Association between Dietary Total Vitamin A, ß-carotene, and Retinol Intake and Risk of cardiometabolic multimorbidity: Results from the China Health and Nutrition Survey, 1997-2015.

Background: The association between vitamin A and single cardiometabolic diseases has been extensively studied, but the relationship between dietary vitamin A intake and the risk of cardiometabolic multimorbidity (CMM) has not been studied. Therefore, the present study was conducted to explore the association with CMM risk by analyzing different sources of vitamin A. Methods: This study utilized 13,603 subjects aged ≥ 18 years from 1997-2015 from the China Health and Nutrition Survey (CHNS). Dietary intake was calculated from 3 consecutive 24-h dietary recalls combined with a house hold food inventory. CMM is defined as the development of at least two cardiometabolic diseases. Results: After a median follow-up of 9.1 years, there were 1050 new cases of CMM. The risk of CMM was significantly lower in those with higher vitamin A intake (Q1 vs Q5 HR 0.66, 95% CI 0.54-0.81). ß-carotene (Q1 vs Q5 HR 0.82, 95% CI 0.66-1.02) and retinol (Q1 vs Q5 HR 0.59, 95%CI 0.48-0.73) intake had a similarly negative correlation. Using restricted cubic spline found an L-shaped relationship between retinol intake and CMM (p non-linear < 0.001). In subgroup analyses, protective effects were stronger for participants aged ≥ 44 years (HR 0.72, 95%CI 0.57-0.92) and for the female group (HR 0.62, 95%CI 0.45-0.84). Conclusion: Dietary vitamin A was a protective factor for CMM, and this effect was stronger in age ≥ 44 years and in the female group. There was a ceiling effect on the protective effect of retinol intake on the risk of CMM.

Association between indoor air pollution and depression: a systematic review and meta-analysis of cohort studies.

OBJECTIVES: Incomplete combustion of solid fuel and exposure to secondhand smoke (SHS) are the primary causes of indoor air pollution (IAP), potentially leading to detrimental effects on individual mental health. However, current evidence regarding the association between IAP and depression remains inconclusive. This study aims to systematically investigate the evidence regarding the association between IAP and the risk of depression. DESIGN: Systematic review and meta-analysis of cohort studies. DATA SOURCES: Two independent reviewers searched PubMed, the Cochrane Library, Web of Science and EMBASE for available studies published up to 13 January 2024. ELIGIBILITY CRITERIA: We included all cohort studies published in English that aimed to explore the relationship between IAP from solid fuel use and SHS exposure and the risk of depression. DATA EXTRACTION AND SYNTHESIS: Two independent reviewers extracted data and assessed the risk of bias. The association between IAP and depression was calculated using pooled relative risk (RR) with 95% CIs. Heterogeneity was assessed using the I2 value, and the effect estimates were pooled using fixed-effects or random-effects models depending on the results of homogeneity analysis. RESULTS: We included 12 articles with data from 61 217 participants. The overall findings demonstrated a significant association between IAP exposure and depression (RR=1.22, 95% CI: 1.13 to 1.31), although with substantial heterogeneity (I2=75%). Subgroup analyses based on pollutant type revealed that IAP from solid fuel use was associated with a higher risk of depression (RR=1.20, 95% CI: 1.13 to 1.26; I2=62%; 5 studies, 36 768 participants) than that from SHS exposure (RR=1.11, 95% CI: 0.87 to 1.41; I2=80%; 7 studies, 24 449 participants). In terms of fuel use, the use of solid fuel for cooking (RR: 1.23, 95% CI: 1.16 to 1.31; I2=58%; 4 studies, 34 044 participants) and heating (RR 1.15, 95% CI: 1.04 to 1.27; I2=65%; 3 studies, 24 874 participants) was associated with increased depression risk. CONCLUSIONS: The findings from this systematic review and meta-analysis of cohort studies indicated an association between exposure to IAP and depression. PROSPERO REGISTRATION NUMBER: CRD42022383285.

Association between dietary inflammatory index and gallstones in US adults.

Introduction: Previous studies have found that diet's inflammatory potential is related to various diseases. However, little is known about its relationship with gallstones. The present study aims to investigate the relationship between dietary inflammatory index (DII) and gallstones. Methods: Data were obtained from the 2003-2020 National Health and Nutrition Examination Survey (NHANES). We used the nearest neighbor propensity score matching (PSM) with a ratio of 1:1 to reduce selection bias. Logistic regression models estimated the association between DII and gallstones. The non-linear relationship was explored with restricted cubic splines (RCS). BMI subgroup stratification was performed to explore further the connection between DII and gallstones in different populations. Results: 10,779 participants were included. Before and after PSM, gallstone group individuals had higher DII scores than non-gallstone group individuals (p < 0.05). Matched logistic regression analysis showed that DII scores were positively correlated with gallstone risk (adjusted OR = 1.14, 95% CI 1.01, 1.29). The stratified analysis showed that this association was stronger in overweight or obese people (adjusted OR = 1.18, 95% CI 1.03, 1.34). RCS analysis suggested that DII and gallstones showed a "J"-shaped non-linear dose-response relationship (p non-linear <0.001). Conclusion: Higher DII score is positively associated with the risk of gallstones, particularly in overweight or obese population, and this relationship is a "J"-shaped non-linear relationship. These results further support that avoiding or reducing a pro-inflammatory diet can be an intervention strategy for gallstone management, particularly in the overweight or obese population.

Higher remnant cholesterol is associated with an increased risk of amnestic mild cognitive impairment: a community-based cross-sectional study.

Background and aims: Amnestic mild cognitive impairment (aMCI) is the most common subtype of MCI, which carries a significantly high risk of transitioning to Alzheimer's disease. Recently, increasing attention has been given to remnant cholesterol (RC), a non-traditional and previously overlooked risk factor. The aim of this study was to explore the association between plasma RC levels and aMCI. Methods: Data were obtained from Brain Health Cognitive Management Team in Wuhan (https://hbtcm.66nao.com/admin/). A total of 1,007 community-dwelling elders were recruited for this project. Based on ten tools including general demographic data, cognitive screening and some exclusion scales, these participants were divided into the aMCI (n = 401) and normal cognitive groups (n = 606). Physical examinations were conducted on all participants, with clinical indicators such as blood pressure, blood sugar, and blood lipids collected. Results: The aMCI group had significantly higher RC levels compared to the normal cognitive group (0.64 ± 0.431 vs. 0.52 ± 0.447 mmol/L, p < 0.05). Binary logistics regression revealed that occupation (P<0.001, OR = 0.533, 95%CI: 0.423-0.673) and RC (p = 0.014, OR = 1.477, 95% CI:1.081-2.018) were associated factors for aMCI. Partial correlation analysis, after controlling for occupation, showed a significant negative correlation between RC levels and MoCA scores (r = 0.059, p = 0.046), as well as Naming scores (r = 0.070, p = 0.026). ROC curve analysis demonstrated that RC levels had an independent predictive efficacy in predicting aMCI (AUC = 0.580, 95%CI: 0.544 ~ 0.615, P < 0.001). Conclusion: Higher RC levels were identified as an independent indicator for aMCI, particularly in the naming cognitive domain among older individuals. Further longitudinal studies are necessary to validate the predictive efficacy of RC.

The mediating role of children's intergenerational support in association between grandparenting and cognitive function among middle-aged and older Chinese: findings from the CHARLS cohort study.

OBJECTIVES: With the world's population increasing in age, there has been a significant rise in the prevalence of cognitive impairment and dementia among individuals. This study aims to investigate the association between grandparenting and cognitive function among middle-aged and older Chinese using data from 2011 to 2018 China Health and Retirement Longitudinal Study (CHARLS). Additionally, the study seeks to explore the potential mediating effect of intergenerational support from children on this relationship, using data from the CHARLS 2011 database. METHODS: 5254 participants were recruited at the baseline survey in CHARLS 2011. Subsequently, a follow-up survey was conducted over 8 years, from CHARLS 2011 to 2018, with 1472 individuals completing the follow-up survey. The CHARLS included surveys on grandparenting and cognitive assessments. Grandparenting was categorized as yes and no. The assessment of cognitive function involved the evaluation of episodic memory and mental intactness. The present study used cross-sectional and longitudinal analyses to examine the relationship between grandparenting and cognitive function. The bootstrap method assessed the mediating effect of children's intergenerational support. RESULTS: The results of both cross-sectional and longitudinal studies indicated a positive association between grandparenting and cognitive function in middle-aged and older Chinese (B = 0.138, p < 0.05; B = 0.218, p < 0.05). Children's emotional and economic support played intermediary roles between grandparenting and cognitive function. CONCLUSION: The results emphasized the significance of policymakers considering the consequences of intergenerational care and family support when formulating and executing social service policies targeted at the middle-aged and older population in China.

Effectiveness of Telecare Interventions on Depression Symptoms Among Older Adults: Systematic Review and Meta-Analysis.

BACKGROUND: Depression is the most common psychiatric disorder among older adults. Despite the effectiveness of pharmacological and psychological therapies, many patients with late-life depression (LLD) are unable to access timely treatment. Telecare has been shown to be effective in addressing patients' psychosocial issues, while its effectiveness in serving patients with LLD remains unclear. OBJECTIVE: This study aimed to evaluate the effectiveness of telecare in reducing depression and anxiety symptoms and improving quality of life (QoL) in patients with LLD. METHODS: Databases including the Cochrane Library, Web of Science, PubMed, Embase, and EBSCO were searched for randomized controlled trials (RCTs) evaluating the effectiveness of telecare for LLD from database establishment to December 28, 2022. RESULTS: A total of 12 RCTs involving 1663 participants were identified in this study. The meta-analysis showed that (1) telecare significantly reduced depressive symptoms in patients with LLD compared to those in usual care (UC; standardized mean difference [SMD]=-0.46, 95% CI -0.53 to -0.38; P<.001), with the best improvement observed within 3 months of intervention (SMD=-0.72, 95% CI -1.16 to -0.28; P<.001); (2) other scales appeared more effective than the Patient Health Questionnaire-9 for LLD in telecare interventions (SMD=-0.65, 95% CI -0.96 to -0.35; P<.001); (3) telecare was more effective than telephone-based interventions for remote monitoring of LLD (SMD=-1.13, 95% CI -1.51 to -0.76; P<.001); (4) the reduction of depressive symptoms was more pronounced in patients with LLD with chronic conditions (SMD=-0.67, 95% CI -0.89 to -0.44; P<.001); (5) telecare was more effective for LLD in Europe and the Americas than in other regions (SMD=-0.73, 95% CI -0.99 to -0.47; P<.001); (6) telecare significantly reduced anxiety symptoms in patients with LLD (SMD=-0.53, 95% CI -0.73 to -0.33; P=.02); and (7) there was no significant improvement in the psychological components of QoL in patients with LLD compared to those receiving UC (SMD=0.30, 95% CI 0.18-0.43; P=.80). CONCLUSIONS: Telecare is a promising modality of care for treatment, which can alleviate depression and anxiety symptoms in patients with LLD. Continued in-depth research into the effectiveness of telecare in treating depression could better identify where older patients would benefit from this intervention.

Chitinases as a potential diagnostic and prognostic biomarker for amyotrophic lateral sclerosis: a systematic review and meta-analysis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the degeneration of motor neurons, and there is currently a lack of reliable diagnostic biomarkers. This meta-analysis aimed to evaluate CHIT1, CHI3L1, and CHI3L2 levels in the cerebrospinal fluid (CSF) or blood and their diagnostic potential in ALS patients. A systematic, comprehensive search was performed of peer-reviewed English-language articles published before April 1, 2023, in PubMed, Scopus, Embase, Cochrane Library, and Web of Science. After a thorough screening, 13 primary articles were included, and their chitinases-related data were extracted for systematic review and meta-analysis. In ALS patients, the CSF CHIT1 levels were significantly elevated compared to controls with healthy control (HC) (SMD, 1.92; 95% CI, 0.78 - 3.06; P < 0.001). CHIT1 levels were elevated in the CSF of ALS patients compared to other neurodegenerative diseases (ONDS) control (SMD, 0.74; 95% CI, 0.22 - 1.27; P < 0.001) and exhibited an even more substantial increase when compared to ALS-mimicking diseases (AMDS) (SMD, 1.15; 95% CI, 0.35 - 1.94, P < 0.001). Similarly, the CSF CHI3L1 levels were significantly higher in ALS patients compared to HC (SMD, 3.16; 95% CI, 1.26 - 5.06, P < 0.001). CHI3L1 levels were elevated in the CSF of ALS patients compared to ONDS (SMD, 0.75; 95% CI, 0.32 - 1.19; P = 0.017) and exhibited a more pronounced increase when compared to AMDS (SMD, 1.92; 95% CI, 0.41 - 3.42; P < 0.001). The levels of CSF chitinases in the ALS patients showed a significant increase, supporting the role of CSF chitinases as diagnostic biomarkers for ALS.

EA-Net: Research on skin lesion segmentation method based on U-Net.

Accurate segmentation of skin lesions is a challenging task because the task is highly influenced by factors such as location, shape and scale. In recent years, Convolutional Neural Networks (CNNs) have achieved advanced performance in automated medical image segmentation. However, existing CNNs have problems such as inability to highlight relevant features and preserve local features, which limit their application in clinical decision-making. This paper proposes a CNN with an added attention mechanism (EA-Net) for more accurate medical image segmentation.EA-Net is based on the U-Net network model framework. Specifically, we added a pixel-level attention module (PA) to the encoder section to preserve the local features of the image during downsampling, making the feature maps input to the decoder more relevant to the ground-truth. At the same time, we added a spatial multi-scale attention module (SA) after the decoding process to increase the spatial weight of the feature maps that are more relevant to the ground-truth, thereby reducing the gap between the output results and the ground-truth. We conducted extensive segmentation experiments on skin lesion images from the ISIC 2017 and ISIC 2018 datasets. The results demonstrate that, when compared to U-Net, our proposed EA-Net achieves an average Dice score improvement of 1.94% and 5.38% for skin lesion tissue segmentation on the ISIC 2017 and ISIC 2018 datasets, respectively. The IoU also increases by 2.69% and 8.31%, and the ASSD decreases by 0.3783 pix and 0.5432 pix, indicating superior segmentation performance. EA-Net can achieve better segmentation results when the original image of skin lesions has an obscure boundary and the segmentation area contains interference factors, which proves that the addition of attention mechanism in the encoder and the application of comprehensive attention mechanism can improve the performance of neural network in the field of skin lesions image segmentation.

Brain tumor feature extraction and edge enhancement algorithm based on U-Net network.

Background: Statistics show that each year more than 100,000 patients pass away from brain tumors. Due to the diverse morphology, hazy boundaries, or unbalanced categories of medical data lesions, segmentation prediction of brain tumors has significant challenges. Purpose: In this thesis, we highlight EAV-UNet, a system designed to accurately detect lesion regions. Optimizing feature extraction, utilizing automatic segmentation techniques to detect anomalous regions, and strengthening the structure. We prioritize the segmentation problem of lesion regions, especially in cases where the margins of the tumor are more hazy. Methods: The VGG-19 network structure is incorporated into the coding stage of the U-Net, resulting in a deeper network structure, and an attention mechanism module is introduced to augment the feature information. Additionally, an edge detection module is added to the encoder to extract edge information in the image, which is then passed to the decoder to aid in reconstructing the original image. Our method uses the VGG-19 in place of the U-Net encoder. To strengthen feature details, we integrate a CBAM (Channel and Spatial Attention Mechanism) module into the decoder to enhance it. To extract vital edge details from the data, we incorporate an edge recognition section into the encoder. Results: All evaluation metrics show major improvements with our recommended EAV-UNet technique, which is based on a thorough analysis of experimental data. Specifically, for low contrast and blurry lesion edge images, the EAV-Unet method consistently produces forecasts that are very similar to the initial images. This technique reduced the Hausdorff distance to 1.82, achieved an F1 score of 96.1%, and attained a precision of 93.2% on Dataset 1. It obtained an F1 score of 76.8%, a Precision of 85.3%, and a Hausdorff distance reduction to 1.31 on Dataset 2. Dataset 3 displayed a Hausdorff distance cut in 2.30, an F1 score of 86.9%, and Precision of 95.3%. Conclusions: We conducted extensive segmentation experiments using various datasets related to brain tumors. We refined the network architecture by employing smaller convolutional kernels in our strategy. To further improve segmentation accuracy, we integrated attention modules and an edge enhancement module to reinforce edge information and boost attention scores.

Association between pro-inflammatory diet and liver cancer risk: a systematic review and meta-analysis.

OBJECTIVE: This systematic review aimed to investigate the association between dietary inflammatory potential and liver cancer to provide evidence regarding scientific dietary health education. DESIGN: Systematic review and meta-analysis. SETTING: A comprehensive literature review was conducted to identify case-control or cohort studies that involved dietary inflammation index (DII)/empirical dietary inflammation pattern (EDIP) and liver cancer in PubMed, EMBASE, Cochrane, and Web of Science databases. Using a combination of DII/EDIP and liver cancer as the search terms, the associations between DII/EDIP and liver cancer were then assessed. PARTICIPANTS: Three case-control studies and two cohort studies were brought into the meta-analysis, with 225 713 enrolled participants. RESULTS: Meta-analysis of categorical variables showed that DII/EDIP in the highest category increased the risk of liver cancer compared to DII/EDIP in the lowest category (relative risk (RR) = 2·35; 95 % CI 1·77, 3·13; P = 0·000) and with low heterogeneity across studies (I2 = 40·8 %, P = 0·119). Meta-analysis of continuous variables showed that significant positive association between liver cancer and DII/EDIP scores (RR = 1·24; 95 % CI 1·09, 1·40; P = 0·001), and no heterogeneity (I² = 0·0 %, P = 0·471). Stratified according to the study design, there was a significant positive association between liver cancer and DII/EDIP scores in both cohort studies (RR = 2·16; 95 % CI 1·51, 3·07; P = 0·000) and case-control studies (RR = 2·75; 95 % CI 1·71, 4·41; P = 0·000). CONCLUSION: The higher the DII/EDIP score, the higher the risk of liver cancer. This finding may have prominent implications for the general population.

Effectiveness of continuity of care in reducing depression symptoms in elderly: A systematic review and meta-analysis.

BACKGROUND: Depression affects 10%-20% of older adults worldwide. The course of late-life depression (LLD) is often chronic, with a poor long-term prognosis. Lower treatment adherence, stigma, and suicide risk lead to significant challenges in the continuity of care (COC) for patients with LLD. Elderly patients with chronic diseases can benefit from COC. As a common chronic disease of the elderly, whether depression can also benefit from COC has not been systematically reviewed. METHODS: Systematic literature search in Embase, Cochrane Library, Web of Science, Ovid, PubMed and Medline. Randomized Controlled Trials (RCTs) on the intervention effects of COC and LLD, published on 12 April 2022, were selected. Two independent researchers made research choices based on consensus. An RCT with COC as an intervention measure for the elderly with depression 60 years old was the inclusion criteria. RESULTS: A total of 10 RCTs involving 1557 participants were identified in this study. The findings showed that: (1) COC significantly reduced depressive symptoms compared to usual care (standardized mean difference [SMD] = -0.47, 95% confidence interval: -0.63 to -0.31), with the best improvement at 3- to 6-month follow-up; (2) The reduction in depressive symptoms was more pronounced for patients with comorbid chronic conditions with LLD (SMD = -0.93, 95% CI: -1.18 to -0.68); (3) COC was more effective than other regions for LLD in Europe and the Americas (SMD = -0.84, 95% CI: -1.07 to -0.61); and (4) COC had a positive impact on the quality of life of patients with LLD (SMD = 0.21, 95% CI: 0.02-0.40). LIMITATIONS: The included studies included several multi-component interventions with widely varying methods. Therefore, it was almost impossible to analyze which of these interventions had an impact on the assessed outcomes. CONCLUSIONS: This meta-analysis shows that COC can significantly reduce depressive symptoms and improve quality of life in patients with LLD. However, when treating and caring for patients with LLD, health care providers should also pay attention to timely adjustments of intervention plans according to follow-up, synergistic interventions for multiple co-morbidities, and actively learning from advanced COC programs at home and abroad to improve the quality and effectiveness of services.

Social media usage of chinese nursing students: Attitudes, motivations, mental health problems, and self-disclosure.

BACKGROUND: Excessive self-disclosure online may risk the reputations, mental health problems, and professional lives of nursing students. This study investigated nursing students' usage of social media, their attitudes towards social media, mental health problems and self-disclosures, and the relationships of these variables. METHODS: A cross-sectional study was conducted online (n = 1054) with questionnaires of Fear of Missing Out (FoMO), Social Media Fatigue (SMF), Students' Uses and Views of Social Media (SUVSM) and self-disclosure in social media which included self-information shown on social media and information viewed by others. RESULTS: Although most of them held positive attitudes towards social media, 17.4% of the participants acknowledged that they had posted inappropriate contents online and 37.6% witnessed improper posts from schoolmates or teachers online. SMF was affected by familiar with relevant regulations on the social media usage (ß = -.10, p < .001), FoMO (ß = .41, p < .001), and SUVSM (ß = .17, p < .001). Additionally, nearly 1/3 participants reported their net-friends could view following information: gender, age, occupation, education level and location. Self- disclosure in social media was positively influenced by education (ß = .10, p < .001), sharing moments or Weibo, etc. (ß = .009, P = 0.009), time spent on social media daily (ß = .11, p < .001), accepting stranger's "friend request" (ß = .06, P = 0.047), FoMO (ß = .14, p < .001) and SMF (ß = .19, p < .001). Furthermore, effect of SUVSM on self-disclosure in social media was mediated by FoMO and SMF. CONCLUSION: Inappropriate contents are posted and witnessed by appreciable proportions of nursing students. Positive attitude towards social media may strengthen FoMO and SMF, which may increase self-disclosure in social media in turn.

Fluorescent Protein Variants Generated by Reassembly between Skeleton and Chromophore.

Fluorescent proteins (FPs) can be used as intrinsic molecular tags to track the dynamic activity in live cells. To obtain variants in an available and massive manner is always a challenge. Here, we adopted a computer-based microarray synthesis method to realize the reassembly between the chromophore and the skeleton. DNAWorks was used to segment the input FP templates into a set of overlapping oligonucleotides (20-43 mer) with a balanced annealing temperature, G + C content, and codon frequency. The constitution of the chromophore was kept in the same section by switching the divided sites during segmentation and the codon was optimized to further keep the balanced parameters. The designed oligonucleotides were synthesized on photo-programmable microfluidic arrays. Sequence analysis and the subsequent conditional induced expression of FPs revealed that oligonucleotides were highly reassembled. Spectra, photostability, and molecular size detection of randomly selected variants showed that they were distinct monomeric proteins that preserved photoactivity. Our study provides an effective means of obtaining FP variants based on a computer-designed parallel synthesis.

Characterization of novel neutralizing mouse monoclonal antibody JM1-24-3 developed against MUC18 in metastatic melanoma.

BACKGROUND: MUC18 is a glycoprotein highly expressed on the surface of melanoma and other cancers which promotes tumor progression and metastasis. However, its mechanism of action and suitability as a therapeutic target are unknown. METHODS: A monoclonal antibody (mAb) (JM1-24-3) was generated from metastatic melanoma tumor live cell immunization, and high-throughput screening identified MUC18 as the target. RESULTS: Analysis of molecular interactions between MUC18 and JM1-24-3 revealed that the downstream signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. JM1-24-3 inhibited melanoma cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo. CONCLUSION: These results confirm that MUC18 is mechanistically important in melanoma growth and metastasis, suggest that the MUC18 epitope identified is a promising therapeutic target, and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent.

Structure-guided evolution of Green2 toward photostability and quantum yield enhancement by F145Y substitution.

Quantum yield is a determinant for fluorescent protein (FP) applications and enhancing FP brightness through gene engineering is still a challenge. Green2, our de novo FP synthesized by microfluidic picoarray and cloning, has a significantly lower quantum yield than enhanced green FP, though they have high homology and share the same chromophore. To increase its quantum yield, we introduced an F145Y substitution into Green2 based on rational structural analysis. Y145 significantly increased the quantum yield (0.22 vs. 0.18) and improved the photostability (t1/2 , 73.0 s vs. 46.0 s), but did not affect the excitation and emission spectra. Further structural analysis showed that the F145Y substitution resulted in a significant electrical field change in the immediate environment of the chromophore. The perturbation of electrostatic charge around the chromophore lead to energy barrier changes between the ground and excited states, which resulted in the enhancement of quantum yield and photostability. Our results illustrate a typical example of engineering an FP based solely on fluorescence efficiency optimization and provide novel insights into the rational evolution of FPs.

Efficient and Low-Cost Error Removal in DNA Synthesis by a High-Durability MutS.

Enzyme-based error correction is a key step in de novo DNA synthesis, yet the inherent instability of error-correction enzymes such as MutS has hindered the throughput and efficiency of DNA synthesis workflows. Here we introduce a process called Improved MICC (iMICC), in which all error-correction steps of oligos and fragments within a complete gene-synthesis cycle are completed in a simple, efficient, and low-cost manner via a MutS protein engineered for high durability. By establishing a disulfide bond of L157C-G233C, full-activity shelf life of E. coli MutS (eMutS) was prolonged from 7 to 49 days and was further extended to 63 days via cellulose-bound 4 °C storage. In synthesis of 10 Cas9 homologues in-solution and 10 xylose reductase (XR) homologues on-chip, iMICC reduced error frequency to 0.64/Kb and 0.41/Kb, respectively, with 72.1% and 86.4% of assembled fragments being error-free. By elevating base accuracy by 37.6-fold while avoiding repetitive preparation of fresh enzymes, iMICC is more efficient and robust than the wild-type eMutS, and it is 6.6-fold more accurate and 26.7-fold cheaper than CorrectASE. These advantages promise its broad applications in industrial DNA synthesis.

High-fidelity de novo synthesis of pathways using microchip-synthesized oligonucleotides and general molecular biology equipment.

Engineering and evaluation of synthetic routes for generating valuable compounds require accurate and cost-effective de novo synthesis of genetic pathways. Here, we present an economical and streamlined de novo DNA synthesis approach for engineering a synthetic pathway with microchip-synthesized oligonucleotides (oligo). The process integrates entire oligo pool amplification, error-removal, and assembly of long DNA molecules. We utilized this method to construct a functional lycopene biosynthetic pathway (11.9 kb encoding 10 genes) in Escherichia coli using a highly error-prone microchip-synthesized oligo pool (479 oligos) without pre-purification, and the error-frequency was reduced from 14.25/kb to 0.53/kb. This low-equipment-dependent and cost-effective method can be widely applied for rapid synthesis of biosynthetic pathways in general molecular biology laboratories.

Identification of hexose kinase genes in Kluyveromyces marxianus and thermo-tolerant one step producing glucose-free fructose strain construction.

In yeast, the hexose assimilation is started at hexose phosphorylation. However, in Kluyveromyces marxianus, the hexokinase (HXK) and glucokinase (GLK) genes were not identified by experiments. Meanwhile, the glucose-free fructose product requires more cost-efficient method. In this study, the KmHXK1 and KmGLK1 genes were functionally identified through gene disruption, over-expression and recombinant enzymes characterization. Both glucose and fructose assimilation ability decreased significantly in KmHXK1 disrupted strain YLM001, however, this ability was not changed obviously in KmGLK1 disrupted strain YLM002. When over-expressing KmGLK1 in YLM001, only the glucose assimilation ability was recovered in obtained strain (YLM005). The kinetic constant analysis of recombinant enzymes also proved that KmHXK1 could phosphorylate glucose (Vmax 553.01 U/mg, Km 0.83 mM) and fructose (Vmax 609.82 U/mg, Km 0.52 mM), and KmGLK1 only phosphorylate glucose with a Vmax of 0.73 U/mg and a Km 4.09 mM. A thermo-tolerant strain YGR003 which produced glucose-free fructose from Jerusalem artichoke tuber in one step was constructed based on the obtained information. The highest production and fastest productivity were 234.44 g/L and 10.26 g/L/h, respectively, which were several folds of the results in previous reports.

Immobilized MutS-Mediated Error Removal of Microchip-Synthesized DNA.

Applications of microchip-synthesized oligonucleotides for de novo gene synthesis are limited primarily by their high error rates. The mismatch binding protein MutS, which can specifically recognize and bind to mismatches, is one of the cheapest tools for error correction of synthetic DNA. Here, we describe a protocol for removing errors in microchip-synthesized oligonucleotides and for the assembly of DNA segments using these oligonucleotides. This protocol can also be used in traditional de novo gene DNA synthesis.

Simultaneous fermentation of glucose and xylose at elevated temperatures co-produces ethanol and xylitol through overexpression of a xylose-specific transporter in engineered Kluyveromyces marxianus.

Engineered Kluyveromyces marxianus strains were constructed through over-expression of various transporters for simultaneous co-fermentation of glucose and xylose. The glucose was converted into ethanol, whereas xylose was converted into xylitol which has higher value than ethanol. Over-expressing xylose-specific transporter ScGAL2-N376F mutant enabled yeast to co-ferment glucose and xylose and the co-fermentation ability was obviously improved through increasing ScGAL2-N376F expression. The production of glycerol was blocked and acetate production was reduced by disrupting gene KmGPD1. The obtained K. marxianus YZJ119 utilized 120g/L glucose and 60g/L xylose simultaneously and produced 50.10g/L ethanol and 55.88g/L xylitol at 42°C. The yield of xylitol from consumed xylose was over 98% (0.99g/g). Through simultaneous saccharification and co-fermentation at 42°C, YZJ119 produced a maximal concentration of 44.58g/L ethanol and 32.03g/L xylitol or 29.82g/L ethanol and 31.72g/L xylitol, respectively, from detoxified or non-detoxified diluted acid pretreated corncob.