Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

Establishment of an indirect competitive immunoassay for the detection of dicamba based on a highly specific nanobody.

Dicamba, a traditional highly effective and low toxicity herbicide, has gained new life with the development of dicamba-tolerant transgenic crops in recent years. However, dicamba is highly volatile and therefore easy to cause drift damage to sensitive crops. The development of efficient and sensitive detection methods is essential for monitoring of trace dicamba in the environment. Nanobody-based immunoassay plays an important role in on-site detection of pesticides. However, now rapid and sensitive immunoassay methods based on nanobody for dicamba detection were lacking. In this study, the nanobodies specifically recognizing dicamba were successfully obtained by immunising camels and phage display library construction, and then an indirect competitive immunoassay based on Nb-242 was constructed with IC50 of 0.93 µg/mL and a linear range of 0.11-8.01 µg/mL. Nb-242 had good specificity with no cross-reactivities against the dicamba analogs other than 2,3,6-trichlorobenzoic acid and the developed immnoassay had a good correlation with the standard HPLC in the spike-recovery studies. Finally, the key amino acid Ala 123, Tyr 55, Tyr 59 and Arg 72 of Nb-242 that specifically recognizing and binding with dicamba were identified by homologous modeling and molecular docking, laying an important foundation for further structural modification of Nb-242. This study has important guiding significance for constructing immunoassay method of dicamba based on nanobody and provides a sensitive, specific, and reliable detection method that is suitable for the detection of dicamba in the environment.

Development of an immunoassay based on a specific antibody for the detection of diphenyl ether herbicide fomesafen.

Fomesafen belongs to the diphenyl ether herbicide, and is widely used in the control of broadleaf weeds in crop fields due to its high efficiency and good selectivity. The residual of fomesafen in soil has a toxic effect on subsequent sensitive crops and the microbial community structure because of its long residual period. Therefore, an efficient method for detecting fomesafen is critical to guide the correct and reasonable use of this herbicide. Rapid and sensitive immunoassay methods for fomesafen is unavailable due to the lack of specific antibody. In this study, a specific antibody for fomesafen was generated based on rational design of haptens and a sensitive immunoassay method was established. The half maximal inhibitory concentration (IC50) of the immunoassay was 39 ng/mL with a linear range (IC10-90) of 1.92-779.8 ng/mL. In addition, the developed assay had a good correlation with the standard UPLC-MS/MS both in the spike-recovery studies and in the detection of real soil samples. Overall, the developed indirect competitive enzyme immunoassay reported here is important for detecting and quantifying fomesafen contamination in soil and other environmental samples with good sensitivity and high reproducibility.

[Comparison of the impact of different fat emulsions on clinical outcomes in preterm infants with varying duration of parenteral nutrition: a randomized controlled multicenter study]. / ä¸¤ç§è„‚è‚ªä¹³å‰‚ä¸åŒè‚ å¤–è¥å »æ—¶é—´å¯¹æ—©äº§å„¿ä¸´åºŠç»“å±€å½±å“çš„æ¯”è¾ƒï¼šä¸€é¡¹éšæœºå¯¹ç §å¤šä¸­å¿ƒç ”ç©¶.

OBJECTIVES: To compare the impact of two types of fat emulsion on clinical outcomes in preterm infants with varying duration of parenteral nutrition (PN). METHODS: Preterm infants meeting the inclusion criteria were randomly assigned to two groups: medium/long-chain triglyceride fat emulsion (referred to as MCT/LCT) group or multi-oil fat emulsion (containing soybean oil, medium-chain triglycerides, olive oil, and fish oil; referred to as SMOF) group. The infants were stratified into groups based on the duration of PN (15-21 days, 22-28 days, and ≥29 days). Clinical characteristics, nutritional status, biochemical indicators, and clinical outcomes were compared between the two groups. RESULTS: Compared with the MCT/LCT group, the SMOF group had lower peak levels of triglyceride during the hospital stay in preterm infants with PN of 15-21 days, 22-28 days, and ≥29 days, respectively (P<0.05). Logistic regression trend analysis showed that with a longer duration of PN, the risk of parenteral nutrition-associated cholestasis (PNAC) and bronchopulmonary dysplasia (BPD) significantly increased in the MCT/LCT group (P<0.05), while the risk of brain injury did not significantly change (P>0.05). In the SMOF group, the risks of PNAC and BPD did not significantly change with a longer duration of PN (P>0.05), but the risk of brain injury significantly decreased (P=0.006). CONCLUSIONS: Compared to MCT/LCT, SMOF have better lipid tolerance. With a longer duration of PN, SMOF does not increase the risks of PNAC and BPD and had a protective effect against brain injury. This suggests that in preterm infants requiring long-term PN, the use of SMOF is superior to MCT/LCT.

Highly efficient expression and secretion of human lysozyme using multiple strategies in Pichia pastoris.

BACKGROUND: Human lysozyme (hLYZ), an emerging antibacterial agent, has extensive application in the food and pharmaceutical industries. However, the source of hLYZ is particularly limited. RESULTS: To achieve highly efficient expression and secretion of hLYZ in Pichia pastoris, multiple strategies including G418 sulfate screening, signal sequence optimization, vacuolar sorting receptor VPS10 disruption, and chaperones/transcription factors co-expression were applied. The maximal enzyme activity of extracellular hLYZ in a shaking flask was 81,600 ± 5230 U mL-1 , which was about five times of original strain. To further reduce the cost, the optimal medium RDMY was developed and the highest hLYZ activity reached 352,000 ± 16,696.5 U mL-1 in a 5 L fermenter. CONCLUSION: This research provides a very useful and cost-effective approach for the hLYZ production in P. pastoris and can also be applied to the production of other recombinant proteins.

Effects of mixed oil emulsion on short-term clinical outcomes in premature infants: A prospective, multicenter, randomized controlled trial.

OBJECTIVE: This study compared the clinical effects of two different lipid emulsions in premature infants with gestational age < 32 weeks (VPI) or birth weight < 1500 g (VLBWI) to provide an evidence-based medicine basis for optimizing intravenous lipid emulsion. METHODS: This was a prospective multicenter randomized controlled study. A total of 465 VPIs or VLBWIs, admitted to the neonatal intensive care unit of five tertiary hospitals in China from March 1, 2021 to December 31, 2021, were recruited. All subjects were randomly allocated into two groups, namely, medium-chain triglycerides/long-chain triglycerides (MCT/LCT) group (n = 231) and soybean oil, medium-chain triglycerides, olive oil, and fish oil (SMOF) group (n = 234). Clinical features, biochemical indexes, nutrition support therapy, and complications were analyzed and compared between the two groups. RESULTS: No significant differences were found in perinatal data, hospitalization, parenteral and enteral nutrition support between the two groups (P > 0.05). Compared with the MCT/LCT group, the incidence of neonates with a peak value of total bilirubin (TB) > 5 mg/dL (84/231 [36.4% vs. 60/234 [25.6%]), a peak value of direct bilirubin (DB) ≥ 2 mg/dL (26/231 [11.3% vs. 14/234 [6.0%]), a peak value of alkaline phosphatase (ALP) > 900 IU/L (17/231 [7.4% vs. 7/234 [3.0%]), and a peak value of triglycerides (TG) > 3.4 mmol/L (13/231 [5.6% vs. 4/234[1.7%]]) were lower in the SMOF group (P < 0.05). Univariate analysis showed that in the subgroup analysis of < 28 weeks, the incidence of parenteral nutrition-associated cholestasis (PNAC) and metabolic bone disease of prematurity (MBDP) were lower in the SMOF group (P = 0.043 and 0.029, respectively), whereas no significant differences were present in the incidence of PNAC and MBDP between the two groups at > 28 weeks group (P = 0.177 and 0.991, respectively). Multivariate logistic regression analysis revealed that the incidence of PNAC (aRR: 0.38, 95% confidence interval [CI]: 0.20-0.70, P = 0.002) and MBDP (aRR: 0.12, 95% CI: 0.19-0.81, P = 0.029) in the SMOF group were lower than that in the MCT/LCT group. In addition, no significant differences were recorded in the incidence of patent ductus arteriosus, feeding intolerance, necrotizing enterocolitis (Bell's stage ≥ 2), late-onset sepsis, bronchopulmonary dysplasia, intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity and extrauterine growth retardation between the two groups (P > 0.05). CONCLUSIONS: The application of mixed oil emulsion in VPI or VLBWI can reduce the risk of plasma TB > 5 mg/dL, DB ≥ 2 mg/dL, ALP > 900 IU/L, and TG > 3.4 mmol/L during hospitalization. SMOF has better lipid tolerance, reduces the incidence of PNAC and MBDP, and exerts more benefits in preterm infants with gestational age < 28 weeks.

Analysis of risk factors for parenteral nutrition-associated cholestasis in preterm infants: a multicenter observational study.

BACKGROUND: It is proposed that the development of parenteral nutrition-associated cholestasis (PNAC) was significantly associated with preterm birth, low birth weight, infection, etc.; however, the etiology and pathogenesis of PNAC are not fully understood. Most of the studies examining PNAC-associated risk factors were single-center studies with relatively small sample sizes. OBJECTIVE: To analyze the risk factors associated with PNAC in preterm infants in China. METHODS: This is a retrospective multicenter observational study. Clinical data on the effect of multiple oil-fat emulsions (soybean oil-medium chain triglycerides-olive oil-fish oil, SMOF) in preterm infants were collected from a prospective multicenter randomized controlled study. A secondary analysis was performed in which preterm infants were divided into the PNAC group and the non-PNAC group based on the PNAC status. RESULTS: A total of 465 cases very preterm infants or very low birth weight infants were included in the study in which 81 cases were assigned to the PNAC group and 384 cases were assigned to the non-PNAC group. The PNAC group had a lower mean gestational age, lower mean birth weight, longer duration of invasive and non-invasive mechanical ventilation, a longer duration oxygen support, and longer hospital stay (P < 0.001 for all). The PNAC group had higher respiratory distress syndrome, hemodynamically significant patent ductus arteriosus, necrotizing enterocolitis (NEC) with stage II or higher, surgically treated NEC, late-onset sepsis, metabolic bone disease, and extrauterine growth retardation (EUGR) compared to the non-PNAC group (P < 0.05 for all). In contrast with the non-PNAC group, the PNAC group received a higher maximum dose of amino acids and fat emulsion, more medium/long-chain fatty emulsion, less SMOF, had a longer duration of parenteral nutrition, lower rates of breastfeeding, higher incidence of feeding intolerance (FI), more accumulated days to achieve total enteral nutrition, less accumulated days of total calories up to standard 110 kcal/kg/day and slower velocity of weight growth (P < 0.05 for all). Logistic regression analysis indicated that the maximum dose of amino acids (OR, 5.352; 95% CI, 2.355 to 12.161), EUGR (OR, 2.396; 95% CI, 1.255 to 4.572), FI (OR, 2.581; 95% CI, 1.395 to 4.775), surgically treated NEC (OR, 11.300; 95% CI, 2.127 ~ 60.035), and longer total hospital stay (OR, 1.030; 95% CI, 1.014 to 1.046) were independent risk factors for the development of PNAC. SMOF (OR, 0.358; 95% CI, 0.193 to 0.663) and breastfeeding (OR, 0.297; 95% CI, 0.157 to 0.559) were protective factors for PNAC. CONCLUSIONS: PNAC can be reduced by optimizing the management of enteral and parenteral nutrition and reducing gastrointestinal comorbidities in preterm infants.

Data augmentation-based statistical inference of diffusion processes.

The identification of diffusion processes is challenging for many real-world systems with sparsely sampled observation data. In this work, we propose a data augmentation-based sparse Bayesian learning method to identify a class of diffusion processes from sparsely sampled data. We impute latent unsampled diffusion paths between adjacent observations and construct a candidate model for the diffusion processes with the sparsity-inducing prior on model parameters. Given the augmented data and candidate model, we investigate the full joint posterior distribution of all the parameters and latent diffusion paths under a Bayesian learning framework. We then design a Markov chain Monte Carlo sampler with non-degenerate acceptance probability on system dimension to draw samples from the posterior distribution to estimate the parameters and latent diffusion paths. Particularly, the proposed method can handle sparse data that are regularly or irregularly sampled in time. Simulations on the well-known Langevin equation, homogeneous diffusion in a symmetric double-well potential, and stochastic Lotka-Volterra equation demonstrate the effectiveness and considerable accuracy of the proposed method.

Factors influencing extrauterine growth retardation in singleton-non-small for gestational age infants in China: A prospective multicenter study.

BACKGROUND: The incidence of extrauterine growth retardation (EUGR) varies considerably in different countries due to the distinct definitions and inclusion criteria of individual studies. Most studies included small for gestational age (SGA) very preterm infants (VPIs), resulting in a higher incidence of EUGR. Experts have suggested the accurate definition of "EUGR" in SGA infants is not "true EUGR". The postnatal growth curve of multiple premature births also differs from that of singletons. As far as we know, there is no study about relationship between singleton-non-SGA preterm infants and EUGR. OBJECTIVES: To analyze the factors influencing EUGR among VPIs who were singleton-non-SGA in China. METHODS: A prospective-multicenter study was conducted in 28 hospitals distributed through China from September 2019 to December 2020. The clinical data on singleton-non-SGA among VPIs were divided into EUGR group (n = 692) and non-EUGR group (n = 912). RESULTS: Compared to non-EUGR group, the mean gestational age (GA), mean birth weight (BW) and percentage of BW in Fenton curve in EUGR group were lower (P < 0.001 for all). The incidence of EUGR among distinct GA groups (classifications of GA < 28weeks, 28-28+6 weeks, 29-29+6 weeks, 30-30+6 weeks and 31-31+6 weeks) and distinct BW groups (classifications of BW<1000 g, 1000-1249 g, 1250-1499 g, 1500-1999g and 2000-2500 g) were statistically significant (P = 0.004 and P <.001). Logistic regression analysis indicated that later addition of human milk fortifier (HMF), later attainment of HMF sufficient fortification, later return to BW, more accumulative days of fasting, longer duration of parenteral nutrition, total duration of oxygen support and moderate/severe bronchopulmonary dysplasia (BPD) were risk factors for the development of EUGR in singleton-non-SGA VPIs (P < 0.001, P = 0.002, P < 0.001, P = 0.002, P = 0.017, P = 0.003 and P = 0.002, respectively). The use of full-course antenatal steroids, greater BW as a percentile of the Fenton curve, breastfeeding initiation and faster average velocity of weight growth effectively protected against EUGR (P = 0.008, P < 0.001, P < 0.001 and P < 0.001, respectively). CONCLUSIONS: The overall incidence of EUGR was 43.1% among singleton-non-SGA VPIs in China. Raising the full-course antenatal steroids usage, reducing the incidence of moderate and severe BPD, attaching importance to the management of enteral nutrition in VPIs and increasing the weight growth velocity can reduce the incidence of EUGR.

Establishment of a Novel Cell Surface Display Platform Based on Natural "Chitosan Beads" of Yeast Spores.

Cell surface display technology, which expresses and anchors proteins on the surface of microbial cells, has broad application prospects in many fields, such as protein library screening, biocatalysis, and biosensor development. However, traditional cell surface display systems have disadvantages: the molecular weight of phage display proteins cannot be too large; bacterial display lacks the post-translational modification process for eukaryotic proteins; yeast display is prone to excessive protein glycosylation and misfolding of multisubunit proteins; and the compatibility of Bacillus subtilis spore display needs to be further improved. Therefore, it is extremely valuable to develop an efficient surface display platform with strong universality and stress resistance properties. Although yeast surface display systems have been extensively investigated, the establishment of a surface display platform using yeast spores has rarely been reported. In this study, a novel cell surface display platform based on natural "chitosan beads" of yeast spores was developed. The target protein in fusion with the chitosan affinity protein (CAP) exhibited strong binding capability with "chitosan beads" of yeast spores in vitro and in vivo. Moreover, this protein display system showed highly preferable enzymatic properties and stability. As an example, the displayed LXYL-P1-2-CAP demonstrated high thermostability and reusability (60% of the initial activity after seven cycles of reuse), high storage stability (75% of original activity after 8 weeks), and excellent tolerance to a concentration up to 75% (v/v) organic reagents. To prove the practicability of this surface display system, the semisynthesis of paclitaxel intermediate was demonstrated and its highest conversion rate was 92% using 0.25 mM substrate. This study provides a novel and useful platform for the surface display of proteins, especially for multimeric macromolecular proteins of eukaryotic origin.

Determination of Pb, Cr, Cd, and As in Aluminum-Plastic Packaging Materials via Inductively Coupled Plasma-Mass Spectrometry with Microwave Digestion.

Microwave digestion was performed to study the pretreatment methods of aluminum-plastic packaging materials (APPMs). Five different digestion reagent combinations and proportions were thoroughly considered. Digestion results indicated that the most suitable reagent combination was sulfuric and nitric acids with the optimal proportion of 1∶7 after the orthogonal experiment. Moreover, the possible reasons of the experimental phenomenon were analyzed. The contents of Pb, Cr, Cd, and As in APPMs were subsequently determined via inductively coupled plasma-mass spectrometry (ICP-MS). The satisfactory linearity of calibration curves was obtained with the linear correlation coefficients above 0.999 5, and the instrument detection limits of Pb, Cr, Cd, and As for the current method were 0.215, 0.067, 0.006 and 0.020 ng·mL-1, respectively. Furthermore, the recoveries of standard addition ranged from 83.8% to 111.6%, and the relative standard deviations ranged from 0.5% to 7.4%. Two independent parallel determination results of Pb, Cr, Cd, and As in APPMs were approaching, and the student's t-test (confidence level, α=0.05) showed that the determination results had no significant differences. In conclusion, the present method exhibited fine linearity, low detection limit, high recovery, and good precision, which can accurately be utilized to analyze Pb, Cr, Cd, and As elements in APPMs or other similar materials.