Biparatopic anti-PCSK9 antibody enhances the LDL-uptake in HepG2 cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target to reduce lipids. In 2020, we reported a chimeric camelid-human heavy chain antibody VHH-B11-Fc targeting PCSK9. Recently, it was verified that VHH-B11 binds one linear epitope in the PCSK9 hinge region. To enhance its druggability, we have developed a novel biparatopic B11-H2-Fc Ab herein. Thereinto, surface plasmon resonance (SPR) confirmed the epitope differences in binding-PCSK9 among VHH-B11, VHH-H2 and the approved Repatha. Additionally, SPR revealed the B11-H2-Fc exhibits an avidity of approximately 0.036 nM for PCSK9, representing a considerable increase compared to VHH-B11-Fc (~ 0.69 nM). Moreover, we found the Repatha and B11-H2-Fc exhibited > 95% PCSK9 inhibition efficiency compared to approximately 48% for the VHH-Fc at 7.4 nM (P < 0.0005). Further, we verified its biological activity using the human hepatoma cells G2 model, where the B11-H2-Fc exhibited almost 100% efficiency in PCSK9 inhibition at only 0.75 µM. The immunoblotting results of low-density lipoprotein cholesterol (LDL-c) uptake assay also demonstrated the excellent performance of B11-H2-Fc on recovering the LDL-c receptor (LDLR), as strong as the Repatha (P > 0.05). These findings provide the first evidence of the efficacy of a novel Ab targeting PCSK9 in the field of lipid-lowering drugs.

The Improved Antineoplastic Activity of Thermophilic L-Asparaginase Tli10209 via Site-Directed Mutagenesis.

Amino acid deprivation therapy (AADT) is a novel anticancer therapy, considered nontoxic and selective. Thermophilic L-asparaginase enzymes display high stability and activity at elevated temperatures. However, they are of limited use in clinical applications because of their low substrate affinity and reduced activity under physiological conditions, which may necessitate an improved dosage, leading to side effects and greater costs. Thus, in an attempt to improve the activity of L-Asn at 37 °C, with the use of a semi-rational design, eight active-site mutants of Thermococcus litoralis DSM 5473 L-asparaginase Tli10209 were developed. T70A exhibited a 5.11-fold increase compared with the wild enzyme in physiological conditions. Double-mutant enzymes were created by combining mutants with higher hydrolysis activity. T70A/F36Y, T70A/K48L, and T70A/D50G were enhanced by 5.59-, 6.38-, and 5.58-fold. The immobilized enzyme applied in MCF-7 breast cancer cells only required one-seventh of the dose of the free enzyme to achieve the same inhibition rate under near-infrared irradiation. This provides a proof of concept that it is possible to reduce the consumption of L-Asn by improving its activity, thus providing a method to manage side effects.

The effect of stir-frying on the aging of oat flour during storage: A study based on lipidomics.

In this study, we used the LC-ESI-MS/MS technique to elucidate the effects of stir-frying on the lipidomics of oat flour before and after storage. We detected 1540 lipids in 54 subclasses; triglycerides were the most abundant, followed by diacylglycerol, ceramide (Cer), digalactosyldiacylglycerol, cardiolipin, and phosphatidylcholine. Principal component analysis and orthogonal least squares discriminant analysis analyses showed that oat flour lipids were significantly different before and after storage in stir-fried oat flour and raw oat flour. After oat flour was stir-fried, most of the lipid metabolites in it were significantly downregulated, and the changes in lipids during storage were reduced. Sphingolipid metabolism and ether lipid metabolism were the key metabolic pathways, and Cer, PC, and lyso-phosphatidylcholine were the key lipid metabolites identified in the related metabolic pathways during oat flour storage. Frying inhibits lipid metabolic pathways during storage of oat flour, thereby improving lipid stability and quality during storage. This study laid the foundation for further investigating quality control and the mechanism of changes in lipids during the storage of oat flour.

Enhanced nitrate removal efficiency and microbial response of immobilized mixed aerobic denitrifying bacteria through biochar coupled with inorganic electron donors in oligotrophic water.

The nitrogen removal characteristics and microbial response of biochar-immobilized mixed aerobic denitrifying bacteria (BIADB) were investigated at 25 °C and 10 °C. BIADB removed 53.51 ± 1.72 % (25 °C) and 39.90 ± 4.28 % (10 °C) nitrate in synthetic oligotrophic water. Even with practical oligotrophic water, BIADB still effectively removed 47.66-53.21 % (25 °C), and 39.26-45.63 % (10 °C) nitrate. The addition of inorganic electron donors increased nitrate removal by approximately 20 % for synthetic and practical water. Bacterial and functional communities exhibited significant temperature and stage differences (P < 0.05), with temperature and total dissolved nitrogen being the main environmental factors. The dominant genera and keystone taxa exhibited significant differences at the two temperatures. Structural equation model analysis showed that dissolved organic matter had the highest direct and indirect effects on diversity and function, respectively. This study provides an innovative pathway for utilizing biochar and inorganic electron donors for nitrate removal from oligotrophic waters.

Selective enhanced cytotoxicity of amino acid deprivation for cancer therapy using thermozyme functionalized nanocatalyst.

BACKGROUND: Enzyme therapy based on differential metabolism of cancer cells has demonstrated promising potential as a treatment strategy. Nevertheless, the therapeutic benefit of reported enzyme drugs is compromised by their uncontrollable activity and weak stability. Additionally, thermozymes with high thermal-stability suffer from low catalytic activity at body temperature, preventing them from functioning independently. RESULTS: Herein, we have developed a novel thermo-enzymatic regulation strategy for near-infrared (NIR)-triggered precise-catalyzed photothermal treatment of breast cancer. Our strategy enables efficient loading and delivery of thermozymes (newly screened therapeutic enzymes from thermophilic bacteria) via hyaluronic acid (HA)-coupled gold nanorods (GNRs). These nanocatalysts exhibit enhanced cellular endocytosis and rapid enzyme activity enhancement, while also providing biosafety with minimized toxic effects on untargeted sites due to temperature-isolated thermozyme activity. Locally-focused NIR lasers ensure effective activation of thermozymes to promote on-demand amino acid deprivation and photothermal therapy (PTT) of superficial tumors, triggering apoptosis, G1 phase cell cycle arrest, inhibiting migration and invasion, and potentiating photothermal sensitivity of malignancies. CONCLUSIONS: This work establishes a precise, remotely controlled, non-invasive, efficient, and biosafe nanoplatform for accurate enzyme therapy, providing a rationale for promising personalized therapeutic strategies and offering new prospects for high-precision development of enzyme drugs.

Theoretical Study on the Mechanism of the Hydrogenation of Azo (N═N) Bonds to Amines Catalyzed by Manganese.

The mechanism of the transition metal manganese complex Mn(PhPNN)(CO)2Br (CA-4) that catalyzed the hydrogenation of the azo (N═N) bond to amines has been investigated using the PBE0 function. The results show that the whole reaction involves three basic processes: (1) the addition of H2 to CA gives IN2, which can hydrogenate the azo (N═N) bond at the later stage; (2) hydrogenation of azobenzene by IN2, which gives 1,2-diphenylhydrazine (PhNHNHPh); and (3) hydrogenation of 1,2-diphenylhydrazine by IN2, which affords aniline (PhNH2). The results suggest that the hydrogenation of CA and hydrogenation of azobenzene by IN2 to afford PhNHNHPh are easy to occur due to the low barriers, and the overall rate-determining step is the formation of IN11 and PhNH2 by breaking the N-N bond in the stage of hydrogenation of 1,2-diphenylhydrazine by IN2, with an energy barrier of 39.1 kcal/mol. The computed results are in good agreement with the experimental results. The mechanism of the azobenzene reaction catalyzed by manganese was analyzed by charge and orbital analysis in detail. The theoretical results provide a deeper understanding of the mechanism and fully explain the experimental facts.

Research on Processability and Transmission Performance of Low Temperature Co-Fired Ceramic Ball Grid Array Packaging Based on Electroless Plating Surface Modification for Microwave Transceiver Circuits.

A microwave transmitter/receiver using the low-temperature co-fired ceramic substrate and ball grid array packaging demonstrates superior properties, including high integration, miniaturization, and high electromagnetic shielding. However, it holds limitations of inadequate hermeticity (that is, gas or moist impermeability), high cost, and low reproducibility. In this work, we aim to overcome these difficulties by introducing a new packing technique. The packaging utilizes an electroless plated Ni/Pd/Au surface, resulting in a significant enhancement of the packaging hermeticity by orders of magnitude, approaching the level of <5 × 10-9 Pa·m3/s. Both Sn63Pb37 and Au80Sn20 solder alloys demonstrate exceptional solderability, attributed to Pd atoms diffusing to the Au layer during soldering at 310 °C. A reliability test of the packaging shows that the shear strength of the solder balls drops after thermal shocks but negligibly affects the hermeticity of the packaging. Furthermore, a meticulously designed internal vertical interconnect structure and I/O interconnections were engineered in the ball grid array packaging, showcasing excellent transmission characteristics within the 10-40 GHz frequency range while ensuring effective isolation between ports.

Fluorescence enhancement of surface plasmon coupled emission by Au nanobipyramids and its modulation effect on multi-wavelength radiation.

Surface plasmon coupled emission (SPCE), a novel surface-enhanced fluorescence technique, can generate directional and amplified radiation by the intense interaction between fluorophores and surface plasmons (SPs) of metallic nanofilms. For plasmon-based optical systems, the strong interaction between localized and propagating SPs and "hot spot" structures show great potential to significantly improve the electromagnetic (EM) field and modulate optical properties. Au nanobipyramids (NBPs) with two sharp apexes to enhance and restrict the EM field were introduced through electrostatic adsorption to achieve a mediated fluorescence system, and the emission signal enhancement was realized by factors over 60 compared with the normal SPCE. It has been demonstrated that the intense EM field produced by the NBPs assembly is what triggered the unique enhancement of SPCE by Au NBPs, which effectively overcomes the inherent signal quenching of SPCE for ultrathin sample detection. This remarkable enhanced strategy offers the chance to improve the detection sensitivity for plasmon-based biosensing and detection systems, and expand the range of applications for SPCE in bioimaging with more comprehensive and detailed information acquisition. The enhancement efficiency for various emission wavelengths was investigated in light of the wavelength resolution of SPCE, and it was discovered that enhanced emission for multi-wavelength could be successfully detected through the different emission angles due to the angular displacement caused by wavelength change. Benefit from this, the Au NBP modulated SPCE system was employed for multi-wavelength simultaneous enhancement detection under a single collection angle, which could broaden the application of SPCE in simultaneous sensing and imaging for multi-analytes, and expected to be used for high throughput detection of multi-component analysis.

Improving the adsorption characteristics and antioxidant activity of oat bran by superfine grinding.

Oat bran (OB) is a by-product of oat, which is rich in ß-glucan. As a new food processing technology, ultrafine powder can improve the surface properties of samples. OB with different grinding times was prepared, and its functional components, physical properties, adsorption properties, and antioxidant properties were evaluated. Results showed that with increased grinding times, the average particle size of OB decreased significantly (p < .05). And the water-holding capacity, swelling capacity, and water solubility index of OB increased significantly (p < .05), whereas the animal and vegetable oil-holding capacities decreased. Oat bran could adsorb cholic acid and glucose, which was related to the time of superfine grinding. In addition, the antioxidant capacity of OB was improved after superfine grinding. Related analysis shows that there was significant positive relationship between ß-glucan, polyphenols and soluble dietary fibers and antioxidant indicators (p < .05). The Fourier transform infrared (FTIR) results showed that the FTIR spectra of OB powder with different crushing times were similar. On the basis of the above analyses, it is suggested that OB prepared by superfine grinding for 5 min had good physical and chemical properties and antioxidant properties and is widely used in food.

New polyketides from the basidiomycetous fungus Pholiota sp.

Two new polyketides, pholiotones B and C (1 and 2), and four known compounds, trichodermatide D (3), vermistatin (4), dehydroaltenuene A (5) and terpestacin (6) were isolated from the crude extract of Pholiota sp. Their structures were identified by NMR and MS spectroscopic data. The absolute configurations of compounds 1 and 2 were elucidated by modified Mosher's method, electronic circular dichroism (ECD) calculations and 13C NMR calculations as well as DP4+ probability analyses. All the compounds were evaluated for their antifungal and cytotoxicity.

Alternarialone A, a new curvularin-type metabolite from the mangrove-derived fungus Alternaria longipes.

Alternarialone A (1), one new curvularin derivative, and two known compounds (2 and 3) were isolated from the crude extract of the mangrove-derived fungus Alternaria longipes. Their structures were elucidated by comprehensive spectroscopic analyses, including MS and NMR spectroscopic data. The absolute configuration of 1 was assigned by 13C NMR calculations and a comparison of electronic circular dichroism (ECD) spectra. All compounds were evaluated for their antibacterial activities against Helicobacter pylori. Compounds 2 and 3 showed antibacterial activities against H. pylori G27 with MIC values of 8 and 16 µg/ml, respectively, while compound 3 also displayed antibacterial activity against H. pylori BHKS159 with the MIC value of 16 µg/ml.

Effects of Pretreatment on the Volatile Composition, Amino Acid, and Fatty Acid Content of Oat Bran.

Pretreatment improves the edible quality of oat bran and prolongs the shelf life, whereas the effect of pretreatments (i.e., steaming(S-OB), microwaving(M-OB), and hot-air drying(HA-OB)) on the flavor characteristics of oat bran is unknown. This study identified volatile composition using HS-SPME/GC−MS and an electronic nose of oat bran. The amino acid compositions were determined by a High-Speed automatic amino acid analyzer and the fatty acids were determined by gas chromatography. The results showed that steaming and microwaving pretreatments enhanced the nutty notes of oat bran. Sixty-four volatile compounds in four oat brans were identified. OB exhibited higher aroma-active compounds, followed by S-OB, and M-OB, and the HA-OB had the lowest aroma-active compounds. Hexanal, nonanal, (E)-2-octenal,1-octen-3-ol, 2-ethylhexan-1-ol, and 2-pentylfuran were the key volatile compositions in oat bran. The aldehyde content decreased and the esters and ketones increased in steamed oat bran. Microwaving and hot air drying increased the aldehyde content and decreased the ester and alcohol content. Steamed oat bran had the lowest levels of total amino acids (33.54 g/100 g) and bitter taste amino acids (5.66 g/100 g). However, steaming caused a significant reduction in saturated fatty acid content (18.56%) and an increase in unsaturated fatty acid content (79.60%) of oat bran (p < 0.05). Hot air drying did not result in an improvement in aroma. The results indicated that steaming was an effective drying method to improve the flavor quality of oat bran.

ECG segmentation algorithm based on bidirectional hidden semi-Markov model.

Accurate segmentation of electrocardiogram (ECG) waves is crucial for cardiovascular diseases (CVDs). In this study, a bidirectional hidden semi-Markov model (BI-HSMM) based on the probability distributions of ECG waveform duration was proposed for ECG wave segmentation. Four feature-vectors of ECG signals were extracted as the observation sequence of the hidden Markov model (HMM), and the statistical probability distribution of each waveform duration was counted. Logistic regression (LR) was used to train model parameters. The starting and ending positions of the QRS wave were first detected, and thereafter, bidirectional prediction was employed for the other waves. Forwardly, ST segment, T wave, and TP segment were predicted. Backwardly, P wave and PQ segments were detected. The Viterbi algorithm was improved by integrating the recursive formula of the forward prediction and backward backtracking algorithms. In the QT database, the proposed method demonstrated excellent performance (Acc = 97.98%, F1 score of P wave = 98.37%, F1 score of QRS wave = 97.60%, F1 score of T wave = 97.79%). For the wearable dynamic electrocardiography (DCG) signals collected by the Shandong Provincial Hospital (SPH), the detection accuracy was 99.71% and the F1 of each waveform was above 99%. The experimental results and real DCG signal validation confirmed that the proposed new BI-HSMM method exhibits significant ability to segment the resting and DCG signals; this is conducive to the detection and monitoring of CVDs.

Gut mucosal microbiota profiles linked to colorectal cancer recurrence.

BACKGROUND: Emerging evidence links gut microbiota to various human diseases including colorectal cancer (CRC) initiation and development. However, gut microbiota profiles associated with CRC recurrence and patient prognosis are not completely understood yet, especially in a Chinese cohort. AIM: To investigate the relationship between gut mucosal microbiota profiles and CRC recurrence and patient prognosis. METHODS: We obtained the composition and structure of gut microbiota collected from 75 patients diagnosed with CRC and 26 healthy controls. The patients were followed up by regular examination to determine whether tumors recurred. Triplet-paired samples from on-tumor, adjacent-tumor and off-tumor sites of patients diagnosed with/without CRC recurrence were analyzed to assess spatial-specific patterns of gut mucosal microbiota by 16S ribosomal RNA sequencing. Next, we carried out bioinformatic analyses, Kaplan-Meier survival analyses and Cox regression analyses to determine the relationship between gut mucosal microbiota profiles and CRC recurrence and patient prognosis. RESULTS: We observed spatial-specific patterns of gut mucosal microbiota profiles linked to CRC recurrence and patient prognosis. A total of 17 bacterial genera/families were identified as potential biomarkers for CRC recurrence and patient prognosis, including Anaerotruncus, Bacteroidales, Coriobacteriaceae, Dialister, Eubacterium, Fusobacterium, Filifactor, Gemella, Haemophilus, Mogibacteriazeae, Pyramidobacter, Parvimonas, Porphyromonadaceae, Slackia, Schwartzia, TG5 and Treponema. CONCLUSION: Our work suggests that intestinal microbiota can serve as biomarkers to predict the risk of CRC recurrence and patient death.

Effects of Oat Complex High-Fiber Formula Powder on the Composition of Intestinal Microbiota and Enzyme Activities in Mice Induced by a High-Fat Diet.

Using oat-corn-konjac extruded mixed powder, oat bran micro powder, skim milk powder, Pueraria whole powder, and pumpkin powder as raw materials, a formula powder with high dietary fiber was prepared, and its effect on obesity in mice with a high-fat diet was investigated. After 7 days of adaptive feeding, the mice were divided into blank group, high-fat diet group, formula powder + high-fat diet group, and weight-loss drug + high-fat diet group. After 8 weeks of treatment, the body weight of mice were observed and measured to determine the composition of tract flora, liver leptin content, insulin content, and activities of AMP-activated protein kinase (AMPK), lipoprotein lipase (LPL), fatty acid synthetase (FAS), sterol-regulatory element-binding proteins (SREBPs), and acetyl CoA carboxylase 1 (ACC1). The results indicated that treatment with the formula powder could reduce the body weight of mice and increase the abundance of Bifidobacterium, Akkermansia, and Romboutsia compared to the group given a high-fat diet. Moreover, the leptin and insulin contents of the experimental group decreased from 5.67 µg/L to 0.12 µg/L and from 12.71 µg/L to 7.13 µg/L, respectively, compared to the control group, which was not significantly different from the blank group (P > 0.05). Also, the activities of AMPK and LPL increased, and the activities of FAS, SREBPs, and ACC1 were significantly decreased (P < 0.05). Some pathogenic bacteria were significantly positively correlated with leptin and FAS and significantly negatively correlated with LPL. Some beneficial bacteria were positively correlated with LPL. Therefore, the formula powder used in this study could reduce the body weight of mice, increase the abundance of some beneficial bacteria in the colonic intestinal microbiota, and improve the activities of enzymes related to lipid metabolism in the liver. This study provides a theoretical reference for the pathway by which high-fiber diet improves liver and intestinal metabolic abnormalities.

Concurrent chemoradiotherapy using gemcitabine and nedaplatin in recurrent or locally advanced head and neck squamous cell carcinoma.

BACKGROUND: Patients with recurrent or locally advanced head and neck squamous cell carcinoma (HNSCC) typically have limited treatment options and poor prognosis. AIM: To evaluate the efficacy and safety of two drugs with potent radio-sensitization properties including gemcitabine and nedaplatin as concurrent chemoradiotherapy regimens in treating HNSCC. METHODS: This single-arm prospective study enrolled patients with HNSCC to receive gemcitabine on days 1 and 8 and nedaplatin on days 1 to 3 for 21 days. Intensity-modulated radiation therapy with a conventional fraction was delivered 5 days per week. Objective response rate (ORR), disease control rate, and toxicity were observed as primary endpoints. Overall survival (OS) and progression free survival were recorded and analyzed as secondary endpoints. RESULTS: A total of 24 patients with HNSCC were enrolled. During the median 22.4-mo follow-up, both ORR and disease control rate were 100%. The one-year OS was 75%, and one-year progression-free survival (PFS) was 66.7% (median PFS was 15.1 mo). Recurrent HNSCC patients had a poorer prognosis than the treatment-naïve patients, and patients who achieved complete response had better survival than those in the PR group (all P < 0.05). The most common grade 1-4 (100%) or grade 3-4 toxicities (75%) were hematological, and the most common grade 3-4 non-hematological toxicity was mucositis in 17 (71%) patients. CONCLUSION: Gemcitabine plus nedaplatin with concurrent chemoradiotherapy is a therapeutic option for HNSCC with predictable tolerability. Considering the high adverse event rate, the optimized dose and schedule must be further explored.

Deficient DNASE1L3 facilitates neutrophil extracellular traps-induced invasion via cyclic GMP-AMP synthase and the non-canonical NF-κB pathway in diabetic hepatocellular carcinoma.

Objective: Diabetic hepatocellular carcinoma (HCC) patients have high mortality and metastasis rates. Diabetic conditions promote neutrophil extracellular traps (NETs) generation, which mediates HCC metastasis and invasion. However, whether and how diabetes-induced NETs trigger HCC invasion is largely unknown. Here, we aimed to observe the effects of diabetes-induced NETs on HCC invasion and investigate mechanisms relevant to a DNA sensor cyclic GMP-AMP synthase (cGAS). Methods: Serum from diabetic patients and healthy individuals was collected. Human neutrophil-derived NETs were isolated for stimulating HCC cell invasion. Data from the SEER and TCGA databases were used for bioinformatics analysis. In HCC cells and allograft models, NETs-triggered invasion was observed. Results: Diabetic HCC patients had poorer survival than non-diabetic ones. Either diabetic serum or extracted NETs caused HCC invasion. Induction of diabetes or NETosis elicited HCC allograft invasion in nude mice. HCC cell invasion was attenuated by the treatment with DNase1. In TCGA_LIHC, an extracellular DNase DNASE1L3 was downregulated in tumor tissues, while function terms (the endocytic vesicle membrane, the NF-κB pathway and extracellular matrix disassembly) were enriched. DNASE1L3 knockdown in LO2 hepatocytes or H22 cell-derived allografts facilitated HCC invasion in NETotic or diabetic nude mice. Moreover, exposure of HCC cells to NETs upregulated cGAS and the non-canonical NF-κB pathway and induced expression of metastasis genes (MMP9 and SPP1). Both cGAS inhibitor and NF-κB RELB knockdown diminished HCC invasion caused by NETs DNA. Also, cGAS inhibitor was able to retard translocation of NF-κB RELB. Conclusion: Defective DNASE1L3 aggravates NETs DNA-triggered HCC invasion on diabetic conditions via cGAS and the non-canonical NF-κB pathway.

Dual functional coatings with antifogging and antimicrobial performances for endoscope lens, via facile adsorption-cross-linking strategy.

Surface fogging causes various inconvenience for human daily life, especially for clinic inspection and medical diagnosis, hence the surfaces with reliable antifogging performances have received tremendous interests. Herein, through a facile adsorption-cross-linking strategy, a dual functional coating with both excellent antifogging/frost-resisting properties and reliable antibacterial activity has been steadily integrated onto varied substrates. A series of copolymers poly(HEAA-co-QAC-co-BP) with UV-initiable BP groups are synthesized, and then are covalently fixed on the substrate surfaces via UV triggered cross-linking reaction. The hydrophilic HEAA units endow the surface with excellent antifogging performance, while the introduced QAC groups bring essential antibacterial activity. ZOI results prove that the antibacterial activity stems from the surface contact-killing of bacteria, without releasing any bactericidal agents. Moreover, the functional surface exhibits remarkable resistance toward non-specific protein adsorption as well as no obvious effect on the hemolysis. The coating with the unique merits of both antifogging and antibacterial properties could find broad applications in antifogging fields, in particular for medical diagnosis, health monitoring, etc.