Effect of no eyeglasses sales on the quality of eye care: an experimental evidence from China.

BACKGROUND: Eye examinations and eyeglasses acquisition are typically integrated into a cohesive procedure in China. We conducted a randomized controlled trial using incognito standardized patient (SP) approach to evaluate the impact of separating eyeglasses sales on the accuracy of final prescription. METHODS: 52 SPs were trained to provide standardized responses during eye examinations, and undergoing refraction by a senior ophthalmologist at a national-level clinical center. SPs subsequently received eye examinations at 226 private optical shops and public hospitals in Shaanxi, northwestern China. The visits were randomly assigned to either control group, where SPs would typically purchase eyeglasses after refraction, or treatment group, where SPs made an advance declaration not to purchase eyeglasses prior to refraction. The dioptric difference between the final prescriptions provided by local refractionists and expert in the better-seeing eye was determined using the Vector Diopteric Distance method, and the completeness of exams was assessed against national standards. Multiple regressions were conducted to estimate the impact of no eyeglasses sales on the accuracy of the final prescription of local refractionists, as well as the completeness of examinations. RESULTS: Among 226 eye exams (73 in public hospitals, 153 in private optical shops), 133 (58.8%) were randomized to control group and 93 (41.2%) to no eyeglasses sales group. The inaccuracy rate of final prescriptions provided by local refractionists (≥ 1.0 D, experts' final prescription as the reference) was 25.6% in control group, while 36.6% in no-sale group (P = 0.077). The likelihood of providing inaccurate final prescriptions was significantly higher in no-sale group compared to control group (OR = 1.607; 95% CI: 1.030 to 2.508; P = 0.037). This was particularly evident in private optical shops (OR = 2.433; 95% CI: 1.386 to 4.309; P = 0.002). In terms of process quality, the no-sale group performed significantly less subjective refraction (OR = 0.488; 95% CI: 0.253 to 0.940; P = 0.032) and less testing SP's own eyeglasses (OR = 0.424; 95% CI: 0.201 to 0.897; P = 0.025). The duration of eye exams was 3.917 min shorter (95% CI: -6.798 to -1.036; P = 0.008) in no-sale group. CONCLUSIONS: Separating eyeglasses sales from optical care could lead to worse quality of eye care. Policy makers should carefully consider the role of economic incentives in healthcare reform.

Fermentation characteristics and microbial community composition of wet brewer's grains and corn stover mixed silage prepared with cellulase and lactic acid bacteria supplementation.

OBJECTIVE: The objective of this study was to investigate how cellulase or/and lactic acid bacteria (LAB) affected the fermentation characteristic and microbial community in wet brewer's grains (WBG) and corn stover (CS) mixed silage. METHODS: The WBG was mixed thoroughly with the CS at 7:3 (w/w). Four treatment groups were studied: i) CON, no additives; ii) CEL, added cellulase (120 U/g fresh matter [FM]), iii) LAB, added LAB (2×106 cfu/g FM), and iv) CLA, added cellulase (120 U/g FM) and LAB (2×106 cfu/g FM). RESULTS: All additive-treated groups showed higher fermentation quality over the 30 d ensiling period. As these groups exhibited higher (p<0.05) LAB counts and lactic acid (LA) content, along with lower pH value and ammonia-nitrogen (NH3-N) content than the control. Specifically, cellulase-treated groups (CEL and CLA) showed lower (p<0.05) neutral detergent fiber and acid detergent fiber contents than other groups. All additives increased the abundance of beneficial bacteria (Firmicutes, Lactiplantibacillus, and Limosilactobacillus) while they decreased abundance of Proteobacteria and microbial diversity as well. CONCLUSION: The combined application of cellulase and LAB could effectively improve the fermentation quality and microbial community of the WBG and CS mixed silage.

Beyond mothers: the crucial role of family caregivers' knowledge on exclusive breastfeeding in rural western China.

BACKGROUND: The exclusive breastfeeding rate in China remains significantly low. Numerous studies have identified the impact of maternal characteristics on exclusive breastfeeding; however, the correlation between primary family caregivers' characteristics, such as health and nutrition knowledge, and exclusive breastfeeding still lacks clarity. The aim of this study is to investigate the association between the health and nutrition knowledge of primary family caregivers and exclusive breastfeeding in rural China. METHODS: In 2019, a cross-sectional study was conducted in two prefectures within the Qinba Mountains area, located in the southern region of Shaanxi province. Data on knowledge of health and nutrition, breastfeeding practices, breastfeeding family support, breastfeeding self-efficacy, and conflict frequency were collected via structured questionnaires from 372 caregiver-infant pairs. Infant feeding practices were assessed based on the caregivers' recall of the previous day (within the 24 h before the interview). The mother was interviewed first, followed by a brief questionnaire for the primary family caregiver, both conducted individually to minimize disruptions from other family members. Univariate and multivariate regression analyses were conducted to explore the correlation between knowledge of mothers and primary family caregivers and exclusive breastfeeding. RESULTS: The exclusive breastfeeding rate for six-month-old infants in the sample was 15.7%. On average, mothers scored 4.6 (SD 1.4) for health and nutrition knowledge, while primary family caregivers scored 3.6 (SD 1.4). Both maternal (OR 1.48; 95% CI 1.16, 1.88) and primary family caregiver's (OR 1.34; 95% CI 1.05, 1.70) health and nutrition knowledge were significantly associated with exclusive breastfeeding. A positive correlation (OR 1.98; 95% CI 1.40, 2.80) existed between the average health and nutrition knowledge of the mother and primary family caregiver and exclusive breastfeeding. The primary family caregiver's health and nutrition knowledge was positively correlated with the practical family support perceived by the mother (OR 1.23; 95% CI 1.02, 1.49) and breastfeeding self-efficacy of the mother (ß = 1.40; 95% CI 0.29, 2.50). CONCLUSIONS: The characteristics of the primary family caregiver play a large role in exclusive breastfeeding. To promote exclusive breastfeeding, interventions should address the needs of the whole family instead of just mothers.

Stop-Flow Lithography for the Continuous Production of Degradable Hydrogel Achiral Crescent Microswimmers.

The small size of robotic microswimmers makes them suitable for performing biomedical tasks in tiny, enclosed spaces. Considering the effects of potentially long-term retention of microswimmers in biological tissues and the environment, the degradability of microswimmers has become one of the pressing issues in this field. While degradable hydrogel was successfully used to prepare microswimmers in previous reports, most hydrogel microswimmers could only be fabricated using two-photon polymerization (TPP) due to their 3D structures, resulting in costly robotic microswimmers solution. This limits the potential of hydrogel microswimmers to be used in applications where a large number of microswimmers are needed. Here, we proposed a new type of preparation method for degradable hydrogel achiral crescent microswimmers using a custom-built stop-flow lithography (SFL) setup. The degradability of the hydrogel crescent microswimmers was quantitatively analyzed, and the degradation rate in sodium hydroxide solution (NaOH) of different concentrations was investigated. Cytotoxicity assays showed the hydrogel crescent microswimmers had good biocompatibility. The hydrogel crescent microswimmers were magnetically actuated using a 3D Helmholtz coil system and were able to obtain a swimming efficiency on par with previously reported microswimmers. The results herein demonstrated the potential for the degradable hydrogel achiral microswimmers to become a candidate for microscale applications.

The compliance of free folic acid supplements among pregnant women in rural areas of Northwestern China: The role of related knowledge.

Background: High prevalence of neural tube defects remains one of the major threats to newborns in rural China. Folic acid supplementation before and during early pregnancy can effectively reduce the risk of neural tube defects. Despite the efforts of the free folic acid mass distribution, the actual usage of folic acid supplements was still suboptimal among rural pregnant women in China. The objective of this study is to investigate if and how knowledge can influence the picking up and intake of the free folic acid supplements distributed by the government. Methods: We collected survey data from 821 pregnant women in rural areas of Shaanxi, China, in March and December of 2021. Face-to-face interviews and questionnaire surveys were conducted with every participant. Univariate and multivariate logistic regression analyses were performed to test the relationship between knowledge and dependent variables. Results: Our study found that there were 76.4% of pregnant women would pick up folic acid supplements distributed by the government and only 44.5% of women would use folic acid before current pregnancy. Awareness of folic acid policy both affects the picking up (OR: 6.708, 95% CI: 4.672-9.632) and periconceptional intake (OR: 1.912, 95% CI:1.326-2.758) of folic acid supplements. Knowledge of health and nutrition in pregnancy showed no significant relationship with the picking up and periconceptional intake of folic acid supplements but was positively associated with the intake duration (Coefficient: 9.278, 95% CI: 2.966-15.591). Conclusion: Despite the relatively high level of picking up, the actual folic acid usage was not ideal among pregnant women in rural areas of China. Folic acid policy awareness was positively associated with the picking up and intake of folic acid before and during conception. Knowledge of health and nutrition about pregnancy was related to a longer duration of folic acid intake but had no impact on the picking up rate and periconceptional intake of folic acid supplements.

High-level expression of Thermomyces dupontii thermo-alkaline lipase in Pichia pastoris under the control of different promoters.

In this study, 15 methanol-inducible and 9 constitutive promoters were used to drive the expression of Thermomyces dupontii lipase (TDL) in Pichia pastoris. Of the 15 methanol-inducible promoters, formaldehyde dehydrogenase promoter (PFLD1) showed the highest efficiency in driving lipase production, followed by alcohol oxidase 1 (PAOX1) and dihydroxyacetone synthase (PDAS1) promoters. The maximum lipase activity of transformants with PFLD1, PAOX1 and PDAS1 promoters in 5-l bioreactor was 27,076, 24,159 and 22,342 U/ml, respectively. For the nine constitutive promoters, glycosyl phosphatidyl inositol-anchored protein promoter (PGCW14) produced the highest amount of lipases in a medium containing glucose or glycerol as the only carbon source, followed by mitochondrial alcohol dehydrogenase isozyme (P0472) and glyceraldehyde-3-phosphate dehydrogenase (PGAP) promoters. The maximum lipase yields in 5-l bioreactors under the control of PGCW14, P0472 and PGAP promoters were 17,353, 15,046 and 14,276 U/ml, respectively. The result of this study not only identifies a few highly efficient promoters for the heterologous expression of TDL in P. pastoris, but also casts some insight into the optimization of protein production in heterologous systems.

Improved Production of Aspergillus usamii endo-ß-1,4-Xylanase in Pichia pastoris via Combined Strategies.

A series of strategies were applied to improve expression level of recombinant endo-ß-1,4-xylanase from Aspergillus usamii (A. usamii) in Pichia pastoris (P. pastoris). Firstly, the endo-ß-1,4-xylanase (xynB) gene from A. usamii was optimized for P. pastoris and expressed in P. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression of xynB-opt, the Vitreoscilla hemoglobin (VHb) gene was transformed to the recombinant strain containing xynB-opt. The results showed that recombinant strain harboring the xynB-opt and VHb (named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHb in 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHb was optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHb reached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins in P. pastoris.

Codon Optimization Significantly Improves the Expression Level of α -Amylase Gene from Bacillus licheniformis in Pichia pastoris.

α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use.

Gene Cloning, High-Level Expression, and Characterization of an Alkaline and Thermostable Lipase from Trichosporon coremiiforme V3.

The present study describes the gene cloning and high-level expression of an alkaline and thermostable lipase gene from Trichosporon coremiiforme V3. Nucleotide analysis revealed that this lipase gene has an open reading frame of 1,692 bp without any introns, encoding a protein of 563 amino acid residues. The lipase gene without its signal sequence was cloned into plasmid pPICZαA and overexpressed in Pichia pastoris X33. The maximum lipase activity of recombinant lipase was 5,000 U/ml, which was obtained in fed-batch cultivation after 168 h induction with methanol in a 50 L bioreactor. The purified lipase showed high temperature tolerance, and being stable at 60 °C and kept 45% enzyme activity after 1 h incubation at 70 °C. The stability, effects of metal ions and other reagents were also determined. The chain length specificity of the recombinant lipase showed high activity toward triolein (C18:1) and tripalmitin (C16:0).

High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris.

A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75°C, while recombinant protein encoded by the "wild type" xylanase gene xyn2, also expressed in Pichia, was 20°C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80°C for 10min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22°C, respectively. The maximum xylanase activity of Mxyn2p was 13,000nkat/mg (9.88g/l) in fed-batch cultivation after 168h induction with methanol in a 50l bioreactor.

Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

High-level expression of pro-form lipase from Rhizopus oryzae in Pichia pastoris and its purification and characterization.

A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca(2+) and inhibited by Hg(2+) and Ag(+). The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0).

Expression of insulin-like growth factor-1 of orange-spotted grouper (Epinephelus coioides) in yeast Pichia pastoris.

IGF-1 plays a key role in development, growth, and metabolism in teleost. Recombinant fish IGF-1 may be a useful tool for both theoretical research and aquaculture applications. However, using the Escherichia coli expression system has several drawbacks for producing quality fish IGF-1 protein. To explore the yeast expression system for generating fish IGF-1 protein, the cDNA coding for the mature orange-spotted grouper IGF-1 peptide without signal peptide and E domain was cloned into the secreting expression organism Pichia pastoris. Tricine-SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rgIGF-1 was secreted into the culture medium, had a molecular weight of 8.7 kDa. The production peaked at 24h of induction and the optimal pH for expression was 5.0. The recombinant protein was purified using a combined ammonium sulfate precipitation with Ni(2+) affinity chromatography. Finally, 17.9 mg of the protein was obtained from 420 ml of the culture supernatant and the purity was about 92.4%. Bioactivity of the rgIGF-1 was confirmed by the ability to stimulate proliferation of embryo cell line of grouper (GP cell line) and MFC-7 cell. The present results suggest that the Pichia pastoris expression system can be used to produce a functional rgIGF-1 for both research and aquaculture application.

The second prolactin receptor in Nile tilapia (Oreochromis niloticus): molecular characterization, tissue distribution and gene expression.

Prolactin (PRL) is one of the most versatile hormones found in the pituitary of vertebrates and exerts its actions through binding to a specific PRL receptor (PRLR). Here we describe the cloning and characterization of a second prolactin receptor (ntPRLR2), isolated from the ovary of Nile tilapia (Oreochromis niloticus). The newly identified PRLR cDNA was 2011 bp in length and encoded 529 amino acids. It shared 31.6% identity in nucleotide sequence and 29.2% in deduced amino acid sequence with the first PRLR identified in Nile tilapia (ntPRLR1). Both of these ntPRLRs resemble the long form mammalian PRLRs. The nominated ntPRLR2 was further confirmed as a real prolactin receptor based on its competence to transactivate the beta-casein and c-fos promoters in the transiently ntPRLR2-transfected HEK293 cells. The ntPRLR2 gene also found to encode a 864-bp short form transcript in the ovary, which was confirmed by Northern blot analysis. A tissue distribution study by real-time PCR revealed that the mRNA of both receptors (ntPRLR1 and ntPRLR2) was widely expressed in different tissues, with an extremely high abundance in the osmoregulatory organs, including the gills, intestine and kidney. ntPRLR1 mRNA was more abundant than ntPRLR2 in the testis, while the reverse expression pattern was found in the ovary. In the ovary, ntPRLR2 mRNA demonstrated a distinct gonadal development-dependent expression profile, with significantly higher levels at a sexual mature stage than at sexual recrudescent and sexual regressed stages. When challenged with estradiol, ntPRLR2 mRNA expression was up-regulated by E2, whereas E2 had no significant effect on ntPRLR1.