Tm Values Research Articles

Computation-Based Design of Salt Bridges in PETase for Enhanced Thermostability and Performance for PET Degradation.

Polyethylene terephthalate (PET) is one of the most widely used plastics, and the accumulation of PET poses a great threat to the environment. IsPETase can degrade PET rapidly at moderate temperatures, but its application is greatly limited by the low stability. Herein, a molecular dynamics (MD) simulations combined with a sequence alignment strategy was adopted to introduce salt bridges into the flexible region of IsPETase to improve its thermal stability. In the designed variants, the Tm values of IsPETaseI168R/S188D and IsPETaseI168R/S188E were 7.4 and 8.7 °C higher than that of the wild type, respectively. The release of products degraded by IsPETaseI168R/S188E was 4.3 times that of the wild type. Tertiary structure characterization demonstrated that the structure of the variants IsPETaseI168R/S188D and IsPETaseI168R/S188E became more compact. Extensive MD simulations verified that a stable salt bridge was formed between the residue R168 and D186 in IsPETaseI168R/S188D, while in IsPETaseI168R/S188E an R168-D186-E188 salt bridge network was observed. These results confirmed that the proposed computation-based salt bridge design strategy could efficiently generate variants with enhanced thermal stability for the long-term degradation of PET, which would be helpful for the design of enzymes with improved stability.

Alcohol-initiated and Tin(II) 2-ethylhexanoate-catalyzed polymerization of l-lactide in bulk – About separate crystallization of cyclic and linear Poly(l-lactide)s

Alcohol-initiated ROPs of l-Lactide were performed at 140 °C in bulk with variation of the initiator/catalyst ratio and time. Lower ratios favor the formation of cycles which upon annealing display a change of the MALDI mass peak distribution towards a new maximum with a “saw-tooth pattern” of the mass peaks representing the cycles. Such a pattern was not observed for the mass peak of the linear chains. The coexistence of these patterns indicate that linear and cyclic poly (l-lactide)s (PLA) crystallize in separate crystals, and that the crystallites of the cycles are made up by extended rings. High Tm and ΔHm values confirm that these extended-ring crystallites represent a thermodynamically optimized form of PLA. Experiments with preformed cyclic and linear PLAs support this interpretation.

Purine DNA Constructs Designed to Expand the Genetic Code: Functionalization, Impact of Ionic Forms, and Molecular Recognition of 7-Deazaxanthine-7-Deazapurine-2,6-diamine Base Pairs and Their Purine Counterparts.

Purine DNA represents an alternative pairing system formed by two purines in the base pair with the recognition elements of Watson-Crick DNA. Base functionalization of 7-deaza-2'-deoxyxanthosine with ethynyl and octadiynyl residues led to clickable side chain derivatives with short and long linker arms. As complementary bases, purine-2,6-diamine or 7-deazapurine-2,6-diamine 2'-deoxyribonucleosides were used. 7-Deaza-7-iodo-2'-deoxyxanthosine served as a starting material for Sonogashira cross-coupling and the p-nitrophenylethyl group for base protection. Phosphoramidite building blocks for DNA synthesis were prepared. Oligonucleotides containing single modifications or runs of three purine base pairs embedded in 12-mer Watson-Crick DNA were synthesized and hybridized with complementary strands with purine- or 7-deazapurine-2,6-diamine located opposite to the xanthine derivatives. The stability of base pairs was evaluated in a comparative study on the basis of DNA melting experiments and Tm values. As 7-deazaxanthine and xanthine nucleosides form anionic forms at neutral pH, duplex stability became pK-dependent, and the system with 7-deazapurine displayed a significant higher stability as that containing xanthine. Alkynyl side chains are well accommodated in the purine-purine helix. Click adducts with pyrene showed that short linker arms destabilize duplexes, whereas long linkers increase duplex stability. CD and fluorescence measurements provide further insights into purine-purine base pairing.

A novel anti-IL-33 antibody recognizes an epitope FVLHN of IL-33 and has a therapeutic effect on inflammatory diseases.

As a crucial member of the Interleukin-1 (IL-1) family, IL-33 plays an indispensable role in modulating inflammatory responses. Here, we developed an effective anti-human IL-33 monoclonal antibody (mAb) named 5H8. Importantly, we have identified an epitope (FVLHN) of IL-33 protein as a recognition sequence for 5H8, which plays an important role in mediating the biological activity of IL-33. We observed that 5H8 significantly suppressed IL-33-induced IL-6 expression in bone marrow cells and mast cells in a dose-dependent manner in vitro. Furthermore, 5H8 effectively relievedHDM-induced asthma and PR8-induced acute lung injury in vivo. These findings indicate that targeting the FVLHN epitope is critical for inhibiting IL-33 function. In addition, wedetected that the Tm value of 5H8 was 66.47℃ and the KD value was 173.0pM, which reflected that 5H8 had good thermal stability and high affinity. Taken together, our data suggest that our newly developed 5H8 antibody has potential as a therapeutic antibody for treating inflammatory diseases.

Identification and Thermostability Modification of the Mesophilic L-asparaginase from Limosilactobacillus secaliphilus.

L-asparaginase (L-ASNase, E.C.3.5.1.1) could effectively inhibit the formation of acrylamide (AA) by hydrolyzing the AA precursor L-asparagine. However, most of the L-ASNases showed a relatively weak thermostability, posing a big threat on the application of enzyme at high processing temperatures. Here, the recombinant L-ASNase from mesophilic bacteria Limosilactobacillus secaliphilus was identified for the first time. The recombinant enzyme exhibited its optimal activity at pH 8.0 and 60 ℃. Additionally, the thermostability of L. secaliphilus L-ASNase was enhanced by site-directed mutagenesis after multiple sequence alignment. Ten mutants were reasonably constructed, among which the single-point mutants L24Y, S55T, and V155S showed more than 1 ℃ elevated Tm value compared to the wild-type enzyme. In addition, the half-life of mutant at 40, 50, and 55 ℃ was 376.7min, 62.1min, and 18.7min, much higher than that of wild-type enzyme. The molecular dynamic simulation showed that compared to the wild-type enzyme, the structural stability of V155S was greatly strengthened due to the lower RMSF and RMSD value as well as a decreased total energy compared to that of the wild-type enzyme. The results were positive and provided some useful information for the thermostability modification of L-ASNase.

Melt granulation for Enhanced Dissolution Rate and Antimicrobial activity of Cefpodoxime Proxetil

The aim of this work was to improve dissolution rate and antibacterial activity of cefpodoxime proxetil (CP) by Pluronic PE 6800 (Poloxamer 188) as eutectic forming hydrophilic polymer. CP was incorporated in the polymer by melt granulation technique. CP was mixed with melted polymer before addition of Avicel, in absence and presence of HPMC E6, with mixing to provide flowable particles. The formulations were characterized using thermal analysis and Fourier transform infrared spectroscopy (FTIR) in addition to dissolution studies. The antibacterial activity of CP against Proteus mirabilis was also explored. FTIR suggested possible hydrogen bonding between CP and polymers. All formulations improved CP dissolution compared to unprocessed CP. This could be attributed to the formation of eutectic species, as reflected by thermal analysis as new endothermic peak at Tm value of 40°C was detected in thermograms of all tested formulations. Microbiological investigations indicated that the optimized formulation offered advantages over pure CP with reduced minimum inhibitory concentration. Additionally, formulated CP decreased the biofilm formation ability among the tested P. mirabilis clinical isolates relative to unprocessed CP. The study introduced melt granulation of CP with poloxamer 188 as a promising tool for enhanced dissolution and augmented activity against Proteus mirabilis with capacity to minimize the biofilm formation.

Establishing a high‐precision real‐time Precipitable Water Vapor model in China with Global Navigation Satellite System and Fifth‐Generation Reanalysis Model data

AbstractThe real‐time high precision of Precipitable Water Vapor (PWV) is widely recognized as crucial for advancing numerical weather prediction and enhancing our understanding of climate change. PWV is usually measured with radiosondes, microwave radiometers, and meteorological satellites. Nevertheless, these instruments have limitations including low temporal or spatial resolutions, high cost and weather dependence. To address these issues, we developed a real‐time PWV grid model for China by integrating the ground‐based Global Navigation Satellite System (GNSS) network with ERA5 reanalysis products. In this study, we explored an alternative method using ERA5 products to construct an accurate Elevation Normalization Factor Model (ENFM) and Tm models. The culmination of our efforts resulted in the creation of a real‐time 1° × 1° PWV grid model for China. To validate the models of Tm and PWV, we compared them against the data obtained from radiosondes in China throughout 2021. The results indicate that the root‐mean‐square error (RMSE) value of the deviation between the Tm derived from our new Tm model, excluding meteorological parameters, and the radiosonde‐derived Tm, is better than 4.22 K. The Tm values of the new Tm model are improved by 15.31% compared to those estimated based on the Bevis model plus HGPT2 temperature model. The RMSE values of the deviations between the new grid PWV and the radiosonde‐derived PWV are less than 3.45 mm. The precision of our new PWV grid model is improved by 37.2% compared to that of the traditional Askne and Nordius model.

Crystalline Arrays of Copper Porphyrin Qubits Based on Ion-Paired Frameworks.

Molecular electronic spin qubits have great potential for use in quantum information science applications because their structure can be rationally tuned using synthetic chemistry. Their integration into a new class of materials, ion-paired frameworks, allows for the formation of ordered arrays of these molecular spin qubits. Three ion-paired frameworks with varying densities of paramagnetic Cu(II) porphyrins were isolated as micron-sized crystals suitable for characterization by single-crystal X-ray diffraction. Pulse-electron paramagnetic resonance (EPR) spectroscopy probed the spin coherence of these materials at temperatures up to 140 K. The crystals with the longest Cu-Cu distances had a spin-spin relaxation time (Tm) of 207 ns and a spin-lattice relaxation time (T1) of 1.8 ms at 5 K, which decreased at elevated temperature because of spin-phonon coupling. Crystals with shorter Cu-Cu distances also had lower T1 values because of enhanced cross-relaxation from qubit-qubit dipolar coupling. Frameworks with shorter Cu-Cu distances exhibited lower Tm values because of the increased interactions between qubits within the frameworks. Incorporating molecular electronic spin qubits in ion-paired frameworks enables control of composition, spacing, and interqubit interactions, providing a rational means to extend spin relaxation times.

Association between N-Terminal Pyrenes Stabilizes the Collagen Triple Helix.

Collagen model peptides featuring the fluorophore pyrene at their N-termini have been synthesized, and their thermal denaturation has been examined using circular dichroism (CD) and fluorescence spectroscopies. Flanking the (Pro-Hyp-Gly)7 core of the peptide monomers at positions 1 and/or 23 in the primary sequence, Lys residues were introduced to ensure water solubility. Triple helices derived from such peptides show a broad excimer emission at ∼480 nm, indicative of interaction between the pyrene units. CD experiments show that the fluorophores enhance helix stability primarily through entropic effects. Unfolding temperatures (Tm) increase by up to 7 °C for systems with N-terminal lysine residues and by up to 21 °C for systems in which the first-position Lys is replaced by Ala. Tm values derived from fluorescence measurements (at 50 μM) typically lie within ∼1 °C of those obtained using CD (at 200 μM). Computational modeling in a water continuum using B3LYP-GD3 and M06-2X functionals predicts that face-to-face association of fluorophores can occur while H-bonding within the [(POG)n]3 assembly is retained. Such parallel stacking is consistent with hydrophobically driven stabilization. Labeling collagen peptides with pyrene is a synthetically simple way to promote triple helicity while providing a means to obtain Tm data on relatively dilute samples.

Structural basis for the development of potential inhibitors targeting FadD23 from Mycobacterium tuberculosis.

Sulfolipid-1 (SL-1) is a lipid that is abundantly found in the cell wall of Mycobacterium tuberculosis (Mtb). MtbFadD23 is crucial in the SL-1 synthesis pathway. Previously, 5'-O-[N-(11-phenoxyundecanoyl)sulfamoyl]adenosine (PhU-AMS) has been shown to be a general inhibitor of fatty-acid-adenylating enzymes (FadDs) in Mtb. However, the fatty acyl-AMP ligase (FAAL) class of FadDs, which includes MtbFadD23, appears to be functionally nonredundant in the production of multiple fatty acids. In this study, the ability of PhU-AMS to bind to MtbFadD23 was examined under in vitro conditions. The crystal structure of the MtbFadD23-PhU-AMS complex was determined at a resolution of 2.64 Å. Novel features were identified by structural analysis and comparison. Although PhU-AMS could bind to MtbFadD23, it did not inhibit the FAAL adenylation activity of MtbFadD23. However, PhU-AMS improved the main Tm value in a differential scanning fluorimetry assay, and a structural comparison of MtbFadD23-PhU-AMS with FadD32 and PA1221 suggested that PhU-AMS blocks the loading of the acyl chain onto Pks2. This study sheds light on the structure-based design of specific inhibitors of MtbFadD23 and general inhibitors of FAALs.

Electron paramagnetic resonance spectroscopic characterization of the human KCNE3 protein in lipodisq nanoparticles for structural dynamics of membrane proteins

One of the major challenges in solubilization of membrane proteins is to find the optimal physiological environment for their biophysical studies. EPR spectroscopy is a powerful biophysical technique for studying the structural and dynamic properties of macromolecules. However, the challenges in the membrane protein sample preparation and flexible motion of the spin label limit the utilization of EPR spectroscopy to a majority of membrane protein systems in a physiological membrane-bound state. Recently, lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have emerged as a membrane mimetic system for investigating the structural studies of membrane proteins. However, its detail characterization for membrane protein studies is still poorly understood. Recently, we characterized the potassium channel membrane protein KCNQ1 voltage sensing domain (KCNQ1-VSD) and KCNE1 reconstituted into lipodisq nanoparticles using EPR spectroscopy. In this study, the potassium channel accessory protein KCNE3 containing flexible N- and C-termini was encapsulated into proteoliposomes and lipodisq nanoparticles and characterized for studying its structural and dynamic properties using nitroxide based site-directed spin labeling EPR spectroscopy. CW-EPR lineshape analysis data indicated an increase in spectral line broadenings with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated an enhanced quality of distance measurements with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles, when compared to proteoliposomes. These results agree with the solution NMR structural structure of the KCNE3 and EPR studies of other membrane proteins in lipodisq nanoparticles. This study along with our earlier studies will provide the reference characterization data that will provide benefit to the membrane protein researchers for studying structural dynamics of challenging membrane proteins.

Watson-Crick Base Pairs with Protecting Groups: The 2-Amino Groups of Purine- and 7-Deazapurine-2,6-Diamine as Target Sites for DNA Functionalization by Selective Nucleobase Acylation.

The recognition of Watson-Crick base pairs carrying nucleobase protecting groups is reported as a new approach for DNA functionalization. The 2-amino groups of purine- and 7-deazapurine-2,6-diamine 2'-deoxyribonucleosides served as molecular targets for this functionalization. The 2-amino group withstands oligonucleotide deprotection with ammonia, whereas all other protecting groups are released after chemical DNA synthesis. On this basis, a method was developed for the selective functionalization of oligonucleotides at the 2-position of purines and 7-deazapurines. Melting experiments and Tm values obtained from hybridization studies revealed that duplexes with protected (2-amino-dA) and (2-amino-7-deaza-dA)-dT base pairs are as stable as their nonprotected counterparts. Mismatch discrimination of protected purine- and 7-deazapurine-2,6-diamine DNA was superior to that of nonprotected DNA. Click functionalization in the minor groove of the DNA double helix became accessible via introduction of heptynoyl protecting groups bearing a terminal triple bond. Click reactions with pyrene azide validated the usability. DNA conjugates with bulky pyrene residues at the 2-position (minor groove) developed the same high stability as those functionalized at the 7-position (major groove). This demonstrates the potential of our new method using protected base pairs for DNA functionalization and paves the way for new DNA labeling strategies.

Poly lactic acid production using the ring opening polymerization (ROP) method using Lewis acid surfactant combined iron (Fe) catalyst (Fe(DS)3)

LASC catalyst synthesis process was carried out using iron metal (Fe) and its characterization properties were tested. There are five stages carried out, namely, the manufacture of the LASC Fe(DS)3 catalyst, lactic acid dehydration, polycondensation of lactic acid into l-lactic acid oligomers (OLLA), depolymerization of oligomers into l-lactide, and the ROP process to convert l-lactide into Polymers. l-lactic acid (PLLA). The analysis used is as follows, Karl-Fisher; Spectroscopy Fourier Transform Infrared (FTIR); and Nuclear Magnetic Resonance (HNMR); Gel Permeation Chromatography (GPC); Thermogravimetry Gravity Analysis (TGA) and X-Ray Diffraction (XRD). Based on the experimental results, the catalyst Fe(DS)3 has a crystallinity value of 24% which indicates an amorphous nature and is easy to react. The value of the thermal stability of the catalyst is at 180 °C where the catalyst is able to process up to a temperature of 180 °C. Each step of the ROP is validated by FTIR analysis which shows that the components of the group are in accordance with the expected product. The comparison of the crystallinity values of PLA produced with FeCl3 and Fe(DS)3 were 37.5 and 49%, respectively. Melting temperature (Tm) values are 111 and 107 °C, respectively, Td values are 327 and 352 °C, and the resulting molecular weights are 23,720 and 20,232 g/mol. Based on the results obtained, it can be seen that the use of a LASC catalyst in the form of Fe(DS)3 is more effective than without using a LASC catalyst.

Mealworm larvae (Tenebrio molitor) and crickets (Acheta domesticus) show high total protein in vitro digestibility and can provide good-to-excellent protein quality as determined by in vitro DIAAS.

Edible insects, such as mealworms (Tenebrio molitor larvae; TM) and crickets (Acheta domesticus; AD), are a sustainable, protein-dense novel food with a favorable amino acid profile, which might be an alternative to animal proteins. To assess the protein quality of TM and AD, we assessed the digestible indispensable amino acid scores (DIAAS), considering individual amino acids and their ileal amino acid digestibility, using an in vitro model based on the INFOGEST digestion protocol. In addition, we evaluated if various processing and food preparation steps influenced the in vitro digestibility of individual amino acids and the in vitro DIAAS values of TM and AD and compared them to chicken breast as a reference of excellent protein quality. The total protein in vitro digestibility ranged from 91 to 99% for TM and from 79 to 93% for AD and was negatively affected by oven-drying and, to a lesser extent, by chitin-reduction. The in vitro DIAAS values were 113, 89, and 92 for chicken, blanched TM, and blanched AD, respectively, when considering the indispensable amino acid (IAA) requirements of young children between 6 months and 3 years. Across different processing and food preparation methods, the in vitro DIAAS values ranged between 59 and 89 for TM and between 40 and 92 for AD, with the lowest values found in chitin-reduced insects. Due to their similarities to chicken regarding protein composition, total protein in vitro digestibility, and in vitro DIAAS values, TM and AD might be an alternative to traditional animal proteins, provided that suitable processing and food preparation methods are applied. Our in vitro DIAAS results suggest that TM and AD can thus be considered good-quality protein sources for children older than 6 months. The DIAAS calculations are currently based on crude protein (total nitrogen × 6.25), resulting in an overestimation of insect protein content, and leading to an underestimation and potential misclassification of protein quality. The in vitro model applied in this study is a valuable tool for product development to optimize the protein quality of edible insects. Further studies are required to assess the in vivo DIAAS of insects in humans.

Computational study of structural, thermoelectric, elastic and optoelectronic properties of new halide perovskites CsRbTeX6 (X = I, Br and Cl) for solar cell applications

Recently the lead-free halide perovskites materials have attracted considerable attention due to their nontoxic nature and outstanding performances. In this work, using density-functional theory (DFT), the structural, thermoelectric, elastic and optoelectronic properties of new series perovskites CsRbTeX6 (X= I, Br, and Cl) have been investigated. The Goldschmidt tolerance factor (τ) and octahedral factors (μ) results show a high structural stability. The computed bond length Te-X and lattice parameter a0 decrease progressively when the size of the X Halide atoms increases. The elastic constants and their derived parameters reveal a brittle behavior, high mechanical stability and anisotropy nature of these materials. The low values of the bulk modulus in the three structures indicate that they are relatively soft materials and that they are amenable to being converted into thin films, which is essential for solar cell applications. The computed values of Tm are 582.49K, 579.36 K and 578.71 K for CsRbTeI6, CsRbTeCl6 and CsRbTeBr6, respectively. Because of their high melting temperature, these materials are more appropriate for high temperature applications. The band structure profile exhibits a semiconductor behavior with indirect band gap of about 2.81 eV, 2.2 eV and 1.51 eV for CsRbTeCl6, CsRbTeBr6 and CsRbTel6 respectively. With such tunable band gaps, the optical performance of these materials has been examined. The results show a very high light absorption in the order of 106 cm−1, a high power conversion efficiency (ƞ) and a low reflectivity in the order 20% in the visible and UV region. Finally, the transport properties reveal a sufficient figure of merit and a high Seebeck coefficient. According to these results, we suggest that the studied perovskites exhibit outstanding performance for thermoelectric and optoelectronic applications.

Rapid and simultaneous identification of Lonicera Japonicae Flos and Lonicerae Flos using MLPA‐HRM and rhPCR‐HRM

AbstractBackgroundLonicera Japonicae Flos (LJF) and Lonicerae Flos (LF), derived from different species of Lonicera, are two different types of traditional Chinese medicine (TCM) according to their differences in composition and effectiveness. Because of the high price, good effectiveness of the medicinal, and functional values, LJF is often adulterated by LF in the market. Therefore, two single nucleotide polymorphism‐based genotyping methods, multiplex ligation‐dependent probe amplification high‐resolution melting (MLPA‐HRM) and RNase H2‐dependant PCR (rhPCR)‐HRM, were established for the identification of LF and LJF.ResultsHighly specific MLPA probes and rhPCR‐primers were designed based on the trnL‐trnF regions. The melting temperature (Tm) of amplicons was derived from the peak of the HRM curve, and the differences in Tm values were used to identify LJF and LF. The two methods have both shown good specificity for identifying LJF and LF without cross‐reaction, and have high sensitivity with the detection limits of 0.1 ng DNA template. For mixed samples, MLPA‐HRM could detect LJF mixed with 10% LF while rhPCR‐HRM could detect 5% LF in mixed samples. The MLPA‐HRM and rhPCR‐HRM were performed on DNA extracted from 54 samples of LJF and LF randomly selected from the medicinal material market, all samples were successfully identified and further verified with DNA barcoding.ConclusionMLPA‐HRM and rhPCR‐HRM methods developed in our study not only provided fast, simple, sensitive, and practical identification of LJF and LF, but also provided technical references for the adulteration identification of other herb‐medicines, they are of great significance to the quality control and safe use of plant medicinal materials.

Syntheses of Cyclic Poly(l‐lactide)s by Means of Zinc‐Based Ring‐Opening Polymerization with Simultaneous Polycondensation (ROPPOC) Catalysts

AbstractRing‐opening polymerizations of l‐lactide are studied in bulk at 140 or 160 °C with zinc n‐hexanoate, zinc 4‐chlorothiophenolate, and zinc pentafluoro thiophenolate (ZnSPF) as catalysts. The reactivity increases in the given order. With all three catalysts a high fraction of cycles is obtained only at polymerization (annealing) times around 7 d. With ZnSPF weight average molecular weights (Mw) up to 178 000, a Tm around 199 °C and a ΔHm around 99 J g−1 w ere achieved. The samples annealed for 4 or 7 d also display a saw tooth pattern of the mass peak distribution in the matrix‐assisted laser desorption/ionization time of flight spectra indicating transesterification reactions across the surface of extended ring crystals. This process optimizes the thermodynamical properties of the crystalline cyclic polylactides and is responsible for the high Tm and ΔHm values.

Optimisation of the machining time required by insole orthotic shoes for patients with clubfoot using the Taguchi and response surface methodology approach

In this study, the application of the computer-aided reverse engineering system (CARE) to the novel design and manufacture of a comfortable insole for a clubfoot patient is presented. The Taguchi method (TM) and response surface methodology (RMS) were used to predict the machining time of the orthotic boot insole during both computer-aided manufacturing (CAM) simulation and computer numerical control (CNC) machining. Taguchi’s experimental design, presented as a matrix orthogonal array L2736, was acquired for controlling parameters, namely tool path strategy (A), spindle speed (B), step-down (C), step-over of the cutter (D), cutter diameter (E), and dimensional tolerance (F) of the insole size. In this method, the model generated by the RMS method evaluates the six parameters influencing the machining time. The objective of this study is to develop a regression model that demonstrates the relationship between the cutting parameters and insole machining time. The optimal parameters are A1B1C3D2E1F2, where A1 denotes raster finishing, B1 denotes a spindle speed of 10,000 rpm, C3 denotes a step-down of 850 mm, D2 denotes a step-over of 0.25 mm, E1 denotes a cutter diameter of 20–35 mm, and F2 deontes a tolerance of 0.75 mm. The experimental and calculated machining time (tm) results were 236 and 125.4 min, respectively. However, the real machining results were 334 and 152.25 min with error values of 46.86% and 54.42%, respectively. Meanwhile, with the tm RMS method, the simulated and calculated machining time results were 189.22 and 236.35 min, whereas the real tm values were 236.52 and 334.86 min with error values of 19.94% and 29.37%, respectively. This research obtains improvements of 19.82% (simulation time) and 29.19% (real-time).

Determining MSI status by a one-step PCR melting curve analysis of Chinese-specific mononucleotide repeats.

e15532 Background: Microsatellite instability (MSI) is a hypermutated phenotype primarily caused by the deficiency of DNA mismatch repair activity. Conventional MSI detection platforms are time consuming and need extra normal tissue. Due to the short DNA sequences of microsatellite loci in MSI samples, the method can be simplified to a one-step approach to differentiate MSI status by observing the melting curve. Herein a novel feasible MSI status detection method based on one step-PCR in single tumor tissue was developed, and validated in colorectal cancer samples. Methods: Specific microsatellite sites shorter than 12 bp in length (feasibility of fluorescence probes) were selected from public databases for analysis. In tumor tissues from 98 CRC patients with known MSI status, microsatellite sites with the highest receiver operating characteristic (ROC)-area under the ROC curve (AUC) were eventually selected for the final PCR panel design. The cut-off value of this assay was defined by the number of melting peaks in a specific Tm value range. An independent set of 199 tumor tissues from CRC patients with known MSI by Sanger sequencing was recruited to validate the performance of this novel PCR MSI kit. Results: The ROC-AUCs of the pre-selected eight mononucleotide repeats were 0.82, 0.93, 0.79, 0.78, 0.98, 0.95, 0.92, and 0.79. When 0 or 1 marker had a melting peak within the specified Tm value range, the sample was identified as "MSS/MSI-low (MSI-L)"; when 2 or more markers had melting peaks within the specified Tm value range, the sample was determined to be "MSI-high (MSI-H)". In the clinical sample set, compared with the results given by conventional MSI detection method, the sensitivity of the 8-gene panel for MSI-H was 99.4% (166/167), the specificity of the 8-gene panel was 100%, and the overall concordance reached to 99.5%. Conclusions: The novel PCR-MSI detection method using 8-gene panel developed in this study was feasible and accessible in clinical practice, and a large-scale clinical validation is in progress. [Table: see text]

Effect of electron beam irradiation on thermal stability and crystallization behavior of flexible copolyester/multiwalled carbon nanotubes nanocomposites

AbstractPoly(butylene adipate‐co‐terephthalate) (PBAT), a biodegradable copolyester, was used as the polymer matrix to prepare nanocomposites with multiwalled carbon nanotubes (MWCNT) by melt‐mixing followed by hot‐pressing. The PBAT/MWCNT nanocomposites were exposed to electron beam (EB) irradiation, and thermal stability, melting and crystallization behavior of irradiated and unirradiated nanocomposites were comparatively investigated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), respectively. TGA results reveal increased thermal stability (up to 17°C) and maximum degradation temperature (Tmax) (up to 15°C) of PBAT/MWCNT nanocomposites, attributed to the high thermal stability of MWCNT and good MWCNT–PBAT interfacial interactions. However, the activation energy for thermal degradation (Ea) decreased with the presence of MWCNT in comparison to neat PBAT regardless of the MWCNT concentration. Both the thermal stability and Tmax of irradiated nanocomposites decreased by 3°C despite the crosslinking which can be attributed to successive minor irradiation‐induced polymer degradation, while Ea remained unchanged. Declined melting temperature (Tm), enthalpy of crystallization, enthalpy of melting and crystallinity of nanocomposites with the presence of MWCNT suggest the formation of less perfect crystals. Meanwhile, their increased glass transition temperature (Tg) and crystallization temperature (Tc) are due to the increased rigidity of PBAT chains and a reduced crystallization process in the presence of MWCNT, respectively. Similarly, reduced crystallinity and values of Tm and Tc of EB‐irradiated nanocomposites by 4.1%, 9.6%, and 7.5%, respectively, signifying the presence of PBAT‐crosslinks resulting in crystal defects.