Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

Enhancement of Carrier Mobility in Multilayer InSe Transistors by van der Waals Integration.

Two-dimensional material indium selenide (InSe) holds great promise for applications in electronics and optoelectronics by virtue of its fascinating properties. However, most multilayer InSe-based transistors suffer from extrinsic scattering effects from interface disorders and the environment, which cause carrier mobility and density fluctuations and hinder their practical application. In this work, we employ the non-destructive method of van der Waals (vdW) integration to improve the electron mobility of back-gated multilayer InSe FETs. After introducing the hexagonal boron nitride (h-BN) as both an encapsulation layer and back-gate dielectric with the vdW interface, as well as graphene serving as a buffer contact layer, the electron mobilities of InSe FETs are substantially enhanced. The vdW-integrated devices exhibit a high electron mobility exceeding 103 cm2 V-1 s-1 and current on/off ratios of ~108 at room temperature. Meanwhile, the electron densities are found to exceed 1012 cm-2. In addition, the fabricated devices show an excellent stability with a negligible electrical degradation after storage in ambient conditions for one month. Electrical transport measurements on InSe FETs in different configurations suggest that a performance enhancement with vdW integration should arise from a sufficient screening effect on the interface impurities and an effective passivation of the air-sensitive surface.

Image fusion guidance for left subclavian artery in situ fenestration during thoracic endovascular repair.

OBJECTIVES: To evaluate the feasibility and clinical benefit of utilizing image fusion for thoracic endovascular repair (TEVAR) with in situ fenestration (ISF-TEVAR). MATERIALS AND METHODS: Between January 2020 and December 2020, we prospectively collected 18 consecutive cases with complex thoracic aortic lesions who underwent image fusion guided ISF-TEVAR. As a control group, 18 patients were collected from historical medical records from June 2019 to December 2019. The fusion group involved the use of 3D fusion of CTA and fluoroscopic images for real-time 3D guidance, and the control group involved the use of only regular fluoroscopic images for guidance. The total contrast medium volume, hand-injected contrast medium volume, overall operative time, radiation dose and fluoroscopy time were compared between the two groups. Accuracy was measured based on preoperative CTA and intraoperative digital subtraction angiography. RESULTS: 3D fusion imaging guidance was successfully implemented in all patients in the fusion group. Hand-injected contrast medium volume and overall operative time were significantly lower in the fusion group than in the control group (p = .028 and p = .011). Compared with the control group, the fusion group showed a significant reduction in time and radiation dose-area product (DAP) for fluoroscopy (p = .004 and p = .010). No significant differences in total radiation dose (DAP) or total contrast medium volume were observed (p = .079 and p = .443). Full accuracy was achieved in 8 cases (44%), with a mean deviation of 2.61 mm ± 3.1 (range 0.0-8.4 mm). CONCLUSIONS: 3D image fusion for ISF-TEVAR was associated with a significant reduction in hand-injected contrast medium, time and radiation exposure for fluoroscopy and overall operative time. The image fusion guidance showed potential clinical benefits towards improved treatment safety and accuracy for complex thoracic endovascular interventions.

Puerarin ameliorates metabolic dysfunction-associated fatty liver disease by inhibiting ferroptosis and inflammation.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is frequently linked to type 2 diabetes mellitus (T2DM), and both conditions exacerbate the progression of the other. However, there is currently no standardized treatment or drug for MAFLD. In this study, A MAFLD animal model through a high-fat diet (HFD) along with administration of streptozotocin (STZ), and palmitic acid (PA)-induced AML12 cells were treated by puerarin. The objective of this study was to assess the therapeutic effect of puerarin, a flavonoid substance that possesses various pharmacological properties, on MAFLD. The results showed that puerarin administration enhanced glucose tolerance and insulin sensitivity, while also mitigating liver dysfunction and hyperlipidemia in MAFLD mice. Moreover, puerarin attenuated oxidative stress levels and inflammation in the liver. Transmission electron microscopy and Western blot analysis indicated that puerarin inhibited ferroptosis in vivo. Further mechanistic investigations revealed that puerarin upregulated SIRT1 expression, increased nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels, and facilitated translocation into the nucleus. The protective effect of puerarin on PA-induced AML12 cells was diminished by the utilization of EX-527 (a SIRT1 inhibitor) and Nrf2 siRNA. Overall, the results demonstrate that puerarin ameliorates MAFLD by suppressing ferroptosis and inflammation via the SIRT1/Nrf2 signaling pathway. The results emphasize the possible medicinal application of puerarin for managing MAFLD.

Convolutional neural network model by deep learning and teaching robot in keyboard musical instrument teaching.

Keyboard instruments play a significant role in the music teaching process, providing students with an enjoyable musical experience while enhancing their music literacy. This study aims to investigate the current state of keyboard instrument teaching in preschool education, identify existing challenges, and propose potential solutions using the literature review method. In response to identified shortcomings, this paper proposes integrating intelligent technology and subject teaching through the application of teaching robots in keyboard instrument education. Specifically, a Convolutional Neural Network model of Deep Learning is employed for system debugging, enabling the teaching robot to analyze students' images and movements during musical instrument play and deliver targeted teaching. Feedback from students who participated in keyboard instrument teaching with the robot indicates high satisfaction levels. This paper aims to diversify keyboard instruments' teaching mode, introduce the practical application of robots in classroom teaching, and facilitate personalized teaching catering to individual students' aptitudes.

A fungal-algal self-flocculation system and its application to treat filter sludge leachate in the sugar industry.

The efficient and economical treatment of wastewater using microalgae has attracted much attention. However, harvesting microalgae cells from treated wastewater remains challenging. In the present study, a Chlorella vulgaris suspension containing filamentous fungi Aspergillus niger and Chaetomium gracile was successfully used to construct a self-flocculating system, with a microalgae flocculation efficiency of 99.6% achieved by gravity sedimentation within 4 h. The diameter of fungi played an important role in determining flocculation efficiency, and the optimal particle size was 10 mm. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) results indicated that the sweeping action of fungal mycelia and the interaction between the functional groups of fungi and the C. vulgaris surface contributed to improve flocculation. Co-cultivation of C. vulgaris and fungi could effectively remove 83.53%, 94.45% and 76.88% of total phosphorus, total nitrogen and chemical oxygen demand, respectively, from the sludge leachate from a sugar mill. The fungal-algal biomass reached 5.75 g/L. Herein, the constructed self-flocculation system had coupled efficient flocculation of C. vulgaris with removal of pollutants from wastewater in a short period of time, and providing a green, pollution-free, low-cost method for simultaneous wastewater treatment and the production of high quality biomass.

Anti-Pan-Rspo Chimeric Protein-Conjugated Albumin Nanoparticle Provides Promising Opportunities in Cancer Targeted Therapy.

Rspos (R-spondins) belong to a family of secreted proteins that causes various cancers via interacting the corresponding receptors. However, targeted therapeutic approaches against Rspos are largely lacking. In this study, a chimeric protein Rspo-targeting anticancer chimeric protein (RTAC) is originally designed, engineered, and characterized. RTAC shows satisfactory anticancer effects through inhibition of pan-Rspo-mediated Wnt/ß-catenin signaling activation both in vitro and in vivo. Furthermore, a conceptually novel antitumor strategy distinct from traditional drug delivery systems that release drugs inside tumor cells is proposed. A special "firewall" nano-system is designed to enrich on tumor cell surface and cover the plasma membrane, rather than undergoing endocytosis, to block oncogenic Rspos from binding to receptors. Cyclic RGD (Arg-Gly-Asp) peptide-linked globular cluster serum albumin nanoparticles (SANP) are integrated as a vehicle for conjugating RTAC (SANP-RTAC/RGD) for tumor tissue targeting. These nanoparticles can adhere to the tumor cell surface and enable RTAC to locally capture free Rspos with high spatial efficiency and selectivity to antagonize cancer progression. Therefore, this approach offers a new nanomedical anticancer route and obtains the "dual-targeting" capability for effective tumor clearance and low potential toxicity. This study presents a proof-of-concept for anti-pan-Rspo therapy and a nanoparticle-integrated paradigm for targeted cancer treatment.

Pt single atoms meet metal-organic frameworks to enhance electrocatalytic hydrogen evolution activity.

The electrochemical hydrogen evolution reaction (HER) effectively produces clean, renewable, and sustainable hydrogen; however, the development of efficient electrocatalysts is required to reduce the high energy barrier of the HER. Herein, we report two excellent single-atom (SA)/metal-organic framework (MOF) composite electrocatalysts (PtSA-MIL100(Fe) and PtSA-MIL101(Cr)) for HER. The obtained PtSA-MIL100(Fe) and PtSA-MIL101(Cr) electrocatalysts exhibit overpotentials of 60 and 61 mV at 10 mA cm-2, respectively, which are close to that of commercial Pt/C (38 mV); they exhibit overpotentials of 310 and 288 mV at 200 mA cm-2, respectively, which are comparable to that of commercial Pt/C (270 mV). Theoretical simulations reveal that Pt SAs modulate the electronic structures of the MOFs, leading to the optimization of the binding strength for H* and significant enhancement of the HER activity. This study describes a novel strategy for preparing desirable HER electrocatalysts based on the synergy between SAs and MIL-series MOFs. Using MIL-series MOFs to support SAs could be valuable for future catalyst design.

An ultra-small fluorescence zero-valent iron nanoclusters selectively kill gram-positive bacteria by promoting reactive oxygen species generation.

A list of the most dangerous bacteria that are multiple-drug resistance has been published by WHO, among which are various Gram-positive bacteria related with serious healthcare and community-associated infection. An effort is called for developing new strategies to combat the resistance, and nanomaterial-based approaches provide an ideal potential to mitigate the antimicrobial resistance as an alternative to antibiotics. Nanoscale zero-valent iron particles exhibited a good antimicrobial activity by triggering Fenton reaction, however, no zero-valent iron nanoclusters are developed as antimicrobial medical materials. In this work, a novel ultra-small zero-valent iron nanoclusters (usZVIN) was synthesized by one-step reduction in aqueous solutions, which exhibited bright red fluorescence at 616 nm. Interestingly, the usZVIN displayed an excellent selectively antibacterial activity against Gram-positive bacteria, and little effects on Gram-negative bacteria. The killing efficiency of usZVIN against S. aureus can reach 100 % with a concentration of 40 µg mL-1 after 1 h incubation, whereas there is no killing effect of usZVIN against E.coli even with a concentration of 900 µg mL-1 for 4 h. The antimicrobial mechanism of usZVIN was demonstrated to be the intracellular reactive oxygen species (ROS) production triggered by usZVIN due to its excellent peroxidase-like activity. Collectively, our findings suggested that usZVIN is a good medical-material candidate for fighting against Gram-positive bacterial infections, especially when we need leave beneficial Gram-negative bacteria intact.

Electronic and Optoelectronic Monolayer WSe2 Devices via Transfer-Free Fabrication Method.

Monolayer transition metal dichalcogenides (TMDs) have drawn significant attention for their potential applications in electronics and optoelectronics. To achieve consistent electronic properties and high device yield, uniform large monolayer crystals are crucial. In this report, we describe the growth of high-quality and uniform monolayer WSe2 film using chemical vapor deposition on polycrystalline Au substrates. This method allows for the fabrication of continuous large-area WSe2 film with large-size domains. Additionally, a novel transfer-free method is used to fabricate field-effect transistors (FETs) based on the as-grown WSe2. The exceptional metal/semiconductor interfaces achieved through this fabrication method result in monolayer WSe2 FETs with extraordinary electrical performance comparable to those with thermal deposition electrodes, with a high mobility of up to ≈62.95 cm2 V-1 s-1 at room temperature. In addition, the as-fabricated transfer-free devices can maintain their original performance after weeks without obvious device decay. The transfer-free WSe2-based photodetectors exhibit prominent photoresponse with a high photoresponsivity of ~1.7 × 104 A W-1 at Vds = 1 V and Vg = -60 V and a maximum detectivity value of ~1.2 × 1013 Jones. Our study presents a robust pathway for the growth of high-quality monolayer TMDs thin films and large-scale device fabrication.

Irreversible biosynthesis of D-allulose from D-glucose in Escherichia coli through fine-tuning of carbon flux and cofactor regeneration engineering.

BACKGROUND: As a rare hexose with low calories and various physiological functions, d-allulose has drawn increasing attention. The current industrial production of d-allulose from d-fructose or d-glucose is achieved via epimerization based on the Izumoring strategy; however, the inherent reaction equilibrium during reversible reaction limits its high conversion yield. Although the conversion of d-fructose to d-allulose could be enhanced via phosphorylation-dephosphorylation mediated by metabolic engineering, biomass reduction and byproduct accumulation remain a largely unresolved issue. RESULTS: After modifying the glycolytic pathway of Escherichia coli and optimizing the whole-cell reaction condition, the engineered strain produced 7.57 ± 0.61 g L-1 d-allulose from 30 g L-1 d-glucose after 24 h of catalysis. By developing an ATP regeneration system for enhanced substrate phosphorylation, the cell growth inhibition was alleviated and d-allulose production increased by 55.3% to 11.76 ± 0.58 g L-1 (0.53 g g-1 ). Fine-tuning of carbon flux caused a 48% reduction in d-fructose accumulation to 1.47 ± 0.15 g L-1 . After implementing fed-batch co-substrate strategy, the d-allulose titer reached 15.80 ± 0.31 g L-1 (0.62 g g-1 ) with a d-glucose conversion rate of 84.8%. CONCLUSION: The present study reports a novel strategy for high-yield d-allulose production from low-cost substrate. © 2023 Society of Chemical Industry.

Whole-cell biotransformation for simultaneous synthesis of allitol and d-gluconic acid in recombinant Escherichia coli.

Allitol and gluconic acid (GA) are important industrial compounds that are preferably produced via bio-production processes. In this research, d-psicose-3-epimerase (DPEase), glucose dehydrogenase (GDH), and ribitol dehydrogenase (RDH) were heterologously expressed in Escherichia coli, realizing the co-production of allitol and GA. Compared to the loss of carbon flux from formate dehydrogenase (FDH), glucose dehydrogenase can produce GA while generating NAD(H). The recombinant strain Ec/pAd-pRrg boosted NADH production to 2.4 µmol/gDCW, 118% higher than with the control strain. Under the optimized conditions, 12.0 g/L allitol and 14.8 g/L GA were produced from 25 g/L d-fructose and 20 g/L d-glucose; i.e., 66.7% and 66.3% higher yields compared to the case of fermentation without optimization, respectively. Furthermore, 42.7 g/L allitol and 56.2 g/L GA can be obtained from pretreated molasses (containing 139.2 g/L d-fructose and 149.1 g/L d-glucose). This work provides a practicable strategy for industrial and efficient co-production of allitol and GA from a cheap raw substrate.

Mesenchymal stromal cells ameliorate diabetes-induced muscle atrophy through exosomes by enhancing AMPK/ULK1-mediated autophagy.

BACKGROUND: Diabetes and obesity are associated with muscle atrophy that reduces life quality and lacks effective treatment. Mesenchymal stromal cell (MSC)-based therapy can ameliorate high fat-diet (HFD) and immobilization (IM)-induced muscle atrophy in mice. However, the effect of MSCs on muscle atrophy in type 2 diabetes mellitus (T2DM) and the potential mechanism is unclear. Here, we evaluated the efficacy and explored molecular mechanisms of human umbilical cord MSCs (hucMSCs) and hucMSC-derived exosomes (MSC-EXO) on diabetes- and obesity-induced muscle atrophy. METHODS: Diabetic db/db mice, mice fed with high-fat diet (HFD), mice with hindlimb immobilization (IM), and C2C12 myotubes were used to explore the effect of hucMSCs or MSC-EXO in alleviating muscle atrophy. Grip strength test and treadmill running were used to measure skeletal muscle strength and performance. Body composition, muscle weight, and muscle fibre cross-sectional area (CSA) was used to evaluate muscle mass. RNA-seq analysis of tibialis anterior (TA) muscle and Western blot analysis of muscle atrophy signalling, including MuRF1 and Atrogin 1, were performed to investigate the underlying mechanisms. RESULTS: hucMSCs increased grip strength (P = 0.0256 in db/db mice, P = 0.012 in HFD mice, P = 0.0097 in IM mice), running endurance (P = 0.0154 in HFD mice, P = 0.0006 in IM mice), and muscle mass (P = 0.0004 in db/db mice, P = 0.0076 in HFD mice, P = 0.0144 in IM mice) in all models tested, with elevated CSA of muscle fibres (P < 0.0001 in db/db mice and HFD mice, P = 0.0088 in IM mice) and reduced Atrogin1 (P = 0.0459 in db/db mice, P = 0.0088 in HFD mice, P = 0.0016 in IM mice) and MuRF1 expression (P = 0.0004 in db/db mice, P = 0.0077 in HFD mice, P = 0.0451 in IM mice). MSC-EXO replicated all these hucMSC-mediated changes (P = 0.0103 for grip strength, P = 0.013 for muscle mass, P < 0.0001 for CSA of muscle fibres, P = 0.0171 for Atrogin1 expression, and P = 0.006 for MuRF1 expression). RNA-seq revealed that hucMSCs activated the AMPK/ULK1 signalling and enhanced autophagy. Knockdown of AMPK or inhibition of autophagy with 3-methyladenine (3-MA) diminished the beneficial anti-atrophy effects of hucMSCs or MSC-EXO. CONCLUSIONS: Our results suggest that human umbilical cord mesenchymal stromal cells mitigate diabetes- and obesity-induced muscle atrophy via enhancing AMPK/ULK1-mediated autophagy through exosomes, with implications of applying hucMSCs or hucMSC-derived exosomes to treat muscle atrophy.

Room-temperature valley transistors for low-power neuromorphic computing.

Valley pseudospin is an electronic degree of freedom that promises highly efficient information processing applications. However, valley-polarized excitons usually have short pico-second lifetimes, which limits the room-temperature applicability of valleytronic devices. Here, we demonstrate room-temperature valley transistors that operate by generating free carrier valley polarization with a long lifetime. This is achieved by electrostatic manipulation of the non-trivial band topology of the Weyl semiconductor tellurium (Te). We observe valley-polarized diffusion lengths of more than 7 µm and fabricate valley transistors with an ON/OFF ratio of 105 at room temperature. Moreover, we demonstrate an ion insertion/extraction device structure that enables 32 non-volatile memory states with high linearity and symmetry in the Te valley transistor. With ultralow power consumption (~fW valley contribution), we enable the inferring process of artificial neural networks, exhibiting potential for applications in low-power neuromorphic computing.

MiRNA-21 promotes differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells by regulating the Ajuba/Isl1 axis pathway.

Introduction: In this study, we aimed to investigate the role of miRNA-21 and the regulating pathway that promotes the differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods: We used miR-21-OE, miR-21-KD, ajuba-OE and ajuba-KD plasmids to infect BMSCs. The expression of miRNA-21, ajuba, Isl1 and cTnI was detected by RT-qPCR, WB and immunofluorescence staining in groups. Results: MiRNA-21 over-expression increased the expression of Isl1, and vice versa. Ajuba over-expression decreased the expression of Isl1, and vice versa. Ajuba negatively regulated the differentiation of BMSCs into cardiomyocyte-like cells. Conclusions: MiRNA-21 could regulate differentiation of bone marrow mesenchymal stem cells (BMSCs) to cardiomyocyte-like cells through the ajuba/Isl1 axis pathway.

Phosphorylation-Driven Production of d-Allulose from d-Glucose by Coupling with an ATP Regeneration System.

d-Allulose is a desirable sucrose substitute with potential applications in food and health care. d-Allulose can be synthesized using d-glucose as a substrate through coupling glucose isomerase with d-allulose 3-epimerase (DAEase); however, the product yield is typically less than 20% at reaction equilibrium and thus limits its use in industrial applications. Here, a 3R-ketose phosphorylation pathway coupled with an adenosine triphosphate (ATP) regeneration system was developed for the efficient synthesis of d-allulose in Escherichia coli using d-glucose as a substrate. The l-rhamnulose kinase (RhaB) was used to break the inherent reaction equilibrium due to its substrate specificity, resulting in increases in d-allulose titer by 69.9% to 4.96 ± 0.49 g/L. By optimizing the whole cell transformation conditions and designing an ATP regeneration module, d-allulose production reached 17.62 ± 0.77 g/L from 30 g/L d-glucose with a final yield of 0.73 g/g without the addition of exogenous ATP. To evaluate the potential industrial application of this multienzyme cascade system, d-allulose was produced from cane molasses (124.16 ± 2.69 g/L glucose equivalent) with a final d-allulose titer of 62.60 ± 3.76 g/L. The present study provides a practical enzymatic approach for the economical synthesis of d-allulose.

Cardiovascular abnormalities and its correlation with genotypes of children with osteogenesis imperfecta.

Background and objectives: Osteogenesis imperfecta (OI) is a rare disorder of abnormal production or modification of type I collagen, which is caused by mutations in COL1A1, COL1A2 or other genes. We investigate the cardiac abnormalities and its correlation with pathogenic mutations in OI children. Methods: A cross-sectional comparative study was completed in a relatively large sample of OI children, who were matched in body surface area (BSA) with healthy controls. All echocardiography was performed by experienced cardiologists using Vivid 7 equipment (GE Medical Systems, Horton, Norway). The resting standard 12-lead electrocardiogram (ECG) were obtained in OI patients by FX-8600 machine. Skeletal phenotypes of OI patients were evaluated, including information of bone fractures, deformities, motility, and bone mineral density (BMD). Pathogenic mutations of OI were detected by a next-generation sequencing panel and confirmed by Sanger sequencing. Results: A total of 69 OI children and 42 healthy children matched in BSA were enrolled. Abnormalities of echocardiography were found in 6 OI children, including enlarged left atrium (n=5), increased internal diameter of the left ventricle (n=1), who all carried the COL1A1 mutation. Mild regurgitation of mitral or tricuspid valves was observed in 26 OI patients. Abnormal ECG manifestations were found in 8 OI children, including deep Q wave, T wave change, premature ventricular complexes, short P-R interval, incomplete bundle branch block and high voltage of left ventricular. Compared with healthy controls, OI children had significant larger values in the main pulmonary artery (1.84 vs 1.60 cm, P < 0.01), left atrial diameter (2.58 vs 2.11 cm, P < 0.001), left ventricular internal dimension at end-diastolic (LVEDd) (3.85 vs 3.50 cm, P < 0.05) and lower left ventricular ejection fraction (LVEF) (68.40% vs 71.74%, P < 0.01). Moreover, OI patients with COL1A1 mutation tended to have greater main pulmonary artery, larger diameters of left atrial and LVEDd, and lower LVEF than healthy controls. COL1A1 mutation was correlated to dilated MPA (ß = 1.557, P < 0.01), LAD (ß = 3.915, P < 0.001), and LVEDd (ß = 2.714, P < 0.01), and decreased LVEF (ß = -3.249, P < 0.01). Conclusions: Cardiovascular alterations were identified in OI children, including increased dimensions of the main pulmonary artery and left chamber, and low LVEF. The cardiovascular abnormalities seemed to be correlated to COL1A1 mutation and defects of type I collagen, which expanded our understandings of the cardiac phenotypes of OI children.

Clarification of Limed Sugarcane Juice by Stainless Steel Membranes and Membrane Fouling Analysis.

The performance of stainless steel membranes with pore sizes of 100 and 20 nm in clarifying limed sugarcane juice was investigated under different operating conditions. An increase in transmembrane pressure (TMP) for the 20 nm membrane from 2 to 5 bar led to an increase in the average flux from 146.6 Lm-2 h-1 to 187.8 Lm-2 h-1 (approximately 9 h). The increase in crossflow velocity from 2 to 5 m/s led to an increase in the average flux from 111.9 Lm-2 h-1 to 158.1 Lm-2 h-1. The increase in temperature from 70 °C to 90 °C caused an increase in the average flux from 132.8 Lm-2 h-1 to 148.6 Lm-2 h-1. Simultaneously, the test produced a high-quality filtered juice with an average of 1.26 units of purity rise. The purity increased with time, and a 99.99% reduction in turbidity and an average 29.3% reduction in colour were observed. In addition, four classic filtration mathematical models and scanning electron microscopy (SEM) analyses suggested that cake formation is the main mechanism for flux decline. Fourier transform infrared (FTIR) spectrometry and energy-dispersive X-ray (EDX) spectrometry indicated that organic fouling is the main foulant. This study demonstrates the potential of stainless steel membranes as filters for the clarification of raw sugarcane juice.

Improved thermostability of D-allulose 3-epimerase from Clostridium bolteae ATCC BAA-613 by proline residue substitution.

d-allulose, a rare sugar that is scarce in nature, exerts several beneficial effects and has commercial potential. d-allulose 3-epimerase (DAEase) plays a vital role in catalyzing the isomerization from d-fructose to d-allulose. However, the industrial application of DAEase for d-allulose production is hindered by its poor long-term thermostability. In the present research, we introduced a proline residue (i) to restrict its spatial conformation and (ii) to reduce the entropy of the unfolded state of DAEase. The t1/2 value of the double-site Clostridium bolteae DAEase mutant Cb-51P/89P was prolonged to 58 min at 55 °C, a 2.32-fold increase compared with wild-type DAEase. The manipulation did not cause obvious changes in the enzymatic properties, including optimum pH, optimal temperature, optimum metal ion, and enzymatic activity. As the accumulation of multiple small effects through proline substitution could dramatically improve the thermostability of the mutant protein, our method to improve the thermostability while roughly retaining the original enzymatic properties is promising.

Clinical features and functions of a novel Lpl mutation C.986A>C (p.Y329S) in patient with hypertriglyceridemia.

OBJECTIVE: To investigate and assess the clinical features and functions of a new lipoprotein lipase (Lpl) gene mutation c.986A>C (p.Y329S) found in hypertriglyceridemia(HTG) patients from a Chinese family. METHODS: Five members of a family with the proband were diagnosed with HTG were investigated, and fasting peripheral blood was collected . The plasma was then used to measure triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein cholesterol (HDL-C), free fatty acids (FFA), and glucose tolerance. Following that, genomic deoxyribonucleic acid (DNA) was extracted from whole-blood samples using the QIAamp whole-blood DNA kit, and the coding exon regions and flanking regions of 95 dyslipidemia-related genes were captured using GenCap liquid-phase target gene capture technology. The activity of LPL and its mutation were then determined using cell assays, and the newly discovered LPL mutant was functionally analyzed. The binding site of fenofibrate and LPL, as well as the mutation, were subjected to predictive analysis. RESULTS: The LPL gene's c.986A>C (p.Y329S) heterozygous mutation was discovered, and patients with the mutation had the typical phenotype of LPL deficiency and weakened LPL activity. Furthermore, this mutant has been treated with fenofibrate, and its triglyceride level is perfectly controlled and stable. The prediction analysis of the fenofibrate and LPL binding sites reveals that the wild-type system, Phe378 contributes most to the binding energy of fenofibrate. In the mutant system, Tyr394, which contributes the most to the binding energy of fenofibrate, the contribution of S329 is greater than that of Y329 (0.9∼0.7 kal/mol) . After Y329 is mutated, the hydrogen bond data of fenofibrate and LPL will also increase to quote H-bond diagrams. CONCLUSIONS: A heterozygous mutation c.986A>C (p.Y329S) in exon 6 of Lpl gene occurs in the proband with familial HTG. Lpl c.986A>C (p.Y329S) mutation weakens the activity of the LPL, which may be the pathogenic mutation of HTG. In addition, The proband has been treated with fenofibrate and the triglyceride level is ideally controlled and stable. The prediction analysis of the fenofibrate and LPL binding site shows that the wild-type system, Phe378 contributes most to the binding energy of fenofibrate. In the mutant system, Tyr394, which contributes the most to the binding energy of fenofibrate, the contribution of S329 is greater than that of Y329 (0.9∼0.7 kal/mol). After Y329 is mutated, the hydrogen bond data of fenofibrate and LPL will also increase, which may be one of the reasons why the mutation has no effect on the therapeutic effect of fenofibrate.