In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production.

BACKGROUND: Biodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement. RESULTS: In this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. Thermomyces lanuginosus lipase (TLL) with high activity and thermostability was obtained using the Pichia pastoris expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt-TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, [Formula: see text] (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt-TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt-TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil. CONCLUSIONS: An integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.

YTHDF2-mediated regulations bifurcate BHPF-induced programmed cell deaths.

N6-methyladenosine (m6A) is a critical regulator in the fate of RNA, but whether and how m6A executes its functions in different tissues remains largely obscure. Here we report downregulation of a crucial m6A reader, YTHDF2, leading to tissue-specific programmed cell deaths (PCDs) upon fluorene-9-bisphenol (BHPF) exposure. Currently, Bisphenol A (BPA) substitutes are widely used in plastic manufacturing. Interrogating eight common BPA substitutes, we detected BHPF in 14% serum samples of pregnant participants. In a zebrafish model, BHPF caused tissue-specific PCDs triggering cardiac and vascular defects. Mechanistically, BHPF-mediated downregulation of YTHDF2 reduced YTHDF2-facilitated translation of m6A-gch1 for cardiomyocyte ferroptosis, and decreased YTHDF2-mediated m6A-sting1 decay for caudal vein plexus (CVP) apoptosis. The two distinct YTHDF2-mediated m6A regulations and context-dependent co-expression patterns of gch1/ythdf2 and tnfrsf1a/ythdf2 contributed to YTHDF2-mediated tissue-specific PCDs, uncovering a new layer of PCD regulation. Since BHPF/YTHDF2-medaited PCD defects were also observed in mammals, BHPF exposure represents a potential health threat.

Increased Internal Carotid Artery Tortuosity is a Risk Factor for Spontaneous Cervicocerebral Artery Dissection.

OBJECTIVE: Spontaneous cervicocerebral artery dissection (sCCD) is an important cause of ischaemic stroke that often occurs in young and middle aged patients. The purpose of this study was to investigate the correlation between tortuosity of the carotid artery and sCCD. METHODS: Patients with confirmed sCCD who underwent computed tomography angiography (CTA) were reviewed retrospectively. Age and sex matched patients having CTA were used as controls. The tortuosity indices of the cervical arteries were measured from the CTA images. The carotid siphon and the extracranial internal carotid artery (ICA) were evaluated according to morphological classification. The carotid siphons were classified into five types. The extracranial ICA was categorised as simple tortuosity, coiling or kinking. Independent risk factors for sCCD were investigated using multivariable analysis. RESULTS: The study included sixty-six patients with sCCD and 66 controls. There were no differences in vascular risk factors between the two groups. The internal carotid tortuosity index (ICTI) (25.24 ± 12.37 vs. 15.90 ± 8.55, respectively; p < .001) and vertebral tortuosity index (VTI) (median 11.28; interquartile range [IQR] 6.88, 18.80 vs. median 8.38; IQR 6.02, 12.20, respectively; p = .008) were higher in the patients with sCCD than in the controls. Type III and Type IV carotid siphons were more common in the patients with sCCD (p = .001 and p < .001, respectively). The prevalence of any vessel tortuosity, coiling and kinking of the extracranial ICA was higher in the patients with sCCD (p < .001, p = .018 and p = .006, respectively). ICTI (odds ratio [OR] 2.964; p = .026), VTI (OR 5.141; p = .009), and Type III carotid siphons (OR 4.654; p = .003) were independently associated with the risk of sCCD. CONCLUSION: Arterial tortuosity is associated with sCCD, and greater tortuosity of the cervical artery may indicate an increased risk of arterial dissection.

Clinical class 1 integron-integrase gene - A promising indicator to monitor the abundance and elimination of antibiotic resistance genes in an urban wastewater treatment plant.

In this study, 295 antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) from the influent, activated sludge (AS), and membrane bioreactor (MBR) permeate were primarily examined in the wastewater treatment plant (WWTP) biweekly over 13 months. The absolute concentrations of ARGs and MGEs respectively ranged from 1.27 × 1010 to 1.94 × 1011 and 8.00 × 109 to 1.24 × 1011 copies/L in the influent, of which were reduced by 2 to 3 orders of magnitude in the permeate. No significant seasonal variation of ARGs and MGEs was found in the WWTP, except that the absolute abundance of ARGs and MGEs in the AS was peaked during spring. The antibiotics affected neither ARGs nor MGEs significantly, suggesting their concentrations may be not high enough to pose a selective pressure. In contrast, the bacterial community had direct effect on the MGEs variation, meanwhile the MGEs influenced the ARG abundance directly. Class 1 integron-integrase gene (intI1), clinical intI1, and Tn21 associated more frequently with ARGs in the AS over long-term, suggesting the potential of them involved in horizontal gene transfer. Both intI1 and clinical intI1 had significantly positive associations with the overall abundance of ARGs, as well as significantly negative relationships with the overall removal rates of ARGs in the MBR. However, the abundances between intI1 and clinical intI1 were significantly different. Meanwhile, clinical intI1 remained rather consistent proportion with the ARG abundance in the AS and permeate, was stronger correlated with human pathogens, and was associated with greater number of ARGs over time. Moreover, clinical intI1 was significantly associated with the removal efficiency of ARGs from all classes. Taken together, clinical intI1 can be adopted as an indicator for the abundance and removal efficiency of ARGs in the WWTP.

How exogenous influent communities and environmental conditions affect activated sludge communities in the membrane bioreactor of a wastewater treatment plant.

In this study, the residual population of influent and activated sludge (AS) communities was defined based on their occurrence frequency and relative abundance through long-term and fine-scale sampling from the membrane bioreactor (MBR) of a wastewater treatment plant (WWTP). There were 481 OTUs defined as the residual OTUs, which taken up 67.90 ±â€¯9.36% of relative abundance in the influent community. Besides, 6.76 ±â€¯5.71% of the residual population migrated to and remained in the AS community. Additionally, the residual populations were more likely to be anaerobes and microaerobes. As the most predominant genus from residual community, the relative abundance of Arcobacter was reduced from 15.78 ±â€¯3.58% in the influent to 1.15 ±â€¯1.35% in the AS. The residues that migrated from the influent have increased the richness and evenness of AS community, as well as the dissimilarities among samples over long-term. The rank-abundance distribution showed identical pattern for the residual species between influent and AS. By adopting the analysis of neutral model, 2766 out of 7491 shared OTUs between influent and AS communities were identified as neutral OTUs, which respectively made up 53.9% and 41.8% of the total relative abundance of influent and AS communities. These indicated that the AS community was to some extent, but not entirely assembled by neutral process. For the residual community in the AS, dissolved oxygen (DO) was positively associated with several aerobic genera, meanwhile influent chemical oxygen demand (COD) had positive relationship with genus Pseudomonas. Last but most importantly, the influent community could not inoculate the nitrifiers in the AS, but instead, was able to inoculate the denitrifiers; as well as enhance the biodiversity and the ability of resisting external disturbance for the AS community in MBR.

Selective effect and elimination of antibiotics in membrane bioreactor of urban wastewater treatment plant.

Analyzing the temporal dynamics of antibiotics, antibiotic resistance genes (ARGs) and the functional community could contribute to the regulation and optimization of wastewater treatment plant (WWTP) operation to achieve better antibiotics and ARGs removal performances during different seasons. However, there is little research in this area. Therefore, in this study, samples were collected from the influent, activated sludge (AS), and permeate of the membrane bioreactor (MBR) in a WWTP located in Beijing, China, biweekly over 13 months, and then analyzed systematically. The removal efficiency for all detected antibiotics through biodegradation and adsorption was 59.25 ±â€¯2.79%, with the highest rate of 64.79 ±â€¯4.68% observed in summer, indicating that the higher temperature in summer may promote biodegradation in MBR. In contrast, the elimination of antibiotics through microfiltration was negligible and unfavorable, with negative overall removal rates. However, a positive rejection rate of 9.48 ±â€¯8.92% was only observed in winter, indicating that a colder temperature might lead to better, but still limited, antibiotics rejection. Sulfonamides (SAs) were more likely to impose a selective pressure on their corresponding ARGs. However, due to the degradability of tetracyclines (TCs) and potential selection of ARGs in wastewater before entering WWTP, there were still TC resistances with non-detectable TCs. Significantly negative relationships were observed between the relative abundance of nitrifying bacteria (Nitrosomonas and Nitrospira) and the concentrations of certain antibiotic classes, indicating that nitrifying bacteria could be involved in the co-metabolic biodegradation of certain antibiotics through enzyme catalyzation during nitrification.

Influence of polyaluminum chloride on microbial characteristics in anaerobic membrane bioreactors for sludge digestion.

In this study, two parallel lab-scale anaerobic membrane bioreactors (AnMBRs), one of which was dosed with polyaluminum chloride (PAC) for membrane fouling control, were operated for treating excess activated sludge collected from a wastewater treatment plant (WWTP). The AnMBRs were inoculated with anaerobic digested sludge collected from an anaerobic digester of another WWTP. The microbial community of digested sludge and cake layer in AnMBRs, as well as that of excess sludge, was analyzed through polymerase chain reaction coupled with denaturing gradient gel electrophoresis (PCR-DGGE) and Illumina MiSeq. The dynamic variation of archaeal community in AnMBRs was not as obvious as that of bacterial community based on the PCR-DGGE results. Under the circumstance of stable operation, Cloacimonetes, Chloroflexi, Bacteroidetes, Proteobacteria, Firmicutes, and Ignavibacteriae were observed as the predominant phyla in digested sludge based on the Illumina results. In addition to that, the cake layer possessed similar predominant phyla with the digested sludge but owned a higher diversity. Furthermore, overlapping bacterial communities were discovered between the excess sludge and digested sludge. However, the abundance of aerobic bacteria was substantially reduced, while the abundance of anaerobic microorganisms like phylum Cloacimonetes and Smithella was enriched in digested sludge over time. Additional PAC dosing, on the one hand, affected the bioavailable substrate, thus further changing the microbial community structure; on the other hand, aluminum itself also affected specific microbial communities. Besides, PAC dosing indirectly influenced the bacterial diversity in AnMBR as well.

Influence of Al³âº addition on the flocculation and sedimentation of activated sludge: comparison of single and multiple dosing patterns.

In this study, the flocculation and sedimentation performance of activated sludge (AS) with single and multiple dosing of trivalent aluminum (Al(3+)) were studied. The AS samples were cultivated in sequencing batch reactors at 22 °C. The dosages of Al(3+) were 0.00, 0.125, 0.5, 1.0, and 1.5 meq/L for single dosing, and 0.1 meq/L for multiple dosing. Under single dosing conditions, as Al(3+) dosage increased, the zeta potential, total interaction energy, and effluent turbidity decreased, whereas the sludge volume index (SVI) increased, indicating that single Al(3+) dosing could enhance sludge flocculation, but deteriorate sedimentation. By comparison, adding an equal amount of Al(3+) through multiple dosing achieved a similar reduction in turbidity, but the zeta potential was higher, while the loosely bound extracellular polymeric substances (LB-EPS) content and SVI remarkably declined. Although the difference in the flocculation performances between the two dosing patterns was not significant, the underlying mechanisms were quite distinct: the interaction energy played a more important role under single dosing conditions, whereas multiple dosing was more effective in reducing the EPS content. Multiple dosing, which allows sufficient time for sludge restructuring and floc aggregation, could simultaneously optimize sludge flocculation and sedimentation.