Exploring feasibility criteria for stereotactic radiosurgical treatment of multiple brain metastases using five linac machines.

PURPOSE: This study aimed to find descriptors that correlates with normal brain dose to determine the feasibility of performing fractionated stereotactic radiosurgery (SRS) for multiple brain metastases (BMs) using five linac machines. METHODS: Thirty-two patients with 1-30 BMs were enrolled. Treatment plans were created using TrueBeam, Novalis Tx, TrueBeam Edge, Halcyon, and Tomotherapy linacs. The sum of all planning target volumes (PTVs) was defined as PTVall, and the brain region excluding PTVall was defined as normal brain. The total surface area (TSA) of the PTV was calculated from the sum of the surface areas of the equivalent spheres for each PTV. Volumes receiving more than 5, 12, and 18 Gy (V5Gy, V12Gy, and V18Gy, respectively) were used for evaluation of normal brain dose. Correlations between normal brain dose and each tumor characteristic (number, PTVall, and TSA) were investigated using the Spearman rank correlation coefficient. RESULTS: Correlations between each characteristic and normal brain dose were statistically significant (p < 0.05) across all machines. The correlation coefficients between each characteristic and V18Gy for the five machines were as follows: tumor number, 0.39-0.60; PTVall, 0.79-0.93; TSA, 0.93-0.99. The fit equations between TSA and V18Gy exhibited high coefficients of determination, ranging from 0.92 to 0.99 across five machines. CONCLUSION: This study devised fractionated SRS plans using for 1-30 BMs across five linac machines to find descriptors for determining SRS feasibility based on normal brain dose. TSA proved to be a promising descriptor of SRS feasibility for treating multiple BMs.

Infliximab Inhibits Colitis Associated Cancer in Model Mice by Downregulating Genes Associated with Mast Cells and Decreasing Their Accumulation.

Inflammatory bowel diseases (IBDs), such as Crohn's disease or ulcerative colitis, can be treated with anti TNF-alpha (TNF-α) antibodies (Abs), but they also put patients with IBDs at risk of cancer. We aimed to determine whether the anti TNF-α Ab induces colon cancer development in vitro and in vivo, and to identify the genes involved in colitis-associated cancer. We found that TNF-α (50 ng/mL) inhibited the proliferation, migration, and invasion of HCT8 and COLO205 colon cancer cell lines and that anti TNF-α Ab neutralized TNF-α inhibition in vitro. The effects of anti TNF-α Ab, infliximab (10 mg/kg) were investigated in mouse models of colitis-associated cancer induced by intraperitoneally injected azoxymethane (AOM: 10 mg/kg)/orally administered dextran sodium sulfate (DSS: 2.5%) (AOM/DSS) in vivo. Infliximab significantly attenuated the development of colon cancer in these mice. Microarray analyses and RT-qPCR revealed that mast cell protease 1, mast cell protease 2, and chymase 1 were up-regulated in cancer tissue of AOM/DSS mice; however, those mast cell related genes were downregulated in cancer tissue of AOM/DSS mice with infliximab. These results suggested that mast cells play a pivotal role in the development of cancer associated with colitis in AOM/DSS mice.

Urgent and emergency endoscopic retrograde cholangiopancreatography for gallstone-induced acute cholangitis and pancreatitis.

Urgent or emergency endoscopic retrograde cholangiopancreatography (ERCP) is indicated for gallstone-induced acute cholangitis and pancreatitis. The technique and optimal timing of ERCP depend on the disease state, its severity, anatomy, patient background, and the institutional situation. Endoscopic transpapillary biliary drainage within 24 h is recommended for moderate to severe acute cholangitis. The clinical outcomes of biliary drainage with nasobiliary drainage tube placement and plastic stent placement are comparable, and the choice is made on a case-by-case basis considering the advantages and disadvantages of each. The addition of endoscopic sphincterotomy (EST) is basically not necessary when performing drainage alone, but single-session stone removal following EST is acceptable in mild to moderate cholangitis cases without antithrombotic therapy or coagulopathy. For gallstone pancreatitis, early ERCP/EST are recommended in cases with impacted gallstones in the papilla. In some cases of gallstone pancreatitis, a gallstone impacted in the papilla has already spontaneously passed into the duodenum, and early ERCP/EST lacks efficacy in such cases, with unfavorable findings of cholangitis or cholestasis. If it is difficult to diagnose the presence of gallstones impacted in the papilla on imaging, endoscopic ultrasonography can be useful in determining the indication for ERCP.

Optimal mode and power output of electrosurgical units for endoscopic papillectomy based on animal experiments and a preliminary clinical study (with videos).

BACKGROUND AND AIMS: The optimal electrosurgical unit (ESU) settings for endoscopic papillectomy (EP) have not been investigated. We conducted animal experiments to determine the optimal endoCUT settings with VIO (Erbe, Tübingen, Germany) ESUs and then conducted a small clinical study. METHODS: Dedicated animal experimental models were created. To investigate the incision force, chicken meat was resected with a snare whose handle was a hung weight. To investigate the coagulation effect, a surgical needle electrode was inserted into a pig liver and energized, and to determine changes over time in the coagulation status, simulated EP was performed using a living pig. These experiments were performed using the knife-setting or snare-setting endoCUT modes and various effect, duration, and interval settings and compared with results for ICC (Erbe) ESUs. Based on the results, we performed EP in a small number of patients. RESULTS: The main factor affecting the incision force was duration. The coagulation effect was related to not only effect but also duration. In the endoCUT mode, knife-setting produced a higher incision force and lower coagulation effect. The nondischarge coagulation effect may cause deep ulceration. Based on the animal experiments, we concluded the ideal ESU setting for EP, "VIO EP mode," is knife-setting with high duration, lowest effect, and low interval settings. In the clinical study, there were no significant adverse events such as bleeding, pancreatitis, or perforation. CONCLUSIONS: "VIO EP mode" seems to afford optimal papillectomy. Larger scale clinical studies are needed to accumulate further data and make clinical comparisons with the ICC ESU.

Efficacy and factors affecting procedure results of short-type single-balloon enteroscopy-assisted ERCP for altered anatomy: a multicenter cohort in Japan.

BACKGROUND AND AIMS: Although single-balloon enteroscopy (SBE)-assisted or short-type SBE (short SBE)-assisted ERCP has been reported as useful in patients with surgically altered anatomy, most studies had small sample sizes or single-center designs. This study aimed to evaluate the efficacy and factors affecting the procedure results of short SBE-assisted ERCP in patients with surgically altered anatomy. METHODS: This multicenter, retrospective study was conducted at 8 tertiary referral care centers in Japan. The data of patients who underwent ERCP-related procedures using short SBE between September 2011 and August 2019 at each facility were analyzed. RESULTS: Overall, 1318 patients were included in this analysis. The enteroscopy (reaching the target site), cannulation, and total procedural success rates were 87.9% (95% confidence interval [CI], 86.1%-89.6%), 87.0% (95% CI, 84.9%-88.8%), and 74.9% (95% CI, 72.5%-77.2%), respectively. Adverse events occurred in 7.7% of patients (95% CI, 6.4%-9.3%). Multiple logistic regression analysis indicated that age (≥75 years), Roux-en-Y reconstruction, pancreatic indication, and malignancy were factors affecting the total procedural failure. CONCLUSIONS: This large-scale study proved that short SBE-assisted ERCP in patients with surgically altered anatomy was effective. Moreover, it clarified factors affecting procedure results. Proficiency with alternative treatment techniques is required in difficult cases. (Clinical trial registration number: UMIN00004045.).

Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-Onset Chronic Pancreatitis.

BACKGROUND & AIMS: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca2+-selective ion channel, in an international cohort of patients and in mice. METHODS: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6mut/mut mice. RESULTS: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10-8). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10-8). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10-9 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10-5). TRPV6mut/mut mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis. CONCLUSIONS: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca2+ balance in pancreatic cells. TRPV6 regulates Ca2+ homeostasis and pancreatic inflammation.

Feasibility of biliary stenting to distal malignant biliary obstruction using a novel designed metal stent with duckbill-shaped anti-reflux valve.

OBJECTIVES: Anti-reflux metal stent (ARMS) has been used to prevent recurrent biliary obstruction (RBO) due to sludge formation or food impaction. However, the most suitable ARMS remains to be identified. This study aimed to evaluate the feasibility, safety, and efficacy of biliary drainage using duckbill-shaped ARMS (D-ARMS). METHODS: This was a multicenter, retrospective observational study conducted at three Japanese tertiary institutions. Patients with distal malignant biliary obstruction who underwent biliary stenting using D-ARMS were eligible to participate. Technical success, functional success, adverse event (AE), and time to RBO (TRBO) were evaluated. RESULTS: Thirty patients underwent biliary stenting using D-ARMS from December 2018 to October 2019. The technical success rate and functional success rate were 93% and 87%, respectively. However, nonvisibility of the markers at the tip of the metal stent or spontaneous extension was occasionally observed, making biliary stenting using D-ARMS difficult. Early AE occurred in 10% of the patients including cholangitis and pancreatitis. During the median postprocedural observation time of 5.1 months (range, 0.8-22.8), RBO occurred in 33% and the median TRBO was 261 days. As for reintervention, D-ARMS could be extracted in 67% of the patients with RBO. However, the stent was torn off on removal in half of the patients. CONCLUSIONS: Although some modifications are still needed, D-ARMS was feasible and safe for biliary drainage, and sufficient TRBO was achieved. Subsequent multi-institutional studies involving a larger number of patients and a longer follow-up period are warranted to validate the present results.

[A case of distal bile duct carcinoma converted to neuroendocrine cancer and proliferated into the pancreas].

A woman in her 50s was referred due to a solid mass in the head of the pancreas. It was diagnosed as a neuroendocrine carcinoma (NEC) by endoscopic ultrasound-guided fine-needle aspiration. Consequently, pancreatoduodenectomy was performed. A well-differentiated adenocarcinoma component was revealed in the resected bile duct, suggesting a relationship with the NEC component in the pancreas. Genetic examination suggested that cholangiocarcinoma, but not coexisting carcinoma, was converted to NEC after the interstitial invasion. Finally, it was diagnosed as the NEC derived from the extrahepatic bile duct, which is rare at about 0.2-2% in gastrointestinal neuroendocrine neoplasms.

[A case of mucinous cystadenoma of the pancreas changed from multilocular to unilocular with cyst in cyst structure with an 8-year follow-up].

A 57-year-old woman presented with multilocular cysts like a bunch of grapes, 30mm in diameter, in the tail of the pancreas. The number of cysts has increased, and each one had grown. Eventually, they turned into a unilocular cyst with a cyst in the cyst structure of about 50mm in diameter. Laparoscopic distal pancreatectomy was performed, and the resected specimen was diagnosed with mucinous cystadenoma. We report the rare morphological change in this case and consider the mechanism of its occurrence based on pathological considerations.

A head-to-toe dimerization has physiological relevance for ligand-induced inactivation of protein tyrosine receptor type Z.

Protein-tyrosine phosphatase (PTPase) receptor type Z (PTPRZ) has two receptor isoforms, PTPRZ-A and -B, containing tandem intracellular PTP-D1 and -D2 domains, with only D1 being active. Pleiotrophin (PTN) binding to the extracellular PTPRZ region leads to inactivation of its PTPase activity, thereby facilitating oligodendrocyte precursor cell (OPC) differentiation and myelination in the central nervous system. However, the mechanisms responsible for PTN-induced PTPRZ inactivation remain unclear. We herein report that the crystal structure of the intracellular region of PTPRZ (PTPRZ-ICR) shows a "head-to-toe"-type dimer conformation, with D2 masking the catalytic site of D1. MS analyses revealed that PTPRZ-ICR proteins remain in monomer-dimer equilibrium in aqueous solution and that a substrate-derived inhibitory peptide or competitive inhibitor (SCB4380) specifically bind to the monomer form in a 1:1 ratio. A D2 deletion (ΔD2) or dimer interface mutation (DDKK) disrupted dimer formation, but SCB4380 binding was maintained. Similar to WT PTPRZ-B, monomer-biased PTPRZ-B-ΔD2 and PTPRZ-B-DDKK variants efficiently dephosphorylated p190RhoGAP at Tyr-1105 when co-expressed in BHK-21 cells. The catalytic activities of these variants were not suppressed by PTN treatment, but were inhibited by the cell-permeable PTPase inhibitor NAZ2329. Of note, the PTN treatment did not enhance OPC differentiation in primary cultured glial cells from ΔD2 or PTPase-inactive PTPRZ-B (CS) mutant knock-in mice. Our results thus indicate that PTN-induced PTPRZ inactivation results from dimer formation of the intracellular tandem PTP domains in a head-to-toe configuration, which is physiologically relevant to the control of OPC differentiation in vivo.

Small intestinal bacterial overgrowth as a cause of protracted wound healing and vitamin D deficiency in a spinal cord injured patient with a sacral pressure sore: a case report.

BACKGROUND: Pressure sores are sometimes refractory to treatment, often due to malnutrition. Small intestinal bacterial overgrowth (SIBO) obstructs absorption in the digestive tract and causes malnutrition. However, little is known about the association between pressure sore wound healing and SIBO. Here, we report a case of a patient with a refractory sacral pressure sore and SIBO. CASE PRESENTATION: A 66-year-old woman who was spinal cord injured 14 years before visiting our hospital presented with the chief complaint of a sacral pressure sore, 10.0 × 6.5 cm in size, which was refractory to treatment. Physical examination showed abdominal distension and emaciation, with a body mass index of 15. Further examination revealed elevated serum alkaline phosphatase (1260 U/L), bilateral tibial fracture, multiple rib fracture, and osteoporosis. We diagnosed the patient with osteomalacia with vitamin D deficiency. Despite oral supplementation, serum levels of calcium, phosphorous, and vitamin D remained low. Also, despite concentrative wound therapy for the sacral pressure sore by plastic surgeons, no wound healing was achieved. Due to a suspicion of disturbances in nutrient absorption, we performed bacterial examination of collected gastric and duodenal fluid, which showed high numbers of bacteria in gastric content (104 E. coli, 105 Streptococcus species, and 105 Neisseria species) and duodenal content (106 E. coli, 104 Candida glabrata). Therefore, we diagnosed the patient with SIBO and started selective decontamination of the digestive tract using polymyxin B sulfate and amphotericin B. After starting treatment for SIBO, the sacral pressure sore began to heal and was nearly healed after 285 days. The patient's serum levels of calcium, phosphorous, vitamin D, and other fat-soluble vitamins also gradually increased after starting treatment for SIBO. CONCLUSION: We report a case of a patient with a refractory sacral pressure sore that healed after starting treatment for SIBO. We conclude that SIBO may be an overlooked cause of malnutrition and poor wound healing in patients with chronic pressure sores.

RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro.

Mg2+ ion stimulates the DNA strand exchange reaction catalyzed by RecA, a key step in homologous recombination. To elucidate the molecular mechanisms underlying the role of Mg2+ and the strand exchange reaction itself, we investigated the interaction of RecA with Mg2+ and sought to determine which step of the reaction is affected. Thermal stability, intrinsic fluorescence, and native mass spectrometric analyses of RecA revealed that RecA binds at least two Mg2+ ions with KD ≈ 2 mM and 5 mM. Deletion of the C-terminal acidic tail of RecA made its thermal stability and fluorescence characteristics insensitive to Mg2+ and similar to those of full-length RecA in the presence of saturating Mg2+. These observations, together with the results of a molecular dynamics simulation, support the idea that the acidic tail hampers the strand exchange reaction by interacting with other parts of RecA, and that binding of Mg2+ to the tail prevents these interactions and releases RecA from inhibition. We observed that binding of the first Mg2+ stimulated joint molecule formation, whereas binding of the second stimulated progression of the reaction. Thus, RecA is actively involved in the strand exchange step as well as bringing the two DNAs close to each other.

Ex vivo assessment of anchoring force of covered biflanged metal stent and covered self-expandable metal stent for interventional endoscopic ultrasound.

BACKGROUND AND AIMS: Endoscopic ultrasound (EUS)-guided transmural drainage using a covered biflanged metal stent (CBFMS) and a conventional tubular biliary covered self-expandable metal stent (CSEMS) has recently been performed by EUS experts. However, appropriate traction force of the sheath to prevent the migration during stent deployment is well unknown. Herein, we assessed the anchoring force (AF) of the distal flange in CBFMSs and CSEMSs. METHODS: The AFs of four CBFMSs (Stents AX, NG, PL, and SX) and six CSEMSs (Stents BF, BP, EG, HN, SP, and WF) were compared in an ex vivo setting. We assessed the AF produced by each stent using an EUS-guided transmural drainage model and an EUS-guided hepaticogastrostomy model consisting of sheet-shaped specimens of the stomach, gelatin gel, and gelatin tubes. RESULTS: For CBFMSs, the maximum AF of Stent AX was significantly higher than those of Stents PL and SX (P < 0.05) in the porcine model. In the gelatin series, all stents except Stent NG showed a nearly similar AF. For CSEMSs, Stents HN, EG, BF, and WF showed gradual AF elevation in the porcine stomach. Stents SP and BP showed a lower AF than the other four stents. For the gelatin setting, the maximum AF of Stents HN, EG, and WF was higher than those of the other stents regardless of the type of specimens. CONCLUSIONS: The significance of the AF and traction distance according to the property of various CBFMSs and CSEMSs could be elucidated using ex vivo models.

Novel peroral direct digital cholangioscopy-assisted lithotripsy using a monorail technique through the overtube in patients with surgically altered anatomy (with video).

Efficacy of cholangioscopy-assisted lithotripsy for difficult stones such as huge stones, multiple large stones and an impacted stone in patients with non-altered anatomy has been reported. Herein, we describe peroral direct digital cholangioscopy (PDCS)-assisted electrohydraulic lithotripsy (EHL) with a new technique in patients with surgically altered anatomy. Five patients received PDCS-assisted EHL with the monorail technique due to failed conventional stone extraction. Balloon enteroscope was removed, leaving the stiff guidewire in the bile duct and an overtube with inflated balloons. The cholangioscope was then inserted into the bile duct over the wire through the overtube. After direct visualization of the stone, PDCS-assisted EHL was carried out. This technique was named the 'monorail technique'. Complete removal of biliary stones in one session was accomplished in four patients and only one case required two sessions. There was no adverse event in any of the cases. PDCS-assisted EHL using the monorail technique was effective and safe for difficult biliary stones in patients with surgically altered anatomy.

Cooperative Binding of KaiB to the KaiC Hexamer Ensures Accurate Circadian Clock Oscillation in Cyanobacteria.

The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data have identified KaiB-KaiC interaction sites; however, KaiB mutations distal from the binding surfaces can impair KaiB-KaiC interaction and the circadian rhythm. Reportedly, KaiB and KaiC exclusively form a complex in a 6:6 stoichiometry, indicating that KaiB-KaiC hexamer binding shows strong positive cooperativity. Here, mutational analysis was used to investigate the functional significance of this cooperative interaction. Results demonstrate that electrostatic complementarity between KaiB protomers promotes their cooperative assembly, which is indispensable for accurate rhythm generation. SasA does not exhibit such electrostatic complementarity and noncooperatively binds to KaiC. Thus, the findings explain KaiB distal mutation effects, providing mechanistic insights into clock protein interplay.

Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome α Subunits.

Eukaryotic proteasomes harbor heteroheptameric α-rings, each composed of seven different but homologous subunits α1-α7, which are correctly assembled via interactions with assembly chaperones. The human proteasome α7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric α6. We comprehensively characterized the oligomeric state of human proteasome α subunits and demonstrated that only the α7 subunit exhibits this unique, self-assembling property and that not only α6 but also α4 can disrupt the α7 double ring. We also demonstrated that mutationally monomerized α7 subunits can interact with the intrinsically monomeric α4 and α6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.

Comparison of patient-specific intensity modulated radiation therapy quality assurance for the prostate across multiple institutions.

AIM: To evaluate the success of a patient-specific intensity modulated radiation therapy (IMRT) quality assurance (QA) practice for prostate cancer patients across multiple institutions using a questionnaire survey. BACKGROUND: The IMRT QA practice involves different methods of dose distribution verification and analysis at different institutions. MATERIALS AND METHODS: Two full-arc volumetric modulated arc therapy (VMAT) plan and 7 fixed-gantry IMRT plan with DMLC were used for patient specific QA across 22 institutions. The same computed tomography image and structure set were used for all plans. Each institution recalculated the dose distribution with fixed monitor units and without any modification. Single-point dose measurement with a cylindrical ionization chamber and dose distribution verification with a multi-detector or radiochromic film were performed, according to the QA process at each institution. RESULTS: Twenty-two institutions performed the patient-specific IMRT QA verifications. With a single-point dose measurement at the isocenter, the average difference between the calculated and measured doses was 0.5 ±â€¯1.9%. For the comparison of dose distributions, 18 institutions used a two or three-dimensional array detector, while the others used Gafchromic film. In the γ test with dose difference/distance-to-agreement criteria of 3%-3 mm and 2%-2 mm with a 30% dose threshold, the median gamma pass rates were 99.3% (range: 41.7%-100.0%) and 96.4% (range: 29.4%-100.0%), respectively. CONCLUSION: This survey was an informative trial to understand the verification status of patient-specific IMRT QA measurements for prostate cancer. In most institutions, the point dose measurement and dose distribution differences met the desired criteria.

Structural Dynamics of the PET-Degrading Cutinase-like Enzyme from Saccharomonospora viridis AHK190 in Substrate-Bound States Elucidates the Ca2+-Driven Catalytic Cycle.

A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca2+ binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca2+ binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond is close to the active site and pointing outward from the active site, respectively, for the two complexes. Ca2+ binding induces the pocket to open, enabling the substrate to access the pocket more easily. Molecular dynamics simulations suggest that a post-reaction state in the engaged form presumably exists between the experimentally observed forms, indicating that the substrate would be cleaved in the engaged form and then requires the enzyme to change to the open form to release the product, a process that Ca2+ can greatly accelerate.

Bifacial Nucleobases for Hexaplex Formation in Aqueous Solution.

Although DNA can form triplex and quadruplex structures through hydrogen bonds, design and preparation of structures with more than five strands is difficult even when artificial nucleic acids are used. Herein we report a hexaplex formed by oligomers of artificial nucleic acids bearing bifacial molecules on d-threoninol. Aminopyrimidine and cyanuric acid derivatives were selected as bases because they have complementary hydrogen bonding patterns. The complex formed by aminopyrimidine and cyanuric acid decamers melted with large hysteresis. Hexaplex formation was indicated by gel electrophoresis, size exclusion chromatography and atomic force microscopy imaging, and proven directly through native mass spectrometry. CD measurements and molecular dynamics simulations indicated that the hexaplex adopts a helical structure. The hexaplex formation was highly dependent on pH and the presence of divalent cations. The hexaplex was stable in aqueous solution, and its unique structure and properties may lead to novel nanostructures, molecular assemblies, metal sensors, and ion channels.

Native mass spectrometry for understanding dynamic protein complex.

Biomolecules have evolved to perform specific and sophisticated activities in a highly coordinated manner organizing into multi-component complexes consisting of proteins, nucleic acids, cofactors or ligands. Understanding such complexes represents a task in earnest for modern bioscience. Traditional structural techniques when extrapolating to macromolecules of ever increasing sizes are confronted with limitations posed by the difficulty in enrichment, solubility, stability as well as lack of homogeneity of these complexes. Alternative approaches are therefore prompted to bridge the gap, one of which is native mass spectrometry. Here we demonstrate the strength of native mass spectrometry, used alone or in combination with other biophysical methods such as analytical ultracentrifugation, small-angle neutron scattering, and small-angle X-ray scattering etc., in addressing dynamic aspects of protein complexes including structural reorganization, subunit exchange, as well as the assembly/disassembly processes in solution that are dictated by transient non-covalent interactions. We review recent studies from our laboratories and others applying native mass spectrometry to both soluble and membrane-embedded assemblies. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.