Search

Multistate Bennett acceptance ratio (MBAR) works as a method to analyze molecular dynamics (MD) simulation data after the simulations have been finished. It is widely used to estimate free-energy changes between different states and averaged properties at the states of interest. MBAR allows us to treat a wide range of states from those at different temperature/pressure to those with different model parameters. Due to the broad applicability, the MBAR equations are rather difficult to apply for free-energy calculations using different types of MD simulations including enhanced conformational sampling methods and free-energy perturbation. In this review, we first summarize the basic theory of the MBAR equations and categorize the representative usages into the following four: (i) perturbation, (ii) scaling, (iii) accumulation, and (iv) full potential energy. For each, we explain how to prepare input data using MD simulation trajectories for solving the MBAR equations. MBAR is also useful to estimate reliable free-energy differences using MD trajectories based on a semi-empirical quantum mechanics/molecular mechanics (QM/MM) model and ab initio QM/MM energy calculations on the MD snapshots. We also explain how to use the MBAR software in the GENESIS package, which we call mbar_analysis, for the four representative cases. The proposed estimations of free-energy changes and thermodynamic averages are effective and useful for various biomolecular systems.

Circadian rhythms influence the transport function of the blood-brain barrier (BBB) and peripheral organs. However, the influence of circadian rhythms on protein expression in the BBB remains to be completely elucidated. Therefore, we aimed to investigate diurnal changes in protein expression in the mouse BBB using quantitative proteomics. Quantitative proteomics showed that the expression of 67, 10, and 20 proteins in the isolated mouse brain capillary fraction changed significantly at zeitgeber time (ZT) 6, 12, and 18, respectively, compared to ZT0. Among them, the levels of 44 proteins were significantly increased at ZT6 and then returned to the same level as ZT0 at ZT12 and ZT18. Gene ontology analysis indicated that the proteins significantly increased at ZT6 were majorly related to translation. The brain capillary endothelial cell-selective proteins sepiapterin reductase and vascular endothelial growth factor receptor 2 showed diurnal variation. In contrast, the expression of ABC transporters, SLC transporters, and receptors associated with receptor-mediated transcytosis, and tight junction proteins did not change within a day. The present findings demonstrated that protein expression related to transport function and physical barrier at the BBB was maintained throughout the day, although the proteins involved in some biological processes exhibited diurnal variation at the BBB.

Tryptophan synthase (TRPS) is a bifunctional enzyme consisting of α and β-subunits and catalyzes the last two steps of l-tryptophan (L-Trp) biosynthesis, namely, cleavage of 3-indole-d-glycerol-3'-phosphate (IGP) into indole and glyceraldehyde-3-phosphate (G3P) in the α-subunit, and a pyridoxal phosphate (PLP)-dependent reaction of indole and l-serine (L-Ser) to produce L-Trp in the β-subunit. Importantly, the IGP binding at the α-subunit affects the β-subunit conformation and its ligand-binding affinity, which, in turn, enhances the enzymatic reaction at the α-subunit. The intersubunit communications in TRPS have been investigated extensively for decades because of the fundamental and pharmaceutical importance, while it is still difficult to answer how TRPS allostery is regulated at the atomic detail. Here, we investigate the allosteric regulation of TRPS by all-atom classical molecular dynamics (MD) simulations and analyze the potential of mean-force (PMF) along conformational changes of the α- and β-subunits. The present simulation has revealed a widely opened conformation of the β-subunit, which provides a pathway for L-Ser to enter into the β-active site. The IGP binding closes the α-subunit and induces a wide opening of the β-subunit, thereby enhancing the binding affinity of L-Ser to the β-subunit. Structural analyses have identified critical hydrogen bonds (HBs) at the interface of the two subunits (αG181-βS178, αP57-βR175, etc.) and HBs between the β-subunit (βT110 - βH115) and a complex of PLP and L-Ser (an α-aminoacrylate intermediate). The former HBs regulate the allosteric, β-subunit opening, whereas the latter HBs are essential for closing the β-subunit in a later step. The proposed mechanism for how the interdomain communication in TRPS is realized with ligand bindings is consistent with the previous experimental data, giving a general idea to interpret the allosteric regulations in multidomain proteins.

Sulfur-embedded polycyclic aromatic compounds have been used as building blocks for numerous organic semiconductors over the past few decades. While the success is based on thiophene-containing compounds, aromatic compounds that contain thiepine, a sulfur-containing seven-membered-ring arene, has been less well investigated. Here we report the synthesis and properties of π-extended pyrrole-fused heteropine compounds such as thiepine and oxepine. A π-extended pyrrole-fused thiepine exhibited a "pitched π-stacking" structure in the crystal, and exhibited a high charge carrier mobility of up to 1.0 cm2 V-1 s-1 in single-crystal field-effect transistors. Nanyang Technological University This work was supported by Nanyang Technological University, Grant-in-Aid for JSPS KAKENHI (Grant No. JP21H04694), and the Mitsubishi Foundation.

The insulin receptor (INSR) mediates insulin signaling to modulate cellular functions. Although INSR is expressed at the blood–brain barrier (BBB), its role in the modulation of BBB function is poorly understood. Therefore, in this study, we aimed to analyze the effect of INSR knockdown on the expression levels of functional proteins at the BBB. We established the INSR-knockdown cell line (shINSR) using human cerebral microvascular endothelial cells (hCMEC/D3). The cellular proteome was analyzed using quantitative proteomics. INSR mRNA and protein expressions were decreased in shINSR cells. The suppression of INSR-mediated signaling in shINSR cells was evaluated. The proteins involved in glycolysis and glycogenolysis were suppressed in shINSR cells. As amyloid-β peptide-related proteins, the expressions of presenilin-1 was increased, and those of the insulin-degrading enzyme and neprilysin were decreased. The expressions of BBB transporters, including the ABCB1/MDR1, ABCG2/BCRP, and SLCO2A1/OATP2A1 were significantly decreased by more than 50% in shINSR cells. The efflux activity of ABCB1/MDR1 was also suppressed. The expressions of the low-density lipoprotein receptor-related protein 1 were significantly increased, and those of the transferrin receptor were significantly decreased in shINSR cells. The expression of claudin-5 was also suppressed in shINSR cells. The present study suggests that INSR-mediated signaling is involved in the regulation of functional protein expression at the BBB and contributes to the maintenance of BBB function. Changes in the expressions of amyloid-β peptide-related proteins may contribute to the development of cerebral amyloid angiopathy via the suppression of INSR-mediated signaling.

Background Some studies have reported that adhesion prevention barriers (APBs) reduce adhesion after abdominal surgery; however, evidence showing that APBs reduce the incidence of postoperative small bowel obstruction (SBO), one of the most serious complications after abdominal surgery, is little. One concern is that APBs are usually applied only under the midline incision, although adhesion can occur at any place in the peritoneum where an incision is made during surgery. INTERCEEDⓇ is an APB that reportedly prevents postoperative SBO after surgery. This study aims to assess the clinical utility of INTERCEEDⓇ for the prevention of SBO after laparoscopic colorectal cancer surgery and determine whether the application site of INTERCEEDⓇ affects the incidence of SBO. Methods/Design This study is a prospective, multicenter, observational study conducted in Japan. The primary end point is the incidence of postoperative SBO 2 years after laparoscopic colorectal cancer surgery. The secondary end points include whether the site of the application of INTERCEEDⓇ affects the incidence of SBO. Each surgeon selects one of the following three procedures: 1) INTERCEEDⓇ is placed only under the midline incision; 2) INTERCEEDⓇ is placed at the site of bowel mobilization and/or lymph node dissection, but not under the midline incision; and 3) INTERCEEDⓇ is placed at both sites. Discussion This is the first study to assess whether the placement of APBs affects the incidence of SBO. The study results may lead to a subsequent randomized study.

Although endoscopic repair is often performed for sigmoid colon volvulus without intestinal necrosis, surgery is the common choice of treatment due to recurrence. With no established treatment, Hartmann’s operation or sigmoid colon resection is often performed. We report a case of a 65-year-old man with transanal ileus tube placement before surgery for sigmoid colon volvulus to prevent recurrence and achieve intestinal decompression followed by Sharon’s operation to achieve one-stage anastomosis. The patient showed good postoperative course, with no recurrence 3 months after surgery. This report discusses the usefulness of the transanal ileus tube and Sharon’s operation for sigmoid colon volvulus without intestinal necrosis along with a review of the literature.

A 68-year-old male was found unconscious in a hot bathtub on a cold winter’s day and was brought to our hospital. On arrival, the patient’s systolic blood pressure was 65 mm hg, heart rate was 134 beats per min, and rectal temperature was 40.5°C. A large volume infusion of acetated Ringer’s solution and vasopressors were administered as an immediate treatment for circulatory failure due to heatstroke. Although the circulatory failure temporarily improved, the blood pressure dropped almost to the point of cardiac arrest. Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) was implemented and the hemodynamics were soon stabilized. Subsequently, VA-ECMO was withdrawn on the third hospital day. The patient recovered enough to feed normally on the 40th hospital day and was transferred to another hospital on the 116th hospital day. Based on this case study, we consider that VA-ECMO implementation before cardiac arrest might be a suitable strategy.

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

Introduction Primary malignant melanoma of the esophagus is a rare and aggressive disease that tends to have a poor response to chemotherapies. Previous studies have indicated that currently available treatment for primary malignant melanoma of the esophagus is insufficient. Here, we describe a case of recurrent primary malignant melanoma of the esophagus successfully treated with the immune checkpoint inhibitor nivolumab. Case presentation An 81-year-old Japanese female presented with a 3-month history of dysphagia. She was medicated for hypertension and sarcoidosis. The patient had no past history of cutaneous, ocular, or other-site melanomas. An esophagoscopy identified a 30-mm giant tumor in the lower esophagus, at a site 30 cm from the incisors. Enhanced computed tomography revealed wall thickening measuring 30 mm in size at the middle-third of the intrathoracic esophagus, with no significant lymph node infiltration or distant metastasis. Esophageal biopsy showed proliferation of large round tumor cells and melanophages. On the basis of these findings, the patient was diagnosed with esophageal malignant melanoma and underwent esophagectomy and lymph node dissection with gastric tube reconstruction. Although the pathological diagnosis was primary malignant melanoma of the esophagus, the patient presented with multiple lymph node and bone metastases 4 months after surgery. Subsequently, treatment with nivolumab 240 mg every 2 weeks was administered as the first-line treatment. Diffusion-weighted imaging with background body signal suppression following eight courses of nivolumab revealed that the multiple lymph node and bone metastases were markedly reduced. The patient received 30 courses of nivolumab and has maintained the partial response. No severe adverse events related to the immunotherapy were recorded. Conclusion The current study suggests that nivolumab may be a viable option for patients with metastatic primary malignant melanoma of the esophagus. Additional evidence from future clinical trials and research is necessary to fully validate these findings.

The first enantioselective carbometalation reaction of azabicycloalkenes has been achieved by iron catalysis to in situ form optically active organozinc intermediates, which are amenable to further synthetic elaborations. The observed chiral induction, along with the DFT and XAS analyses, reveals the direct coordination of the chiral phosphine ligand to the iron centre during the carbon–carbon and carbon–metal bond forming step. This new class of iron-catalysed asymmetric reaction will contribute to the synthesis and production of bioactive molecules.

This article proposes a mechatronic system design for atomic force microscopes (AFMs) in order to realize a large range and high control bandwidth of the vertical probe motion by a flexure-guided voice coil actuator. The first mechanical resonant frequency of the actuator is decreased to 67 Hz for a large motion, whereas the second resonant frequency is maximized to 1.15 kHz. The frequency band between the resonances enables two-degree-of-freedom control with a position sensor to realize a control bandwidth of 881 Hz, which is 13 times higher than the first resonance that is the limitation of conventional AFMs. The closed-loop actuator achieves a positioning resolution of 1.7 nm and a nonlinearity of 0.02% for a motion range of 700 $\mu$ m. Consequently, the actuator realizes a range-bandwidth product of 617 kHz $\cdot \mu$ m, breaking through a theoretical limit of piezoelectric actuators (567 kHz $\cdot \mu$ m). The high performance of the actuator is further confirmed by 400 $\mu$ m topography measurement in the constant force mode with a bandwidth of 197 Hz. The achieved resolution of the topography measurement is 2.9 nm, and it is demonstrated by successfully imaging nanostructures on a CD-ROM disk.

The Ritter reaction, Brønsted- or Lewis acid-mediated amidation of alkene or alcohol with nitrile via a carbocation, represents a classical method for the synthesis of tertiary amides. Although analogous reactions through a vinyl cation or a species alike may offer a route to enamide, an important synthetic building block as well as a common functionality in bioactive compounds, such transformations remain largely elusive. Herein, we report a Ritter-type trans-difunctionalization of alkynes with a trivalent iodine electrophile and nitrile, which affords β-iodanyl enamides in moderate to good yields. Mediated by benziodoxole triflate (BXT), the reaction proves applicable to a variety of internal alkynes as well as to various alkyl- and arylnitriles. The benziodoxole group in the product serves as a versatile handle for further transformations, thus allowing for the preparation of various tri- and tetrasubstituted enamides that are not readily accessible by other means., Ritter-type trans-selective iodo(iii)amidation of internal alkynes with benziodoxole triflate and various nitriles has been achieved for the stereocontrolled synthesis of multisubstituted enamides.

To achieve highly precise and linear scanning motion by a hybrid reluctance actuator (HRA), this article proposes a flux-controlled mode that uses regulated magnetic flux as the control input for the actuator operation and evaluates its performance in comparison with the conventional current-controlled mode. In the conventional case, HRAs exhibit magnetic nonlinearities (e.g., hysteresis) and position-dependent force that can make the system unstable. A model-based analysis reveals that they are included in the variable magnetic flux of an HRA. Thus, they are captured by flux estimation and rejected by flux feedback control for high-quality scanning motion. For the estimation, sensor fusion with a current monitor and a search coil is used. Proportional integral (PI) controllers are used for the flux feedback control, as well as for current feedback control of the benchmarking current-controlled mode. During scanning, feedforward control is used to compensate linear dynamics. When sine motions are experimentally tested at 60–300 Hz, the current-controlled mode exhibits a nonlinearity between 6% and 23%, which is decreased to less than 5% by the flux-controlled mode. For a $\pm$ 75 $\mathrm{\mu }$ m triangular motion at 100 Hz, the flux-controlled mode decreases the tracking error by a factor of 19 to 3.2 $\mathrm{\mu }$ m, successfully demonstrating its high-quality linear scanning motion.

In hybrid reluctance actuators, the achievable closed-loop system bandwidth is affected by the eddy currents and hysteresis in the ferromagnetic components and the mechanical resonance modes. Such ...

Sequencing the entire RNA molecule leads to a better understanding of the transcriptome architecture. SMARTer (Switching Mechanism at 5′-End of RNA Template) is a technology aimed at generating full-length cDNA from low amounts of mRNA for sequencing by short-read sequencers such as those from Illumina. However, short read sequencing such as Illumina technology includes fragmentation that results in bias and information loss. Here, we built a pipeline, UNAGI or UNAnnotated Gene Identifier, to process long reads obtained with nanopore sequencing and compared this pipeline with the standard Illumina pipeline by studying the Saccharomyces cerevisiae transcriptome in full-length cDNA samples generated from two different biological samples: haploid and diploid cells. Additionally, we processed the long reads with another long read tool, FLAIR. Our strand-aware method revealed significant differential gene expression that was masked in Illumina data by antisense transcripts. Our pipeline, UNAGI, outperformed the Illumina pipeline and FLAIR in transcript reconstruction (sensitivity and specificity of 80% and 40% vs. 18% and 34% and 79% and 32%, respectively). Moreover, UNAGI discovered 3877 unannotated transcripts including 1282 intergenic transcripts while the Illumina pipeline discovered only 238 unannotated transcripts. For isoforms profiling, UNAGI also outperformed the Illumina pipeline and FLAIR in terms of sensitivity (91% vs. 82% and 63%, respectively). But the low accuracy of nanopore sequencing led to a closer gap in terms of specificity with Illumina pipeline (70% vs. 63%) and to a huge gap with FLAIR (70% vs 0.02%). Electronic supplementary material The online version of this article (10.1007/s10142-020-00732-1) contains supplementary material, which is available to authorized users.

For highly precise motion of a galvanometer scanner that tracks a periodic motion reference, learning control significantly decreases the tracking error. To achieve higher quality motion by reducing the angular sensor noise, this paper investigates inversion-based iterative control (IIC) that can learn only at the fundamental and harmonic frequencies of the periodic motion reference. This enables to separate the compensable tracking error from the noise to be eliminated during learning in the frequency domain. The analysis in the paper reveals a tradeoff for the noise reduction in the IIC design, and this paper proposes an equation to quickly tune a design parameter in the tradeoff for better performance. Furthermore, the effectiveness of the IIC algorithm is experimentally demonstrated for a galvanometer scanner. When the galvanometer scanner tracks a 20 Hz triangular motion of ± 10 degrees, the IIC successfully decreases the residual tracking error by 41 % to 2.83 ×10-4 deg, by utilizing the noise reduction.

Various quantities related to the relative openness constant of a linear operator are studied. An estimate of the relative openness constant of the product of two linear operators is also obtained.

The first example of a cyclophane bearing a nitrogen-containing buckybowl was synthesized via sequential 1,3-dipolar cycloaddition and palladium-catalyzed intramolecular cyclization. The key to the successful synthesis is the strain-induced 1,3-dipolar cycloaddition of a polycyclic aromatic azomethine ylide to the K-region of [7](2,7)pyrenophane. The resulting π-extended azacorannulenophane exhibits intriguing structural and physical properties, including unique variation of bowl depth, extraordinarily high-field chemical shifts in its 1 H NMR spectrum, a decreased HOMO-LUMO gap, and a red shift in the absorption/emission spectrum, when compared to those of the parent azacorannulene. These characteristics are derived from both the π-extension to the polycyclic aromatic system in the cyclophane structure and the increased curvature enforced by the seven-carbon aliphatic chain. Nanyang Technological University Submitted/Accepted version This work was supported by the U.S. National Science Foundation, the Natural Sciences and Engineering Research Council (N.S.E.R.C.) of Canada (G.J.B.) and Nanyang Technological University (NTU; S.I.).

Podocalyxin (PODXL) is a highly sialylated transmembrane protein that is expressed on the luminal membrane of brain microvascular endothelial cells. To clarify the role of PODXL in the blood-brain barrier (BBB), the present study aimed to investigate the effect of PODXL-knockdown on protein expression, especially the expression of ABCB1/MDR1, in human microvascular endothelial cells (hCMEC/D3). By quantitative proteomics, gene ontology enrichment with differentially expressed proteins showed that PODXL-knockdown influenced the immune response and intracellular trafficking. Among transporters, the protein expression of ABCB1/MDR1 and ABCG2/BCRP was significantly elevated by approximately 2-fold in the PODXL-knockdown cells. In the knockdown cells, the efflux activity of ABCB1/MDR1 was significantly increased, while its mRNA expression was not significantly different from that of the control cells. As receptors and tight junction proteins, levels of low-density lipoprotein receptor-related protein 1 and occludin were significantly increased, while those of transferrin receptor and claudin-11 were significantly decreased in the knockdown cells. The present results suggest that PODXL functions as a modulator of BBB function, including transport, tight junctions, and immune responses. Furthermore, PODXL post-transcriptionally regulates the protein expression and efflux activity of ABCB1/MDR1 at the BBB, which may affect drug distribution in the brain.

Polycyclic aromatic molecules are promising functional materials for a wide range of applications, especially in organic electronics. However, their largely hydrophobic nature has impeded further applications. As such, imparting high solubility/hydrophilicity to polycyclic aromatic molecules leads to a breakthrough in this research field. Herein, we report the synthesis of diazapentabenzocorannulenium, a cationic nitrogen-embedded buckybowl bearing a central imidazolium core, by a bottom-up strategy from polycyclic aromatic azomethine ylide. X-ray crystallography analyses have revealed a bowl-shaped molecular structure that is capable of forming charge-segregated one-dimensional columns by bowl-in-bowl packing. In addition to its fluorescence capabilities and high dispersibility in water, the molecule was found to selectively localize in the mitochondria of various tumor cells, showing potential as viable mitochondria-selective fluorescent probes. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) B. X. acknowledges the financial support from MOE Tier 1 RG6/20, A*Star SERC A1983c0028, and A20E5c0090.

A new synthetic approach to π-extended imidazolium salts is developed based on 1,3-dipolar cycloaddition of polycyclic aromatic azomethine ylides with imidoyl chlorides, where the key to the successful conversion is the addition of cesium fluoride as an additive. Experimental and theoretical studies revealed that this cycloaddition proceeds via a stepwise mechanism rather than a concerted mechanism. This present method provides an efficient method to synthesize a variety of imidazolium-containing polycyclic aromatic compounds. Submitted/Accepted version

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold as well as the number of quantified transmembrane proteins compared to an in-solution digestion (ISD) method. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10000 cells using the ISD method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.