPH. Apart from the previously known regions, structure-guided mutational studies have identified several residues that contribute to the fluorescence of mBFP, including two aromatic residues (F97 and Y157) near the nicotinamide moiety of the bound NADPH. A single histidine mutation at Y157 (Y157H) has conferred more stabilized, time-independent fluorescence even in the presence of substrates. Furthermore, we discovered another SDR protein that can also emit blue fluorescence. These results open a new possibility for the development of BFP as a stable cellular reporter for widespread use, independent of subcellular environments.

Structural basis for the selective addition of an oxygen atom to cyclic ketones by Baeyer-Villiger monooxygenase from Parvibaculum lavamentivorans.

Baeyer-Villiger monooxygenase (BVMO) catalyzes insertion of an oxygen atom into aliphatic or cyclic ketones with high regioselectivity. The BVMOs from Parvibaculum lavamentivorans (BVMOParvi) and Oceanicola batsensis (BVMOOcean) are interesting because of their homologies, with >40% sequence identity, and reaction with the same cyclic ketones with a methyl moiety to give different products. The revealed BVMOParvi structure shows that BVMOParvi forms a two-domain structure like other BVMOs. It has two inserted residues, compared with BVMOOcean, that form a bulge near the bound flavin adenine dinucleotide in the active site. Furthermore, this bulge is linked to a nearby α-helix via a disulfide bond, probably restricting access of the bulky methyl group of the substrate to this bulge. Another sequence motif at the entrance of the active site (Ala-Ser in BVMOParvi and Ser-Thr in BVMOOcean) allows a large volume in BVMOParvi. These minute differences may discriminate a substrate orientation in both BVMOs from the initial substrate binding pocket to the final oxygenation site, resulting in the inserted oxygen atom being in different positions of the same substrate.

Structures of Zymomonas 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase with and without a Substrate Analog at the Phosphate-Binding Loop.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apo-structure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

Clinical Results of Arteriovenous Fistulas Constructed Using Autologous Vessels in End-Stage Renal Disease Patients on Hemodialysis.

BACKGROUND: For hemodialysis patients with end-stage renal disease (ESRD), it is important to construct an efficient vascular access with a superior patency rate. This study investigated the factors influencing the efficiency of arteriovenous fistulas (AVFs) constructed using an autologous vessel and evaluated the necessity of ultrasonography as a preoperative tool for AVF construction. METHODS: A retrospective analysis was performed of 250 patients in whom an AVF was constructed using an autologous vessel due to ESRD at our institution from January 2009 to April 2016. RESULTS: The 1-, 3-, and 5-year patency rates for all subjects were 87.6%, 85.6%, and 84.4%, respectively. The patients who underwent a preoperative evaluation of their vessels via ultrasonography had better patency rates than those who did not. Superior patency rates were found in patients under 65 years of age or with an anastomotic vein diameter of 3 mm or more. The 1-year patency rate and the diameter of the anastomotic vein showed a positive relationship. CONCLUSION: Ultrasonography is strongly recommended for AVF construction, and efforts should be made to increase the patency rate in patients over 65. Superior clinical results can be expected when an AVF is made using an autologous vessel with an anastomotic vein diameter of at least 3 mm.

Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA.

Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central ß-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the ß-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5'-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.

Surgical Outcomes of Forearm Loop Arteriovenous Fistula Formation Using Tapered versus Non-Tapered Polytetrafluoroethylene Grafts.

BACKGROUND: Tapered grafts, which have a smaller diameter on the arterial side, have been increasingly used for arteriovenous fistula (AVF) formation. We compared the outcomes of 4-6-mm tapered and 6-mm straight forearm loop arteriovenous grafts. METHODS: A total of 103 patients receiving forearm loop arteriovenous grafts between March 2005 and March 2015 were retrospectively analyzed and separated into 2 groups (group A, 4- to 6-mm tapered grafts, n=78; group B, 6-mm straight grafts, n=25). In each group, complications and patency rates after surgery were assessed. RESULTS: Clinical characteristics and laboratory results, except for cerebrovascular disease history (group A, 7.7%; group B, 28.0%; p=0.014), were similar between the groups. No significant differences were found for individual complications. Kaplan-Meier survival analysis revealed no significant differences in 1-year, 3-year, and 5-year patency rates between groups (61.8%, 44.9%, and 38.5% vs. 62.7%, 41.1%, and 35.3%, respectively). CONCLUSION: We found no significant differences in complication and patency rates between the tapered and straight graft groups. If there are no differences in complication and patency between the two graft types, tapered grafts may be a valuable option for AVF formation in light of their other advantages.

A Case of Severe Thoracoabdominal Impalement by a Steel Bar.

A 53-year-old man arrived at the trauma center with a steel bar penetrating from the epigastrium to the right scapula. He was hypotensive and hypoxic, and immediate resuscitation and basic evaluation were performed. An emergency operation was performed due to an unstable hemodynamic state. Multiple injuries were confirmed in the right lower lobe, posterior chest wall, diaphragm, and liver lateral segment. Right lower lobectomy and liver lateral sectionectomy were performed following removal of the bar. The patient recovered without additional hemorrhage after the surgery, and was transferred to a rehabilitation institution with periodic follow-up.

A Case of Recurrent Aortic Rupture Associated with Klebsiella pneumoniae Pericarditis Treated by Two Separate Aortic Operations.

A 49-year-old female presented with severe dyspnea. She was diagnosed with cardiac tamponade combined with ascending aortic pseudoaneurysm and rupture, which was caused by Klebsiella pneumoniae infection. This extremely rare condition was managed by an emergency pericardiostomy and two separate aortic operations. Antibiotics active for the K. pneumoniae isolate were used throughout. The patient was well for nine months after discharge and continues to be followed up for signs of possible reinfection.

Trapped Stent in the Left Coronary Sinus in a Myocardial Infarction Patient.

Stent entrapment is a very rare complication of percutaneous coronary intervention. The interventional approach could be a treatment strategy. However, if it does not work, surgical treatment should be considered. Here, we report a case of surgical treatment of stent entrapment in the left coronary sinus of a 53-year-old male patient.

An unusual case of superior vena cava syndrome caused by the intravascular invasion of an invasive thymoma.

Superior vena cava syndrome (SVCS) is usually caused by extrinsic compression or invasion of the superior vena cava (SVC) by malignant tumors involving mediastinal structures. Although thymomas are well-known causes of SVCS, cases of SVCS caused by malignant thymomas protruding into adjacent vessels draining the SVC with thrombosis have been very rarely reported worldwide. We experienced a 39-year-old female patient with SVCS that developed after the direct invasion of the left brachiocephalic vein (LBCV) and SVC by an anterior mediastinal mass with a high maximum standardized uptake value on the chest computed tomography (CT) and positron emission tomography-CT. Based on these results, she underwent en bloc resection of the tumor, including removal of the involved vessels, and was eventually diagnosed as having a type B2 thymoma permeating into the LBCV and SVC. We present this case as a very rare form of SVCS caused by an invasive thymoma.

Floating thrombus in aortic arch.

Floating thrombi in the aortic arch are very rare and an unusual source of systemic embolism. Herein, a case of a 3-cm thrombus in the aortic arch is reported. It was a floating, highly mobile thrombus attached to the lesser curvature of the aortic arch. The patients had a hypercoagulable disorder induced by protein C and S deficiency. The thrombus was operatively removed with a favorable outcome.