Ancestral sequence reconstruction of the prokaryotic three-domain laccases for efficiently degrading polyethylene.

Biodegradation of polyethylene (PE) plastics is environmentally friendly. To obtain the laccases that can efficiently degrade PE plastics, we generated 9 ancestral laccases from 23 bacterial three-domain laccases through ancestral sequence reconstruction. The optimal temperatures of the ancestral laccases were between 60 °C-80 °C, while their optimal pHs were at 3.0 or 4.0. Without substrate pretreatment and mediator addition, all the ancestral laccases can degrade low-density polyethylene (LDPE) films at pH 7.0 and 60 °C. Among them, Anc52, which shared low sequence identity (18 %-41.7 %) with the reported PE-degrading laccases, was the most effective for LDPE degradation. After the catalytic reactions at 90 °C for 14 h, Anc52 (0.2 mg/mL) induced clear wrinkles and deep pits on the PE film surface detected by scanning electron microscope, and its carbonyl and hydroxyl indices reached 2.08 and 2.42, respectively. Then, we identified the residues 203 and 288 critical for PE degradation through site-directed mutation on Anc52. Moreover, Anc52 be activated by heat treatment (60 °C and 90 °C) at pH 7.0, which gave it a high catalytic efficiency (kcat/Km= 191.73 mM-1·s-1) and thermal stability (half-life at 70 °C = 13.70 h). The ancestral laccases obtained here could be good candidates for PE biodegradation.

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

A CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci in Aspergillus niger.

Aspergillus niger is a highly versatile fungal strain utilized in industrial production. The expression levels of recombinant genes in A. niger can be enhanced by increasing the copy number. Nevertheless, given the prolonged gene editing cycle of A. niger, a "one-step" strategy facilitating the simultaneous integration of recombinant genes into multiple genomic loci would provide a definitive advantage. In our previous study, a visual multigene editing system (VMS) was designed to knock out five genes, employing a tRNA-sgRNA array that includes the pigment gene albA and the target genes. Building upon this system, hybrid donor DNAs (dDNAs) were introduced to establish a clustered regularly interspaced short palindromic repeats (CRISPR)-based multiplex integration toolkit. Firstly, a CRISPR-Cas9 homology-directed repair (CRISPR-HDR) system was constructed in A. niger by co-transforming the CRISPR-Cas9 plasmid (with a highly efficient sgRNA) and the dDNA, resulting in precise integration of recombinant xylanase gene xynA into the target loci (the ß-glucosidase gene bgl, the amylase gene amyA, and the acid amylase gene ammA). Subsequently, the length of homology arms in the dDNA was optimized to achieve 100% editing efficiency at each of the three gene loci. To achieve efficient multiplex integration in A. niger, the CRISPR plasmid pLM2 carrying a sgRNA-tRNA array was employed for concurrent double-strand breaks at multiple loci (bgl, amyA, ammA, and albA). Hybrid dDNAs were then employed for repair, including dDNA1-3 (containing xynA expression cassettes without selection markers) and dDNAalbA (for albA knockout). Among the obtained white colonies (RLM2'), 23.5% exhibited concurrent replacement of the bgl, amyA, and ammA genes with xynA (three copies). Notably, the xynA activity obtained by simultaneous insertion into three loci was 48.6% higher compared to that obtained by insertion into only the bgl locus. Furthermore, this multiple integration toolkit successfully enhanced the expression of endogenous pectinase pelA and Candida antarctica lipase CALB. Hence, the combined application of VMS and the CRISPR-HDR system enabled the simultaneous application of multiple selection markers, facilitating the rapid generation in the A. niger cell factories.

Exploring the Transmembrane Behaviors of Dietary Flavonoids under Intestinal Digestive Products of Different Lipids: Insights into the Structure-Activity Relationship In Vitro.

This study aimed to investigate the transmembrane transport behavior and structure-activity relationships of various dietary flavonoids in the presence of dietary lipids derived from different sources in vitro. Results revealed that the digestion products of soybean oil (SOED) and lard (LOED) augmented the apparent permeability coefficients of most dietary flavonoids, and SOED exhibited higher transport compared with LOED. The structural properties of flavonoids and the potential interactions between fatty acids in these digestion products and flavonoids may influence the outcomes. 3D quantitative structure-activity relationship analyses revealed that incorporating small-volume groups at position 8 of the A-ring augmented the transmembrane transfer of flavonoids in the LOED system compared with the control group. By contrast, the integration of hydrophobic groups at position 5 of the A-ring and hydrogen bonding acceptor groups at position 6 of the A-ring enhanced the transmembrane transportation of flavonoids in the SOED system. Molecular dynamics simulations revealed that the SOED system may facilitate the interactions with flavonoids to form more stable and compact fatty acid-flavonoid complexes compared to the LOED system. These findings may provide valuable insights into flavonoid absorption to facilitate the development and utilization of functional foods or dietary supplements based on dietary flavonoids.

Transmembrane behaviors and quantitative structure-activity relationship of dietary flavonoids in the presence of intestinal digestive products from different carbohydrate sources based on in vitro and in silico analysis.

Bioavailability plays a key role for flavonoids to exert their bioactivities. This study investigated the transmembrane transport behavior and structure-activity of dietary flavonoids. Results showed that the apparent permeability coefficients of some flavonoids could be significantly increased when digestion products from rice flour (RD) or wheat flour (WD) are present (p < 0.05), especially in the WD, potentially due to higher reducing sugar (p < 0.05). 3D-QSAR revealed that the hydrogen bond acceptor groups at positions 5 and 6 of ring A, small-volume groups at position 3', hydrophobic groups at position 4', and large-volume groups at position 5' of ring B increased the transmembrane transport of flavonoids in the WD. A hydrogen bond donor group at position 4' of ring B enhanced the transmembrane transport of flavonoid compounds in the RD. These findings contribute to our comprehensive understanding of flavonoid absorption within the context of intestinal carbohydrate digestion.

Comparative study of binding interactions between different dietary flavonoids and soybean ß-conglycinin and glycinin: Impact on structure and function of the proteins.

The mechanisms underlying the interaction between different dietary flavonoids and soybean ß-conglycinin (7S) and glycinin (11S) were comparatively investigated, and the alterations in conformation and function of the complexes were further evaluated. Among the 23 flavonoids studied, 3 flavonoids with glycosides with the top three ranked T Scores in molecular docking analysis-phlorizin, luteoloside, and vitexin-4'-O-glucoside-with the highest binding affinity to 7S and 11S and quenched their intrinsic fluorescence in a static manner. The binding interactions of these flavonoids to 7S and 11S were structure dependent. Hydrophobic forces played important roles in interactions between 7S and both luteoloside and vitexin-4'-O-glucoside, whereas the binding of phlorizin to 7S, and of the three flavonoids to 11S, was driven by hydrogen bonding and van der Waals forces. The binding of these flavonoids interfered with the microenvironment around tyrosine and tryptophan, thereby altering the secondary structures of 7S and 11S. The binding of the three flavonoids enhanced solubility, emulsifying properties, thermal stability, and antioxidant capacity of 7S and 11S. The use of flavonoids could facilitate the design of soybean protein-based products with desirable functional properties.

Screening of Oxygenated Aromatic Compounds for Potential Antifungal Activity against Geotrichum citri-aurantii through Structure-Activity Relationship Analysis.

Sour rot caused by Geotrichum citri-aurantii (G. citri-aurantii) is responsible for huge economic losses during citrus fruit storage. However, the availability of chemical fungicides for controlling this disease is rather limited. In the present study, the antifungal activities of 25 oxygenated aromatic compounds against the mycelial growth of G. citri-aurantii were determined, and their corresponding structure-activity relationships were illustrated. Salicylaldehyde (pMIC = 2.689) possessed the strongest inhibitory effect on G. citri-aurantii growth, followed by thymol (pMIC = 2.478) and o-phthalaldehyde (pMIC = 2.429). Molecular electrostatic potential and molecular orbital analysis showed that the antifungal efficiency of test compounds was determined by the number and location of hydroxyl and aldehyde groups and the length of the ester chain. All compounds were selected for quantitative structure-antifungal activity relationship (QSAR) analysis. A three-dimensional-QSAR model of G. citri-aurantii inhibitors was established and demonstrated good predictive capability [comparative molecular field analysis, q2 = 0.532, optimum number of components (ONC) =10, R2 = 0.996, F = 560.325, standard error of estimation (SEE) = 0.034, and two descriptors; comparative similarity index analysis, q2 = 0.675, ONC = 6, R2 = 0.989, F = 263.354, SEE = 0.054, and five descriptors]. QSAR analysis showed that substitution at position 1 with hydrophilic and electron-withdrawing groups produced a hydrogen donor and thus improved the antifungal activity. In contrast, substitution at positions 4 or 5 with hydrophilic and electron-donating groups decreased its antifungal activity. These findings can provide theoretical guidance for preparing effective antifungal drugs for controlling sour rot in citrus.

Dietary Flavonoids Alleviate Inflammation and Vascular Endothelial Barrier Dysfunction Induced by Advanced Glycation End Products In Vitro.

The aim of this study was to compare the protective effects of three dietary flavonoids (apigenin-7-O-glucoside (A7G), isorhamnetin-3-O-rutinoside (I3R), and cyanidin-3-O-glucoside (C3G)) on advanced glycation end products (AGEs)-induced inflammation and vascular endothelial dysfunction. Furthermore, the potential mechanisms of varied effects of those three dietary flavonoids were analyzed by molecular docking analysis. Results showed that C3G (40 µM) achieved the best inhibition on inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in AGEs-induced RAW264.7 cells, followed by I3R, and A7G was the weakest. The molecular docking results also showed that C3G exhibited the closest binding with the receptor for AGE. However, I3R (40 µM) demonstrated the best effect in improving endothelial dysfunction in AGEs-induced EA.hy926 cells, followed by C3G, and A7G was the weakest, as evidenced by the molecular docking results of flavonoids with profilin-1. This work may provide knowledge and helpful suggestions regarding the benefits of dietary flavonoids in diabetic vascular complications.

Interfering effects on the bioactivities of several key proteins of COVID-19/variants in diabetes by compounds from Lianqiao leaves: In silico and in vitro analyses.

Diabetes is considered to be one of the diseases most associated with COVID-19. In this study, interfering effects and potential mechanisms of several compounds from Lianqiao (Forsythia suspensa (Thunb.) Vahl) leaves on the bioactivities of some key proteins of COVID-19 and its variants, as well as diabetic endothelial dysfunctions were illuminated through in vitro and in silico analyses. Results showed that, among the main ingredients in the leaves, forsythoside A showed the strongest docking affinities with the proteins SARS-CoV-2-RBD-hACE2 of COVID-19 and its variants (Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617)), as well as neuropilin-1 (NRP1), and SARS-CoV-2 main protease (MPro) to interfere coronavirus entering into the human body. Moreover, forsythoside A was the most stable in binding to receptors in Delta (B.1.617) system. It also has good antiviral activities and drug properties and has the strongest binding force to the RBD domain of COVID-19. In addition, forsythoside A reduced ROS production in AGEs-induced EA.hy926 cells, maintained endothelial integrity, and bound closely to protein profilin-1 (PFN1) receptor. This work may provide useful knowledge for further understanding the interfering effects and potential mechanisms of compounds, especially forsythoside A, from Lianqiao leaves on the bioactivities of key proteins of COVID-19/variants in diabetes.

Effects of Different Dietary Flavonoids on Dipeptidyl Peptidase-IV Activity and Expression: Insights into Structure-Activity Relationship.

The inhibitory effects of 30 dietary flavonoids on dipeptidyl peptidase-IV (DPP-IV) were investigated to illustrate their quantitative structure-activity relationship (QSAR) and further explore their inhibition at the cellular level. Results of in vitro experiment show that isorhamnetin-3-O-glucoside (IC50, 6.53 ± 0.280 µM) had the strongest inhibition followed by cyanidin-3-O-glucoside (IC50, 8.26 ± 0.143 µM) and isorhamnetin-3-O-rutinoside (IC50, 8.57 ± 0.422 µM). A 3D QSAR model [comparative molecular field analysis, q2 = 0.502, optimum number of components (ONC) = 3, R2 = 0.983, F = 404.378, standard error of estimation (SEE) = 0.070, and two descriptors; comparative similarity index analysis, q2 = 0.580, ONC = 10, R2 = 0.999, F = 1617.594, SEE = 0.022, and four descriptors] indicates that the DPP-IV inhibition of flavonoid was facilitated by crucial structural factors. Position 3 of ring C favored bulky, hydrogen bond acceptors and hydrophilic and electron-donating substituents. The presence of minor and electron-withdrawing groups at position 4' of ring B and positions 5 and 7 of ring A could improve DPP-IV inhibition. Moreover, the three flavonoids mentioned above could effectively suppress DPP-IV activity and expression in Caco-2 cells. This work may supply new insights into dietary flavonoids as DPP-IV inhibitors for controlling blood glucose.

Ligustrazine Inhibits Cartilage Endplate Hypertrophy via Suppression of TGF-ß1.

CEP hypertrophy is one of the characteristics of intervertebral disc degeneration (IDD). LIG exerts a protective effect on IDD in animal model. The effect of LIG on CEP hypertrophy is further investigated in the present study. Cells were isolated from hypertrophic samples obtained from patients during vertebral fusion surgery. Cellular proliferation and the expression of type I collagen (Col I) and TGF-ß1 were tested. In the bipedal rats, the edges of the CEP and the sizes of noncartilaginous outgrowth, as well as the expression of osteogenic markers, Col1a, ALP, Runx2, and TGF-ß1, were detected. Within two passages, the condensed hypertrophic CEP cells exhibited osteogenic capacity by bony-like nodules and ALP positive staining, along with increased expression of Col I and TGF-ß1. LIG inhibited proliferation of CEP cells and downregulated the expression of Col I and TGF-ß1 in vitro. Furthermore, LIG attenuated CEP hypertrophy on the lumbar spine of bipedal rats by reducing Col1a, ALP, Runx2, and TGF-ß1 mRNA expression and TGF-ß1 distribution in vivo. We concluded LIG exerted a preventive effect on CEP hypertrophy via suppression of TGF-ß1 levels. This information could be used to develop alternative therapeutic methods to treat spinal CEP hypertrophy.

Fibrohistiocytoma combined with an aneurysmal bone cyst at T7 in a 63-year-old woman.

We present a case of spinal tumor, with fibrohistiocytoma combined with aneurysmal bone cyst (ABC) at the pedicle and transverse process of T7 in a 63-year-old female. ABC is a rare skeletal tumor and spinal ABC is extremely rare. Fibrohistiocytoma is a type of primary benign bone tumor. ABC is also a rare bone tumor that most often occurs in the pelvis. The combined lesion of two primary bone benign tumors is relatively rare in clinic. In addition, fibrohistiocytoma and ABC are widely confused with other giant cell containing tumors of the bone. X-rays, magnetic resonance imaging (MRI) and positron emission tomography-computed tomography (PET-CT) scans were performed and assessed. Finally, the diagnosis was confirmed by pathological tests. The patient underwent surgery and had an extremely good recovery. The correct diagnosis of a spine tumor is important when determining the surgical procedure.

Posterior internal fixation plus vertebral bone implantation under navigational aid for thoracolumbar fracture treatment.

The aim of this study was to investigate the method of posterior thoracolumbar vertebral pedicle screw reduction and fixation combined with vertebral bone implantation via the affected vertebral body under navigational aid for the treatment of thoracolumbar fractures. The efficacy of the procedure was also measured. Between June 2005 and March 2011, posterior thoracolumbar vertebral pedicle screw reduction and fixation plus artificial bone implantation via the affected vertebral pedicle under navigational aid was used to treat 30 patients with thoracolumbar fractures, including 18 males and 12 females, ranging in age from 21 to 57 years. Compared with the values prior to surgery, intraspinal occupation, vertebral height ratio and Cobb angle at the follow-up were significantly improved. At the long-term follow-up, the postoperative Cobb angle loss was <1° and the anterior vertebral body height loss was <2 mm. Posterior thoracolumbar vertebral pedicle screw reduction and fixation combined with vertebral bone implantation via the affected vertebral body under navigational aid may increase the accuracy and safety of surgery, and it is an ideal method of internal implantation. Bone implantation via the affected vertebral body may increase vertebral stability.

Anterior Z-plate and titanic mesh fixation for acute burst thoracolumbar fracture.

STUDY DESIGN: A retrospective study. OBJECTIVE: To evaluate the clinical outcome, effectiveness, and security of the surgical management of acute thoracolumbar burst fracture with corpectomy, titanic mesh autograft, and Z-plate fixation by anterior approach. SUMMARY OF BACKGROUND DATA: Many surgical methods were adopted to treat acute burst thoracolumbar fracture. But the optimal surgical management remains controversial. METHODS: A retrospective review of a consecutive series of 48 patients with thoracolumbar burst fracture treated with anterior corpectomy, titanic mesh autograft, and Z-plate internal fixation was carried out. Preoperative clinical and radiographic data of all cases were originally collected. Surgical indications were motor neurologic deficit and thoracolumbar column instability. Twenty-two patients (45.8%) with acute thoracolumbar burst fractures presented with a neurologic deficit. The postoperative recovery of neural function, restoration of anterior cortex collapse, kyphotic angle, and spinal canal compromise were observed. RESULTS: The preoperative kyphotic angle was improved to a mean of 5.6°, radiographic height restored to 95.8% of the adjacent normal levels, and canal compromise was 0%. None of the patients had neurologic deterioration. Mean follow-up time was 32.4 months (range, 24-47 months). All 22 patients with neurologic deficit demonstrated at least one Frankel grade improvement on final observation, with 16 (73%) patients had accomplished complete neurologic recovery. Forty-six (96%) patients reported minimal or no pain at final follow-up observation, and 40 (83%) patients who had been working before injury returned to original work. CONCLUSION: The authors considered spinal cord decompression with anterior corpectomy and stability reconstruction with titanic mesh autograft and Z-plate fixation at same time in one incision as an effective technique for unstable thoracolumbar burst fracture with and without neurologic deficit.

Long-term outcomes of two different decompressive techniques for lumbar spinal stenosis.

STUDY DESIGN: A prospective study to evaluate the outcomes of 2 different decompressive techniques for lumbar spinal stenosis. OBJECTIVE: To explore a more effective and less invasive decompression technique without instrument and fusion for lumbar spinal stenosis. SUMMARY OF BACKGROUND DATA: The traditional surgical decompression of spinal stenosis involves laminectomy or unilateral laminotomy. Even in unilateral laminotomy cases, 85.3% had an excellent-to-fair operative result, and the incidence of complications was 9.8%. Although the addition of instrumentation does not increase the complication rate, but compared to the efficiency, the higher costs was controversial. Minimal invasion and destabilization are recommended. METHODS: This prospective study included 152 consecutive patients, sequentially divided into 2 groups, underwent Windows technique (group A) and decompressive laminectomy (group B) by 2 groups of surgeons. RESULTS: The evaluation of the back pain, leg pain, walking tolerance, and neurologic recovery were performed before surgery and after surgery. In group A, at the final evaluation, the overall results were good to excellent in 89% (68/76) of the patients, fair 11% (8/76), and poor 0%. In group B, at the final evaluation, the overall results were good to excellent in 63% (48/76) of the patients, fair 30% (23/76), and poor 7% (5/76). CONCLUSION: Degenerative spinal stenosis can be decompressed adequately with preserving the posterior elements. The "Windows technique" laminoforaminotomy, which obtained satisfactory long-term outcomes with few complications and low cost, can be a standard procedure for the surgical treatment of the degenerative spinal stenosis even with slight congenital spinal stenosis.

Radiographic evaluation of selective anterior thoracolumbar or lumbar fusion for adolescent idiopathic scoliosis.

According to Lenke classification of adolescent idiopathic scoliosis (AIS), patients with type 5 curve in which the structural major curve is thoracolumbar or lumbar curve with nonstructural proximal thoracic and main thoracic curves, could be surgically treated with selective anterior thoracolumbar or lumbar (TL/L) fusion. This study retrospectively analyzed the radiographies of selective anterior TL/L fusion in 35 cases of AIS with Lenke type 5 curve. Segmental fixation with a single rigid rod through anterior thoracoabdominal approach was applied in all patients. Measurements of scoliosis curve in preoperative, immediate postoperative and follow-up radiographies were analyzed. The average follow up time was 36 months (24-42 months). The average preoperative Cobb angle of the TL/L curve was 45.6 degrees and improved into 9.7 degrees immediate postoperatively, with 79.7% curve correction. In addition, the minor thoracic curve decreased from 29.7 degrees preoperatively to 17.6 degrees postoperatively, with a spontaneous correction of 41.5%. During the follow-up, a loss of 4.6 degrees correction was found and the average Cobb angle of TL/L increased to 14.4 degrees . Also, the minor thoracic curve increased to average 20.1 degrees with a loss of 2.4 degrees correction. Trunk shift deteriorated slightly immediate postoperatively and improved at the follow-up. The lowest instrumented vertebra (LIV) tilt was improved significantly and maintained its results at the follow-up. During the follow-up, the coronal disc angle immediately above the upper instrumented vertebra (UIVDA) and below the LIV (LIVDA) aggravated, while the sagittal contours of T5-T12 and T10-L2 were well maintained. The lumbar lordosis of L1-S1 and the sagittal Cobb angle of the instrumented segments were reduced slightly postoperatively and at the follow-up. There were no major complications or pseudarthrosis. The outcomes of this study show that selective anterior thoracolumbar or lumbar fusion with solid rod instrumentation is effective for surgical correction of AIS with Lenke type 5 curve. The TL/L curve, minor thoracic curve, and LIV title can be improved significantly, with good maintenance of sagittal contour. However, the UIVDA and LIVDA aggravate postoperatively when the trunk rebalances itself during follow-up. The degeneration of LIV disc warrants longer-term follow-up.